Molecular characterization of the HIV-1 Vpu protein and its role in antagonizing the cellular restriction factor BST-2/tetherin both in vitro and in vivo

PDF

Download

Open document

Flip pages

Download a page range

Download transcript

Copy asset link

Request this asset

Request accessible transcript

Transcript (if available)

Content

MOLECULAR CHARACTERIZATION OF THE HIV-1 VPU PROTEIN AND
ITS ROLE IN ANTAGONIZING THE CELLULAR RESTRICTION FACTOR
BST-2/TETHERIN BOTH IN VITRO AND IN VIVO

by
Kevin G. Haworth

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
i

Table of Contents

Acknowledgments......................................................................................................... iii

Abstract .......................................................................................................................... v

List of Tables ................................................................................................................ vii

List of Figures ............................................................................................................. viii

Abbreviations ................................................................................................................ x

Chapter 1: Introduction ................................................................................................ 1
1.1 Overview of HIV .................................................................................................. 1
1.1.1 General HIV virology and pathogenesis ................................................ 1
1.1.2 HIV origin and classification .................................................................. 3
1.1.3 HIV genome and proteins ...................................................................... 5
1.1.4 Virion structure .................................................................................... 12
1.1.5 HIV replication and lifecycle ................................................................ 14
1.1.6 Current therapeutic treatment of HIV................................................... 18
1.1.7 Perspectives on future treatment and cures ........................................ 21

1.2 Tetherin and other cellular restriction factors .................................................... 23
1.2.1 Overview of restriction factors ............................................................. 23
1.2.2 Identification of restriction actors ......................................................... 25
1.2.3 Tetherin: Restriction of viral budding ................................................... 25
1.2.4 APOBEC3G: Viral hypermutation ........................................................ 29
1.2.5 Trim5 : Preventing viral uncoating ....................................................... 30
1.2.6 SAMHD1: Depletion of nucleotide pool ............................................... 30

1.3 Humanized mouse models of infection ............................................................. 32
1.3.1 Rodents as models of infection ........................................................... 32
1.3.2 Creation of humanized mice ................................................................ 33
1.3.3 HIV infection of humanized mice ......................................................... 34

Chapter 2: Development of a High Throughput Screen for Novel Inhibitors of
Tetherin Counteraction by HIV-1 Vpu ........................................................................ 37
2.1 Abstract ............................................................................................................. 37
2.2 Introduction ....................................................................................................... 38
2.3 Materials and Methods ...................................................................................... 41
2.4 Results .............................................................................................................. 45
2.3 Discussion......................................................................................................... 63

Chapter 3: Characterizing the Functional Role of Tetherin Restriction During an
HIV-1 Infection in vivo ................................................................................................ 68
3.1 Abstract ............................................................................................................. 68
ii

3.2 Introduction ....................................................................................................... 69
3.3 Materials and Methods ...................................................................................... 72
3.4 Results .............................................................................................................. 79
3.3 Discussion......................................................................................................... 95

Chapter 4: Concluding Remarks and Future Perspectives ................................. 105
4.1 Concluding Remarks ....................................................................................... 105
4.2 Future Perspectives ........................................................................................ 107
4.2.1 Pharmaceutical targeting of accessory proteins including Vpu ......... 107
4.2.2 HIVs manipulating tetherin expression for interferon modulation ...... 109
4.2.3 Additional anti-viral properties of tetherin through signaling
cascades ................................................................................................ 111
4.2.4 Tetherin as a candidate for gene therapy .......................................... 113

Appendix .................................................................................................................... 116
Appendix A: Inhibition of viral budding by the small molecule AME ..................... 116
Appendix B: Tetherin expression is temporally increased on infected cells in
culture ................................................................................................... 119

References ................................................................................................................. 122

iii

Acknowledgments

First and foremost, I need to thank my graduate mentor Dr. Paula Cannon.
Without her support and mentorship this thesis would not have been possible. She has
been an excellent thesis director, not only providing me with funding to carry out these
projects, but also the guidance and feedback which made me a better scientist. I also
need to thank the additional members of the Cannon lab, especially Dr. Lopez, Dr.
Yang, and Dr. Exline, who have provided their support both through teaching, advising,
and even entertaining me throughout my graduate studies. They have provided
immeasurable support both professionally in the laboratory, and personally in life.
I would also like to thank my undergraduate mentor Dr. Olve Peersen at
Colorado State University for encouraging my further education in pursuing a doctoral
degree. Thanks to the time spent working in his research lab surrounded by graduate
students and post-docs, I knew that was the path I wanted to pursue upon completion of
my Bachelors of Science degree. It was in his lab that I started on my scientific journey
and first learned how to become a researcher.
Next I would like to thank my parents, Bob and Kathy Haworth, who instilled in
me the love of learning and the necessity of education, which are among the best
lessons a parent can teach. Thanks to their love and support, I had the opportunity to
pursue my higher education. I also need to think all other friends and family throughout
my entire educational journey for all the encouragement and help they provided. These
formative interactions, discussion, and ideas have shaped who I have become and
focused the path I have taken.
iv

I would also like to thank the support staff from both the Programs in Biomedical
and Biological Sciences and the Department of Molecular Microbiology and Immunology
in the Keck School of Medicine for their assistance in administrative matters and the
coordination of graduate studies.
Finally, none of this would have been possible without the constant love and
support from my wife, Sarah. Throughout my five years as a graduate student and
through all the associated high and low moments, she has been my anchor. No matter
the challenges, failures, or successes I faced, they were bearable or more enjoyable
thanks to her. She has provided endless love, encouragement, and support, and I can’t
imagine my life without her. Thank you.

v

Abstract

The cellular restriction factor BST-2/tetherin exerts a late stage anti-viral activity against
enveloped viruses, retaining newly formed virions at the cell surface and effectively
lowering virus output from infected cells. It is also a key player in the regulation of
interferon production through binding to ILT7 on plasmacytoid dendritic cells, which also
impacts the virus-host interaction. A number of pathogenic viruses have been found to
express counter strategies to tetherin, with the human immunodeficiency virus 1 using
Vpu. This suggests that a strong selective pressure exists for HIV to block tetherin
expression.

This potent antagonism of tetherin by Vpu could pose as a novel target for development
of additional therapeutic compounds to combat viral infection. The accessory proteins
of HIV are not the main targets for current HAART therapy of infected individuals. New
compounds targeting these proteins could dramatically aid in the fight against HIV
infection, especially considering these proteins are responsible for mediating cellular
conditions, permitting efficient viral replication and dissemination.

Current systems to investigate tetherin countermeasures are limited since in vitro cell
cultures do not adequately recapitulate certain aspects of viral replication or innate
immune activation. Additionally, in vivo testing using SIV or SHIV derivatives in monkey
models of infection can be misrepresentative of HIV infections in humans. We are using
humanized mice as a small animal model to study HIV-1 infections in vivo. The mice are
created by engraftment of NOD/SCID/IL2Rγ
-/-
mice with human hematopoietic stem
vi

cells, resulting in the development of mature human CD4+ T cells that support infection
by HIV-1.

We have created a series of Vpu deficient viruses in the NL4-3 backbone, using either a
null mutant or a specific point mutation (A
18
H) that blocks tetherin antagonism without
affecting other functions of Vpu, such as CD4 degradation. Interestingly, neither of
these Vpu mutations had any effect on virus replication that was apparent in the Jurkat-
based JLTRG reporter cell line, but cause decreased replicative fitness in PBMC
cultures. By infecting humanized mice with these viruses which vary in their ability to
counteract tetherin, we were able to gain a better understanding on the role of tetherin
restriction during the course of an in vivo infection. Together, these studies were meant
to better characterize the interaction between tetherin and its viral antagonist Vpu by
both determining the significance of restriction during infection, and discovering novel
ways to disrupt this interaction through therapeutic intervention.

vii

List of Tables

Table 2.1 Overview of chemical libraries screened.................................................. 57

Table 2.2 Summary of compound hit rate from HTS screen .................................... 63

Table 3.1 Summary of Vpu’s requirement in different infection systems ................. 96

viii

List of Figures

Chapter 1

Figure 1.1 Phylogenetic analysis of HIV-1 and HIV-2 and related SIV strains ........... 4

Figure 1.2 Organization of the HIV-1 genome ........................................................... 6

Figure 1.3 Structure of a mature HIV-1 particle ....................................................... 13

Figure 1.4 Overview of the HIV replication lifecycle ................................................. 15

Figure 1.5 Illustration of current therapeutic targets during the HIV-1 lifecycle ........ 20

Figure 1.6 Restriction factors of HIV-1 and the viral proteins responsible for their
counteraction .......................................................................................... 24

Figure 1.7 Tetherin localizes to the cell surface and provides a physical barrier to
viral release ............................................................................................ 27

Figure 1.8 Vpu enhances virus release by counteraction of surface tetherin ........... 28

Figure 1.9 Human cells are present in immune compartments of NSG mice ........... 35

Figure 1.10 HIV-1 infection profile in humanized mice ................................................ 36

Chapter 2

Figure 2.1 Tetherin transmembrane domain sufficient for reporter construct ........... 47

Figure 2.2 Detection of tetherin reporter specifically on the cell surface .................. 48

Figure 2.3 Dual expression vectors ensure overlap in protein expression ............... 52

Figure 2.4 Optimization of 384-well plate screening conditions ............................... 54

Figure 2.5 Statistical validation of the screening assay ............................................ 56

Figure 2.6 Overview of cell plating for screening chemical libraries ......................... 58

Figure 2.7 Illustration of hit compound identification ................................................. 60

Figure 2.8 Individual object analysis indicates non-cellular fluorescence ................. 62

ix

Figure 2.9 Examples of wells which contained abnormalities which affected the
assay readout ......................................................................................... 67

Chapter 3
Figure 3.1 Mutant Vpus deficient in enhancing virus release .................................... 80
Figure 3.2 Anti-tetherin activity is dispensable in JLTRG cell line ............................ 82

Figure 3.3 Mutant viruses exhibit a Vpu dependent phenotype in PBMCs .............. 86

Figure 3.4 Vpu deficient virus’s exhibit lower viral load and delayed CD4 depletion in
humanized mice ....................................................................................... 88
Figure 3.5 All NL4.3
Vpu(A18H)
viruses isolated from mice mutated the histidine residue
at position 18 ........................................................................................... 92

Figure 3.6 Reversions of the 18
th
residue from histidine restore Vpu anti-tetherin
activity ..................................................................................................... 94

Figure 3.7 Full length sequencing results from the JLTRG infections .................... 102

Figure 3.8 Full length sequencing results from the PBMC infections ..................... 103

Figure 3.9 Full length sequencing results from the splenocyte co-culture
infections ............................................................................................... 104

Chapter 4

Figure 4.1 AME treatment induces non-specific decrease in viral budding ............ 118

Figure 4.2 Tetherin is temporally up-regulated on surface of infected cells ........... 121

x

List of Abbreviations

AIDS Acquired Immunodeficiency Syndrome
AME amphotericin B methyl ester
APOBEC3G apolipoprotein B mRNA-editing, enzyme-catalytic,
polypeptide like 3G (A3G)
ARRRP AIDS Research and Reference Reagent Program
AZT azidothymidine (Zidovudine)
BiFC bimolecular fluorescence complementation
BRET bioluminescence resonance energy transfer
BST-2 bone marrow stromal antigen 2
-TrCP beta-transducin repeat containing protein
CA Capsid protein
CCR5 C-C chemokine receptor type 5
CD4 cluster of differentiation 4
CD45 cluster of differentiation 45 (leukocyte common antigen)
CDD Collaborative Drug Discovery
CMV cytomegalovirus
CRISPER clustered regularly interspaced short palindormic repeats
CTD c-terminal domain
CXCR4 C-X-C chemokine receptor type 4
CypA cyclophilin A
DAPI 4’6-diamidino-2-phenylindolel
DCAF DDB1 and CUL4 associated factors
DDB1 DNA damage-binding protein 1
DMEM Dulbecco’s modified eagle medium
xi

DNA deoxyribonucleic acid
dNTP deoxyribonucleotide triphosphate
EIAV Equine Infectious Anemia Virus
ELISA enzyme-linked immunosorbent assay
Env Envelope protein
ER endoplasmic reticulum
ESCRT endosomal sorting complexes required for transport
FBS fetal bovine serum
FRET Förster resonance energy transfer
Gag group-specific antigen
GFP green fluorescent protein
GPI glycophosphatidylinositol
HAART highly active anti-retroviral therapy
HIV Human Immunodeficiency Virus
HSC hematopoietic stem cell
HTS high throughput screen
huCD45 human CD45
IFN  interferon alpha
IL2 interleukin 2
ILT7 immunoglobulin-like transcript 7
IN Integrase protein
IRES internal ribosome entry site
IRF-1 interferon regulatory transcription factor 1
IRF-3 interferon regulatory transcription factor 3
JLTRG Jukat-LTR-GFP
xii

kb kilobase
kD kilodalton
LTR long terminal repeat
m7
G 5’ 7-methylguanosine
MA Matrix protein
MHC major histocompatibility complex
mRNA messenger RNA
MSSR Molecular Screening Shared Resources
mTG miniature-tetherin GFP
mTHT miniature-tetherin HaloTag
NC Nucleocapsid protein
Nef Negative factor
NF- B nuclear factor kappa B
NK natural killer cells
N-MLV N-tropic Murine Leukemia Virus
NNRTI non-nucleoside reverse transcriptase inhibitor
NOD nonobese diabetic
NRTI nucleoside reverse transcriptase inhibitor
NSG NOD/SCID/IL2r
null

PBMC peripheral blood mononuclear cells
PBS phosphate buffered saline
PCR polymerase chain reaction
PIC pre-integration complex
PIP2 phosphatidylinositol 4,5-bisphosphate
PolyA polyadenylation
xiii

PR Protease protein
PRR pattern recognition receptor
Rev Regulator of virion
RNA ribonucleic acid
RRE Rev response element
RT Reverse Transcriptase protein
RT-PCR reverse transcription followed by PCR
SAMHD1 sterile alpha motif and histidine/aspartic acid domain-
containing protein 1
SCID severe combined immunodeficiency
SHIV combination between SIV and HIV components
siRNA small interfering RNA
SIV Simian Immunodeficiency Virus
Skp1 S-phase kinase-associated protein 1
SP-1 specificity protein 1
TAR Trans-activation response element
Tat Trans-activator of transcription
TGN trans-Golgi network
Trim5a tripartite motif-containing protein 5 alpha
Vif Viral infectivity factor
VLP virus like particle
Vpr Viral protein R
Vpu Viral protein U
Vpx Viral protein X
vRNA viral RNA
xiv

WGA wheat germ agglutinin
Z’ Z prime score
ZFN zinc finger nuclease

1

Chapter 1: Introduction

1.1 Overview of the Human Immunodeficiency Virus

1.1.1 General HIV virology and pathogenesis
The Human Immunodeficiency Virus (HIV) was first isolated from patients in 1983 (1, 2),
and has since been identified as the infectious agent that causes Acquired
Immunodeficiency Syndrome (AIDS). Patients suffering from AIDS exhibit a
progressive failure of their immune system (3, 4), leaving them vulnerable to additional
opportunistic pathogens usually resulting in death. HIV accomplishes this through
infection of several cell types present in the immune system, including CD4+
lymphocytes and macrophages (5-7), which are responsible for identification and
elimination of invading foreign pathogens in healthy individuals.

As of 2011, more than 34 million people are living with AIDS worldwide (Center for
Disease Control [CDC], 2011), with approximately 69% of cases occurring in sub-
Saharan Africa, where nearly 1 out of every 20 adults is HIV positive (CDC). In 2011
alone, more than 1.7 million people died from AIDS relate illnesses and over 2.5 million
new cases were diagnosed, the equivalent of 300 people every hour (UNAIDS 2011
report). Since the initial transmission of the virus into the human population, more than
60 million people have become infected with HIV resulting in at least 30 million deaths
worldwide (CDC).

2

HIV is predominantly transmitted through bodily fluids with the most prevalent routes
being unprotected sex and intravenous drug use (CDC). Additional routes of infection
include mother to child during childbirth, and blood transfusion from infected individuals,
although this has been dramatically reduced through donor blood screening (CDC).
Upon initial infection with HIV, some individuals can experience flu like symptoms (8, 9)
as the virus rapidly reaches high titer levels, while others may be asymptomatic during
this period. After peak of viral replication, the immune system suppresses the initial
infection, and the levels of virus circulating in the blood may fall below detectable levels.
During this time however, the virus is not cleared from the body, and instead continues
low level replication. This asymptomatic period of infection can last anywhere from one
to 15 years without treatment (10-12), however the virus can still be readily transmitted
during this time, resulting in many new infections from unaware individuals.

Despite this prolonged asymptomatic period, the virus eventually overcomes
suppression by the immune system, resulting in a second peak of viremia, and
corresponding decline in immune cell number and function. In the late states of the
disease, often decades after the initial infection event, the individual’s immune system
becomes compromised, leaving the patient vulnerable to infection by opportunistic
pathogens the body would normally be able to combat, ultimately leading to death. It is
for these reasons that HIV/AIDS has become one of the most serious and expensive
diseases currently plaguing the human population today.

3

1.1.2 HIV origin and classification
HIV is a lentivirus and a member of the Retroviridae family of viruses. Retroviruses are
classified by their unique way of transmitting their viral genome as single stranded
positive sense RNA molecules. Upon cellular entry, the RNA is reverse transcribed into
a double stranded DNA molecule by viral enzymes carried within the particle, and this
DNA is then integrated into the host genome. Retroviruses are unique for this
conversion of RNA into DNA during their infection cycle. Additionally, the members of
the genus Lentiviridae, which includes both HIV-1 and HIV-2, are capable of infecting
non-dividing cells. Lentiviruses achieve this ability through a unique set of accessory
and regulatory proteins within their genomes not contained in other simpler retroviruses.

Both HIV-1 and HIV-2 are thought to have originated in Western Africa from cross
species transmissions by simian immunodeficiency viruses (SIV) which primarily infect
non-human primates (13) (Figure 1.1). Normally, humans are resistant to SIV infection
with viral suppression by the immune system within weeks of infections. These
successful transmissions of SIV likely occurred from the hunting and subsequent
butchering of infected primates, allowing for numerous sequential infections of humans
with SIV. This prolonged exposure to the virus allowed for adaptation for efficient
replication within the new human hosts. HIV-1 most likely arose from several unique
jumps of the SIV strains that infected the chimpanzee subspecies Pan troglodytes
troglodytes (SIVcpz) (14-16) and the gorilla subspecies Gorilla gorilla gorilla (SIVgor)
(17). HIV-2 entered the human population from a cross species transmission from the
sooty mangabey Cercocebus atys atys (SIVsm) (18-21).
4

HIV-1 can further be broken down into several subgroups including M (major), O
(outlier), N (non-major, non-outlier), and P (putative). Each of these groups is thought
to be an independent transmission event of an SIV, and these groups are classified
based upon genome structure and protein expression. HIV-1 group M is the dominant
virus in the human population and the primary cause of the global HIV/AIDS epidemic.

Figure 1.1: Phylogenetic analysis of HIV-1 and HIV-2 and related SIV strains.
Human infections with HIV-1 occurred through multiple cross-species transmission of
the SIV strains infecting chimpanzees while HIV-2 arose from SIV strains infecting
sooty mangabey monkeys. SIV strains are shown in blue, HIV-1 in red, and HIV-2 in
green. Figure is adapted from Wertheim et al. (2009).

HIV-2 virus cluster
arising from SIV
strains infecting
sooty mangabey
HIV-1 virus
cluster arising
from SIV
strains
infecting
chimpanzees
5

The remaining groups are mainly confined to small regions or populations within Africa.
HIV-1 group P is a relatively recent discovery with on a handful of cases being
diagnoses in individuals in Cameroon (22). The remainder of this text will be focused
on HIV-1 group M.

1.1.3 HIV genome and proteins
The HIV-1 genome, which is approximately 9.7Kb, consists of 9 open reading frames,
and is flanked on either side by identical regions known as long terminal repeats (LTR)
(23) (Figure 1.2). These LTR regions are essential for the activities of reverse
transcription and protein expression (24). Each LTR is comprised of a U3, R, and U5
sequence element and this region serves as the viral enhancer and transcription factor
binding site for viral replication (23). Successful transcription is initiated in the 5’ LTR
promoter and proceeds through the entirety of the viral genome, and terminates at the
3’ LTR regions polyadenylation (polyA) signal. The viral genome is subsequently
capped with a 5’ 7-methylguanosine (
m7
G) residue and 3’ polyA tail (25).
The HIV genome is present in the viral particle as two copies of a single stranded
positive sense RNA molecule. Once a cell is successfully infected by HIV, the genome
exists as an integrated DNA copy within the host’s genome. Once integrated, the viral
genome is known as a provirus. The nine open reading frames of HIV-1 gives rise to 16
different proteins which can be classified into 4 major functional groups: structural,
enzymatic, regulatory, and accessory proteins.

6

Structural Proteins
The structural proteins are encoded by the gag gene and are initially translated as one
large polypeptide (Gag) which is then subsequently processed to its functional
components. These include three major components: the p24 capsid (CA), p17 matrix
(MA), and p7 nucleocapsid (NC), as well as the smaller proteins: p6, p2, and p1, which
is involved in virion assembly, stability, and proteolytic processing of the Gag
polyprotein (26, 27). The p24 CA protein comprises the central core of the viral particle
which contains the two viral RNA (vRNA) copies. The RNA is coated by the NC protein,
which provides stability and prevents genome degradation. The vRNA is directed to the
viral capsid through interactions with the NC protein which recognizes and binds the psi

Figure 1.2: Organization of the HIV-1 genome. Illustration of the 9 open reading
frames encoding all proteins for HIV-1. The three poly proteins are further broken
down into their individual domains as well as the unique regions within the LTR
sequence.

Pol
Vif
Nef
Gag
Vpr
Rev
Tat
Vpu
Env
Core genes
Regulatory genes
Accessory genes
U3 U5 R
MA CA NC p6
PR RT IN
gp120 gp41
Gag
Pol
Env
LTR LTR
7

( ) packaging signal present in each full length vRNA transcript (28-30). The MA
portion mediates the localization of Gag to lipid rafts present on the cell surface though
its interaction with the lipid component phosphatidylinositol-[4,5]-bisphosphate (PIP2)
(31, 32). Once at the cell surface, MA interacts with the envelope protein during viral
assembly and budding (33, 34).

While the Gag protein components comprise the internal structure of the virus, the
envelope protein (Env) provides the external viral components of the particle. The env
gene is also expressed as a large polypeptide (gp160) and subsequently processed into
two subunits, gp120 and gp41 (35). This cleavage occurs by the cellular furin protease
(36). After processing, the two subunits remain covalently bound and traffic to the cell
surface for incorporation into the viral particle. Interactions between the membrane
anchored gp41 with the MA protein provide an assembly platform for particle formation.
The gp120 component contains the receptor recognition domains of the virus, allowing
for interaction with the primary receptor CD4 and co-receptors CCR5/CXCR4. The gp41
subunit contains the fusion peptide sequence. In addition to decorating the exterior of
viral particles, the Env protein also functions in mediating degradation of CD4 molecules
within infected cells (37, 38). This prevents surface CD4 expression which could
interfere with the viral budding process or illicit an immune response.

Enzymatic Proteins
The functional processes of the virus are encoded in the pol gene of HIV, which
overlaps with the 3’ end of the viral gag gene. Pol expression is dependent on a -1
8

frameshift during translation of Gag, resulting in a fraction of Gag-Pol polyprotein
precursors (39). This Gag-Pol construct is then processed through proteolytic cleavage
from activity residing in the protease domain (40, 41). The viral Pol polyprotein contains
three different catalytic domains which are subsequently cleaved into unique proteins:
the viral protease (PR), reverse transcriptase (RT), and integrase (IN). After this
dissociation from Gag, PR proceeds to cleave the Gag precursor into its smaller,
functional subunits as previously described. Pol then continues its own maturation
through further cleavage to yield the RT and IN subunits. This process occurs after viral
formation and budding are completed. The RT enzyme performs the conversion of
vRNA into double stranded DNA through the process of reverse transcription which is
discussed later. Once completed, the proviral DNA is incorporated into the host
genome by IN though induction of a double stranded break (42).

Regulatory Proteins
HIV-1 expresses two proteins which are responsible for efficient regulation and
expression of the viral genome. The first of these is the trans-activator of transcription
(Tat) which promotes productive elongation of HIV-1 transcripts (43). This activity is
essential due to a complex secondary structure, referred to as the Tat activated region
(TAR), which is present in the RNA of HIV-1 directly downstream of the transcription
initiation location (44, 45). Expression of Tat causes recruitment of cellular transcription
factors to this site which promote efficient transcription of the viral genome, and will be
discussed in detail later in this text. These full length transcripts are then targets for the
cellular messenger RNA (mRNA) splicing machinery which gives rise to numerous
9

different transcript variants, and is essential for complete expression of all viral proteins
(46). In order to ensure nuclear export of all splice variant mRNAs as well as the
complete unspliced vRNA for viral incorporation, the regulator of virion protein (Rev) is
required (47). Rev recognizes and binds to the Rev-response element (RRE) within the
vRNA (48, 49), facilitating its export from the nucleus through interactions with cellular
factors, and allowing for the various RNA products to be translated.

Accessory Proteins
A distinguishing trait of members in the lentiviridae genus that is not true of other
retroviruses is the expression of their so-called accessory genes: Vif, Vpr, Vpu, and
Nef. While the major proteins comprised in the gag, pol, and env genes encompass the
core elements of HIV replication and assembly, these accessory genes function in a
variety of ways to manipulate the cellular environment for the virus’s advantage. A
common theme among HIV accessory proteins is their use of a range of ubiquitin ligase
complexes to link specific protein targets to the cellular degradation pathways. Through
their interactions with a variety of cell surface and regulatory proteins, HIVs accessory
proteins profoundly influence the viral lifecycle, permitting efficient persistence and
dissemination of the virus.

Early studies demonstrated that the viral infectivity factor (Vif) was required in certain
cells to ensure that progeny viruses produced were fully infectious (50, 51). In certain
cells types, dubbed ‘non-permissive’, Vif deleted viruses produced particles which were
1000 times less infectious than Vif containing viruses (50, 52-54). This loss of infectivity
10

was determined to occur during the reverse transcription step in the newly infected
cells. Further studies showed this reduction in RT activity was due to the incorporation
of a cellular protein into the virus particles. This protein, apoplipoprotein B mRNA
editing enzyme catalytic peptide 3G (APOBEC3G, A3G) was found to interact with the
viral RNA and the nucleocapsid of the assembling virus (55). During reverse
transcription of the vRNA in newly infected cells, A3G deaminates cytosine residues in
the synthesized DNA strands, converting them into uracil residues (56). This leads to a
high number of guanosine to adenosine mutations in the coding strand of HIV, resulting
in genome instability. The Vif protein mediates the degradation of A3G by interacting
with the cellular ubiqutination machinery and targeting A3G for proteosomal degradation
(55, 57, 58).

The viral protein R (Vpr) is actively incorporated into viral particles through interactions
with the Gag polyprotein, indicating that Vpr plays an important role during the initial
phases of infection (59, 60). Vpr facilitates the transport of the pre-integration complex
(PIC) of HIV to the nuclear envelope of the infected cell (61, 62). Through interactions
with nuclear pore and nuclear transport proteins, Vpr mediates the import of the PIC into
the nucleus for integration into the host chromosome. This activity contributes to
lentiviruses ability to infect non-dividing cells, while other retroviruses require a mitotic
event for nuclear entry. Vpr has also been shown to interact with several transcriptional
regulatory proteins to enhance transcription of both HIV and other cellular genes (63-
65). Additionally, Vpr leads to the subsequent arrest of infected cells in the G
2
phase of
mitosis though manipulation of the DNA damage response pathway (66). Vpr
11

accomplishes this by facilitating the degradation of a key regulatory component of this
pathway, DCAF1, via the cullin4A-DDB1 ubiquitin ligase complex (67-69).

The viral protein U (Vpu) is the smallest accessory protein, comprised of only 86 amino
acids, and contains an N-terminal transmembrane domain. Early research on HIV
indicated that Vpu played at least two important functions for viral replication. One of
these is the degradation of CD4 receptor molecules which are retained in the
endoplasmic reticulum (ER) though interactions with newly synthesized Env proteins
(70). This function is important to allow Env subunits to efficiently traffic to the cell
surface for incorporation into assembling virions. Vpu accomplishes this degradation
through interactions with the -TrCP/cullin1/Skp1 ubiquitin ligase complex, leading to
CD4 ubiquitination and subsequent degradation (71). Vpu was also found to
dramatically enhance virus release from infected cells (72, 73). In the absence of Vpu
expression, fully assembled HIV-1 particles were observed to be tethered to the surface
of cells, and this activity was exacerbated through interferon-  stimulation (74). It was
discovered that the protein responsible for this activity was the bone marrow stromal cell
antigen 2 (BST-2), now called tetherin (75, 76). The mechanism by which tetherin
achieves this restriction will be further described in the next section. Vpu is the viral
factor responsible for interacting with tetherin, retaining it in the trans-Golgi network
(TGN) (77, 78), and mediating its degradation through the -TrCP ubiquitin ligase
complex (79-82).

12

The Nef protein is one of the first proteins expressed by the virus upon HIV infection.
Nef contains an N-terminal myristoylation signal which localizes it to plasma
membranes where it acts to down-regulate several receptors from the cell surface (83).
The most prominent of these targets are the CD4 and MHC class I proteins (84, 85).
Nef accomplishes these activities by different methods, with CD4 being directly targeted
for clathrin-mediated endocytosis (85), while MHC class I molecules are retained in the
TGN (84). Through reduced surface expression of these two immune receptors, HIV
prevents superinfection of cells and evades immune surveillance and targeted cell
killing of infected cells. Several studies have shown the lack of requirement for Nef
during infections of immortalized T cell lines, however its expression is essential during
in vivo infections as demonstrated by Nef deletion studies in primate models of SIV
infection (86-88).

1.1.4 Virion structure
The HIV-1 virion is approximately 120nm in diameter (Figure 1.3). It is an enveloped
virus, meaning its outer layer is derived from the plasma membrane of the infected cell
the virion budded from. This outer membrane layer contains the Env protein subunits
which multimerize to form trimeric structures, and these Env ‘spikes’ are heavily
glycosylated (89) helping to mask the virus from immune detection and prevent efficient
antibody recognition. Interior to the outer membrane is the lattice of viral MA proteins
which interacts with the gp41 subunit of Env and forms an additional layer around the
viral core. Approximately 1500 p24 CA molecules comprise the conical core of the virus
particle containing the two vRNA copies (90). These RNA strands, approximately 9.7kb
13

each, are bound by the nucleocapsid protein, preventing their degradation by nucleases
and ensuring their incorporation into the virion. The viral enzymes are also bound to the
RNA genome, ensuring efficient reverse transcription upon infection. Also housed
within the viral particle are the accessory proteins Vpr, Vif and Nef.

Figure 1.3: Structure of a mature HIV-1 viral particle. Relative location of all the
components of the viral particle are shown after proteolytic cleavage of the Gag and
Pol polyproteins by the protease domain of Pol. The outer lipid layer contains the
trimeric envelope spikes and is derived from the host’s cellular lipid membrane.
Internal to the lipid membrane is the viral matrix protein stabilizing the viral envelope.
The capsid protein lattice forms around the two viral RNA genome copies which are
bound by the nucleocapsid portions of Gag. Enzymatic proteins are present in the
particle, some of which are bound to the viral RNA. Virally packaged accessory
proteins are also found throughout the particle. Figure is adapted from the
NIH:NIAID public domain.

Protease
Lipid Membrane
Nucleocapsid
Viral RNA Genome
Reverse
Transcriptase
Envelope
gp120
gp41
Capsid
Matrix
Vif, Vpr, Nef and p7
Integrase
14

1.1.5 HIV replication and life cycle

Binding and Entry
HIV-1 infects cells of the immune systems which express the cellular receptor CD4.
This includes both macrophages and several T-cell subsets. The infection is initiated by
the binding of the gp120 subunit of Env to a CD4 receptor on the cell surface (Figure
1.4). This binding to CD4 induces conformational changes in the viral envelope which
allows for the binding of an additional co-receptor molecule: CCR5 (91). HIV is capable
of altering this co-receptor usage at later times during infection of the host to bind the
CXCR4 molecule (92), which is primarily expressed on T-cells. This tropism switch is
brought about by modifications in Env surface residues. This specificity in co-receptor
usage provides an additional classification for HIV as either an R5 (CCR5 using) or X4
(CXCR4 using) virus. Once bound to the required receptors, the gp120 subunits
undergoes and additional conformation change which exposes the gp41 subunit of Env
(93), allowing the insertion of the fusion peptide into the cellular membrane. A final
conformation change brings the viral membrane in close enough proximity to the cellular
membrane of the target cell, resulting in membrane fusion and release of the viral
content into the cytoplasm.

Once fusion occurs, the viral reverse transcriptase begins the process of converting the
viral RNA into genomic DNA. The enzyme first uses the positive strand of RNA as a
template for negative strand DNA synthesis, temporarily creating an RNA-DNA
intermediate. This intermediate is then used for positive strand DNA synthesis, with the

15

Figure 1.4: Overview of the HIV-1 replication lifecycle. Overview of the major
steps in viral lifecycle are shown. Upon receptor/co-receptor binding, the viral
membrane fuses to the cellular membrane through multiple conformational changes
in the Env protein, releasing the conical core into the cytoplasm. Reverse
transcription of the viral genome is followed by nuclear import and integration into the
host chromosome where it can either be actively transcribed, or lay dormant for
extended periods of time. Upon induction of transcription, RNA transcripts are
exported from the nucleus giving rise to all the components for viral assembly and
budding. The outer lipid layer contains the trimeric envelope spikes and is derived
from the host’s cellular lipid membrane. Internal to the lipid membrane is the viral
matrix protein surrounding the viral core composed of the capsid protein and
encasing the viral RNA. Particles also contain the viral enzymatic and some
accessory proteins. After budding, proteolytic processing catalyzed by the viral
protease leads to viral maturation.

Receptor binding
Fusion
Capsid uncoating
Reverse
transcription
Nuclear
import
Integration
Transcription
Viral protein
translation
RNA/Gag
interaction
Assembly
Budding
Maturation
Infectious Particle
CD4
CCR5/CXCR4
Nucleus
ssRNA
dsDNA
16

RNA strand being degraded during the process, resulting in a double stranded DNA
product. This process of reverse transcription is not 100% efficient and is prone to
errors, giving rise to random mutation events (94). These mutations provide the virus
with a mechanism for rapid adaptation to altered conditions which could otherwise
inhibit viral replication, such as therapeutic inhibition. During the reverse transcription
process, the viral capsid in association with IN and Vpr is transported through the
cytoplasm to the nucleus of the cell. This collection of proteins and DNA which
comprises the PIC, is actively transported across the nuclear envelope though a Vpr
mediated mechanism (61, 62). Once within the nucleus, the viral genome is inserted
into the host chromosome by a double-strand break facilitated by the IN enzyme. This
inserted viral genome is called a provirus, and it provides the template for all viral
transcription.

HIV Transcription
The integrated provirus is transcribed through interactions of the LTR region with
several cellular transcription factors including NF B, IRF-1, and SP-1 (95, 96). These
transcription factors bind within the U3 region of the LTR and recruit the RNA
polymerase II (97), leading to viral transcription. As previously mentioned, these early
transcription events are hindered by the TAR secondary structure element present in
the transcribed RNA shortly after initiation. In the absence of Tat expression,
repression elements permit recruitment of negative elongation factors, which prevent
the necessary phosphorylation events in the C-terminal domain (CTD) of RNA
polymerase II, resulting in premature dissociation from the DNA (98). Upon sufficient
17

accumulation of Tat, it binds to the TAR element and recruits positive transcription
elongation factors, permitting efficient transcription of the full length HIV genome (99).
The entire HIV genome is initially transcribed as a single RNA transcript. This transcript
contains several splice donor and acceptor sites and is a substrate for the host cellular
splicing machinery (46). RNA splicing naturally occurs within eukaryotic cells, and is a
mechanism by which an RNA molecule is made to express several different isoforms of
a protein from one gene. This occurs through the specific removal of sequences from
the initial RNA transcript, and depending on the size and frequency of these events,
alternate protein expression occurs. HIV utilizes RNA splicing in varying degrees to
produce its array of viral proteins. An initial high frequency of splicing events limit the
number of viral proteins expressed from the mRNA primarily to Tat, Rev, and Nef.
However upon accumulation of sufficient Rev levels, alternatively spliced and unspliced
transcripts are able to be exported from the nucleus due to the presence of the RRE
element. These alternatively spliced transcripts give rise to the remaining accessory
proteins and Env expression (100). The unspliced transcripts code for both Gag and
Pol expression and comprise the vRNA for incorporation into budding virions.

With the accumulation of the viral components in the cytoplasm of infected cells, the
virus begins assembling new virions at the cell surface. Interactions of the Gag
polyprotein with the  packaging signal present in the vRNA recruits the viral genome to
sites of virus assembly (28-30). Additional Gag constructs begin to multimerize through
Gag-Gag and Gag-RNA interactions (101-103), and this Gag oligomeriation initiates the
budding process. In order to complete budding, the plasma membrane between the
18

protruding virion and the cell must be severed. To accomplish this, HIV recruits the
cellular ESCRT machinery (104), which aids in constricting and severing the neck of the
virion, releasing it from the cell membrane. Once budding is completed, the virus
undergoes maturation through the proteolytic processing of incorporated Gag and Gag-
Pol polyproteins (105). This processing is facilitated by the Pol protease domain and
cleaves the precursor proteins into their functional subunits leading to a fully infectious
virus particle.

1.1.6 Current therapeutic treatment of HIV
There are no known cures for HIV infection. Current treatment of infected individuals
requires strict adherence to a specific regiment of a drug cocktail which prevents
efficient viral replication. Due to the high cost of these drugs, their use is generally
limited to wealthier and developed countries. Additionally, these drugs and can also
have serious side effects (106). Viral rebound happens quickly upon cessation of
treatment, indicating that despite being able to repress viral replication below the limits
of detection, viral persistence continues in a pool of latently infected cells. This rebound
results from HIVs integration into the host genome, which precludes total elimination of
the virus from the host without the killing of all cells containing an integrated provirus.
Exactly what comprises this pool of infected cells that is capable of long term survival
despite anti-viral therapy is still the subject of intense investigation. This section will
provide a brief overview of HIV therapeutics starting at the discovery of the first inhibitor
to the current highly active anti-retroviral therapy (HAART).

19

As with the treatment of any viral infection, the ideal targets for therapeutic intervention
are either enzymatic reactions or activities that are specific to the viral lifecycle. This
helps to limit the cytotoxic effect the compounds have on normal cellular processes.
For HIV, there are several such targets for inhibitory compounds (Figure 1.5). Several
of these steps already are the target of anti-viral compounds, and the combination of
several potent inhibitors has lead to the possibility of HIV infected patients to expect a
relatively normal lifespan despite their chronic infection.

The very first therapeutic agent approved for the treatment of HIV was the nucleoside
analog inhibitor azidothymidine (AZT) (107). AZT was approved by the FDA for
treatment of HIV infected individual on March 20, 1987, just 4 years after the initial
discovery and characterization of the virus. The timeline between the discovery of AZTs
anti-viral effects to its approval by the FDA is the shortest of any therapeutic in recent
history, demonstrating the serious need of a treatment for this rapidly spreading
disease. Nucleoside reverse transcriptase inhibitors (NRTI) are nucleotide analogs
which act during reverse transcription. Their random incorporation during DNA strand
synthesis leads to premature termination of transcription and the failure of HIV to
complete genome conversion from RNA to DNA. Daily AZT doses were standard
procedures for infected patients and initial results of viral inhibition were promising.
However, upon extended treatment using AZT alone, the emergence of resistant
mutants proved problematic (108, 109). This adaptation by the virus to resist AZT
treatment underscored the need for a broad spectrum inhibition of the virus using a
combination therapy approach.
20

Figure 1.5: Illustration of current therapeutic targets during the HIV-1 life cycle.
(A) Current therapeutic drugs generally target one of three specific viral events or
processes. (1) Viral receptor binding and fusion with CCR5 blocking compounds. (2)
Reverse transcription and integration inhibitors. (3) Proteolytic processing of Gag and
Gag/Pol polyproteins into function subunits. Currently no drugs target the assembly
and budding phase of the virus (4). Figure adapted from the NCBI retrovirus
database: www.ncbi.nlm.gov/retroviruses. (B) Table outlining examples of drugs
targeting these specific activities.

1
2
3
4
Classes of Drugs Mechanism of Action Drug Examples
CCR5 antagonists Competitively bind to CCR5 on
human cells
Maraviroc
Enfuvirtide
Nucleoside reverse
transcription inhibitors
(NRTI)
Nucleotide analogs whose
incorporation during DNA strand
synthesis causes premature
termination
Tenofovir
Emtricitabine
Lamivudine
Abacavir
Non-nucleoside reverse
transcription inhibitors
(NNRTI)
Bind allosteric sites of the RT
enzyme and preventing activity.
Non-competitive inhibition
Nevirapine
Delavirdine
Efavirenz
Rilpivirine
Integrase inhibitors Bind to and inhibit proviral
integration by disruption of
catalytic metal ions required by
Integrase
Raltegravir
Elvitegravir
Maturation inhibitors Disrupts cleavage of Gag and
Gag/Pol polyproteins, preventing
viral maturation
Atazanavir
Darunavir
A
B
21

HIV lacks a proofreading function in its RT enzyme, which results in a relatively high
mutation rate for the virus (94). This results in countless minor variations between
viruses circulating within one patient, and increasing the odds that the virus will obtain
resistance to a single anti-viral drug through natural selection advantage. To
circumvent this problem of escape mutants, a combination cocktail of drugs is used in
all current treatment protocols for HIV infected individuals. These combination therapy
approaches, known as HAART, generally utilize three drugs: 2 NRTI drugs and either a
protease or integrase inhibitor. By providing multiple drugs which target several unique
functions within HIV enzymes, the emergence of resistant mutations can be dramatically
reduced. Despite the successes of combination therapy, it mostly remains available
only to patients in economically wealthy and developed countries, and the side effects
of these potent drugs can be severe (106). For these reasons, additional therapeutic
agents are needed which can help to minimize unwanted side effects, increase patient
compliance, and be economically viable for worldwide distribution.

1.1.7 Perspective on future treatments and cures
In 2009, a collection of scientists in Germany published a paper documenting the first
‘functional cure’ of an HIV infected individual (110). This patient, known as the Berlin
Patient, was undergoing a bone marrow transplant for leukemia treatment, and as a
marrow donor, the medical team chose an individual who was homozygous for a
mutation in the CCR5 gene. This mutation, known as CCR5- 32, is a naturally
occurring mutation predominantly within the European Caucasian population, and is
thought to have arisen during the 14
th
century in response to the Black Death (111).
22

Approximately 5-14% of individuals from Northern European descent contain at least
one copy of this gene mutation. Individuals carrying this mutation, which is a deletion of
32 nucleotides resulting in a prematurely truncated protein, exhibit no deleterious effects
in their T-cell development or function (112). However, the presence of a homozygous
CCR5- 32 genotype is associated with a strong resistance to HIV infection (113-115),
due to the requirement of CCR5 co-receptor binding by the virus.

The physicians performing the bone marrow transplant for the Berlin Patient were aware
of the CCR5- 32 mediated HIV resistance, and by choosing a donor homozygous for
this mutation, attempted to treat his infection in parallel with the leukemia. After
recovery from the transplant, all circulating CD4+ T-cells in the patient’s blood were
CCR5- 32, and after more than two years without anti-retroviral treatment, no
detectable levels of HIV are present his blood or organs. While this specific method of
treatment is unrealistic for all patients due to the high risk and cost of bone marrow
transplants, it raises the possibility of a genetic based cure for HIV as opposed to a
therapeutic based approach. Several research groups are now attempting to replicate
this finding by using gene therapy based approaches to functionally delete CCR5
expression in hematopoietic stem cells (HSC). If such a deletion could be obtained, it’s
conceivable for HIV infected individuals to receive a transplant of these modified cells to
help in controlling their viral infection.

23

1.2 Tetherin and Other HIV-1 Cellular Restriction Factors

1.2.1 Overview of restriction factors
Since multi-cellular organisms are constantly under the threat of viral infection, over the
course of evolution they have developed mechanisms to combat this threat. These anti-
viral activities often reside in proteins which have specifically evolved to inhibit unique
steps in the lifecycles of invading pathogens. These proteins which contain anti-viral
properties are known as restriction factors due to their role in restricting viral replication
and dissemination. In response to these restriction factors, viruses often evolve specific
counteractions to circumvent inhibition. This phenomenon of viral/host co-evolution can
be observed through phylogenetic analysis of different HIV/SIV strains and the specific
host species they infect. For example, a viral protein from an SIV may counteract a
rhesus macaque restriction factor but be inefficient at counteracting the human
homolog. Likewise, a human restriction factor which is capable of inhibiting HIV may
exhibit no anti-viral activity against an SIV from chimpanzees. Restriction factors are
often induced by the interferon-  pathway (INF ) which is a common cellular response
to infections. INF  induced genes are both key regulators in detecting and suppressing
infection and are consequently common targets for viral proteins. This section will
provide a brief introduction into the major human restriction factors which are known to
inhibit HIV replication (116) (Figure 1.6).

24

Figure 1.6: Restriction factors of HIV-1 and the viral proteins for their
counteraction. The general HIV lifecycle is depicted along with several restriction
factor known to inhibit retroviral infections. Restriction factors are shown in red while
viral proteins which counteract them are shown in green. HIV-1 encodes Vpu, HIV-2
encodes Vpx, and both encode Vif. HIV is resistant to human Trim5  due to specific
residue mutations present in the viral capsid protein. Figure is adapted from Yan and
Chen (2012).

Tetherin
Trim5 
APOBEC3G
SAMHD1
Vpu
Vif
Vpx
25

1.2.2 Identification of restriction factors
Since the functional roles of most human proteins are yet to be determined, the most
prevalent way of identifying a restriction factor is through the study of viral proteins
themselves. By manipulation of the viral genome and observing the functional
consequences on viral replication, it is possible to identify cellular interaction partners of
individual genes. For example, if a virus deleted for a specific gene exhibits a strong
phenotype in one cell type but not another, a broad analysis of the difference in
transcription and protein expression between the two cell types can identify candidates
involved in the difference. Through this method of reverse engineering, it is possible to
identify cellular factors which interact with and potentially alter the function or infection
capability of the virus.

1.2.3 Tetherin: Restriction of viral budding
Early studies on the Vpu protein indicated that it contributed to enhancing viral release
from infected cells (72, 73). When certain cell lines or primary human cells are infected
with a Vpu deficient virus, significantly lower viral yield was detected in the supernatant
when compared with wild type virus (72, 73). This was further demonstrated by electron
microscopy, where fully matured virus particles were tethered to the cell surface (75,
76). These particles were still infectious and could be released by protease treatment,
indicating the physical tether to be a cellular protein. In 2008, two independent groups
published that the bone marrow stromal cell antigen 2 (BST-2) was the cellular
restriction factor responsible for this activity (75, 76), and coined it ‘tetherin’.

26

Tetherin accomplishes this restriction of viral budding due to unusual structural
characteristics. Tetherin contains two membrane anchors within its sequence, an N-
terminal transmembrane region, and a C-terminal glycosylphosphatidylinisotol (GPI)
anchor, with an extracellular coiled-coil in between these two domains (117, 118)
(Figure 1.7). Both of these anchoring regions are essential for tetherin restriction with
the GPI anchor thought to localize the protein to lipid rafts (117). This localization is
important as lipid rafts are the primary location of HIV particle assembly and budding
(119). The direct physical mechanism by which tetherin accomplishes this restriction is
still unknown but likely occurs through the insertion of one membrane anchoring domain
into the outer envelope of the budding virion and one remaining in the cellular
membrane (75, 76, 120). This results in tetherin physically retaining particles on the cell
surface, where they can subsequently be endocytosed and degraded.

Since its identification, tetherin has been shown to restrict a wide range of enveloped
viruses, and consequently different viruses have evolved measured of counteracting
this restriction (121-124). For different lentiviruses, this anti-tetherin function resides in
various proteins (75, 76, 125-130). In the majority of SIVs, the Nef protein interacts with
a short amino acid sequence in the cytoplasmic tail of primate tetherin (127, 130),
allowing for surface down-regulation. This specificity also correlates between species.
For example, Nef from SIV
mac
(127) and SIV
cpz
(129) exhibits activity against rhesus
macaque and chimpanzee tetherin respectively, but not human tetherin which lacks the
required sequence. During the cross species transmission from non-human primates to
humans, anti-tetherin activity for HIV-1 group M viruses switched to the Vpu protein
27

(Figure 1.8). Vpu interacts with tetherin through the transmembrane region of both
proteins (125, 131-135), which results in tetherin degradation (79-82, 136).

Figure 1.7: Tetherin localizes to the cell surface and provides a physical barrier
to virus release. (A) Schematic overview of tetherin topology which includes an N-
terminal cytoplasmic domain, extracellular coiled-coil domain, and terminates in a C-
terminal GPI anchor. (B) Electron microscope image of Vpu viruses attempting to
bud from an infected cell (adapted from Van Damme, 2008). (C) Confocal images of
the two dominant cellular localizations of tetherin on the cell surface and in the trans-
Golgi network.

“Tethered” virions in Vpu deleted HIV-1
Tetherin Localization
Cell Surface Intracellular
Extracellular
Cytoplasm
A B
C
N-terminus
C-terminus
GPI anchor
coild-coil
28

Figure 1.8: Vpu enhances virus release by counteraction of surface tetherin.
(A) Schematic overview of tetherin restriction of HIV budding in the presence or
absence of Vpu. When Vpu is present, transmembrane interactions allow Vpu
mediated retention of tetherin in the trans-Golgi network and an increase in viral
budding. (B) Western blot analysis of HeLa cells either transfected with empty
vector, or proviral clone DNA encoding either the wild type NL4.3 or a clone deficient
in Vpu expression (NL4.3
Vpu
). Cell lysates and supernatants harvested and
analyzed for protein expression with indicated antibodies. (C) HeLa cells were
transfected with Vpu and stained for surface expression of tetherin prior to
permeabilization and intracellular Vpu staining. White arrow indicates loss of surface
tetherin due to Vpu antagonism.

Surface tetherin Vpu
Lysates
Virus
NL4.3
NL4.3
Vpu
-
p24
Vpu
p24
Extracellular
Cytoplasm
Vpu
TGN
tetherin
‘Tethered’ virion
- Vpu + Vpu
A B
C
29

HIV-2, which lacks a Vpu gene, has acquired anti-tetherin function in its Env protein,
which interacts with the ectodomain of tetherin and mediates its surface removal though
specific contact residues in each protein (78, 126).

The fact that such a broad range of viruses have independently acquired means of
counteracting tetherin implies that its role in preventing efficient viral release from cells
is an important cellular defense. The interaction between HIV-1 Vpu and human
tetherin will be the focus of the majority of this thesis.

1.2.4 APOBEC3G: Viral hypermutation
The role of APOBEC3G (A3G) in HIV restriction arose through studies characterizing
the viral protein Vif. Researchers observed that Vif expression was required for HIV
replication in a variety of cell types including primary CD4+ T-cells and various
immortalized cell lines, but it was dispensable in others (50, 52-54). When Vif deficient
viruses were produced in Vif-dependent cell types, the viral particles exhibited
dramatically reduced infectious capability when compared to virus produced in Vif-
independent cells, despite similar levels of viral yield (50, 51). This finding indicated the
presence of a restriction factor which acted upon the early stages of viral infection in the
target cell. Through cell fusion experiments and mRNA expression profiles, A3G was
identified as the cellular restriction factor causing this reduction in infectious potential
(55).

30

APOBEC3G is a cytosine deaminase and converts deoxycytosine residues into
deoxyuracil within DNA sequences (56). This activity results in the mutation from
guanine to adenosine in the complementary DNA strand. A3G exerts this activity during
viral reverse transcription, leading to hypermutation of the viral genome, resulting in the
disruption of coding regions and genome instability (137-139). In A3G expressing cells,
the protein is incorporated into the viral particle due to interactions with the vRNA and
NC proteins (140-142). HIV-1 prevents this incorporation into the virion by expressing
Vif, which interacts with A3G and links it to the cellular ubiquitination pathway leading to
its degradation (55, 57, 58).

1.2.5 Trim5 : Preventing viral uncoating
The existence of a broadly acting anti-viral factor against retroviruses was predicted due
to the restriction of a range of different retroviruses in mammalian cells (143). This
factor was eventually identified as Trim5  by a screen of rhesus macaque genes which
could inhibit HIV replication (144). The human homolog of Trim5  is unable to restrict
HIV despite exhibiting activity against a wide range of other retroviruses such as Murine
Leukemia Virus (N-MLV) and Equine Infectious Anemia Virus (EIAV) (145-148). The
opposite is also true with non-human primate Trim5  efficiently restricting HIV
replication, but exhibiting weak activity against SIV (144). In general, it appears that
Trim5  genes from individual host species are adapt at exerting broad restriction of viral
replication, except against the viruses endemic within their own species. This is most
likely results from the acquisition of mutations which result in resistance to the Trim5 
proteins present in their natural hosts.
31

Trim5  has been shown to restrict viral replication through binding the capsid core upon
its entry into the cell. The exact mechanism by which Trim5  accomplishes this
remains unknown, but it is thought to form a lattice-like structure around the viral core
(149, 150), preventing its transport to the nucleus and mediating its degradation through
the proteosomal pathway. Since Trim5  proteins are able to recognize a wide range of
viral capsids, this indicates a possible structural recognition motif instead of specific
residue interactions. HIV is thought to have evolved specific alterations in its CA protein
to prevent recognition by human Trim5 , evading its restriction potential. A naturally
occurring chromosomal rearrangement in both owl monkeys (151) and some macaques
(152-156) has created a Trim5  isoform which contains the chaperone protein
cyclophilin A (CypA) fused to the C-terminal end. This hybrid protein, Trim5 -CypA, is
not present in the human genome, however it is capable of binding the HIV capsid (155,
157), which raises the option of using such a protein as an anti-HIV gene therapy
candidate.

1.2.6 SAMHD1: Depletion of nucleotide pool
In the past few years, a new restriction factor has been identified which acts to deplete
the pool of available nucleotides within a cell, preventing HIV reverse transcription from
efficiently completing. This protein is the sterile alpha motif (SAM) and histidine/aspartic
acid (HD) domain-containing protein 1, or SAMHD1 (158, 159). The function of this
protein is to maintain a low level of deoxynucleoside triphosphates (dNTP) available in
the cytoplasm of a cell by cleaving off the triphosphate group (160). In doing so,
SAMHD1 is thought to help control the pool of dNTPs available for reverse transcription.
32

A recent report shows that indeed, SAMHD1 expression in myeloid cells maintains the
dNTP pool below the concentration threshold required for HIV RT activity (161),
preventing proviral DNA production and incorporation into the host chromosome.

As with other retraction factors, HIV overcomes this restriction through the targeted
degradation of SAMHD1. Currently this activity has only been observed in the Vpx
protein of HIV-2 (158, 159). Vpx is incorporated into the virion of HIV-2 particles (162,
163), and thus upon delivery into a cell is capable of increasing the dNTP pool available
to the virus and circumventing the restriction imposed by SAMHD1. Whether or not
HIV-1 also exhibits activity against SAMHD1 is yet to be determined. It has been
hypothesized that since HIV-1 primarily infects T lymphocytes that it may not require a
factor to counter SAMHD1 restriction. However, recent evidence has implicated
SAMHD1 activity in CD4+ cells resulting in HIV-1 restriction (164), implying there may
be a gene encoded by HIV-1 to counter this restriction.

1.3 Humanized Mouse Model of HIV Infection

1.3.1 Rodents as models of infection
Due to the obvious ethical dilemmas of conducting HIV research experimentation on
human subjects, alternative models of infection were necessary. The most obvious
animal model for the study of lentiviral infections are non-human primates due the high
similarity and ancestral lineage of HIV from SIV. It is even possible to create hybrid
forms of these viruses (SHIV), allowing for further characterization of specific proteins
33

and functions between the various strains. However, despite these benefits, non-
human primates remain extremely expensive and require large facilities, limiting their
use to only well funded institutes and universities.

The past decade has seen significant advancement in the development of rodent as
models of infection. Rodents are an attractive alternative to non-human primates due to
their shorter reproductive timeframe, lower cost of maintenance, and smaller space
requirements. With the sequencing of several rodent genomes (165), genetic
modification provided a realistic opportunity to create species which were able to
sustain human cellular replication. Such advancements have allowed for the creation of
humanized mice for the study of diseases ranging from diabetes to cancer and HIV.

1.3.2 Creation of humanized mice
A critical step in the use of rodent models for human diseases was achieved with the
creation of the severe combined immunodeficiency (SCID) mouse (166, 167). These
mice are defective in their DNA recombination and repair machinery, resulting in non-
functional V(D)J recombination, and ultimately limiting the adaptive immune system
within the animal. This alteration allowed for the xenotropic engraftment of human
tissues and stem cells, creating a unique environment for in vivo studies. Another
significant advancement in this field was the creation of the non-obese diabetic (NOD)
strain which exhibits reduced IL-2 activity and lack of the complement function of innate
immunity.

34

The combination of these mutations in the NOD/SCID models leads to an absence of
mature T and B cells, and significant reduces the immune development of theses mice
(168-170). When combined with the mutation knocking out the interleukin 2 receptor
gamma chain (IL2r ), the resulting mouse model contains a severely limited immune
system, and is capable of sustain prolonged human tissue engraftment through
reduction in graft rejection due to loss of natural killer (NK) cell function (171). These
NOD/SCID/IL2r 
-/-
(NSG) mice can support human hematopoesis through the injection
of human HSCs (172). Once engrafted, HSCs in these mice give rise to a functional
human immune system, including CD4+ T cell development, and these cells populate
the major lymphoid organs of the animal, including bone marrow, spleen, thymus, and
lymph nodes (173) (Figure 1.9). Additional mouse models exist, including the Rag1
null

model (170, 174), however the remainder of this thesis will be focused around the NSG
stain of mice.

1.3.3 HIV infection of humanized mice
NSG mice are not only capable of sustaining human engraftment and hematopoesis,
but are also able to be used as models of HIV infection (167, 175). These mice
recapitulate many of the pathogenic events associated with HIV including CD4+ T-cell
depletion in the peripheral circulation and lymphoid tissues (176) as well as high viral
titers in the blood plasma (Figure 1.10). These mice also provide an excellent tool for
investigating anti-HIV treatments ranging from siRNA or antibody based therapies (177)
to genetic modification using zinc-finger nucleases (ZFN) (178).

35

Figure 1.9: Human cells are present in immune compartments of NSG mice.
(A) Example of FACS gate analysis of blood from mice engrafted with human cells.
Human cells are identified in blood by the human marker CD45, and subsequent
lineage analysis is performed on this population to identify lymphocyte subsets. (B)
Human cells are also found in numerous lymphoid organs including bone marrow,
spleen, and thymus.

A
28%
SSC
CD45
Blood
51%
SSC
CD19
B cells
17%
28%
CD8
CD4
T cells
53%
SSC
CD45
Spleen
32%
SSC
CD45
Thymus
B
36

Figure 1.10: HIV-1 infection profile in humanized mice. (A) General timeline and
event description for creation, infection, and analysis of humanized mice. (B) When
engrafted mice are left uninfected, overall levels of human cells increase in the
circulation over time (black line). When the mice are challenged with HIV, the levels
of human cells decrease throughout the course of infection (red line). (C) This same
trend can also be observed in CD4+ T cell levels over time. (D) HIV viral loads
typically peak between 4-6 weeks post infection depending on virus strain used for
infection. Persistent viral levels can usually be detected in the blood for the duration
of the infection.

FL35 - Engraftment Levels (Avg)
8 10 12 14 16 18 20 22
0
20
40
60
80
100
Mock
Legend
Age in Weeks of Mice
% Engraftment CD45+
FL35 - Engraftment Levels (Avg)
8 10 12 14 16 18 20 22
0
20
40
60
80
100
Mock
Legend
Age in Weeks of Mice
% Engraftment CD45+
Mock
HIV
Age of Mice (weeks)
% Human Cells
CD4+ Cell Levels
CD4 Levels (Avg)
8 10 12 14 16 18 20 22
0
20
40
60
80
100
Legend
Legend
Age in Weeks of Mice
% CD4+ Cells
Age of Mice (weeks)
CD4 Levels
Average Viral Load
12 14 16 18 20 22
1.0  10
2
1.0  10
3
1.0  10
4
1.0  10
5
1.0  10
6
1.0  10
7
1.0  10
8
Weeks Post Infection
HIV-1 Copies/mL
HIV Levels
FL35 - Engraftment Levels (Avg)
8 10 12 14 16 18 20 22
0
20
40
60
80
100
Mock
Legend
Age in Weeks of Mice
% Engraftment CD45+
HIV
Age of Mice (weeks)
HIV-1RNA Copies/mL
Human Cell Levels
FL35 - Engraftment Levels (Avg)
8 10 12 14 16 18 20 22
0
20
40
60
80
100
Mock
Legend
Age in Weeks of Mice
% Engraftment CD45+
Mock
HIV
B C
C
Engraftment
Irridation
HSC Injection
First Bleed
Second Bleed
HIV Challenge
Analysis at each timpoint:
1) CD4+ cells – FACS
2) Viral load – qRT-PCR
Sacrifice &
Necropsy
A
37

Chapter 2: Development of a High-Throughput Screen for
Novel Inhibitors of Tetherin Counteraction by HIV-1 Vpu

2.1 Abstract

The Human Immunodeficiency Virus 1 (HIV-1) infects cells of the immune system
including CD4+ lymphocytes and macrophages, ultimately leading to cell death and
progression to AIDS. Numerous host restriction factors have evolved to combat such
viral infections and limit their ability to successfully replicate and infect new cells.
Similarly, viruses like HIV-1 have also acquired means of counteracting these restriction
factors, and restoring replication efficiency. One such cellular restriction factor is called
BST-2 or tetherin. Tetherin is an interferon inducible cell surface protein which is
constitutively expressed on a subset of cells within the immune system. Tetherin is
capable of blocking productive budding and release of a wide range of enveloped
viruses from the cell surface, helping to mitigate the spread of infection. HIV-1 counters
this restriction through its Vpu protein, restoring efficient virus release. We hypothesize
that factors like Vpu could prove to be good targets for new therapeutic molecules to
disrupt the viral life-cycle. These late stages of virus assembly and release for HIV-1
are not targeted by any drugs on the market today.
38

2.2 Introduction

2.2.1 Identification of a novel restriction factor
Recently, a new HIV restriction factor was identified simultaneously by two independent
research groups, and was found to be responsible for restricting efficient viral release
from Vpu-deficient viruses (75, 76). This factor was the bone marrow stromal cell
antigen 2 (BST-2), also coined “tetherin” due to its effect of tethering newly formed
virions to the cell surface of infected cells. Tetherin is a transmembrane protein with a
unique topology. It consists of a short N-terminal cytoplasmic tail region, followed by a
transmembrane domain and large extracellular coiled-coil region, and terminates with a
GPI anchor which is thought to localize it to lipid rafts on the cell surface (117).

After the identification of tetherin, it was discovered to be counteracted by a diverse
range of viral proteins, including the HIV-1 Vpu protein, the envelope protein from HIV-2
(75, 126), Kaposi’s sarcoma-associated virus K5 protein (123) and the Ebola
Glycoprotein (122). Additionally, several different SIV genes, including SIV Nef and
Vpu, also contain anti-tetherin activity against their respective host species tetherins
(127, 129, 130). The presence of such factors in a variety of viruses suggests that the
ability to block tetherin is an important characteristic of successful human pathogens.

39

2.2.2 HIV-1 Vpu re-localizes tetherin from the cell surface to the trans-Golgi
network
As previously stated, it was known that the HIV-1 accessory protein Vpu was required
for efficient virion release from infected cells (72, 179), yet how Vpu accomplished this
remained a mystery. The 16kD Vpu protein consists of 81 amino acids with a
transmembrane domain at its N-terminus and is conserved among all HIV-1 and some
SIV viruses but is absent in HIV-2. Aside from its ability to enhance virus release, Vpu
was known to be responsible for the degradation of the surface antigen CD4 through
the ubiquitin-associated pathways utilizing -TrCP (71). It is also thought to potentially
form multimeric complexes which function as ion channels similar to Influenza’s M2
protein (180). Tetherin acts at the plasma membrane by blocking the release of
enveloped viruses such as HIV and physically tethering them to the cell surface (79,
120). These trapped viruses were shown to be fully matured virions, capable of causing
subsequent infections when physically or enzymatically stripped from the cell surface.
This indicates tetherin doesn’t block virus assembly but rather prevents the release of
viruses from the surface (74, 181).

Previous investigations from our lab and others have strongly correlated the anti-
tetherin activity of HIV-1 Vpu with an ability to remove tetherin from the cell surface (76,
78). In the presence of Vpu, both endogenous and exogenous tetherin is relocalized
from the cell surface to a peri-nuclear compartment that was identified as the trans-
Golgi network (TGN). Vpu is also thought to degrade trapped tetherin through either the
lysosomal (82) or proteosomal pathways (80). The direct mechanism behind this
40

altered localization is still not fully understood, however Vpu’s ability to interact with
tetherin has been mapped to its transmembrane domain (125, 133, 134). This is
exemplified by a chimeric protein our lab characterized where the transmembrane
domain of tetherin was replaced with the transmembrane domain from the transferrin
receptor. This hybrid protein was determined to be insensitive to Vpu, and potently
restrict virus release from infected cells (182). Additionally, Vpu containing a
randomized transmembrane domain also lost its ability to interact with tetherin and
downregulate it from the cell surface (183). These two findings underline the
importance of the transmembrane domain of both proteins for the ability of Vpu to
remove tetherin from the cell surface and enhance virus release.

2.2.3 Vpu-tetherin interaction as a novel therapeutic target
With the realization that a broad range of viruses have evolved anti-tetherin factors and
exhibit specific evolutionary pressure to do so, we hypothesized that these anti-tetherin
factors like Vpu might represent good drug targets. Since Vpu acts at a late stage of
the HIV life cycle, it is difficult to use standard high throughput screens (HTS) to identify
inhibitors of these events, and no known anti-HIV drugs act at this stage. In part, this
difficulty arises from the lack of simple reporter-based assays that recapitulate these
late stage events, and instead, such screens often require cumbersome and time-
consuming culture supernatant purification procedures. Instead of using Vpu’s ability to
enhance virus release from cells as a readout of Vpu activity, we instead decided to
exploit Vpu’s property of removing tetherin from the cell surface for designing a high
throughput screen.
41

We hypothesized that surrogate tetherin-based reporters could be designed that
recreate the interaction between Vpu and tetherin, and which could form the basis of a
HTS for inhibitors that block Vpu activity. Such a system would allow for analysis of
small molecule drugs without the need for a cellular lysis step or supernatant
concentration of virus, rapidly decreasing the time and materials needed to determine
effectiveness of screened drugs.

2.3 Materials and Methods

2.3.1 Cell lines
HeLa cells were obtained from the American Type Culture Collection and maintained in
D10 medium: Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech, Herndon, VA)
supplemented with 10% fetal bovine serum (FBS) (Denville Scientific, Metuchen, NJ).

2.3.2 Plasmids
An expression plasmid for human tetherin/BST-2 (pCMV6-XL5-Bst2) was obtained from
Origene (Rockville, MD). The pcDNA-Vphu (Vphu) encodes a human codon-optimized
form of HIV-1 Vpu (119) and was provided by Klaus Strebel (NIH). The miniature
tetherin GFP reporter (mTG) comprises residues 1-50 of tetherin followed by a two
glycine residue linker, and fused to an EGFP reporter (minus the Met start codon) (182).
The plasmid pFC14A expresses the HaloTag fusion protein and was obtained from
Promega (Madison, WI). To create the miniature tetherin HaloTag construct (mTHT),
the tetherin stalk from mTG was combined with the HaloTag protein (minus the Met
42

start codon) from pFC14A by PCR overlap, and reinserted into the parental pFC14A
vector. To create the Vphu-IRES-mTHT plasmid, primers were designed to amplify
Vphu from pcDNA-Vphu, and containing a SgfI and SacII restriction sites on the 5’ and
3’ end respectively. The IRES sequence of Encephalomyocarditis virus was cloned
using specific primers and combined with mTHT by PCR overlap, and containing an
XbaI restriction site at its 3’ end. This construct was then inserted into the original
pFC14A backbone using SgfI and XbaI restriction enzymes. The HcRed-IRES-mTHT
vector, the HcRed monomer gene, contained in a plasmid kindly provided by Paul
Spearmen (Emory University) (184) was cloned into the SgfI and SacII restriction sites
of the Vphu-IRES-mTHT vector.

2.3.3 Western blots
Detection of the tetherin construct mTHT was detected by the Western blotting of HeLa
cell lysates using a 1:1,000 dilution of monoclonal rabbit anti-HaloTag serum
(Promega). The viral protein Vpu was detected using 1:1,000 dilution of rabbit antisera
raised against HIV-1
NL4.3
Vpu (ARRRP; from Frank Maldarelli and Klaus Strebel).
Secondary detection was done using HRP-conjugated goat anti-rabbit IgG (1:12,000)
(Santa Cruz Biotechnology, Santa Cruz, CA). Specific bands were detected by ECL (GE
Healthcare, Pittsburg, PA).

2.3.4 Confocal
HeLa cells were transfected with specific expression plasmids in 10-cm dishes, and 18
to 24 hours later were seeded into 2 well chamber slides (Thermo Scientific, Waltham,
43

MA). The cells were incubated for an additional 24 h at 37°C and processed by ligand
and antibody staining. Cells were fixed with 4% paraformaldehyde (Electron
Microscopy Sciences, Hatfield, PA) for 20 min at room temperature, washed three times
in phosphate buffered saline ([PBS] Mediatech, Herndon, VA), and permeabilized for 20
min in 0.1% Triton X-100 at room temperature, followed by three additional PBS
washes. For analysis of cell surface tetherin, cells were first placed at 4°C for 20 min,
incubated with fresh D10 plus antibodies at 4°C for 30 min, washed with PBS, and then
fixed in 4% paraformaldehyde. Endogenous tetherin was detected using a polyclonal
mouse anti-BST-2 antibody ([clone MaxPab H00000684-B02P], Abnova, Taipei City,
Taiwan), at a 1:250 dilution. Mouse anti-GFP monoclonal antibody ([clone 3E6], Life
Technologies, Carlsbad, CA) was used at a 1:500 dilution. Vpu was detected using
rabbit HIV-1
NL4.3
Vpu antiserum (AIDS Research and Reference Reagent Program,
[ARRRP]) at a 1:1,000 dilution. The conjugated secondary antibodies used were donkey
anti-mouse Alexa Fluor 488 and donkey anti-rabbit Alexa Fluor 594 (Life Technologies).
The mTHT protein was labeled with HaloTag-AlexaFluor488 (Promega) at a 1:5,000
dilution. Processed cells were mounted in Prolong Gold antifade reagent with 4,6-
diamidino-2-phenylindole (DAPI) (Life Technologies). Images were acquired with an
Ultraview ERS laser spinning disk confocal imaging system (PerkinElmer) at 100x
magnification and processed using Volocity software (Improvision; PerkinElmer) and
Adobe Photoshop Creative Suite 2.

44

2.3.5 Microplate reading and high content imaging
HeLa cells were transfected with specific expression plasmids in 10cm dishes and 18-
24 hours later, harvested by tripsin (Mediatech) incubation and resuspended in D10.
Cells were counted and diluted to appropriate concentrations with D10 media and
seeded into 96 well tissue culture plates (BD Biosciences, San Jose, CA) at specified
cell numbers using a multi-channel pipette, for 24 hours at 37 ⁰C. Cell were processed
live by removing media from wells and adding 30 L of D10 containing a 1:10,000
dilution of the HaloTag-AlexaFluor488 ligand for 15-20 min. Media was removed and
cells washed three times in PBS, then fixed with 4% paraformaldehyde. Plates were
analyzed using an EnVision Multilabel Plate Reader (Perkin Elmer, Waltham, MA). For
high content imaging, cellular membranes and nuclei were labeled with wheat germ
agglutin-AlexaFluor647 (WGA) and Dapi respectively with the Image-iT LIVE kit (Life
Technologies) according to manufactures protocols. Images were taken using a BD
Pathway 435 High-Content Bioimager (BD Biosciences) and using Atto Vision software
for analysis (BD Biosciences).

2.3.6 UCLA-MSSR screening protocol
HeLa cells were transfected in 18.5cm dishes using either the screening vector (Vphu-
IRES-mTHT) or control vector (HcRed-IRES-mTHT) and after 24 hours harvested by
tripsin incubation and diluted in D10 media containing 1% penicillin/streptomycin (JR
Scientific, Woodland, CA). Multiple plates of each transfected construct were combined
into one homogenous cell mixture and counted by trypan blue exclusion. Prior to cell
addition, 20 L of media was dispensed to all wells of a 384-well plates (Greiner Bio-
45

One, Monroe, NC) using a MultiDrop 384 (Thermo Scientific) and 1 L of either
compounds or DMSO controls were pinned into all wells by a Biomek FX workstation
(Beckman Coulter, Indianapolis, IN). After compound addition, 30 L of cells were
seeded into all wells using the MultiDrop 384, with rows 1-2 of all plates receiving
control vector transfected cells and remaining rows (3-24) receiving screening vector
transfected cells. Cells were incubated with compound containing media for 48 hours at
37 ⁰C, and then processed for surface expression of mTHT. For processing, media was
aspirated down to 20 L using an ELx405 Plate Washer (BioTek, Winooski, VT) and
10 L of media containing 1:10,000 dilution of HaloTag-AlexaFluor488 ligand was
dispensed to all wells and cells were incubated at 37 ⁰C for 10 min. After labeling,
media was aspirated and all wells were washed 3x in PBS and fixed in 4%
paraformaldehyde for imaging. Cells were imaged using an Acumen Explorer (TTP
Labtech, Cambridge, MA) and analyzed using Acumen Explorer Software.

2.4 Results

2.4.1 Developing a functional cell based tetherin reporter system
Tetherin is an integral membrane protein with a short cytoplasmic tail followed by a
transmembrane domain, an extracellular coiled coil domain, and a C-terminal GPI
anchor (117, 118). Our lab has previously characterized a truncated version of tetherin
that contains only the first 50 amino acids (182), which comprises to the first membrane
spanning domain of the protein, fused to a green fluorescent protein (GFP) reporter
(Figure 2.1A). While this mutant lacks restriction capabilities due to the missing GPI
46

anchor, it retains the ability localize to the cell surface and be down-regulated by Vpu
(182). As with native tetherin, this mini-tetherin GFP (mTG) is concentrated in the trans-
Golgi network (TGN) when co-expressed with Vpu (Figure 2.1A). The mTG reported
also allows for visualization of both cell surface and intracellular tetherin pools without
requiring a permeabiliztion step. This provides an efficient reporter system to visualize
cellular tetherin distribution in the presence of anti-tetherin factors.

This mTG reporter construct was analyzed for its ability to be used in a cell based high-
throughput fluorescent reporter system. The mTG construct was expressed in HeLa
cells both in the presence or absence of human codon optimized Vpu (Vphu) in a 96-
well plate format and analyzed using a Perkin Elmer EnVision fluorescent plate reader
(Figure 2.1B). Cells were washed with PBS and fixed prior to analysis. While a
significant increase in signal was observed with mTG over background (mock) cells,
there was very little decrease in the presence of Vphu. This observation likely resulted
from continued fluorescence of the GFP construct despite being removed from the cell
surface. Being internally re-localized to the TGN is not sufficient to prevent excitation
and emission from the GFP construct.

2.4.2 Alternate reporter constructs for surface expression
With the realization that the mTG construct retained the natural localization and Vpu
counteraction of regular tetherin, additional reporter constructs for the tetherin stalk
were investigated. The HaloTag reporter technology offered an attractive system, in
47

which the reporter construct could be covalently labeled with a fluorescent ligand which
was cell impermeable. The HaloTag enzyme is an inactivate 33kD hydrolase which is

Figure 2.1: Tetherin transmembrane domain sufficient for reporter construct.
(A) Schematic illustrating wild type tetherin topology and the miniature tetherin GFP
(mTG) reporter construct. Tetherin localizes to cell surface and is internalized by
Vphu. Due to antibody properties, the surface stain is lost when cells are
permeablized for internal staining. The mTG also localizes to cell membrane and
TGN and is down regulated by Vphu, illustrated by yellow co-localization color stain.
(B) Representation of plate layout for fluorescent reads. Graph illustrates the
average and standard deviation of total fluorescent signal for all 32 wells of each
condition, and is representative of n=3 experiments.

+Vpu +Vpu
tetherin Vphu
tetherin
mTG Vphu
mTG
Non-permeabilized Permeabilized
A
B
1
B
C
D
E
F
G
H
2 3 4 5 6 7 8 9 10 11 12
A
Mock mTG mTG
+ Vphu
relative fluorescence units
2x10
4
4x10
4
6x10
4
8x10
4
10x10
4
0
B
Total fluorescence of mTG
96-well plate layout
tetherin
Amino
Acids 1-50
Extracellular
Cytoplasm
mini-tetherin
GFP (mTG)
EGFP
48

Figure 2.2: Detection of tetherin reporter specifically on cell surface.
(A) Schematic overview of miniature tetherin HaloTag (mTHT) reporter as compared
to wild type tetherin. (B) HeLa cells transfected with mTHT in the presence or
absence of Vphu. Cells were then stained directly with the fluorescent HaloTag
ligand for surface expression, or permeabilized prior to staining for detection of
internalized tetherin reporters. (C) Tetherin is only detected on the cell surface in the
absence of Vpu expression as illustrated in graph showing mean fluorescent intensity
of surface fluorescence. Each value is calculated from 32 wells and is representative
of n=3 experiments.

Non-Permeabilized Permeabilized
mTHT mTHT +Vphu
mTHT Vphu mTHT Vphu
mTHT mTHT
A
Surface fluorescence of mTHT
tetherin
Amino
Acids 1-50
Extracellular
Cytoplasm
mini-tetherin
HaloTag (mTHT)
B
C
Mock mTHT mTHT
+Vphu
250
500
750
1000
1250
0
relative fluorescence units
Z’ = 0.71
49

capable of specifically and permanently binding the HaloTag ligand for direct
visualization of the reporter. This construct was cloned onto the tetherin reporter stalk
to create a mini-tetherin HaloTag (mTHT) (Figure 2.2A).

This construct was analyzed for its ability to re-capitulate the natural localization of
tetherin, as well as its ability to be recognized and down-regulated by Vpu. For
visualization of mTHT, the cell impermeable ligand containing an AlexaFluor488 was
used. Cells were transfected with the mTHT reporter either in the presence of absence
of Vphu and analyzed for the localization of mTHT by confocal microscopy. Cells were
either treated with detergent to permeabilize them for internal staining, or left untreated
to test for specificity of mTHT surface staining and membrane leakage (Figure 2.2B).
The non-permeable ligand was able to specifically detect mTHT when localized on the
surface in the absence of Vphu, and exhibited no signal when co-expressed with Vphu,
despite the fact expression of mTHT was still apparent when the cells were
permeabilized with detergent prior to ligand staining.

The mTHT was next tested identically to the mTG construct for its ability to exhibit a
decrease in signal when in the presence of Vphu using a fluorescent plate reader
(Figure 2.2C). Cells were stained live with the fluorescent ligand prior to washing and
fixation. Similar to the mTG reporter, a significant increase in signal was detected with
mTHT expression over mock control cells. However when the mTHT protein was
expressed in the presence of Vphu, the signal was reduced to mock levels, indicating a
robust down-regulation of mTHT from the cell surface and subsequent minimal
50

detection when stained with the fluorescent ligand. This assay is appealing due to a
gain of signal being measured for positive hits instead of a loss of signal which often
times can result from cell dysfunction or death.

As a measure of significance, the Z prime score (Z’) was calculated. The Z’ score is the
most common value that screening facilities use in determining that positive drug hits
are statistically significant over background signals. The Z’ score is calculated to
ensure that 99% of data points fall within 3 standard deviations of the mean and follows
the equation Z' = 1-(3 
pos
+ 3 
neg
)/(|3 
pos
- 3 
neg
|) with  being the standard deviation
and  the mean of all samples within each control group. Scores are given on a scale
of 0 to 1.0 with acceptable scores being greater than 0.5 and ideally closer to 1.0. A Z’
score of 0.5 indicates a separation equivalent to 12 standard deviations between your
positive and negative signals. The score for this assay was calculated at 0.71.

2.4.3 Production of a dual expression plasmid
With the validation of the mTHT reporter construct as a readout for our fluorescent
based screening assay, we next sought to create dual expression plasmids containing
both the tetherin reporter and Vphu proteins. Such a dual expression plasmid would
allow for both an easy transfection system, and prevent any cell from receiving only one
construct, minimizing possible false positive hits during screening. To accomplish dual
expression, the mTHT open reading frame was inserted under the control of an internal
ribosome entry site (IRES) immediately downstream of the Vphu stop codon. This
entire transcript was placed under control of a CMV promoter to ensure adequate
51

expression of the full length messenger RNA (mRNA). This plasmid (henceforth
referred to as “screening vector”) was then tested for its ability to express both full
length proteins in HeLa cells, and allow for their detection by confocal microscopy
(Figure 2.3A). The difference in translational efficiencies between the CMV promoter
and IRES was ideal since a higher ratio of Vphu:tetherin expression is beneficial for
complete and efficient cell surface tetherin removal.

An additional control vector was created which lacks Vphu expression to serve as a
positive control vector in a high throughput setting (henceforth referred to as “control
vector”). Since IRES promoters function more efficiently when an open read frame is
present upstream of the promoter, the Vphu coding region was replaced with an HcRed
monomer fluorescent protein. This allows for quick fluorescent detection of expression
while providing minimal bleed-through into the AlexaFluor488 channel for mTHT
detection. In this plasmid, mTHT achieves its usual cellular distribution across the
plasma membrane and the TGN (Figure 2.3B). These dual expression plasmids were
also tested to ensure retention of statistical significance (Figure 2.3C). Correct protein
expression was checked for both plasmids by Western blot (data not shown).

52

Figure 2.3: Dual expression vectors ensure overlap in protein expression. (A)
Plasmids were created to contain both the Vphu and mTHT open reading frames on
a single RNA transcript under the control of a CMV promoter (screening vector). The
Vphu gene is located on the 5’ of the transcript, followed by an IRES sequence
controlling translation of mTHT. (B) An additional vector was also created with
contains an HcRed fluorescent protein in place of Vphu (control vector). (C) Graph
illustrates fluorescent signal difference between the two constructs with associated Z’
score of 0.69.

CMV IRES mTHT Vphu
A
B
Screening Vector
Mock Control
Vector
Screening
Vector
500
relative fluorescence units
Mock
Control
Screening
0
100
200
300
400
500
400
300
200
100
0
C
CMV IRES mTHT HcRed
Control Vector
Z’ = 0.69
mTHT Vphu Merge
mTHT HcRed Merge
53

2.4.4 Adaptation to microwell format and screen optimization
Using the dual expression plasmids to express our mTHT reporter and Vphu protein, we
next sought to optimize screening conditions to maximize surface detection of mTHT
while minimizing staining of internalized reporters. Previous experiments have been
performed using an epi-fluorescent plate reader which determined the total fluorescent
signal produced from individual wells. In order to provide a higher specificity in mTHT
detection on the cell surface, we switched to a high-content imaging system. These
instruments utilize a high powered fluorescent microscope to image each well of a 96-
or 384-well plate on a well-by-well basis. These images can then be analyzed with
computer software to determine localization of fluorescent signals in different cellular
compartments. When this technology is combined with the selective surface labeling of
the mTHT reporter, it is possible to only measure the fluorescent intensity which is
localized to the cell surface of cells within individual wells.

HeLa cells were chosen for the screen due to their large cell size and high expression of
the mTHT reporter on the cell surface. Cells were transfected with either the screening
or control dual expression vectors, and seeded in 384-well plates over a range of cell
concentrations (Figure 2.4A). After 48 hours, cells were processed with the HaloTag
ligand, washed and fixed for imaging and analysis. Wells with identical cell numbers
were compared for surface mTHT expression between the control and screening
vectors. The Z’ score for each pair was calculated to determine the optimal cell
numbers for screening conditions. The condition with 3000 cells/well provided the

54

highest Z’ score of 0.644 over the range of cell numbers screened (Figure 2.4B). This
number was used for all subsequent optimization and analysis.

With the cell number for screening optimized, a final statistical calculation plate to
validate the assay was analyzed. This plate consisted of half the wells of a 384-well
plate containing the positive control vector and the remaining half the screening vector
containing Vphu. Transfected cells were seeded at a density of 3000 cells/well in total
of 50 L media containing 1% DMSO to mimic drug addition. After 48 hours, cells were

Figure 2.4: Optimization of 384 well plate screening conditions. (A) Plate image
representing fluorescent intensities of all wells of a 384 well plate. Cells transfected
with either control or screening vectors were seeded at varying concentrations
between 1000-5000 cells per well and analyzed for optimal signal separation
between the conditions. Several rows contain no cells (-) for additional controls.
(B) Graph illustrating the comparison of the different cell concentrations analyzed.
Numbers at top of graph are the Z’ scores calculated for each pair, with 3000
cells/well providing the most significant score of conditions tested.
Cell Number Optimization
control vector screening vector
- - - -
1000
2000
3000
4000
5000
1000
2000
3000
4000
5000
- - - -
A
0.00
100.00
200.00
300.00
400.00
500.00
600.00
700.00
1000 2000 3000 4000 5000
cells per well
number of green cells in field
0.338 0.389 0.644 0.526 0.617
Z’ score
cell #/well
B
Surface Fluorescence of Cells
55

processed and analyzed for cell surface expression of mTHT. Representative well
images are shown for both control and screening wells (Figure 2.5A). Wells were
individually analyzed for number and intensity of labeled cells using the Accuman
Explorer (TTP Labtech) software. Cells were identified using both object size and
relative fluorescent intensity. Each object was subjected to these criteria and final
analysis was performed only on adhered and intact cells in each well. Example image
of gating strategy and inclusion/exclusion of objects is shown (Figure 2.5B). Final
statistical analysis of all 192 wells of each condition gave a Z’ score of 0.63. This
finding demonstrated that the established assay was rigorous enough for conducting a
high throughput screen with and that DMSO did not provide an inhibitory effect on the
cells or assay.

2.4.6 Screening performed against a variety of drug libraries
With the assay validated using a fluorescent based high content imaging system, we
proceeded to test compound libraries. In collaboration with the Molecular Screening
Shared Resource (MSSR) core at The University of California, Los Angeles, we
screened the assay against several drug libraries containing approximately 100,000
small molecules over a three week period. An overview of several of these libraries is
provided (Table 2.1). Plate layouts were designed to contain 32 wells of positive
controls, 32 wells of negative controls (screening vector with DMSO alone) and up to
320 drug compounds (Figure 2.6).

56

Figure 2.5: Statistical validation of the screening assay. (A) Full plate and
representative well images from validation plate. Half of a 384 well plate was seeded
with screening vector and second half with control vector. Z’ score calculated at 0.63
(B) Zoomed in analysis images of representative wells of both screening and control
vector. Each object within a well is individually analyzed for size and relative intensity
to identify intact cells from background objects or debris. Objects which pass the
validation characteristics are colored cyan while non-cell objects are colored grey.

57

Table 2.1: General overview of libraries tested in this high throughput screen
including brief description and number of the molecules contained in each library.

Library Characteristics Compounds
Prestwick
Chemical library
FDA approved and off-patent
compounds
1,125
NIH Clinical
Collection
Small molecules that have a
history of use in human clinical
trials, highly drug-like with known
safety profiles
600
Microsource
Spectrum
collection
biologically active and structurally
diverse compounds of known
drugs, experimental bioactives,
and pure natural products
2,000
Lead-Like set Unique and diverse lead-like
structures
20,000
Druggable
compound set
Druggable, targeted at kinases,
proteases, ion channels and G-
protein coupled receptors
8,350
Custom libraries Chemically diverse small
molecules
56,200
58

When screening multiple plates of compounds at once, it was necessary to ensure a
homogenous distribution of the pooled cells so all wells of each plate received an
equivalent cell mixture. In order to ensure sufficient cells for multiple 384-well plates, up
to eight 18.5cm plates individually transfected with either control or screening vector
were harvested and pooled together to create the cell mixture. This mixture was then
used in seeding all 384-well plates to be screened each day. In brief, approximately
1 L of drug compound or DMSO controls were pinned into 20 L of media by a Biomek
FX liquid handler. After compound addition, 30 L of the transfected cell mixture was

Figure 2.6: Overview of cell plating for screening chemical libraries.
(A) Multiple transfected plates were combined to one cell mixture and plated as
depicted. (B) The number of wells that each plate had for each of the conditions
described. (C) Example image of plate readout from the high content imager. The
brighter the well, the higher the number of positive cells.
control vector
Rows 1-2
Positive Cnt.
(DMSO)
Rows 23-24
Positive Cnt.
(DMSO)
Rows 3-22
Drug Compounds
(Drug in DMSO)
screening vector
Condition Plasmid
Wells per
Plate
+ Control Control Vector 32
- Control Screening Vector 32
Unknown Screening Vector 320
A B
C
59

dispensed into all wells for a final volume of 50 L and 3000 cells/well. This protocol
was repeated until all compound libraries had been screened.

2.4.7 Multiple approaches used to analyze data ‘hits’
Upon completion of the screening and acquisition process for all of the compound
plates, the data was uploaded to the Collaborative Drug Discovery (CDD) database for
subsequent analysis. Analysis of possible hit compounds was performed using two
methods of classification. First, using software provided through the CDD, large scale
data analysis was performed across all compound plates, by setting positive control
wells as 100% signal level and comparing all wells to this reference value. This
provides a range of efficacy for compounds from no activity (signal between 0-15% as
established by negative control wells), up to high activity (signal >70%). This form of
analysis provides global comparison between all library compounds (Figure 2.7).
The data was then subjected to a second round of analysis on a plate-by-plate basis.
For each plate, the Z’ score was calculated as well as the mean and standard deviation
of wells in each subgroup (positive control, negative control, and compounds). Hit
compounds were identified as any well which fell outside of three standard deviations
from the mean of all compound wells. This analysis ensures that any positive hit falls
outside the intrinsic background noise of each plate, and also serves to validate the
assay on a plate-by-plate basis by ensuring that the positive controls also fall outside
this range. By combining these two methods of analysis, a total of 44 compounds (hit
rate of 0.04%) were identified as candidates for further characterization.

60

2.4.8 Secondary cherry pick screen of hit compounds
The 44 compounds identified as possible candidates in the preliminary screen were
further subjected to additional rounds of screening for verification. All 44 compounds
were re-plated on a master ‘cherry-pick’ plate to streamline the secondary screen and
provide direct comparison between the compounds. This cherry-pick plate was

Figure 2.7: Illustration of hit compound identification. A selection of data from 20
individual plates is shown in graph at left with each vertical column representing one
plate containing 384 data points, indicated as individual dots. Red dots represent
positive controls, blue dots represent negative controls, and black dots represent
screened compounds. By this method, hits were determined by a signal increase
corresponding to >50% of positive controls. Representative images of each condition
(positive, negative, and’ hit’) are shown, including the structural composition of one
drug determined as a positive hit.

100
0
200
% of positive signal
Plate #
1 20
positive well image
compound well image
negative well image
compound structure
61

rescreened in triplicate using the preliminary screening protocol. In addition to the
triplicate plates, one more plate was made to serve as a control for compound auto-
fluorescence. This additional plate received no HaloTag ligand during the processing
step and was washed, fixed, and imaged alongside the three labeled plates (Figure
2.8A).

Analysis of the secondary screen plates revealed the majority of the compounds
identified in the preliminary screen were auto-fluorescent compounds themselves, as
evidenced by their strong fluorescent signal even in the unlabeled control plate. Several
other compounds did not repeat the activity observed in the initial screen, indicating a
false positive hit (Figure 2.9 [end of section]). However, 5 compounds did appear to
repeat their activity and were not positive on the auto-fluorescent plate. These
compounds were further investigated on a cell-by-cell basis within each well to ensure
appropriate cellular conditions such as size and intensity of signal. When compared
back to positive control wells and the shape and cellular distribution of signal intensity,
the remaining 5 compounds were not consistent with intact labeling of the mTHT
reporter (Figure 2.8B). The shape of identified objects was not cellular in distribution
with the vast majority being elongated streaks. These objects also exhibited a
fluorescent intensity 8- to 10-fold higher than positive control cells, indicating they were
not cellular in nature. Ultimately none of the rescreened 44 compounds exhibited
specific activity of returning the mTHT reporter to the cell surface (Table 2.2).

62

Figure 2.8: Individual object analysis indicates non-cellular fluorescence.
(A) Graphical representation of fluorescent signal from each well of the cherry pick
plate. Only top three rows of compounds rows contained drug. Green wells are
control vector wells, red wells are negative control wells. Three plates were
fluorescently labeled and one left unlabeled as control. Left graph is representative
of the three labeled plates and right graph is the unlabeled plate, indicating high auto-
fluorescent signal from many compound wells. (B) Representative well image of a
positive control well. Topographic image illustrates the fluorescent intensity over the
cell surface from one fluorescent object within the well. Typical range for cell surface
fluorescence from labeled mTHT molecules is 1000-3000 units. (C) Image of
example compound which initially was classified as a positive hit by all parameters.
Upon evaluation of individual objects within the well, the shape and average
fluorescent intensity do not indicate typical cellular behavior.
0
1000
2000
3000
4000
relative fluorescence Plate stained with fluorescent ligand
0
1000
2000
3000
4000
relative fluorescence
Control plate left unstained
A
2000
17000
well image representative object profile
positive control well example drug well
C
Intensity Intensity
B
63

2.5 Discussion

The restriction factor BST-2/tetherin has been shown to be effective at inhibiting efficient
virus budding of a wide range of enveloped viruses. Tetherin accomplishes this by
providing a physical link between the outgoing viral particle and the cell membrane,
eventually leading to the endocytosis and degradation of the virus. Several viruses
which are targets of tetherin restriction have evolved proteins to counter this anti-viral
activity. For HIV-1, this activity resides in the Vpu protein. Through transmembrane
interactions with tetherin, Vpu mediates the surface removal and degradation of
tetherin. Reliving this restriction permits efficient viral budding. Numerous studies have
demonstrated the potency of this restriction in cell culture, and a better understanding of
tetherin restriction during in vivo infections is the subject of several studies.

Table 2.2: Summary of compound hit rates at different stages of analysis.

# of
Compounds
Percent of Total
Initial Drug Library 121,120 100.00%
Initial Hit List 44 0.04%
Of the 44 hit
compounds
Auto-fluorescent 23 0.02%
Abnormalities 16 0.015%
False Positives 5 0.005%
End Result Hits 0 0.00%
64

The accessory proteins of HIV are largely responsible for mediating the cellular
environment and priming the cell for efficient viral replication. These proteins carry out
essential function for viral replication in vivo such as causing cell cycle arrest,
preventing detection by the immune system, and deregulating the interferon anti-viral
responses. Since these proteins are unique to the viral genome, they are excellent
targets for therapeutic intervention, helping to mitigate efficient viral replication once a
cell becomes infected. Through mediating disruption of the budding process,
specifically by targeting the anti-tetherin activity of Vpu, it could be possible to slow
down dissemination of the virus through an infected host.

Currently no therapeutic compounds target the viral assembly and budding stages of
replication, and these might represent excellent targets for intervention. Additional anti-
viral drugs are always needed since extremely high costs and side effects of current
anti-retroviral therapy can often prevent continued treatment. In this study we sought to
design and perform a high throughput drug screen for the identification of novel
compounds which could inhibit the anti-tetherin activity of HIV-1 Vpu. If such a drug
could be identified which was effective at inhibiting the anti-tetherin activity of Vpu and
restoring viral restriction, it could open the door to drug screens against additional
proteins which antagonize tetherin in other enveloped viruses.

The cell based high throughput drug screen described here was based on a tetherin
reporter construct which allowed for specific detection when present on the cell surface.
The assay was designed this way in order to identify compounds that not only disrupt
65

the Vpu-tetherin interaction, but restore the surface expression of tetherin. Such
surface expression is essential since tetherin’s anti-viral activity is exerted at the site of
viral budding. This provides a superior based assay over a purely interaction based
assay such as förster resonance energy transfer (FRET) or bi-molecular fluorescence
complementation (BiFC) assays, which would be insufficient in determining the
functional location of tetherin. Since the readout of the assay is based on a gain-of-
signal function, it also helps to eliminate false positive hits due to non specific effects or
toxic compounds.

Initial characterization of the assay established a statistically significant assay,
indicating a robust platform for screening drug libraries. After optimization and
validation of the reporter assay, several chemical libraries were screened in
collaboration with the Molecular Screening Shared Resources core at the University of
California, Los Angeles. These libraries contained over one hundred thousand highly
diversified small molecule compounds. Criteria for identification of hit compounds were
based up either restoration of 50-70% of the positive control signal, or compounds
which fall outside three standard deviations from the signal mean from each plate.
Each wells signal was calculated both as a number of objects within the well which were
cellular in size and morphology, as well as the relative fluorescent signal from the
identified objects. Such objects were determined by the Acuman analysis software.
This method provided both an intra- and inter-plate hit compound identification and
validation of the assay.

66

The initial screen yielded a low hit rate of 0.04% which was well below the general
target of 0.1-1%. Despite this low hit rate, the 44 compounds which were identified
were re-screened on triplicate plates to both validate their initial finding, and to
determine the relative efficacy between them. Compounds which were auto-florescent
were identified and discounted from this secondary screen by a fourth plate which was
unprocessed with the fluorescent ligand. Of the initial 44 compounds, only 5 repeated
their activity from the primary screen once auto-fluorescent compounds were excluded.
However even these 5 compounds did not exhibit specific activity in restoring tetherin
surface expression. Ultimately, no compounds were identified in this screen to inhibit
Vpu activity and restore tetherin to the cell surface.

The major shortcoming of this assay was the lack of a validated positive control for
disruption of the Vpu-tetherin interaction. Such a control would have provided a good
proof of principle for the concept of the assay, and allowed for a more accurate control
during the screening process. However, such a compound does not currently exits,
which was one of the main reasons for the design and execution of this screen. Despite
the failure of this screen to identify novel inhibitors of the HIV-1 Vpu protein, the assay
maintained statistical significance throughout and still represents an excellent platform
for screening additional libraries or alternative inhibitory compounds such as small
proteins or synthetic peptides. Additionally, the methodology presented here was one
of the first to use such a detection system for drug identification using a cellular based
screening assay. This technology could certainly be adapted for other systems which
require the detection of a protein of interest in specific cellular locations, such as nuclear
67

translocations or alterations in surface expression of cellular receptors. Perhaps future
screens will successfully identify inhibitors of HIV accessory proteins like Vpu, and
these drugs will prove to be a source for additional tools in the progress towards an
affordable, tolerable, and effective treatment of HIV/AIDS.

Figure 2.9: Examples of wells which contained abnormalities affecting assay
readout. Several different events occurred during the screen within drug containing
wells which led to false positive hit identification. Examples of these included but not
limited to: (A) cellular foci formation (B non-cellular contamination such as small hair
or dust particles (C) drug crystals due to solubility issues (D) and drug auto-
fluorescence.

Cellular Foci Large Objects
Drug Crystals Auto-fluorescence
A B
C D
68

Chapter 3: Characterizing the Functional Role of Tetherin
Restriction During an HIV-1 Infection in vivo

3.1 Abstract

BST-2/tetherin exerts a late stage anti-viral activity against enveloped viruses, retaining
newly formed virions at the cell surface. A number of pathogenic viruses express anti-
tetherin factors, including the Vpu protein from HIV-1, suggesting that a strong selective
pressure exists for these viruses to block tetherin activities. Initial reports of accessory
proteins demonstrated their function was often dispensable in cell cultures while
phenotypic differences were observed in primary tissue infections using viruses
deficient in these genes. With the determination of Vpu in providing the antagonism of
tetherin to increase efficient viral budding, the significance of this activity has since been
the subject of intense investigation. In this report, we take advantage of an in vivo
infection system using humanized NOD/SCID/IL2R 
null
mice to address the significance
that tetherin restriction imposes on a spreading HIV-1 infection. We observed reduced
viremia and delayed CD4 cell depletion in mice infected with viruses specifically lacking
anti-tetherin activity in their Vpu protein. These viruses were subsequently capable of
reverting to re-acquire anti-tetherin activity though targeted evolution during the in vivo
infection of the humanized mice. Tetherin restriction provides a significant block to
efficient HIV-1 replication in vivo and exerts strong evolutionary pressure on the virus to
retain and anti-tetherin function within its genome.
69

3.2 Introduction

BST-2/tetherin is an interferon inducible transmembrane protein that exerts a late stage
anti-viral activity against enveloped viruses, retaining newly formed virions at the cell
surface (75, 76). A number of pathogenic viruses express anti-tetherin proteins and
have developed their own strategies to overcome tetherin suggesting that a strong
selective pressure exists for these viruses to block tetherin activity (121-124). For HIV-1,
this anti-tetherin activity resides in the Vpu protein (75, 76), which removes tetherin from
the site of virus budding at the cell surface (76, 183) and sequesters it in the trans-Golgi
network (TGN) (77, 78). Vpu accomplishes this though a transmembrane interaction
with tetherin which has been reported to result in degradation of the sequestered
tetherin, either through protesomal (79, 80) or lysosmal (81, 82, 136) pathways.
Phylogenic analysis of HIV and its SIV counterparts also indicates that at each cross-
species transmission of the virus, anti-tetherin activity also evolved to optimally counter
the new host tetherin (79, 129, 133, 185).

The interaction between Vpu and tetherin has been mapped to the transmembrane
domains of both proteins (125, 127, 131-135) with a presumed alanine face residing in
Vpu thought to be crucial for this activity (186). Disruption of this interaction domain
through substitution of a single alanine residue at position 18 with histidine (A
18
H) in the
HIV-1 NL4.3 backbone prevents efficient Vpu interactions with tetherin (185, 187). This
single point mutation allows for specific targeting of Vpu anti-tetherin activity. This is
important since Vpu also carries out other important viral functions including the
70

degradation of cellular CD4 (70) and NK-cell T-cell B-cell antigen (NTB-A) (188). Vpu
was also reported to degrade interferon regulatory factor 3 (IRF3) (189, 190)
presumably preventing efficient anti-viral responses within infected cells, although a
recent report refutes this finding (191). This multi-functional nature of Vpu complicates
the analysis of any experiment using a Vpu deleted HIV-1 aiming to study the effects of
Vpu on any single aspect of virus replication. Similar to Vpu, tetherin also functions in
other roles aside from viral restriction. These include the regulation of interferon
production through an interaction with immunoglobulin-like transcript 7 (ILT7) on
plasmacytoid dendritic cells (192), as well as modulating the nuclear factor B (NFB)
pathway (193-195), both of which may play a more signification role in vivo and can
complicate analysis during the course of an infection.

Several initial reports characterized Vpu as an accessory protein that had little or no
requirement during in vitro infection studies (73, 196, 197). Some studies have shown
impairment of virus replication in primary human peripheral blood mononuclear cells
(PBMCs) (198), while other studies in derived cell lines conclude Vpu expression is
dispensable for efficient viral spread (73, 196, 197). While dramatic effects of tetherin
restriction in preventing viral egress has been observed in transient transfection assays
using HeLa and HEK293T cells (129, 182), the role of restriction in spreading virus
infections may be more. Some of these findings can be attributed to levels of tetherin in
the various cell types (183) but it remains true that this effect can be difficult to
characterize in vitro.

71

A recent study by Sato et al. addressed the possible role of Vpu in HIV pathogenesis
during an in vivo infection (199). Using the HIV-1
AD8
virus, which has been shown to
partially compensate for Vpu deficiencies in its envelope protein (200), the authors
determined that Vpu activity is critical during the initial phases of infection, however this
requirement was lost over time in response to the accumulation of productively infected
cells. We were interested in addressing a similar question, specifically the role that
tetherin could be playing during the course of an in vivo infection utilizing a virus in
which the only deficiency in Vpu is its ability to interact with tetherin. In contrast to Sato
et al. (199), our study presented here uses lower infectious doses of virus and permitted
the infection to progress over the course of 20 weeks allowing for better
characterizations of the difference in viral kinetics. Additionally, our use of a longer time
course allows for evaluation of any evolutionary pressure exerted upon the virus
through sequence analysis.

We show that preventing efficient counteraction of tetherin leads to considerably
delayed viral replication and T-cell depletion in vivo using a humanized mouse model of
infection (NOD/SCID/IL2r 
null
, [NSG]). Additionally the virus was capable of regaining
an anti-tetherin phenotype with just one passage through this animal model of infection.

72

3.3 Materials and Methods

3.3.1 Cell lines and primary cells
HeLa and 293T cells were obtained from the American Type Culture Collection and
maintained in D10 medium: Dulbecco’s modified Eagle’s medium (DMEM) (Mediatech,
Herndon, VA) supplemented with 10% fetal bovine serum (FBS) (Denville Scientific,
Metuchen, NJ). Jurkat GFP reporter cells (JLTRGs) (201, 202) were obtained through
the AIDS Research and Reference Reagent Program (ARRRP), from Olaf Kutsch, and
maintained in R10 medium: RPMI-1640 (Mediatech) supplemented with 10% FBS and
1% penicillin/streptomycin (JR Scientific, Woodland, CA). GHOST X4/R5 cells (AAARP,
from Vineet N. KewalRamani and Dan R. Littman) were maintained in D10
supplemented with 1% penicillin/streptomycin, 500 g/mL G418 (Sigma-Aldrich, St.
Louis MO), 100 g/mL hygromycinB (Sigma-Aldrich), and 1 g/mL puromycin (Sigma-
Aldrich). Human peripheral blood mononuclear cells (PBMC) were obtained from
AllCells (Emeryville, CA), depleted of CD8+ T cells using a positive selection CD8
microbead isolation kit (Miltenyi Biotech, Gladbach, Germany), and cultured in
supplemented R10 medium containing 5 g/mL phytohaemagglutinin (PHA) (Sigma-
Aldrich) and 50 g/mL interlukin-2 (IL2) (R&D Systems, Minneapolis, MN). PMBCs were
thawed and activated for 4-5 days in media prior to use in sustained infections.

3.3.2 Plasmids
Plasmid pHIV-1-pack expresses HIV-1 Gag-Pol and Rev and generates HIV-1 virus like
particles (VLPs) (203). (203). Plasmid pCMV-Vpu (Vpu) encodes the Vpu protein from
73

NL4.3 (203), while pcDNA-Vphu (Vphu) encodes a human codon-optimized form of
HIV-1 Vpu (119). Point mutants in Vphu (A
18
H, A
18
C, A
18
L, A
18
Y) were created using
splice overlap PCR in the pcDNA-Vphu backbone. GFP expression plasmid (copGFP)
was obtained from System Biosciences (Mountain View, CA). Proviral clones HIV-1
NL4.3

(pNL4.3) was obtained through ARRRP, from Malcolm Martin (204), pNL-U35 (131,
205) was obtained from ARRRP by Klaus Strebel, and pNL4.3
Vpu
has been previously
described (Yang 2011). Proviral clone pNL4.3
Vpu(A18H)
was generated using splice
overlap PCR to change the nucleotide sequence of the 18
th
residue in Vpu from GCA to
CAC.

3.3.3 Production and analysis of HIV-1 virus and VLPs for virus release assays
HIV-1 virus/VLPs were generated in HeLa cells by transient transfection using Turbofect
(Thermo Scientific, Waltham, MA) in 10-cm plates with Vphu or associated mutants
(2 g) and either plasmid pHIV-1 pack (8 g) to make VLPs or 5 g proviral clones to
make HIV-1 virus, as described previously (185), with pSA91 used as empty vector.
Supernatants and cell lysates were harvested 24 hours post transfection and analyzed
by Western blotting using rabbit HIV-1
SF2
p24 antiserum (ARRRP) at a 1:3,000 dilution
followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000)
(Santa Cruz Biotechnology, Santa Cruz, CA). Specific bands were visualized using the
enhanced chemiluminescence (ECL) detection system (Amersham International,
Arlington Heights, IL) and quantified using the public domain NIH ImageJ software as
previously described (182). The fold-enhancement of VLP release was calculated as the
ratio of p24-reacting bands in supernatants:lysates, and normalized to the pHIV-1-pack
74

only control. The percent virus release was calculated by normalizing all values to the
pNL4.3 only control. Statistical significance of data was determined using one-way
analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test from
GraphPad Prism (GraphPad Software, La Jolla, CA).

3.3.4 Western blotting
Tetherin was detected in cell lysates using a polyclonal rabbit anti-BST-2 serum
(1:20,000) (ARRRP; from Klaus Strebel), followed by HRP-conjugated goat anti-rabbit
IgG (1:10,000) (Santa Cruz Biotechnology). Vpu was detected using a 1:3,000 dilution
of rabbit anti-Vpu antiserum (ARRRP, from Frank Maldarelli and Klaus Strebel) and
HIV-1 p24 by rabbit HIV-1
SF2
p24 antiserum (ARRRP) at a 1:3,000 dilution followed by
HRP-conjugated goat anti-rabbit IgG (1:10,000) (Santa Cruz Biotechnology). Actin was
detected in cell lysates using mouse anti-actin monoclonal antibody (clone C4) at a
1:3,000 dilution (Roche Applied Sciences, Indianapolis, IN) followed by HRP-conjugated
goat anti-mouse IgG (1:10,000) (Sigma-Aldrich).

3.3.5 Production of HIV-1 stocks
Virus stocks were produced in 293T cells by transient transfection using TurboFect
(Thermo Scientific) and 12 g of proviral plasmid. Supernatants were harvested at 48
hours, filtered through 0.45 m filters, and dispensed into single use doses and frozen at
-80°C. Viruses were quantified by p24 ELISA (Zeptometrix, Buffalo, NY) and by
GHOST cell titer (Morner, 1999) to determine infectious units per mL (IU/mL). Titering
was performed per GHOST cell line protocol obtained through ARRRP.
75

3.3.6 Tetherin surface removal assay
HeLa cells were transiently transfected in 6 well plates with either Vphu or mutants
(1 g) and the copEGFP expression plasmid (200ng) using Turbofect (Thermo
Scientific). Cells were harvested at 24 hours and stained in PBS +1% FBS using an
anti-tetherin-PE conjugated antibody at a 1:20 dilution (eBioscience, San Diego, CA) for
15 minutes at room temperature according to manufactures protocol. Cells were
analyzed for tetherin surface expression by flow cytometry. Statistical significance of
data was determined using one-way analysis of variance (ANOVA) followed by
Dunnett’s multiple comparison test from GraphPad Prism 5 (GraphPad).

3.3.7 Infections of JLTRG cells and human PBMCs
JLTRGs were infected with virus stocks at 100ng/mL HIV-1 p24. Every 3-4 days cells
were analyzed for expression of GFP by flow cytometry. For subsequent passages
25 L of normalized virus was used to infect fresh JLTRGs. Infections of PBMCs were
performed identical to JLTRGs and every 4-5 days 2mL were harvested and
replenished with 2mL of media containing fresh PBMCs. Harvested media was pelleted
with cells and virus supernatant frozen for future analysis. Supernatants were assayed
for viral content using p24 ELISA (Zeptometrix). For multiple rounds of PBMC
infections, PBMCs from 3 unique donors were mixed to prevent donor variability and
used for the remaining passages.

76

3.3.8 Hematopoietic stem cell isolation and NSG mouse transplantation
Human CD34+ hematopoietic stem cells (HSC) were isolated from fetal livers obtained
from Advanced Bioscience Resources, INC (ABR, Alameda, CA). Tissue was disrupted
and incubated with 1mg/mL Collagenase/Dispase (Roche Applied Sciences) for 15min
at 37 ⁰C. Cells were isolated by passing the disrupted tissue through a 70 m filter. Red
blood cells were lysed in BD Pharm Lyse (BD Biosciences, San Jose, CA), with CD34+
cells being isolated using CD34 MACS microbeads (Miltenyi) according to
manufacturer’s instructions with an additional purification step using a second column.
NOD.Cg-Prkdc scid Il2rg tm1Wj/Szj (NOD/SCID/IL2r 
null
, NSG) mice were obtained from
Jackson Laboratories (Bar Harbor, ME). Neonatal mice received 150 cGy radiation,
then 2-4 hours later 1x10
6
CD34+ HSCs in 1% heparin (Celgene, Summit, NJ) via
intrahepatic injection. Mice were monitored for engraftment levels of human CD45+
cells and development of T cells and B cells at 8 and 12 weeks post engraftment.

3.3.9 Mouse infections and analysis of human cells in tissue
Mice were infected at 12 weeks of age with 5x10
4
IU of HIV-1 in 200 Lby intraperitoneal
infection. Every 2 weeks post-infection starting at week 4, mice were anesthetized by
inhalation of 2.5% isoflourane and blood was collected retro-orbitally until 21 weeks
post-infection when mice were sacrificed. The spleen and thymus were harvested for
analysis. Organs were dissociated by passage through a 70 m filter (BD Biosciences),
and washed with PBS. Whole blood and tissue cell suspensions were blocked for 20
minutes at room temperature in FBS (Denville) and stained with appropriate FACS
antibodies for 15 minutes at room temperature. Stained whole blood and tissues were
77

fixed and red blood cells were removed by incubation in BD FACS Lysing Solution (BD
Biosciences). Lysis buffer was removed by dilution with PBS prior to analysis by flow
cytometry.

HIV-1 levels in peripheral blood were determined by extracting viral RNA from mouse
plasma at each blood draw using a viral RNA isolation kit (Qiagen, Germantown, MD)
followed by Taqman One-Step RT-PCR (Life Technologies, Carlsbad, CA) using a
primer and probe set targeting the HIV-1 LTR region, as previously described (178,
206). Reactions were performed and analyzed on a 7500 Fast Realtime PCR System
(Life Technologies).

3.3.10 Co-cultures of splenocytes and JLTRG cells
After necropsy and organ isolation, 2.5x10
6
JLTRG cells were incubated with
splenocytes from infected mice in R10 medium. Cells were amplified in culture, and
every 2-3 days, a fraction of cells were analyzed for expression of GFP by flow
cytometry. Supernatants were harvested at each time point and frozen at -80°C for viral
sequence characterization and use in subsequent infections. Viral RNA was isolated
from co-cultures using the viral RNA isolation kit (Qiagen) and samples frozen at -80°C
for sequence analysis.

3.3.11 Flow cytometry
Stained cells were acquired on a FACS Canto II (BD Biosciences) and analyzed using
FlowJo software v7.6.5 (Tree Star Inc., Ashland, OR). For tetherin surface removal in
78

HeLa cells, 20,000 events were recorded. Analysis was performed on the viable cell
population, and tetherin expression was analyzed on GFP expressing cells using single
color controls. Infected JLTRG cells were washed twice with PBS prior to fixation with
4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Gates were
established to contain ≤0.3% GFP positive cells in mock infections. Blood samples were
stained using mouse antibodies at a 1:20 dilution for CD4-FITC (RPA-T4), CD8-PE
(RPA-T8), CD19-APC (HIB19), and CD45-PerCP (TUI16) (BD Bioscience). Up to
10,000 events were recorded for viable cell populations and gated based on
fluorescence minus one controls as previously described (178).

3.3.12 Viral sequencing analysis
Isolated viral RNA was reverse transcribed using Superscript III Reverse Transcriptase
(Life Technologies). The Vpu open reading frame was amplified with specific primers
(NL4.3 5749-5769 F [5’-GCTACTGAATTCTGCAACAACTGCTGTTTATCC-3’] and
NL4.3 7249-7227 R [5’-GCTACTGCTAGCTATCTGTTTTAAAGTGGCATTCC-3’]) using
High Fidelity Accuprime Taq Polymerase (Life Technologies). Primers also contained
either an EcoRI or NheI restriction sequence and 6 nucleotide cap. PCR products were
cloned into the pCR2.1 TOPO Cloning Vector (Life Technologies) for subsequent
sequencing. Alternatively, genomic DNA was harvested from infected cell cultures using
a mammalian cell DNA isolation kit (Qiagen) and PCR products were cloned using the
specific primers previously described and cloned into pCMV6-XL5 vector (OriGene,
Rockville, MD) for sequencing.

79

3.4 Results

3.4.1 Vpu defective viruses are deficient at enhancing viral release from HeLa
cells.
HIV-1 Vpu promotes virus release through the cell surface down regulation (183) and
subsequent degradation of tetherin (79, 81). Vpu accomplishes this, in part, due to an
interaction with tetherin within the transmembrane domains of the two proteins (125,
127, 133-135). A single amino acid change at position 18, from alanine to histidine,
within the transmembrane domain of Vpu abolishes its interaction with tetherin without
disrupting any of Vpu’s other functions (185, 187). To further characterize the
importance of Vpu counteraction of tetherin, we verified that the A
18
H mutation behaved
as previously described in a proviral background.

The proviral clone NL4.3 was altered to create several Vpu mutants: the alanine at
position 18 in Vpu was changed to histidine (NL4.3
Vpu(A18H)
), NL4.3
Vpu(U35)
contains a
truncated Vpu reading frame consisting of the first 35 amino acids (131, 205), and a
Vpu null virus, (NL4.3
Vpu
) was created by deleting the first 10 nucleotides of the Vpu
coding frame (Figure 3.1A). These proviral clones were analyzed for their ability to
efficiently release virus when transfected into HeLa cells, which endogenously
expresses tetherin. As expected, Vpu deficient viruses were inefficiently released into
the supernatant (Figure 3.1B). However, when wild-type Vpu was provided in trans by
co-transfection, the ability to enhance virus release was restored to varying degrees.
Interestingly, the Vpu
A18H
protein expressed from the proviral clone exhibited reduced
80

Figure 3.1: Mutant Vpu’s deficient in enhancing virus release. (A) Schematic of
NL4.3 Vpu domains and the various mutant or deletion Vpus characterized in this
chapter. (B) HeLa cells were transfected with either a control CMV expression
plasmid (mock) or an HIV-1 (pNL4.3) proviral clone (8mg). Wild-type Vpu was
provided in trans (2mg) to proviral clones containing a deficient Vpu. Cell lysates and
pelleted virus supernatant were analyzed by Western blot using the indicated
antibodies. Percent virus release was made relative to wild type NL4.3 virus
released into supernatant (100%). Graph illustrates mean plus standard deviation for
n=3 independent experiments, p < 0.01(**) p < 0.001(***).

+
A
NH
2
COOH
Transmembrane Helix 1 Helix 2
No Start Codon
NL4.3
NL4.3
Vpu(A18H)
NL4.3
Vpu(U35)
NL4.3
Vpu
COOH
A
18
H
NH
2
COOH NH
2
B
lysates
virus
NL4.3
pNL4.3
Vpu(U35)
pNL4.3
Vpu(A18H)
tetherin
pNL4.3
Vpu
p55
p24
Vpu
p24
(n=3)
% virus release
Relative Virus Release from HeLa Cells
0
20
40
60
80
100
% of Wild Type Virus
- - - + - - + Vpu
Mock
HeLa
*** *** ***
**
40
60
80
0
20
100
81

expression levels in cell lysates when compared to wild-type Vpu, possibly due to mis-
localization and aberrant degradation (185). Additionally, when Vpu is provided to the
NL4.3
Vpu(A18H)
proviral clone, it fails to reach wild-type levels of virus release despite
similar levels of Vpu expression, indicating a possible dominant negative effect by the
A
18
H variant.

3.4.2 Anti-tetherin activity is dispensable in JLTRG cell line.
Since clear differences were observed in the various Vpu’s abilities to enhance virus
release in HeLa cells, we transitioned to analyzing a spreading infection of Jurkat cells,
a human T cell line. We used a specific line of Jurkat cells (JLTRG) which express GFP
upon HIV-1 infection (201, 202). The JLTRG cells were infected with low levels of each
HIV-1 Vpu mutant virus (100 ng/mL p24) and at the indicated times after infection a
sample of cells were analyzed for the percent GFP positive by flow cytometry (Figure
3.2A). Interestingly, no appreciable difference was observed in the growth rate or total
percent of cells infected between the wild-type NL4.3 virus and the three Vpu mutants.
This finding was in contrast to the dramatic decrease in virus production from HeLa cells
(Figure 3.1B), however it is consistent with a varied requirement for accessory proteins
in cell culture infections.

Since Vpu mutant viruses were capable of replicating to wild-type levels in JLTRG cells,
we next wanted to determine if multiple infection cycles of the viruses would provide
increased evolutionary pressure to reacquire anti-tetherin activity. To test this, we
permitted the virus to complete several infection cycles in JLTRG cells. Low levels of
82

A
days post infection
JLRTG cells
(rep of 3)
%infected (GFP+)
2 4 6 8 10 12 14
0
10
20
30
40
50
60
B
days post infection
%infected (GFP+)
2 4
0
20
40
60
80
6 8 10 12 14 2 4 6 8 10 2 4 6 8 10 12 14 16 18 20 22
Passage 1 Passage 2 Passage 3
12
C
Transmembrane
e
Helix 1 Helix 2
NH
2
COOH
NL4.3
NL4.3
Vpu(A18H)
COOH
A
18
H
NH
2
x
W
22
Stop
83%
M
1
I
17%
x
x
I
17
M
9%
M
1
T
18%
x x
E
47
K
9%
M
1
I
45%
E
59
K
9%
x
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
Mock
NL4.3
U35
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
JLTRG Infection Group 2
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
0
10
20
30
40
50
60
Mock
NL4-3
U35
A18H
Mock +IFNa
NL4-3 +IFNa
U35 +IFNa
A18H +IFNa
Days Post Infection
% Infected (GFP+) Cells
Mock
NL4.3
U35
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
83

each virus was used (100 ng/mL p24) and harvests performed every 2-3 days to assay
GFP expression by flow cytometry (Figure 3.2B). Once infections peaked and fell to
persistent steady state levels, the next round of infection was initiated by a transfer of
infected cells to a fresh population of JLTRG cells allowing for a new infection cycle to
begin. This was repeated so that the virus was replicating in culture for a total of 48
days.

Once the third passage of the virus had fallen to steady state infection levels (Day 22),
viral RNA was harvested from the supernatant for sequence analysis. A 1500 base pair
fragment encompassing the Vpu reading frame and the first 1000 nucleotides of Env
was analyzed for any changes which occurred during the course of infection. Several
clones were sequenced from each virus (Figure 3.7 [end of section]) and an overview
of observed Vpu mutations is summarized (Figure 3.2C). Sequence analysis indicates
Figure 3.2: Anti-tetherin activity is dispensable in JLTRG cell line. (A) JLTRG
cells were infected with equivalent viral loads of parental NL4.3 or indicated mutant.
Infections were harvested every 2-3 days and assayed for GFP expression by flow
cytometry. Data shown is representative of n=3 independent experiments. (B)
JLTRG cells were infected with the parental or mutant viruses and allowed to
undergo a full infection cycle. A portion of cells were harvested every 2-3 days for
analysis of GFP expression by flow cytometry. Upon reaching steady state levels of
infection, a fraction of cells from each virus were used to seed fresh cultures. This
was repeated an additional time. At day 22 post infection of the third infection cycle
(day 48 from beginning of infection) viral RNA was extracted from the supernatant
and subjected to RT-PCR for sequence analysis. (C) Schematic representation of
amino acid mutations observed. Mutations are highlighted in each sequence as
black ‘X’ marks with accompanying amino acid changes including position and
frequency of observation in clones sequenced. Number of clones analyzed for each
virus were: NL4.3 (6), A
18
H (11), and U35 (8).

84

a lack of targeted evolution in both mutant viruses to regain anti-tetherin activity within
the Vpu gene, with the majority of mutations observed in single a clone. On the
contrary, both viruses which express a full length Vpu protein exhibited a tendency to
self-delete expression of Vpu. After three rounds of passage, wild-type NL4.3 had
created a truncated Vpu by introducing a stop codon at amino acid 22, which occurred
in 5 of 6 clones (83%). The NL4.3
Vpu(A18H)
virus had deleted Vpu in 7 of 11 clones (64%)
by mutating the start codon. This start codon deletion was achieved by two unique
nucleotide changes (M
1
T and M
1
I) indicating two independent mutagenesis events. No
nucleotide changes were detected in the truncated NL4.3
Vpu(U35)
clones. Previous
experiments demonstrated very little mutagenesis during one round of infection (data
not shown) suggesting these mutation were selected for during the course of multiple
rounds of infection. This lack of anti-tetherin activity could be resulting from a reduced
requirement due to low tetherin expression in these cells.

3.4.3 Viruses exhibit phenotype in PBMCs which express higher tetherin levels.
With the finding that the mutant Vpu viruses replicated as efficiently as wild type virus in
JLTRG cells, we wanted to test them in a cell type which naturally expresses higher
levels of tetherin since previous reports have indicated that Jurkat cells express
relatively low levels of tetherin (183). We confirmed these results by comparing tetherin
expression profiles in both HeLa and JLTRG cells as well as human PBMCs (Figure
3.3A). When actin levels were equalized, the PBMCs displayed the highest level of
tetherin expression, corresponding to about a 13-fold increase over the JLTRG cells.

85

Since the higher levels of tetherin expression in PBMCs may impose a greater inhibition
of virus production than the levels found in JLTRG cells, we infected PBMCs from
several unique donors. Whole PBMCs were infected with 100ng/mL p24 of each virus.
In PBMCs, a decrease in the growth kinetics of the HIV-1 Vpu mutant viruses was
observed (Figure 3.3B). In three unique donors, our NL4.3
Vpu(A18H)
point mutant and
NL4.3
Vpu
mutant exhibited delayed replication kinetics and reduced peaks of viral titer.
Noting this difference, we next determine the effect of multiple passages of the virus
though human PBMCs. Due to the phenotypic difference of the viruses in these cells,
we expected a higher evolutionary pressure to regain Vpu function. In order to account
for variability between PBMC donors, three unique donor PBMCs were obtained and
depleted of CD8+ T cells to prevent possible cross-reactivity from different HLA
genotypes. These cells were mixed together to create a pool of PBMCs used for the
duration of the infection. PBMCs were infected as previously described with low levels
of HIV-1 (Figure 3.3C). Virus supernatants were harvested between days 18-20 which
correlated to the peak viral load and assayed for viral content. Fresh PBMCs were
infected with equivalent levels of p24 from peak time points to initiate the next
replication cycle. As previously observed, both the NL4.3
Vpu(A18H)
and NL4.3
Vpu
viruses
replicated at lower efficiency when compared to wild type NL4.3, indicating continued
restriction on efficient viral replication.

After three infection cycles through PBMCs totaling 57 days, viral RNA was harvested
and for sequence analysis (Figure 3.8 [end of section]). Similar to the JLTRG
infections, majority of mutations observed in the population were unique events.
86

However, unlike in the JLTRGs, the only Vpu mutation observed was in a single

Figure 3.3: Mutant viruses exhibit a Vpu dependent phenotype in PBMCs. (A)
Western blot analysis of tetherin levels on both JLTRG cells and human PBMCs in
culture as compared to HeLa cells. (B) PBMCs were infected with equivalent levels
of virus (100ng/mL). Supernatants were harvested every 3-4 days and assayed for
virus release by p24 ELISA (pg/mL). Data shown is representative of n=3
independent experiments with unique cell donors. (C) Human PBMCs pooled from 3
unique donors were infected with either parental NL4.3 or indicated mutant.
Supernatants were harvested every 3-4 days and analyzed for their p24 content by
ELISA. At peak viremia, 18-20 days post infection, virus harvested from the cells and
normalized for p24 content, and used to initiate a new infection in fresh culture of
PBMCs. This passage protocol was repeated for a total of 57 days of virus
replicating in.

0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
-
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
0
50000
100000
150000
200000
250000
300000
Mock
NL4.3
A18H
DelVpu
Passage 2
0 1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20
0
50000
100000
150000
200000
250000
Mock
NL4.3
A18H
DelVpu
Data 1
JLTRG
PBMC 1
PBMC 2
0
2
4
6
8
10
12
PBMCs
days post infection
2.0x10
5
1.5x10
5
1.0x10
5
0.5x10
5
0
0 2 4 6 8 10 12 14 16
HIV-1 p24 (pg/mL)
2.5x10
5
18 20
B
C
days post infection
2 4 6 8 101214 2 4 6 8 10 2 4 6 8 101214161820 1214161820 1618
HIV-1 p24 (pg/mL)
Passage 1 Passage 2 Passage 3
2.0x10
5
1.5x10
5
1.0x10
5
0.5x10
5
3.0x10
5
2.5x10
5
0
actin
PBMC
Donor A
PBMC
Donor B
tetherin
JLTRG
A
(rep of 3)
25
75
37
0
2
4
6
8
10
PBMC
Donor B
relative tetherin levels
37
50
kD
12
JLTRG
PBMC
Donor A
Mock
NL4.3
Vpu
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
Mock
NL4.3
Vpu
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
87

NL4.3
Vpu(A18H)
clone and consisted of an early termination codon, 5 amino acids short of
the normal stop codon. This overall decrease in mutation events could possibly be due
to a lack of plasticity in the PBMC infection conditions. Perhaps in primary human cells,
pressure to adhere to a strict amino acid sequence is greater than in immortalized cell
lines, were the virus has wider range of tolerated amino acid composition.

3.4.4 Viruses deficient in anti-tetherin activity replicated at lower levels and
demonstrated delayed CD4 loss in vivo.
Our lab has previously characterized a humanized mouse model, and confirmed it is
capable of sustaining ongoing HIV infections (178). These NSG mice are capable of
developing a multi-lineage immune system will fully functional T cells and B cells and
provides a more complete model for studying the intricacies of the virus-host interaction
(207, 208). Using this model, we examined the mutant Vpu HIV-1 viruses for any affect
the Vpu-tetherin interaction may have on virus infections in a system more relevant to
natural infections than tissue culture models.

Once mice attained engraftment levels >20% human CD45+ (huCD45+) cells and >15%
CD4+ T cells in the peripheral blood, they were infected by intraperitoneal injection with
5x10
4
IU of virus. Infected mice were bled every two weeks to assay for viral load and
circulating lymphocyte levels. Percent huCD45+ cells in the peripheral blood of
uninfected (mock) NSG mice steadily increases over time (Figure 3.4A). Mice infected
with HIV-1 NL4.3 exhibit a rapid decrease in overall huCD45+ cells circulating in the
peripheral blood. Both NL4.3
Vpu(A18H)
and NL4.3
Vpu
viruses exhibited a delayed
88

4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
1.0  10
2
1.0  10
3
1.0  10
4
1.0  10
5
1.0  10
6
1.0  10
7
Mock
NL4.3
A18H
DelVpu
0 1 2 3 4 5 6 7 8 9 101112131415161718192021
0
20
40
60
80
100
Mock
NL4.3
A18H
DelVpu
0 1 2 3 4 5 6 7 8 9 101112131415161718192021
0
20
40
60
80
100
Mock
NL4.3
A18H
DelVpu
Spleen
Thymus
0.1
1
10
100
NL4-3
A18H
DelVpu
Mock
NL4.3
U35
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
B A
C
HIV-1 Copies/mL
1x10
2
1x10
3
1x10
4
1x10
5
1x10
6
1x10
7
4 6 8 10 12 14 16 18 20
weeks post infection
weeks post infection
%CD45+ in blood
100
80
60
40
20
0
0 2 4 6 8 10 12 14 16 18 20
%CD4 / (CD4+CD8)
100
80
60
40
20
0
4 6 8 10 12 14 16 18 20
weeks post infection
0 2
E
0.1
0
%CD4+ in CD45+ population
spleen thymus
10
100
4 6 8 10 12 14 16 18 21 0
A
18
H
3 Alive 2 Alive 1 Alive 0 Alive
survival chart per week post infection
Vpu
NL4.3
Mock
NL4.3
U35
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
Mock
NL4.3
U35
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
NL4.3
U35
A
18
H
Passaging of Virus in Jurkat (JLTRG) Cells
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
New Passage
1-1
2-1
3-1
4-1
5-1
6-1
7-1
8-1
9-1
10-1
11-1
New Passage
1-2
2-2
3-2
4-2
5-2
6-2
7-2
8-2
9-2
10-2
11-2
12-2
13-2
14-2
15-2
16-2
17-2
18-2
19-2
20-2
21-2
22-2
0
20
40
60
80
Mock
NL4-3
U35
A18H
Days Post Infection
% Infected (GFP+) Cells
89

depletion of huCD45+ cells in the blood, with levels lower than that of mock mice, but
appreciably higher than NL4.3 infected mice at most time points. By week 18 post
infection, all mock infected mice succumbed to high engraftment levels presumably
leading to graft vs. host (GvH) disease and had to be sacrificed, as shown in a chart
illustrating the survival of mice at each time point (Figure 3.4B).
In addition to overall engraftment levels, we monitored for the depletion of CD4+
lymphocytes in the peripheral blood, a well characterized hallmark of HIV/AIDS
progression (Figure 3.4C). The percentage of CD4+ T cells in uninfected animals
remained constant, between 60-80% of total human lymphocytes in the blood for
throughout the infection, while NL4.3 infected mice displayed a rapid and pronounced
loss of CD4+ cells starting at 6 weeks post infection. Unlike huCD45+ levels, the two
mutant viruses exhibited different phenotypes relative to CD4+ levels with NL4.3
Vpu

infected mice maintaining levels similar to uninfected controls and NL3.4
Vpu(A18H)

Figure 3.4: Vpu deficient viruses exhibit lower viral load, delayed CD4
depletion in humanized mice. Mice were bled at week 8 and 12 post birth to
analyze engraftment levels and cell lineage development. Mice were infected at 12
weeks of age with 5x10
4
IU by i.p. injection and bled every 2 weeks post infection.
Peripheral blood was assayed for cellular expression of the human marker CD45, as
well as T-cell lineage markers CD4 and CD8. (A) Graph showing the percent of
human (huCD45+) cells in blood at indicated times. Error bars indicate standard
deviation of mice in each viral group. (B) Survival chart indicating the number of
mice alive at each bleed. (C) Relative CD4+ levels of each group are represented as
a percentage of total T cells (CD4+ and CD8+) in the blood. (D) Viral RNA was
isolated from the mouse plasma and analyzed using quantitative RT-PCR with
primers targeting the HIV-1 LTR. Error bars represent the standard deviation for all
mice in each viral group. (E) At sacrifice, lymphoid organs were harvested for
analysis of CD4 depletion. Shown is the percent of cells which were CD4+ in the
human cell population (huCD45+) for each virus. Mean represented with horizontal
bar.

90

infected mice demonstrating a loss in CD4+ cells, though this loss was delayed when
compared to wild-type NL4.3. This data is in agreement with the requirement of a
functional Vpu gene in the pathogenesis of HIV in vivo which is often not observed
during in vitro infections.

The amount of viral RNA circulating in the blood was determined at each time point by
quantitative RT-PCR to determine the effect that Vpu mutations had on viral load in the
peripheral blood (Figure 3.4D). While the wild-type NL4.3 virus persisted at relatively
high levels despite the loss of circulating CD4+ cells in the blood, both mutant viruses
demonstrated reduced levels of replication, anywhere from 10- to over 100-fold lower
titers. This discrepancy persisted until 14-16 weeks post infection at which point the
titer of mutant viruses was equivalent to wild-type NL4.3. Presumably this resulted from
wild-type animals having significantly reduced the pool of infected cells capable of
producing virus, as evidenced by the low huCD45+ and CD4+ levels in the blood. The
difference between NL4.3 and the NL4.3
Vpu(A18H)
mutant indicates an important role for
tetherin in helping to mitigate the viral levels circulating in the blood and delaying T cell
depletion.

When the viral loads of the Vpu mutant viruses were became equivalent with wild type
levels, and CD4 depletion was observed in the NL4.3
(VpuA18H)
infected mice, animals
were sacrificed to analyze huCD45+ and CD4+ levels in the lymphoid organs, and to
harvest virus for sequencing. Both the spleen and thymus were assayed for evidence
of CD4+ cell depletion (Figure 3.4E). As seen in the blood, both NL4.3 and
91

NL4.3
Vpu(A18H)
infected mice exhibited extremely low CD4+

levels in both organs, while
the NL4.3
Vpu
mice showed appreciably higher levels. Taken together, the data
collected from the NL4.
3Vpu(A18H)
virus indicates that, while dispensable for cell culture
infection, Vpu counteraction of tetherin is necessary for efficient HIV-1 replication in the
context of an intact immune system in vivo.

3.4.5 Vpu A
18
H point mutation was target for specific evolution in vivo.
The late rise in virus titers and depletion of CD4+ cells observed in the HIV-1 Vpu
mutant viruses could be attributable to several factors, one of which is acquisition of
compensatory mutations allowing for reacquisition of a functional Vpu. Since the
NL4.3
Vpu(A18H)
mutant virus mutation was localized to one amino acid residue which
abrogated anti-tetherin activity, we were particularly interested in determining the
sequence of this region for any mutations that would confer a tetherin specific revertant
phenotype.
To accomplish this, at time of sacrifice, splenocytes from infected mice were incubated
with JLTRG cells to amplify virus populations present within the mice. This co-culture
step allowed for an amplification of isolated virus in a system where replication was
independent of Vpu’s ability to counteract tetherin. For all co-culture infections, each
initiated a spreading infection in the recipient JLTRG cells (Figure 3.5A).

At peak of viral replication (day 9) post co-culture, cells were harvested and genomic
DNA isolated for sequencing of incorporated provirus in the cell population. A segment
of the integrated HIV genome was cloned using the same methods as used for infected
92

JLTRG/PBMC culture experiments (Figure 3.9 [end of section]). A summary of the
sequences obtained from the co-cultures is shown (Figure 3.5B). All of sequences
cloned from NL4.3
Vpu(A18H)
infected mice (32/32 sequences) contained additional
changes at the 18
th
amino acid removing the histidine residue. This alteration was
observed in both mice infected with this virus. The majority of clones sequenced from
mouse 2 contained a tyrosine at position 18, representing a single nucleotide change
(CAC to TAC) from the initial histidine residue. A second nucleotide change at this
codon occurred (TAC to TGC) further changing the amino acid from tyrosine to
cysteine. These sequential mutations could indicate continued pressure on the virus to
reacquire a reversion phenotype. This would not be surprising since like histidine,
tyrosine contains a bulky ring as its side chain which could severely disrupt the
interaction domain between Vpu and tetherin.

Figure 5: All NL4.3
Vpu(A18H)
viruses isolated from mice mutated the histidine
residue at position 18. (A) At the time of sacrifice, a portion of harvested
splenocytes from each mouse were incubated with JLTRG cells. Co-cultures were
harvested every 2-4 days and analyzed for GFP expression by flow. (B) Sequence
analysis of NL4.3
Vpu(A18H)
viruses isolated from the mice and amplified in JLTRG cells.
Nucleotide sequences shown for codons 16-20 of Vpu compared to wild-type NL4.3.
Corresponding amino acid changes listed including number of clones containing the
indicated sequences.

JLTRG/Splenocyte CoCulture
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
20
40
60
80
Mock
FL42-2
FL42-9
FL44-5
FL44-11
FL44-12
FL44-17
Days Post "Infection" Mixed
% GFP positive
days post infection
80
60
40
20
0
2 4 6 8 10 12
%infected (GFP+)
0
A B
Uninfected
NL4.3 mouse 1
NL4.3 mouse 2
A
18
H mouse 1
A
18
H mouse 2
Vpu mouse 1
Vpu mouse 2
I I A I V
ATAATAGCAATAGTT
......CAC......
......CTC......
......TAC......
......TAC......
......TGC......
NL4.3
A
18
H
Mouse 1
Mouse 2
Leu (12/16)
Tyr (4/16)
Tyr (13/15)
Cys (2/15)
amino acid
nucleotide
18 19 20 16 17
JLTRG/Splenocyte CoCulture
1 2 3 4 5 6 7 8 9 10 11 12 13 14
0
20
40
60
80
Mock
FL42-2
FL42-9
FL44-5
FL44-11
FL44-12
FL44-17
Days Post "Infection" Mixed
% GFP positive
93

3.4.6 Vpu A
18
H reacquired anti-tetherin activity through one passage in
humanized mice.
The changes observed in sequences isolated from mice infected with NL4.3
Vpu(A18H)
may
have been the result of evolutionary pressure to acquire a means for counteracting
tetherin. In order to address this question, we made the equivalent changes in a codon-
optimized Vpu expression plasmid (Vphu) and tested for their abilities to remove
tetherin from the cell surface and enhance virus release.

HeLa cells were transfected with either an empty control expression plasmid or HIV-1
Gag-Pol expression cassette capable of forming budding virus like particles (VLPs), in
the presence of the panel of Vphu mutants. Cell lysates and pelleted supernatants
were harvested and analyzed by Western blot (Figure 3.6A). In the absence of Vphu
expression only a small amount of VLPs are detected in the supernatant. As expected,
Vphu expression rescued virus release while Vphu
A18H
did not. However, all three
reversion mutants observed in the mice exhibited a significant increase in their ability to
counteract tetherin. Two of the mutants, the lysine and cysteine reversions, were able
to enhance virus release as efficiently as Vphu while the tyrosine mutant exhibited an
intermediate phenotype. This finding could further strengthen the evidence that
evolutionary pressure to counteract tetherin continued to drive the accumulation of
changes at the 18
th
amino acid until a functional replacement was obtained.

Since Vpu enhances virus release due to its ability to remove tetherin from the cell
surface, we next sought to determine if the revertant Vpu’s regained this activity. HeLa
94

Figure 6: Reversion of 18
th
residue from histidine restores Vpu’s anti-tetherin
activity. (A) HeLa cells were transfected with HIV-1 Gag-Pol expression cassette
and were accompanied by either an empty expression plasmid or the indicated
mutant Vphu. Analysis of VLPs was performed by western blot of lysates and
pelleted supernatants, and blots probed with indicated antibodies. Graph illustrates
mean plus standard deviation for n=3 independent experiments, p < 0.05(*) p <
0.001(***). (B) HeLa cells were transfected with indicated Vphu expression plasmid,
harvested after 24 hours, stained for surface tetherin expression, and analyzed by
flow cytometry. Graph shows average and standard deviation of surface tetherin
levels from n=3 independent experiments, p < 0.001(***). FACS plots shown are
representative of the n=3 experiments. Unstained control is shown as solid gray,
mock control as solid black line, and Vpu’s as dotted lines.

A
p24
p55
tetherin
Vpu
p24
lysates
VLPs
Vphu
A
18
H
A
18
C
A
18
L
A
18
Y
Mock
B
HeLa
(n=3)
Mock Vphu A18H A18C A18L A18Y
0
5
10
15
Fold Enhancement
Vphu
A
18
H
A
18
C
A
18
L
A
18
Y
5
0
10
15
fold enhancement
Mock
***
*
Unstained
Mock
Vphu
A18H
A18C
A18L
A18Y
0
500
1000
1500
2000
Surface Bst-2 MFI
2000
1500
1000
500
0
Vphu
A
18
H
A
18
C
A
18
L
A
18
Y
Mock
Unstained
(n=3)
tetherin MFI
***
Vphu
A
18
C
Mock
Vphu
Unstained
A
18
H
A
18
L A
18
Y
tetherin
tetherin
% of max % of max
95

cells were transfected with either en empty control vector or Vphu mutants and stained
to asses for their ability to remove tetherin surface expression (Figure 3.6B). Again, all
three reversion mutants exhibited significantly increased ability to remove tetherin from
the cell surface when compared to the original A
18
H mutant. Similar to the virus release
results, the lysine and cysteine mutants were as efficient as Vphu at removing tetherin
while the tyrosine mutant had a phenotype intermediate between Vphu and Vphu
A18H
.

Taken together, both the reduced viral pathogenesis and reversion of the Vpu
A18H

mutant to a phenotype capable of counteracting tetherin provides strong evidence that
this ability is necessary for efficient HIV replication and spread in vivo. This further
implicates tetherin restriction as contributing a significant anti-viral role during an in vivo
infection, and capable of exerting strong evolutionary pressure on the virus. This
pressure can be seen though the reacquisition of functional anti-tetherin activity during a
single passage in humanized mice (Table 3.1).

3.5 Discussion

Since the identification of tetherin as the interferon inducible restriction factor
responsible for preventing matured virions budding from infected cells (75, 76),
numerous reports have characterized this ability to restrict virus release. The interplay
between tetherin, and the HIV-1 Vpu protein responsible for its counteraction, has been
studied for its mechanism and significance in the viral life cycle. While tetherin’s role
and potency have been well characterized in immortalized cells lines like HeLa and
96

HEK 293 cells, its function during actual infections in cell culture using T cell lines has
been less clear. While some reports demonstrate that tetherin is capable of restriction
viral dissemination during spreading infections in cell culture (209), others claim the
opposite, stating that any restriction of free viral particles is overcome by increased
direct cell-cell spread (210, 211). To address this discrepancy in observations
regarding tetherin’s potential role during an infection, we took advantage of an in vivo
model of HIV infection.

In order to characterize tetherin’s function during a spreading infection, we first needed
to characterize mutant viruses which lacked the ability to interact with, and subsequently
down-regulate and degrade tetherin. Previous studies have characterized a Vpu
deficient clone (pNL-U35)(131) of the lab strain NL4.3 and its inability enhance virus
release from cells. Additionally, a separate group identified a specific point mutation

Table 3.1: Summary of Vpu requirement and functionality in different cell lines or
model systems.

Jurkat PBMC huMice
Tetherin
Levels
+ +++ n/a
Replication Yes Yes Yes
Vpu
Phenotype
No Yes Yes
Reacquire
anti-Tetherin
activity
Unnecessary No
Yes
100% of A
18
H
97

(A
18
H in the NL4.3 Vpu reading frame) within Vpu’s transmembrane domain which
abolishes its anti-tetherin activity (187). This point mutant is thought to disrupt the
putative interaction domain in Vpu consisting of a face of three alanine residues
followed by a tyrosine (185, 187). Utilizing this series of viruses, we sought to
determine the physiological relevance of tetherin restriction, and the evolutionary
pressure exerted upon the virus in clones which lack the ability to counteract tetherin
restriction.

We first sought to characterize our panel of Vpu mutants in cell culture to better
understand their replication kinetics, and determine what potential pressure was exerted
on the viruses to re-acquire anti-tetherin activity. Our initial findings in a Jurkat report
line was somewhat surprising. All viruses which contained mutations in Vpu exhibited
no phenotypic difference from the parental NL4.3 virus. This finding seemed to
corroborate with previous studies which have found anti-tetherin activity dispensable for
infection in cell culture (73, 196, 197, 210). It’s conceivable that large number of
tethered virus particles on the surface of infected cells actually promotes direct cell-to-
cell spread of the virus through virological synapses as previous studies have
hypothesized (210, 211).

When we then analyzed the sequence of these viruses after passaging them though
multiple infection cycles in JLTRG cells, we observed numerous instances where the
virus self-deleted the Vpu open reading frame. This was especially prevalent in the
NL4.3
Vpu(A18H)
virus where 63% of clones sequenced exhibited a mutation in the initiating
98

methionine codon of Vpu. This mutation arose not once but twice during the infection
as two different single point mutations were detected. This Vpu deletion was also
observed in the parental virus in 83% of clones sequenced. These findings possibly
indicate a loss of requirement for a functional Vpu protein during infections of this cell
type. This would be in agreement with both the fact that our true NL4.3
Vpu
clone
replicates as efficiently as parental NL4.3 in these cells. Another explanation could also
be the endogenously low levels of tetherin expression within this cells line, mitigating
the need for functional anti-tetherin activity.

Since no phenotypic difference was observed in an immortalized cell line between the
viruses, we next wanted to determine the viral characteristics in primary human cells.
We infected isolated human PBMCs from several unique donors, and unlike the
JLTRGs, the Vpu deficient viruses exhibited impairment in their replication capabilities
in these cells. In all three mutant viruses, we observed a decrease in the viral levels
present in the supernatant, as well as delayed replication kinetics. We next wanted test
to if there existed an evolutionary pressure to regain tetherin counteraction in cells
where a phenotypic difference was observed. Sequence analysis of the viruses
revealed the A
18
H mutation was still present in all of the clones sequenced after three
passages of the virus, and only 8% of clones exhibited an early termination at the 76
th

residue, 5 residues short of the full length protein. Additionally, there was a general
decrease in the overall mutation events detected during the PBMC infections when
compared to a similar timeframe in the JLTRGs. This could possibly indicate an overall
lack of mutational leniency in primary cells as opposed to immortalized cell lines.
99

With the mutant viruses characterized in multiple cell culture conditions, we next wanted
to determine the effect of tetherin restriction on a spreading infection in vivo. We
utilized the NSG mouse model, which support the development of a functional immune
system (207) and is capable of maintaining a sustained HIV infection (178, 208). Mice
which were engrafted with CD34+ HSCs as neonates were infected with the Vpu mutant
viruses, and monitored over the course of 21 weeks for the progression of viral
infection. Individuals which were infected with the mutant viruses NL4.3
Vpu
and
NL4.3
Vpu(A18)
, exhibited not only a decrease in plasma viral levels but a delay in the loss
of circulating CD4 T cells in the peripheral blood. By 12 weeks post infection, less than
10% of circulating T cells were CD4+ in animals infected with the parental NL4.3 virus.
In contrast, animals infected with NL4.3
Vpu
and NL4.3
Vpu(A18)
maintained higher CD4+ T
cells levels (56% and 36% respectively) at this same time point. These higher levels of
circulating CD4 cells persisted in the Vpu infected mice for the duration of the
experiment, while the levels in A
18
H infected mice didn’t fall below 20% until 18 weeks
post infection. Similarly, viral levels in the plasma of the mutants did not reach parental
NL4.3 levels until 14-16 weeks post infection, at which point NL4.3 levels were
decreasing most likely due to exhaustion of infected cells within the animals.

Sequence analysis of the virus populations isolated from the NL4.3
Vpu(A18H)
infected
animals showed a clear selection for viruses which targeted the histidine at position 18
for further mutagenesis. This was dramatically apparent as all sequences (31/31) from
animals infected with the A
18
H virus no longer retained the histidine residue. Instead, all
virus clones contained a leucine, cysteine, or tyrosine at the 18
th
position, all of which
100

are capable of restoring anti-tetherin activity to Vpu at varying degrees. While the
leucine and cysteine residues appear to have regained wild type Vpu activity, the
tyrosine mutant exhibited partial restoration of the activity. The cysteine codon
observed in the virus arose from a second mutagenesis event of the tyrosine codon,
most likely since the initial change from histidine to tyrosine was insufficient for the virus
to regain regular Vpu anti-tetherin activity. This presents strong evidence for the
selection of viruses which contain anti-tetherin activity in their genome. It is conceivable
that the CD4 depletion observed in the mice infected with the A
18
H correlated with the
reacquisition of a functional Vpu gene. Meanwhile, the Vpu viruses, having a much
more severe mutational hindrance to regain a Vpu open reading frame, exhibited no
such ability.

Previously, a separate study involving the use of Vpu deficient HIV was performed using
a similar mouse model (199). In their study, the authors used very high infectious doses
of the HIV-1
AD8
virus, and its corresponding Vpu deleted virus, to assess the role of
tetherin in modulating the initial burst phase of infection. They concluded that tetherin
did exert partial restriction of the virus, but only in a short period post infection, and that
by 14-21 days, any effect of tetherin restriction was overcome by the virus. Additionally,
the virus which was harvested from the animals after the 21 days was demonstrated to
still be deficient in tetherin counteraction thought ex vivo culturing. While interesting,
the doses of virus provided to the animals was significantly higher than that which would
occur in an actual infection, and previous studies have shown that the envelope protein
in the HIV-1
AD8
strain contains some activity in enhancing virus release from infected
101

cells (200). In contrast, our study focuses on the long term effect of tetherin restriction
on viral replication and pathogenesis, as well as determining if in viruses that lack anti-
tetherin activity, there is substantial pressure to reacquire this function. In our model,
we demonstrated a delay in viral pathogenesis in Vpu mutant viruses, and a strong
selection pressure to maintain anti-tetherin activity within the viral genome.

102

Supplementary Figure 3.1: Full analysis of viral sequences isolated from
JLTRG cultures. A portion of the viral genome spanning the Vpu reading frame and
the N-terminal region of Env was isolated from each infection for sequence analysis.
Individual nucleotide changes are indicated by black x marks within each virus. If the
mutation resulted in an amino acid change, the original residue, position number, and
new residue is shown. The frequency of each mutation within the number of clones
sequenced is also shown.

NL4.3
Vpu(A18H)
M
1
T/I
18%-45%
I
17
M
9%
E
47
K
9%
E
59
K
9%
A
48
T
9%
V
120
I
9%
T
138
I
9%
S
162
N
9%
V
182
E
9%
C
194
R
9%
N
195
D/L
9%
T
196
A
9%
P
204
S
9%
T
276
I
9%
N
299
Y
9%
A18H
NL4.3
Vpu ORF 5’ of Env ORF
env
tat
rev
vpr
vif
vpu
NL4.3 Genome Schematic
5757 6628
NL4.3
Vpu(U35)
Q
326
Stop
13%
P
204
A/S/L
13%-74%-13%
JLTRG
x x x x x x x
x x x x
x x x x xx x x x x x x x x x x x
W
22
Stop
83%
M
1
I
17%
x
V
84
I
17%
S
143
N
17%
R
296
S
17%
V
316
I
17%
NL4.3 Nucleotide #
103

Supplemental Figure 3.2: Full analysis of viral sequences isolated from PBMC
culture. A portion of the viral genome spanning the Vpu reading frame and the N-
terminal region of Env was isolated from each infection for sequence analysis.
Individual nucleotide changes are indicated by black x marks within each virus. If the
mutation resulted in an amino acid change, the original residue, position number, and
new residue is shown. The frequency of each mutation within the number of clones
sequenced is also shown.

NL4.3
NL4.3
Vpu(A18H)
Vpu ORF 5’ of Env ORF
NL4.3
Vpu
N
160
K
9%
S
164
I
73%
Y
40
H
13%
env
tat
rev
vpr
vif
vpu
NL4.3 Genome Schematic
5757 6628 NL4.3 Nucleotide #
A18H
G
23
R
8%
W
35
Stop
8%
W
69
G
8%
V
89
Q
8%
A
217
T
8%
F
275
I
8%
W
76
Stop
8%
T
49
N
13%
E
64
D
13%
PBMC
x x x x x
x x x x
x x x x x x x x x x x
104

Supplemental Figure 3.3: Full analysis of viral sequences isolated from
infected mice. A portion of the viral genome spanning the Vpu reading frame and
the N-terminal region of Env was isolated from each JLTRG co-culture infection for
sequence analysis. Individual nucleotide changes are indicated by black x marks
within each virus. If the mutation resulted in an amino acid change, the original
residue, position number, and new residue is shown. The frequency of each
mutation within the number of clones sequenced is also shown.

NL4.3
NL4.3
Vpu(A18H)
Mouse 1
Vpu ORF 5’ of Env ORF
Mouse 2
Mouse 1
Mouse 2
NL4.3
Vpu
Mouse 1
Mouse 2
S
143
N K
130
E R
146
K S
162
I/N K
135
N Q
308
H G
71
R
17% 17% 33% 17% 17% 100% 33%
N
160
S V
182
E T
295
I Q
308
H
40% 60% 20% 80%
A
58
S
33%
V
120
A
50%
H
18
L/Y
75%-25%
V
25
I
25%
E
28
K
6%
E
47
K
6%
G
53
D
6%
V
120
A
94%
S
144
N
6%
R
166
K
6%
A
279
V
13%
S
304
R
19%
Q
308
H
19%
A
7
T
7%
H
18
C/Y
87%-13%
E
47
K
7%
A
48
T
27%
M
147
I
7%
A
217
V
7%
V
68
I
7%
env
tat
rev
vpr
vif
vpu
NL4.3 Genome Schematic
5439 6628
Mouse Viral Sequencing
x x x x x x x x x
x x x x
x x x x
x x
x x x x x x x x x x x x x x
x x x x x x x x x
NL4.3 Nucleotide #
105

Chapter 4: Concluding Remarks and Future Perspectives

4.1 Concluding Remarks

With the worldwide epidemic of HIV/AIDS exhibiting no signs of resolution in the near
future, a better understanding of the basic biology of the virus, as well as the intricate
interactions it has with the human immune system is essential. Such studies not only
help to further the understanding and treatment options of the disease, but also help to
elucidate innate cellular functions within human cells. By studying functions of the HIV
accessory proteins, a greater understanding of normal cellular activities has been
achieved. The Vpu protein from HIV is no exception, having resulted in the
identification and initial characterization for the restriction of enveloped viruses by
tetherin.

Essentially all previous studies involving tetherin’s anti-viral function and Vpu’s
subsequent counteraction have been based on cell culture systems of infection using
either immortalized cell lines or primary human cells. While these tissue culture
systems are useful in their ability to manipulate variables and determine specific
functions and pathways affected, they cannot address the significance of these
functions during actual patient infections. Despite the fact that tetherin provides potent
restriction of viruses deficient in their Vpu protein in culture, it remained to be
determined if this activity is required during in vivo infections. This study was one of the
first to investigate the significance of Vpu and its role during such infections.
106

Additionally, the drug screen presented here, which was performed against Vpu with the
goal of restoring tetherin restriction, is one of the first targeting this virus-host
interaction.

The differences observed between the culture infections for the various HIV mutants
underscores the importance of understanding the model system being used, and
requires certain findings to be taking in the context of the system. If JLTRG cells were
the only cell type to be investigated for Vpu function and requirement, a very different
conclusion would have been drawn. This is consistent with the concept that the
accessory proteins of HIV are dispensable under certain conditions of cell culture
infections. Indeed, the observation that Vpu was deleted from both wild type and
mutant proviruses in JLTRG cultures reinforces this concept. It is possible that this
deletion arose due to an increase in dissemination of the virus through direct cell-cell
contacts with tethered virions providing an ideal method of transmission through the
culture (210, 211).

Through the use of our humanized mouse model of infection, tetherin restriction was
determined to provide a significant impasse for efficient viral replication. This barrier
was significant enough that on two independent occasions, Vpu was targeted for
mutation to regain efficient anti-tetherin activity. Using such a model of infection
provided a more relevant environment to investigate the role of Vpu during the infection.
These reversion mutants provide strong evidence for the requirement of a functional
anti-tetherin factor within the HIV genome. It would have been interesting if a reversion
107

would have been observed in the Vpu infections as well. Since HIV-2 uses its Env
protein and SIV uses Nef for tetherin counteraction (78, 126), it is feasible that provided
enough pressure by tetherin, and without a relatively easy “fix” of removing an inhibitory
amino acid from Vpu, that HIV-1 might also have adopted this technique. Unfortunately
such an adaptation was not seen in these experiments which may require a longer
period of time to be achieved.

There is little doubt that future studies of HIV and other viruses will yield even more anti-
viral factors which participate in innate immunity, and a broader understanding of the
human immune system. These findings will continue to not only better characterize the
interactions between HIV and the human population, but hopefully will continue to teach
us more about the broader fields of virology and immunology, and the myriad of
diseases resulting from the intersection of the two.

4.2 Future Perspectives and Implications

4.2.1 Pharmaceutical targeting of accessory proteins including Vpu
Great advancements have been made in anti-retroviral drugs during the past decade for
the treatment of HIV infected patients. However all current drugs in use today target a
narrow range of functions within the viral lifecycle, with the majority being focused on
the core catalytic functions within the Gag and Pol genes. Additional drugs also target
the binding of HIV to the CCR5 co-receptor through CCR5 antagonists. No drugs in
current HAART therapy target the functions of the accessory proteins for HIV. These
108

proteins therefore represent ideal targets for additional therapeutic intervention since
they are both unique to the virus and carry out numerous functions essential for efficient
viral replication. Preventing their functions in evading detection by the immune system
and modulating the internal environment for viral replication could provide even further
repression of viral replication.

While these accessory proteins do represent good targets for novel drug discovery, as
with all drugs, care will need to be taken not to affect normal cellular processes. This
may prove to be a difficult challenge since often times the mechanism of action for
these proteins is through hijacking the cellular machinery, often times the ubiquitin
degradation pathways to carry out their function. Therapeutic intervention will need to
specifically target the viral protein interaction with the associated proteins, without
affecting the cellular proteins themselves. By targeting accessory protein function, it
may be possible to block a wide range of their activities with only a few small molecule
inhibitors. Vpu is a good example as it has been shown to degrade numerous cellular
proteins through a conserved mechanism. Perhaps by blocking either its recognition of
the cellular proteins, or its interaction with the ubiquitin pathway, it may be possible to
prevent Vpu functions.

Although the performed drug screen summarized in this document did not result in the
identification of any lead compounds, the concept of the target should still be valid. This
is confirmed in the data presented here that inhibition of the anti-tetherin activity of Vpu
does provide a hindrance to viral replication and CD4 depletion in vivo. Several other
109

groups have recently published reports of high throughput screens designed to identify
inhibitors of the Vpu-tetherin axis. Their methods also utilize cell based screen
procedures with either an ELISA detection assay for tetherin’s presence on the cell
surface (212), or a BRET based assay with Vpu and tetherin each tagged on their
cytoplasmic side with one of the reporter proteins (213). Both groups have shown their
assays to be capable of providing significant Z’ scores, so it will be interesting to see
what drugs are identified by subsequent screening of compound libraries.

One of the initial goals of this project was to begin the process of assessing any
candidate drugs identified in our screen to show activity against Vpu within the
humanized mouse model. This model would provide an excellent readout for the
efficacy and function of the drug and could be compared back to clones such as Vpu.
This form of experimental trial will no doubt be the next step in any new anti-retroviral
compound and will provide excellent insight into the validity of drugs discovered though
such high throughput screens.

4.2.2 HIV manipulating tetherin expression for IFN modulation
Vpu has been proposed to reduce cell surface expression of tetherin for additional
reasons aside from enhancing virus release. This counteraction could arise from
tetherin’s innate function in the immune system of helping to regulate interferon
production by dendritic cells. This function occurs through tetherin’s interaction with
ILT7 on dendritic cells (192), the primary produces of interferon, signaling them to
decrease interferon production. Since tetherin is an interferon induced gene, it is
110

thought that this provides a negative feedback loop for the interferon pathway, and a
signal to shutdown production preventing dangerous levels of cellular activation and
inflammation, although a recent paper has called this finding into question (214).
Through Vpu mediated degradation of tetherin, HIV could disrupt this feedback
mechanism and lead to persistently high interferon levels.

The mechanisms behind the interplay between interferon and HIV are not fully
understood. Vpu has recently been hypothesized to degrade the cellular interferon
regulator factor 3 (IRF3)(189, 190) which in part responsible for activating interferon
production within infected cells. Interestingly, the promoter region of tetherin also
contains an IRF binding domain (215), indicating that IRF activation could also lead to
tetherin up-regulation in cells. This could be an additional means for the virus to
regulate tetherin expression at the transcriptional level, not only mediating its
degradation, but also decreasing new protein synthesis.

Interferon de-regulation and chronic immune activation is a known sign of AIDS disease
progression and expedites immune system failure and T cell depletion (216-218). At
the same time, interferon production is an essential activator of immune function which
leads to an increase in the host’s ability to combat viral infection. Perhaps HIV is
straddling both sides of this pathway, hijacking interferon production enough to benefit
viral dissemination but not to levels which would become detrimental to replication. So
by increasing systemic IFN levels, HIV could recruit additional activated T cells to the
site of infection, increasing the pool of infectable cells, while meanwhile preventing the
111

currently infected cells from efficiently reacting to the increase in interferon. This
concept was recent tested in an LCMV mouse model of infection, where persistent
activation of the interferon pathways leads to viral persistence. The authors
demonstrated that blocking chronic IFN signaling preventing chronic immune activation,
restored tissue architecture, and enabled viral clearance from the animals (219). This
concept is further discussed in Appendix B at the end of this document.

4.2.3 Additional anti-viral properties of tetherin through signaling cascades
Recent studies have demonstrated that tetherin is capable of participating in the
activation of the NF B signaling cascade (193-195). This signaling pathway is
significant for HIV infection for a few reasons. NF B is a transcription factor that is
involved in the reaction of cells to invading pathogens, and plays a crucial role in early
innate immune responses within infected cells (220). Several pathways lead to the
activation of NF B, which in turn acts to initiate transcription of a variety of genes to
combat infection. HIV also contains an NF B binding sequence in its LTR region (95,
96), which allows the virus to take advantage of the signaling pathway to increase viral
transcription and protein synthesis. HIV is also thought to lessen or prevent the anti-
viral properties in response to NF B activation, but these mechanisms are not entirely
understood.

The cytoplasmic tail of tetherin contains a motif which is thought to interact with TRAF6
(195), an upstream regulator of NF B activation. TRAF6 hypothesized to be recruited
to the sites of viral budding and tetherin aggregation through these interactions, where it
112

is then activated to propagate a signaling cascade. In this scenario, tetherin acts as a
sensing receptor of viral budding to alert the cell of viral infection and initiate appropriate
anti-viral responses through NF B activation. This would not only activate the
appropriate responses to counteract infection within the cell, but also alert cells in the
surrounding area to the viral presence, and aid in recruitment of additional cells to the
area to combat infection.

Tetherin could further act as a sensor for infections by facilitating the endocytosis of
tethered virions. These tetherin virions are recycled into the cells through tetherin’s
interaction with the AP2/clathrin mediated endocytosis machinery which leads to
endosome formation (221). These endosomes have a high concentration of pattern
recognition receptors (PRR) which are responsible for detecting the presence of
bacterial or viral pathogens. Upon detection these receptors activate the interferon
pathways to combat infection. In this sense, tetherin can act to enhance the detection
of pathogens such as HIV by increasing their uptake into these endocytic vesicles.

Temporally, Vpu expression would occur prior to cell surface accumulation of Gag for
viral assembly and budding. This would allow the virus to circumvent not only the
restrictive properties of tetherin, but also prevent the initiation of these signaling
cascades. Disruption of Vpu mediated tetherin degradation could allow for an increase
in these signaling processes, and ultimately increase the natural cellular defenses for
fighting infection. Therapeutic intervention targeted to Vpu could therefore not only
113

assist in slowing viral spread through restriction of budding virions, but also increase the
hosts immune defenses to aid in fighting the infection.

4.2.4 Tetherin as a candidate for gene therapy
Recent advancements in the field of genetic engineering such as zinc finger nucleases
(ZFN) (222) and the Cas9/CRISPR (223) system have made it possible not only to
disrupt gene function within progenitor cells such as HSCs, but also insert exogenous
coding sequences. This insertion occurs through the creating of a double stranded
DNA break followed by the homologous recombination process of the endogenous
cellular repair machinery. Such technology has made it possible to insert DNA
sequences that can code for new gene expression leading to the possibility of genetic
based therapy for a multitude of diseases. As previously mentioned, this method is
currently being investigated for the treatment of HIV though the knock-out of the CCR5
gene in human HSCs leading to cells which are highly resistant to HIV infection (178).
In addition to disrupting the coding region of CCR5, several research groups including
ours are testing the possibility of inserting additional genes at this locus which could
further help to control HIV replication. Such genes, considered anti-HIV factors, could
provide an active resistance to the virus on top of the passive resistance imparted by
CCR5 disruption.

This concept has already been proven in cell culture using immortalized T cell lines and
a lentiviral based vector system to deliver these anti-HIV genes (224). Though the use
of lentiviral vectors could prove problematic based upon their random integration into
114

the genome of the host cells, if this insertion could targeted to a specific ‘safe-harbor’
locus, such engineering could be safe and reliable. Currently several such anti-HIV
factors are being evaluated for their ability to control infection in cultures. Candidate
genes include proteins such as APOBEC3G and Trim5  which can be designed to
resist HIV mediated evasion through mutagenesis and hybridization with alternate
species genes. Tetherin can also function in this capacity as well. Chimeric proteins
between human and rhesus macaque tetherin have been demonstrated to be resistant
to HIV counteraction (127, 133). Additionally, even a fully artificial tetherin, composed
of similar domains to tetherin but a hybrid of 3 unique proteins, has been shown to have
activity against HIV and other enveloped viruses (120), albeit to a lesser extent than
natural tetherin.

Taken together, these advancements indicate that some form of tetherin based gene
therapy may be possible for the treatment of HIV and possibly other enveloped viruses
which are susceptible to tetherin restriction. Obviously several barriers will need to be
overcome prior to the use of this technology. The biggest of these challenges is the
development of techniques to reliably increase the successful recombination events
without leading to oncogenic transformation. Current methods result in a very low
amount of modified cells, with less than 1-3% of human HSCs exhibiting successful
integration of an exogenous gene. It remains to be proven than the insertion of such
candidate genes, such as a tetherin resistant to viral counteraction, will not alter the
cellular functions of the modified cells. This is of particular concern for any gene which
contains non-human sequences, as this increases the risk of developing an auto-
115

immune response to these exogenous proteins. While there certainly are challenges for
the use of genetic based therapies for HIV and other diseases, these recent scientific
advancements provide promising evidence that such treatment may be possible in the
not too distant future.

116

Appendix A: Inhibition of viral budding by the small
molecule AME
A.1 AME implicated in preventing virus release
In the search for specific controls for the development of the anti-Vpu based high
throughput screen, we came across reports of the anti-viral activities of the small
molecule amphotericin B methyl ester (AME). Another research group published
findings which implemented AME in inhibiting Vpu mediated enhancement of virus
release (225). AME is a cholesterol binding compound, and acts to deplete cellular
membrane of their cholesterol content. Since lipid rafts are a site of high cholesterol
concentration and are preferentially used by HIV for assembly and budding of new viral
particles, it makes sense that AME could affect the budding process. In order to
determine the possibility of using this compound as a positive control for hits in our high
throughput screen, we first needed to determine if AME achieved this affect in a Vpu
dependent manner.

A.2 AME does not inhibit viral budding in a Vpu-dependent manner
In order to address the question of AME activity, an enhancement of virus release assay
was performed as previously described in the methods section of Chapter 3 (section
3.3.3). HeLa or 293T cells were transfected with the HIV-1 Gag-Pol expression
cassette in the presence of two different factors known to enhance virus release: HIV-1
Vpu and the envelope protein from HIV-2 (ROD10). If AME was indeed affecting virus
budding in a Vpu-dependent manner, we would expect to see a decrease in
117

supernatant viral particles in the presence of the compound only in cells transfected with
Vpu. After transfection, media containing 10 M of AME was added to the appropriate
cells, and 24 hours later the supernatant and cell pellets were harvested for analysis by
Western Blot (Figure 4.1). In order to determine the specific effect of drug treatment,
each transfection condition containing AME was compared to the identical condition
without compound addition. In each case, AME treatment reduced viral budding,
regardless of the presence of an anti-tetherin factor, indicating the effect is a non-
specific inhibition. While interesting, this finding indicates that AME is not a good
candidate for a positive control compound causing inhibition of Vpu’s anti-tetherin
activity for a high throughput drug screen.

118

Figure 4.1: AME treatment induces non-specific decrease in viral budding.
HeLa (A) or 293T (B) cells were transfected with an HIV-1 Gag/Pol expression
plasmid in the presence or absence of anti-tetherin factors in duplicate. Half the cells
were cultured in regular D10 media, while the second half received AME containing
media. Cells and supernatant were harvested and analyzed by Western Blot. Graph
illustrates the reduction in viral budding by AME when compared to the equivalent
transfection condition without AME treatment.

293T AME v2
Core
Core+AME
Bst2
Bst2+AME
Vpu
Vpu+AME
Rod10
Rod10+AME
0
25
50
75
100
HeLa AME v2
Core
Core+AME
Vpu
Vpu+AME
Rod10
Rod10+AME
0
25
50
75
100
p55
p24
p24
+
lysates
virus
core
p55
p24
p24
- - + - + AME
HeLa
core +Vpu
core +ROD10
+
core
- - + - + AME
core
core +Vpu
- +
core +ROD10
+ - + - + + tetherin + +
293T
reduction from AME
100
75
50
25
0
-71%
-57%
-70%
+ - - + - + AME
100
75
50
25
0
+ - - + - + AME - +
reduction from AME
-23%
-56%
-41%
-36%
A B
119

Appendix B: Tetherin expression is temporally
increased on infected cells in culture
B.1 Method: Intracellular staining of infected cells for analysis by flow cytometry
Infected PBMCs were harvested and blocked in 100% FBS for 15 minutes at room
temperature. Cells were pelleted and resuspended in PBS +1%FBS containing a 1:20
dilution of both an anti-tetherin (PE conjugated)(eBiosciences) and CD4 (V450
conjugated)(BD Biosciences) antibodies for 15 minutes at room temperature. Cells
were then fixed and permeablized using BD Fix/Perm solution (BD Biosciences)
according to the manufactures protocol. Cells were resuspended in 100 L of wash
buffer containing a 1:20 dilution of an anti-p24 (FITC conjugated)(Beckman Coulter)
antibody for 30 minutes at room temperature. After three washes cells were fixed in 4%
paraformaldehyde (EMS) and analyzed by flow cytometry as described in chapter 3
(section 3.3.11).

B.2 Tetherin is temporally increased on HIV infected cells
Human PBMCs were infected with either NL4.3 or NL4.3
Vpu(A18H)
as described
previously in chapter 3 (section 3.3.7). Every 4 days, cells were removed and analyzed
by flow cytometry for infection by intracellular staining for p24 capsid. Tetherin and CD4
levels were also analyzed to determine the surface expression of each of each protein
in the infected and uninfected cell populations within each culture. At day 8 post
infection, sufficient infected cells were present to compare protein expression profiles
between p24 +/- cells (Figure 4.2A). At this time point, both NL4.3 and NL4.3
Vpu(A18H)

120

infected (p24+) cells exhibited an increase in surface tetherin levels when compared to
either uninfected cells in the same culture, or mock infected control cells (Figure 4.2B).
Interestingly, an increase in tetherin levels on the uninfected (p24-) cells in the cultures
was not observed. This could indicate the up-regulation on infected cells was caused
by innate cellular sensing of viral infection instead of a global response to interferon
levels in the culture medium. CD4 surface down-regulation was also confirmed for both
viruses, and consistent with HIV infection.

B.3 Tetherin expression could be manipulated by Vpu to mediate innate
interferon responses
The observation that tetherin levels were specifically increased on p24+ infected cell in
culture, at least at this time point, could result from the innate cellular pathways
designed to detect and combat viral infection. This could result in from an increase in
tetherin expression in response to anti-viral signals pathways such as IFN , leading to
an increase in tetherin localized to the surface. This increase is prevented by HIV’s
expression of Vpu, resulting in maintaining a steady state of low tetherin expression.
When the ability to antagonize tetherin is disrupted as in Vpu
(A18H)
, this increase in
tetherin expression is not efficiently countered, and leads to the higher surface
expression detected in our infections. Interesting, uninfected (p24-) cells within the
infected cultures do not exhibit this increase in tetherin expression. This implies that the
anti-viral response is specific to infected cells and not does occur from a systemic anti-
viral signaling event, and infected cells are detecting HIV infection through some form of
innate viral sensing not yet fully understood or appreciated.
121

Figure 4.2: Tetherin is temporally up-regulated on the surface of infected cells.
(A) FACS plots of PBMC cultures either uninfected or infected with the indicated
virus. Gates shown represent either infected (p24+) or uninfected (p24-) cells within
each culture and percent infected is indicated. (B) Histogram plots show the surface
expression of either tetherin or CD4 on both the infected and uninfected cells in each
culture. Mock levels are shown as shaded grey and NL4.3 and A18H infected
cultures are shown by red and green bars respectively. Graphs illustrate the mean
fluorescent intensity of either tetherin or CD4 on the p24+ or p24- cells in each
culture, and percentage of p24+ cells in culture is indicated above each bar.

tetherin
CD4
Mock
NL4.3
A18H
p24
Mock
p24 - p24 +
p24
NL4.3
p24 - p24 +
surface tetherin surface CD4
SSC
uninfected
p24- cells
infected
p24+ cells
Bst2 Thesis
Mock
NL4.3
A18H
0
500
1000
1500
2000
2500
p24-
p24+
surface tetherin MFI
2500
2000
1500
1000
500
0
Mock NL4.3 NL4.3
A18H
25.6
10.7
CD4 Thesis
Mock
NL4.3
A18H
0
200
400
600
800
1000
p24-
p24+
surface CD4 MFI
25.6
10.7
1000
800
600
400
200
0
Mock NL4.3 NL4.3
A18H
uninfected
p24- cells
infected
p24+ cells
Mock
NL4.3
A18H
CD4 Thesis
Mock
NL4.3
A18H
0
200
400
600
800
1000
p24-
p24+
CD4 Thesis
Mock
NL4.3
A18H
0
200
400
600
800
1000
p24-
p24+
p24
A18H
p24 - p24 +
10.7%
A
B
25.6% 0.2%
122

References

1. Gallo RC, Sarin PS, Gelmann EP, Robert-Guroff M, Richardson E, Kalyanaraman VS, Mann D,
Sidhu GD, Stahl RE, Zolla-Pazner S, Leibowitch J, Popovic M. 1983. Isolation of human T-cell
leukemia virus in acquired immune deficiency syndrome (AIDS). Science 220:865-867.

2. Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, Dauguet C, Axler-Blin
C, Vezinet-Brun F, Rouzioux C, Rozenbaum W, Montagnier L. 1983. Isolation of a T-
lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).
Science 220:868-871.

3. Clerici M, Stocks NI, Zajac RA, Boswell RN, Lucey DR, Via CS, Shearer GM. 1989. Detection of
three distinct patterns of T helper cell dysfunction in asymptomatic, human immunodeficiency
virus-seropositive patients. Independence of CD4+ cell numbers and clinical staging. The Journal
of clinical investigation 84:1892-1899.

4. Phillips AN, Lee CA, Elford J, Janossy G, Timms A, Bofill M, Kernoff PB. 1991. Serial CD4
lymphocyte counts and development of AIDS. Lancet 337:389-392.

5. Cates W, Jr., Bowen GS. 1989. Education for AIDS prevention: not our only voluntary weapon.
American journal of public health 79:871-874.

6. Nicholson JK, Cross GD, Callaway CS, McDougal JS. 1986. In vitro infection of human monocytes
with human T lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV).
Journal of immunology 137:323-329.

7. Gartner S, Markovits P, Markovitz DM, Kaplan MH, Gallo RC, Popovic M. 1986. The role of
mononuclear phagocytes in HTLV-III/LAV infection. Science 233:215-219.

8. Gaines H, von Sydow M, Pehrson PO, Lundbegh P. 1988. Clinical picture of primary HIV
infection presenting as a glandular-fever-like illness. Bmj 297:1363-1368.

9. Tindall B, Barker S, Donovan B, Barnes T, Roberts J, Kronenberg C, Gold J, Penny R, Cooper D.
1988. Characterization of the acute clinical illness associated with human immunodeficiency
virus infection. Archives of internal medicine 148:945-949.

10. Bacchetti P, Moss AR. 1989. Incubation period of AIDS in San Francisco. Nature 338:251-253.

11. Downs AM, Ancelle-Park RA, Costagliola D, Rigaut JP, Brunet JB. 1991. Transfusion-associated
AIDS cases in Europe: estimation of the incubation period distribution and prediction of future
cases. Journal of acquired immune deficiency syndromes 4:805-813.

12. Lifson AR, Buchbinder SP, Sheppard HW, Mawle AC, Wilber JC, Stanley M, Hart CE, Hessol NA,
Holmberg SD. 1991. Long-term human immunodeficiency virus infection in asymptomatic
homosexual and bisexual men with normal CD4+ lymphocyte counts: immunologic and virologic
characteristics. The Journal of infectious diseases 163:959-965.
123

13. Wertheim JO, Worobey M. 2009. Dating the age of the SIV lineages that gave rise to HIV-1 and
HIV-2. PLoS computational biology 5:e1000377.

14. Gao F, Bailes E, Robertson DL, Chen Y, Rodenburg CM, Michael SF, Cummins LB, Arthur LO,
Peeters M, Shaw GM, Sharp PM, Hahn BH. 1999. Origin of HIV-1 in the chimpanzee Pan
troglodytes troglodytes. Nature 397:436-441.

15. Peeters M, Honore C, Huet T, Bedjabaga L, Ossari S, Bussi P, Cooper RW, Delaporte E. 1989.
Isolation and partial characterization of an HIV-related virus occurring naturally in chimpanzees
in Gabon. Aids 3:625-630.

16. Peeters M, Fransen K, Delaporte E, Van den Haesevelde M, Gershy-Damet GM, Kestens L, van
der Groen G, Piot P. 1992. Isolation and characterization of a new chimpanzee lentivirus (simian
immunodeficiency virus isolate cpz-ant) from a wild-captured chimpanzee. Aids 6:447-451.

17. Van Heuverswyn F, Li Y, Neel C, Bailes E, Keele BF, Liu W, Loul S, Butel C, Liegeois F, Bienvenue
Y, Ngolle EM, Sharp PM, Shaw GM, Delaporte E, Hahn BH, Peeters M. 2006. Human
immunodeficiency viruses: SIV infection in wild gorillas. Nature 444:164.

18. Chen Z, Luckay A, Sodora DL, Telfer P, Reed P, Gettie A, Kanu JM, Sadek RF, Yee J, Ho DD,
Zhang L, Marx PA. 1997. Human immunodeficiency virus type 2 (HIV-2) seroprevalence and
characterization of a distinct HIV-2 genetic subtype from the natural range of simian
immunodeficiency virus-infected sooty mangabeys. Journal of virology 71:3953-3960.

19. Chen Z, Telfier P, Gettie A, Reed P, Zhang L, Ho DD, Marx PA. 1996. Genetic characterization of
new West African simian immunodeficiency virus SIVsm: geographic clustering of household-
derived SIV strains with human immunodeficiency virus type 2 subtypes and genetically diverse
viruses from a single feral sooty mangabey troop. Journal of virology 70:3617-3627.

20. Gao F, Yue L, White AT, Pappas PG, Barchue J, Hanson AP, Greene BM, Sharp PM, Shaw GM,
Hahn BH. 1992. Human infection by genetically diverse SIVSM-related HIV-2 in west Africa.
Nature 358:495-499.

21. Hirsch VM, Olmsted RA, Murphey-Corb M, Purcell RH, Johnson PR. 1989. An African primate
lentivirus (SIVsm) closely related to HIV-2. Nature 339:389-392.

22. Vallari A, Holzmayer V, Harris B, Yamaguchi J, Ngansop C, Makamche F, Mbanya D, Kaptue L,
Ndembi N, Gurtler L, Devare S, Brennan CA. 2011. Confirmation of putative HIV-1 group P in
Cameroon. Journal of virology 85:1403-1407.

23. Klaver B, Berkhout B. 1994. Comparison of 5' and 3' long terminal repeat promoter function in
human immunodeficiency virus. Journal of virology 68:3830-3840.

24. Temin HM. 1982. Function of the retrovirus long terminal repeat. Cell 28:3-5.

25. Valsamakis A, Schek N, Alwine JC. 1992. Elements upstream of the AAUAAA within the human
immunodeficiency virus polyadenylation signal are required for efficient polyadenylation in
vitro. Molecular and cellular biology 12:3699-3705.
124

26. Accola MA, Hoglund S, Gottlinger HG. 1998. A putative alpha-helical structure which overlaps
the capsid-p2 boundary in the human immunodeficiency virus type 1 Gag precursor is crucial for
viral particle assembly. Journal of virology 72:2072-2078.

27. Hill MK, Shehu-Xhilaga M, Crowe SM, Mak J. 2002. Proline residues within spacer peptide p1
are important for human immunodeficiency virus type 1 infectivity, protein processing, and
genomic RNA dimer stability. Journal of virology 76:11245-11253.

28. Berkowitz RD, Goff SP. 1994. Analysis of binding elements in the human immunodeficiency virus
type 1 genomic RNA and nucleocapsid protein. Virology 202:233-246.

29. Berkowitz RD, Ohagen A, Hoglund S, Goff SP. 1995. Retroviral nucleocapsid domains mediate
the specific recognition of genomic viral RNAs by chimeric Gag polyproteins during RNA
packaging in vivo. Journal of virology 69:6445-6456.

30. Dannull J, Surovoy A, Jung G, Moelling K. 1994. Specific binding of HIV-1 nucleocapsid protein
to PSI RNA in vitro requires N-terminal zinc finger and flanking basic amino acid residues. The
EMBO journal 13:1525-1533.

31. Ono A, Ablan SD, Lockett SJ, Nagashima K, Freed EO. 2004. Phosphatidylinositol (4,5)
bisphosphate regulates HIV-1 Gag targeting to the plasma membrane. Proceedings of the
National Academy of Sciences of the United States of America 101:14889-14894.

32. Leung K, Kim JO, Ganesh L, Kabat J, Schwartz O, Nabel GJ. 2008. HIV-1 assembly: viral
glycoproteins segregate quantally to lipid rafts that associate individually with HIV-1 capsids and
virions. Cell host & microbe 3:285-292.

33. Wyma DJ, Kotov A, Aiken C. 2000. Evidence for a stable interaction of gp41 with Pr55(Gag) in
immature human immunodeficiency virus type 1 particles. Journal of virology 74:9381-9387.

34. Cosson P. 1996. Direct interaction between the envelope and matrix proteins of HIV-1. The
EMBO journal 15:5783-5788.

35. Willey RL, Bonifacino JS, Potts BJ, Martin MA, Klausner RD. 1988. Biosynthesis, cleavage, and
degradation of the human immunodeficiency virus 1 envelope glycoprotein gp160. Proceedings
of the National Academy of Sciences of the United States of America 85:9580-9584.

36. Hallenberger S, Bosch V, Angliker H, Shaw E, Klenk HD, Garten W. 1992. Inhibition of furin-
mediated cleavage activation of HIV-1 glycoprotein gp160. Nature 360:358-361.

37. Buonocore L, Rose JK. 1990. Prevention of HIV-1 glycoprotein transport by soluble CD4 retained
in the endoplasmic reticulum. Nature 345:625-628.

38. Crise B, Buonocore L, Rose JK. 1990. CD4 is retained in the endoplasmic reticulum by the human
immunodeficiency virus type 1 glycoprotein precursor. Journal of virology 64:5585-5593.

39. Jacks T, Power MD, Masiarz FR, Luciw PA, Barr PJ, Varmus HE. 1988. Characterization of
ribosomal frameshifting in HIV-1 gag-pol expression. Nature 331:280-283.
125

40. Swanstrom R, Wills JW. 1997. Synthesis, Assembly, and Processing of Viral Proteins. In Coffin
JM, Hughes SH, Varmus HE (ed.), Retroviruses, Cold Spring Harbor (NY).

41. Dunn BM, Goodenow MM, Gustchina A, Wlodawer A. 2002. Retroviral proteases. Genome
biology 3:REVIEWS3006.

42. Whitcomb JM, Hughes SH. 1992. Retroviral reverse transcription and integration: progress and
problems. Annual review of cell biology 8:275-306.

43. Dayton AI, Sodroski JG, Rosen CA, Goh WC, Haseltine WA. 1986. The trans-activator gene of
the human T cell lymphotropic virus type III is required for replication. Cell 44:941-947.

44. Ruben S, Perkins A, Purcell R, Joung K, Sia R, Burghoff R, Haseltine WA, Rosen CA. 1989.
Structural and functional characterization of human immunodeficiency virus tat protein. Journal
of virology 63:1-8.

45. Brigati C, Giacca M, Noonan DM, Albini A. 2003. HIV Tat, its TARgets and the control of viral
gene expression. FEMS microbiology letters 220:57-65.

46. Purcell DF, Martin MA. 1993. Alternative splicing of human immunodeficiency virus type 1
mRNA modulates viral protein expression, replication, and infectivity. Journal of virology
67:6365-6378.

47. Pollard VW, Malim MH. 1998. The HIV-1 Rev protein. Annual review of microbiology 52:491-
532.

48. Malim MH, Hauber J, Le SY, Maizel JV, Cullen BR. 1989. The HIV-1 rev trans-activator acts
through a structured target sequence to activate nuclear export of unspliced viral mRNA. Nature
338:254-257.

49. Cochrane AW, Chen CH, Rosen CA. 1990. Specific interaction of the human immunodeficiency
virus Rev protein with a structured region in the env mRNA. Proceedings of the National
Academy of Sciences of the United States of America 87:1198-1202.

50. Gabuzda DH, Lawrence K, Langhoff E, Terwilliger E, Dorfman T, Haseltine WA, Sodroski J. 1992.
Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.
Journal of virology 66:6489-6495.

51. Simon JH, Malim MH. 1996. The human immunodeficiency virus type 1 Vif protein modulates
the postpenetration stability of viral nucleoprotein complexes. Journal of virology 70:5297-5305.

52. von Schwedler U, Song J, Aiken C, Trono D. 1993. Vif is crucial for human immunodeficiency
virus type 1 proviral DNA synthesis in infected cells. Journal of virology 67:4945-4955.

53. Madani N, Kabat D. 1998. An endogenous inhibitor of human immunodeficiency virus in human
lymphocytes is overcome by the viral Vif protein. Journal of virology 72:10251-10255.

126

54. Simon JH, Gaddis NC, Fouchier RA, Malim MH. 1998. Evidence for a newly discovered cellular
anti-HIV-1 phenotype. Nature medicine 4:1397-1400.

55. Sheehy AM, Gaddis NC, Choi JD, Malim MH. 2002. Isolation of a human gene that inhibits HIV-1
infection and is suppressed by the viral Vif protein. Nature 418:646-650.

56. Harris RS, Petersen-Mahrt SK, Neuberger MS. 2002. RNA editing enzyme APOBEC1 and some of
its homologs can act as DNA mutators. Molecular cell 10:1247-1253.

57. Mehle A, Strack B, Ancuta P, Zhang C, McPike M, Gabuzda D. 2004. Vif overcomes the innate
antiviral activity of APOBEC3G by promoting its degradation in the ubiquitin-proteasome
pathway. The Journal of biological chemistry 279:7792-7798.

58. Stopak K, de Noronha C, Yonemoto W, Greene WC. 2003. HIV-1 Vif blocks the antiviral activity
of APOBEC3G by impairing both its translation and intracellular stability. Molecular cell 12:591-
601.

59. Singh SP, Tungaturthi P, Cartas M, Tomkowicz B, Rizvi TA, Khan SA, Kalyanaraman VS,
Srinivasan A. 2001. Virion-associated HIV-1 Vpr: variable amount in virus particles derived from
cells upon virus infection or proviral DNA transfection. Virology 283:78-83.

60. Paxton W, Connor RI, Landau NR. 1993. Incorporation of Vpr into human immunodeficiency
virus type 1 virions: requirement for the p6 region of gag and mutational analysis. Journal of
virology 67:7229-7237.

61. Jenkins Y, McEntee M, Weis K, Greene WC. 1998. Characterization of HIV-1 vpr nuclear import:
analysis of signals and pathways. The Journal of cell biology 143:875-885.

62. Kulkosky J, BouHamdan M, Geist A, Pomerantz RJ. 1999. A novel Vpr peptide interactor fused
to integrase (IN) restores integration activity to IN-defective HIV-1 virions. Virology 255:77-85.

63. Agostini I, Navarro JM, Rey F, Bouhamdan M, Spire B, Vigne R, Sire J. 1996. The human
immunodeficiency virus type 1 Vpr transactivator: cooperation with promoter-bound activator
domains and binding to TFIIB. Journal of molecular biology 261:599-606.

64. Cohen EA, Dehni G, Sodroski JG, Haseltine WA. 1990. Human immunodeficiency virus vpr
product is a virion-associated regulatory protein. Journal of virology 64:3097-3099.

65. Wang L, Mukherjee S, Jia F, Narayan O, Zhao LJ. 1995. Interaction of virion protein Vpr of
human immunodeficiency virus type 1 with cellular transcription factor Sp1 and trans-activation
of viral long terminal repeat. The Journal of biological chemistry 270:25564-25569.

66. Zimmerman ES, Sherman MP, Blackett JL, Neidleman JA, Kreis C, Mundt P, Williams SA,
Warmerdam M, Kahn J, Hecht FM, Grant RM, de Noronha CM, Weyrich AS, Greene WC,
Planelles V. 2006. Human immunodeficiency virus type 1 Vpr induces DNA replication stress in
vitro and in vivo. Journal of virology 80:10407-10418.

127

67. Belzile JP, Duisit G, Rougeau N, Mercier J, Finzi A, Cohen EA. 2007. HIV-1 Vpr-mediated G2
arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase. PLoS pathogens 3:e85.

68. DeHart JL, Zimmerman ES, Ardon O, Monteiro-Filho CM, Arganaraz ER, Planelles V. 2007. HIV-1
Vpr activates the G2 checkpoint through manipulation of the ubiquitin proteasome system.
Virology journal 4:57.

69. Hrecka K, Gierszewska M, Srivastava S, Kozaczkiewicz L, Swanson SK, Florens L, Washburn MP,
Skowronski J. 2007. Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell
cycle. Proceedings of the National Academy of Sciences of the United States of America
104:11778-11783.

70. Willey RL, Maldarelli F, Martin MA, Strebel K. 1992. Human immunodeficiency virus type 1 Vpu
protein induces rapid degradation of CD4. Journal of virology 66:7193-7200.

71. Margottin F, Bour SP, Durand H, Selig L, Benichou S, Richard V, Thomas D, Strebel K, Benarous
R. 1998. A novel human WD protein, h-beta TrCp, that interacts with HIV-1 Vpu connects CD4 to
the ER degradation pathway through an F-box motif. Molecular cell 1:565-574.

72. Klimkait T, Strebel K, Hoggan MD, Martin MA, Orenstein JM. 1990. The human
immunodeficiency virus type 1-specific protein vpu is required for efficient virus maturation and
release. Journal of virology 64:621-629.

73. Sakai H, Tokunaga K, Kawamura M, Adachi A. 1995. Function of human immunodeficiency virus
type 1 Vpu protein in various cell types. The Journal of general virology 76 ( Pt 11):2717-2722.

74. Neil SJ, Sandrin V, Sundquist WI, Bieniasz PD. 2007. An interferon-alpha-induced tethering
mechanism inhibits HIV-1 and Ebola virus particle release but is counteracted by the HIV-1 Vpu
protein. Cell host & microbe 2:193-203.

75. Neil SJ, Zang T, Bieniasz PD. 2008. Tetherin inhibits retrovirus release and is antagonized by HIV-
1 Vpu. Nature 451:425-430.

76. Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, Johnson MC, Stephens EB, Guatelli
J. 2008. The interferon-induced protein BST-2 restricts HIV-1 release and is downregulated from
the cell surface by the viral Vpu protein. Cell host & microbe 3:245-252.

77. Dube M, Roy BB, Guiot-Guillain P, Binette J, Mercier J, Chiasson A, Cohen EA. 2010.
Antagonism of tetherin restriction of HIV-1 release by Vpu involves binding and sequestration of
the restriction factor in a perinuclear compartment. PLoS pathogens 6:e1000856.

78. Hauser H, Lopez LA, Yang SJ, Oldenburg JE, Exline CM, Guatelli JC, Cannon PM. 2010. HIV-1 Vpu
and HIV-2 Env counteract BST-2/tetherin by sequestration in a perinuclear compartment.
Retrovirology 7:51.

79. Goffinet C, Allespach I, Homann S, Tervo HM, Habermann A, Rupp D, Oberbremer L, Kern C,
Tibroni N, Welsch S, Krijnse-Locker J, Banting G, Krausslich HG, Fackler OT, Keppler OT. 2009.
128

HIV-1 antagonism of CD317 is species specific and involves Vpu-mediated proteasomal
degradation of the restriction factor. Cell host & microbe 5:285-297.

80. Mangeat B, Gers-Huber G, Lehmann M, Zufferey M, Luban J, Piguet V. 2009. HIV-1 Vpu
neutralizes the antiviral factor Tetherin/BST-2 by binding it and directing its beta-TrCP2-
dependent degradation. PLoS pathogens 5:e1000574.

81. Mitchell RS, Katsura C, Skasko MA, Fitzpatrick K, Lau D, Ruiz A, Stephens EB, Margottin-Goguet
F, Benarous R, Guatelli JC. 2009. Vpu antagonizes BST-2-mediated restriction of HIV-1 release
via beta-TrCP and endo-lysosomal trafficking. PLoS pathogens 5:e1000450.

82. Iwabu Y, Fujita H, Kinomoto M, Kaneko K, Ishizaka Y, Tanaka Y, Sata T, Tokunaga K. 2009. HIV-
1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane
interactions leading to lysosomes. The Journal of biological chemistry 284:35060-35072.

83. Giese SI, Woerz I, Homann S, Tibroni N, Geyer M, Fackler OT. 2006. Specific and distinct
determinants mediate membrane binding and lipid raft incorporation of HIV-1(SF2) Nef.
Virology 355:175-191.

84. Collins KL, Chen BK, Kalams SA, Walker BD, Baltimore D. 1998. HIV-1 Nef protein protects
infected primary cells against killing by cytotoxic T lymphocytes. Nature 391:397-401.

85. Wildum S, Schindler M, Munch J, Kirchhoff F. 2006. Contribution of Vpu, Env, and Nef to CD4
down-modulation and resistance of human immunodeficiency virus type 1-infected T cells to
superinfection. Journal of virology 80:8047-8059.

86. Deacon NJ, Tsykin A, Solomon A, Smith K, Ludford-Menting M, Hooker DJ, McPhee DA,
Greenway AL, Ellett A, Chatfield C, Lawson VA, Crowe S, Maerz A, Sonza S, Learmont J, Sullivan
JS, Cunningham A, Dwyer D, Dowton D, Mills J. 1995. Genomic structure of an attenuated quasi
species of HIV-1 from a blood transfusion donor and recipients. Science 270:988-991.

87. Schindler M, Munch J, Kutsch O, Li H, Santiago ML, Bibollet-Ruche F, Muller-Trutwin MC,
Novembre FJ, Peeters M, Courgnaud V, Bailes E, Roques P, Sodora DL, Silvestri G, Sharp PM,
Hahn BH, Kirchhoff F. 2006. Nef-mediated suppression of T cell activation was lost in a lentiviral
lineage that gave rise to HIV-1. Cell 125:1055-1067.

88. Gotz N, Sauter D, Usmani SM, Fritz JV, Goffinet C, Heigele A, Geyer M, Bibollet-Ruche F, Learn
GH, Fackler OT, Hahn BH, Kirchhoff F. 2012. Reacquisition of Nef-mediated tetherin antagonism
in a single in vivo passage of HIV-1 through its original chimpanzee host. Cell host & microbe
12:373-380.

89. Leonard CK, Spellman MW, Riddle L, Harris RJ, Thomas JN, Gregory TJ. 1990. Assignment of
intrachain disulfide bonds and characterization of potential glycosylation sites of the type 1
recombinant human immunodeficiency virus envelope glycoprotein (gp120) expressed in
Chinese hamster ovary cells. The Journal of biological chemistry 265:10373-10382.

90. Briggs JA, Simon MN, Gross I, Krausslich HG, Fuller SD, Vogt VM, Johnson MC. 2004. The
stoichiometry of Gag protein in HIV-1. Nature structural & molecular biology 11:672-675.
129

91. Jones PL, Korte T, Blumenthal R. 1998. Conformational changes in cell surface HIV-1 envelope
glycoproteins are triggered by cooperation between cell surface CD4 and co-receptors. The
Journal of biological chemistry 273:404-409.

92. Berger EA, Doms RW, Fenyo EM, Korber BT, Littman DR, Moore JP, Sattentau QJ,
Schuitemaker H, Sodroski J, Weiss RA. 1998. A new classification for HIV-1. Nature 391:240.

93. Dimitrov AS, Jacobs A, Finnegan CM, Stiegler G, Katinger H, Blumenthal R. 2007. Exposure of
the membrane-proximal external region of HIV-1 gp41 in the course of HIV-1 envelope
glycoprotein-mediated fusion. Biochemistry 46:1398-1401.

94. Preston BD, Poiesz BJ, Loeb LA. 1988. Fidelity of HIV-1 reverse transcriptase. Science 242:1168-
1171.

95. Sgarbanti M, Remoli AL, Marsili G, Ridolfi B, Borsetti A, Perrotti E, Orsatti R, Ilari R, Sernicola L,
Stellacci E, Ensoli B, Battistini A. 2008. IRF-1 is required for full NF-kappaB transcriptional
activity at the human immunodeficiency virus type 1 long terminal repeat enhancer. Journal of
virology 82:3632-3641.

96. Sune C, Garcia-Blanco MA. 1995. Sp1 transcription factor is required for in vitro basal and Tat-
activated transcription from the human immunodeficiency virus type 1 long terminal repeat.
Journal of virology 69:6572-6576.

97. Peterlin BM, Price DH. 2006. Controlling the elongation phase of transcription with P-TEFb.
Molecular cell 23:297-305.

98. Kao SY, Calman AF, Luciw PA, Peterlin BM. 1987. Anti-termination of transcription within the
long terminal repeat of HIV-1 by tat gene product. Nature 330:489-493.

99. Cujec TP, Cho H, Maldonado E, Meyer J, Reinberg D, Peterlin BM. 1997. The human
immunodeficiency virus transactivator Tat interacts with the RNA polymerase II holoenzyme.
Molecular and cellular biology 17:1817-1823.

100. Stoltzfus CM, Madsen JM. 2006. Role of viral splicing elements and cellular RNA binding
proteins in regulation of HIV-1 alternative RNA splicing. Current HIV research 4:43-55.

101. Jouvenet N, Simon SM, Bieniasz PD. 2009. Imaging the interaction of HIV-1 genomes and Gag
during assembly of individual viral particles. Proceedings of the National Academy of Sciences of
the United States of America 106:19114-19119.

102. Ivanchenko S, Godinez WJ, Lampe M, Krausslich HG, Eils R, Rohr K, Brauchle C, Muller B, Lamb
DC. 2009. Dynamics of HIV-1 assembly and release. PLoS pathogens 5:e1000652.

103. Kutluay SB, Bieniasz PD. 2010. Analysis of the initiating events in HIV-1 particle assembly and
genome packaging. PLoS pathogens 6:e1001200.

130

104. Dussupt V, Javid MP, Abou-Jaoude G, Jadwin JA, de La Cruz J, Nagashima K, Bouamr F. 2009.
The nucleocapsid region of HIV-1 Gag cooperates with the PTAP and LYPXnL late domains to
recruit the cellular machinery necessary for viral budding. PLoS pathogens 5:e1000339.

105. Wiegers K, Rutter G, Kottler H, Tessmer U, Hohenberg H, Krausslich HG. 1998. Sequential steps
in human immunodeficiency virus particle maturation revealed by alterations of individual Gag
polyprotein cleavage sites. Journal of virology 72:2846-2854.

106. Montessori V, Press N, Harris M, Akagi L, Montaner JS. 2004. Adverse effects of antiretroviral
therapy for HIV infection. CMAJ : Canadian Medical Association journal = journal de l'Association
medicale canadienne 170:229-238.

107. Mitsuya H, Weinhold KJ, Furman PA, St Clair MH, Lehrman SN, Gallo RC, Bolognesi D, Barry
DW, Broder S. 1985. 3'-Azido-3'-deoxythymidine (BW A509U): an antiviral agent that inhibits the
infectivity and cytopathic effect of human T-lymphotropic virus type III/lymphadenopathy-
associated virus in vitro. Proceedings of the National Academy of Sciences of the United States
of America 82:7096-7100.

108. Richman DD. 1990. Susceptibility to nucleoside analogues of zidovudine-resistant isolates of
human immunodeficiency virus. The American journal of medicine 88:8S-10S.

109. Wainberg MA, Brenner BG, Turner D. 2005. Changing patterns in the selection of viral
mutations among patients receiving nucleoside and nucleotide drug combinations directed
against human immunodeficiency virus type 1 reverse transcriptase. Antimicrobial agents and
chemotherapy 49:1671-1678.

110. Hutter G, Nowak D, Mossner M, Ganepola S, Mussig A, Allers K, Schneider T, Hofmann J,
Kucherer C, Blau O, Blau IW, Hofmann WK, Thiel E. 2009. Long-term control of HIV by CCR5
Delta32/Delta32 stem-cell transplantation. The New England journal of medicine 360:692-698.

111. Sabeti PC, Walsh E, Schaffner SF, Varilly P, Fry B, Hutcheson HB, Cullen M, Mikkelsen TS, Roy J,
Patterson N, Cooper R, Reich D, Altshuler D, O'Brien S, Lander ES. 2005. The case for selection
at CCR5-Delta32. PLoS biology 3:e378.

112. Bai J, Gorantla S, Banda N, Cagnon L, Rossi J, Akkina R. 2000. Characterization of anti-CCR5
ribozyme-transduced CD34+ hematopoietic progenitor cells in vitro and in a SCID-hu mouse
model in vivo. Molecular therapy : the journal of the American Society of Gene Therapy 1:244-
254.

113. Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, Goedert JJ, Buchbinder
SP, Vittinghoff E, Gomperts E, Donfield S, Vlahov D, Kaslow R, Saah A, Rinaldo C, Detels R,
O'Brien SJ. 1996. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion
allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter
AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE
Study. Science 273:1856-1862.

131

114. Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup
RA, Landau NR. 1996. Homozygous defect in HIV-1 coreceptor accounts for resistance of some
multiply-exposed individuals to HIV-1 infection. Cell 86:367-377.

115. Samson M, Libert F, Doranz BJ, Rucker J, Liesnard C, Farber CM, Saragosti S, Lapoumeroulie C,
Cognaux J, Forceille C, Muyldermans G, Verhofstede C, Burtonboy G, Georges M, Imai T, Rana
S, Yi Y, Smyth RJ, Collman RG, Doms RW, Vassart G, Parmentier M. 1996. Resistance to HIV-1
infection in caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene.
Nature 382:722-725.

116. Yan N, Chen ZJ. 2012. Intrinsic antiviral immunity. Nature immunology 13:214-222.

117. Kupzig S, Korolchuk V, Rollason R, Sugden A, Wilde A, Banting G. 2003. Bst-2/HM1.24 is a raft-
associated apical membrane protein with an unusual topology. Traffic 4:694-709.

118. Ohtomo T, Sugamata Y, Ozaki Y, Ono K, Yoshimura Y, Kawai S, Koishihara Y, Ozaki S, Kosaka
M, Hirano T, Tsuchiya M. 1999. Molecular cloning and characterization of a surface antigen
preferentially overexpressed on multiple myeloma cells. Biochemical and biophysical research
communications 258:583-591.

119. Nguyen DH, Hildreth JE. 2000. Evidence for budding of human immunodeficiency virus type 1
selectively from glycolipid-enriched membrane lipid rafts. Journal of virology 74:3264-3272.

120. Perez-Caballero D, Zang T, Ebrahimi A, McNatt MW, Gregory DA, Johnson MC, Bieniasz PD.
2009. Tetherin inhibits HIV-1 release by directly tethering virions to cells. Cell 139:499-511.

121. Jouvenet N, Neil SJ, Zhadina M, Zang T, Kratovac Z, Lee Y, McNatt M, Hatziioannou T, Bieniasz
PD. 2009. Broad-spectrum inhibition of retroviral and filoviral particle release by tetherin.
Journal of virology 83:1837-1844.

122. Kaletsky RL, Francica JR, Agrawal-Gamse C, Bates P. 2009. Tetherin-mediated restriction of
filovirus budding is antagonized by the Ebola glycoprotein. Proceedings of the National Academy
of Sciences of the United States of America 106:2886-2891.

123. Mansouri M, Viswanathan K, Douglas JL, Hines J, Gustin J, Moses AV, Fruh K. 2009. Molecular
mechanism of BST2/tetherin downregulation by K5/MIR2 of Kaposi's sarcoma-associated
herpesvirus. Journal of virology 83:9672-9681.

124. Sakuma T, Noda T, Urata S, Kawaoka Y, Yasuda J. 2009. Inhibition of Lassa and Marburg virus
production by tetherin. Journal of virology 83:2382-2385.

125. Gupta RK, Mlcochova P, Pelchen-Matthews A, Petit SJ, Mattiuzzo G, Pillay D, Takeuchi Y,
Marsh M, Towers GJ. 2009. Simian immunodeficiency virus envelope glycoprotein counteracts
tetherin/BST-2/CD317 by intracellular sequestration. Proceedings of the National Academy of
Sciences of the United States of America 106:20889-20894.

132

126. Le Tortorec A, Neil SJ. 2009. Antagonism to and intracellular sequestration of human tetherin by
the human immunodeficiency virus type 2 envelope glycoprotein. Journal of virology 83:11966-
11978.

127. Jia B, Serra-Moreno R, Neidermyer W, Rahmberg A, Mackey J, Fofana IB, Johnson WE,
Westmoreland S, Evans DT. 2009. Species-specific activity of SIV Nef and HIV-1 Vpu in
overcoming restriction by tetherin/BST2. PLoS pathogens 5:e1000429.

128. Sauter D, Schindler M, Specht A, Landford WN, Munch J, Kim KA, Votteler J, Schubert U,
Bibollet-Ruche F, Keele BF, Takehisa J, Ogando Y, Ochsenbauer C, Kappes JC, Ayouba A,
Peeters M, Learn GH, Shaw G, Sharp PM, Bieniasz P, Hahn BH, Hatziioannou T, Kirchhoff F.
2009. Tetherin-driven adaptation of Vpu and Nef function and the evolution of pandemic and
nonpandemic HIV-1 strains. Cell host & microbe 6:409-421.

129. Yang SJ, Lopez LA, Hauser H, Exline CM, Haworth KG, Cannon PM. 2010. Anti-tetherin activities
in Vpu-expressing primate lentiviruses. Retrovirology 7:13.

130. Zhang F, Wilson SJ, Landford WC, Virgen B, Gregory D, Johnson MC, Munch J, Kirchhoff F,
Bieniasz PD, Hatziioannou T. 2009. Nef proteins from simian immunodeficiency viruses are
tetherin antagonists. Cell host & microbe 6:54-67.

131. Strebel K, Klimkait T, Maldarelli F, Martin MA. 1989. Molecular and biochemical analyses of
human immunodeficiency virus type 1 vpu protein. Journal of virology 63:3784-3791.

132. Schubert U, Bour S, Ferrer-Montiel AV, Montal M, Maldarell F, Strebel K. 1996. The two
biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable
structural domains. Journal of virology 70:809-819.

133. McNatt MW, Zang T, Hatziioannou T, Bartlett M, Fofana IB, Johnson WE, Neil SJ, Bieniasz PD.
2009. Species-specific activity of HIV-1 Vpu and positive selection of tetherin transmembrane
domain variants. PLoS pathogens 5:e1000300.

134. Rong L, Zhang J, Lu J, Pan Q, Lorgeoux RP, Aloysius C, Guo F, Liu SL, Wainberg MA, Liang C.
2009. The transmembrane domain of BST-2 determines its sensitivity to down-modulation by
human immunodeficiency virus type 1 Vpu. Journal of virology 83:7536-7546.

135. Kobayashi T, Ode H, Yoshida T, Sato K, Gee P, Yamamoto SP, Ebina H, Strebel K, Sato H,
Koyanagi Y. 2011. Identification of amino acids in the human tetherin transmembrane domain
responsible for HIV-1 Vpu interaction and susceptibility. Journal of virology 85:932-945.

136. Douglas JL, Viswanathan K, McCarroll MN, Gustin JK, Fruh K, Moses AV. 2009. Vpu directs the
degradation of the human immunodeficiency virus restriction factor BST-2/Tetherin via a
{beta}TrCP-dependent mechanism. Journal of virology 83:7931-7947.

137. Conticello SG, Thomas CJ, Petersen-Mahrt SK, Neuberger MS. 2005. Evolution of the
AID/APOBEC family of polynucleotide (deoxy)cytidine deaminases. Molecular biology and
evolution 22:367-377.
133

138. Harris RS, Liddament MT. 2004. Retroviral restriction by APOBEC proteins. Nature reviews.
Immunology 4:868-877.

139. Holmes RK, Malim MH, Bishop KN. 2007. APOBEC-mediated viral restriction: not simply editing?
Trends in biochemical sciences 32:118-128.

140. Schafer A, Bogerd HP, Cullen BR. 2004. Specific packaging of APOBEC3G into HIV-1 virions is
mediated by the nucleocapsid domain of the gag polyprotein precursor. Virology 328:163-168.

141. Svarovskaia ES, Xu H, Mbisa JL, Barr R, Gorelick RJ, Ono A, Freed EO, Hu WS, Pathak VK. 2004.
Human apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G) is
incorporated into HIV-1 virions through interactions with viral and nonviral RNAs. The Journal of
biological chemistry 279:35822-35828.

142. Zennou V, Perez-Caballero D, Gottlinger H, Bieniasz PD. 2004. APOBEC3G incorporation into
human immunodeficiency virus type 1 particles. Journal of virology 78:12058-12061.

143. Bieniasz PD. 2003. Restriction factors: a defense against retroviral infection. Trends in
microbiology 11:286-291.

144. Stremlau M, Owens CM, Perron MJ, Kiessling M, Autissier P, Sodroski J. 2004. The cytoplasmic
body component TRIM5alpha restricts HIV-1 infection in Old World monkeys. Nature 427:848-
853.

145. Hatziioannou T, Perez-Caballero D, Yang A, Cowan S, Bieniasz PD. 2004. Retrovirus resistance
factors Ref1 and Lv1 are species-specific variants of TRIM5alpha. Proceedings of the National
Academy of Sciences of the United States of America 101:10774-10779.

146. Keckesova Z, Ylinen LM, Towers GJ. 2004. The human and African green monkey TRIM5alpha
genes encode Ref1 and Lv1 retroviral restriction factor activities. Proceedings of the National
Academy of Sciences of the United States of America 101:10780-10785.

147. Perron MJ, Stremlau M, Song B, Ulm W, Mulligan RC, Sodroski J. 2004. TRIM5alpha mediates
the postentry block to N-tropic murine leukemia viruses in human cells. Proceedings of the
National Academy of Sciences of the United States of America 101:11827-11832.

148. Yu Q, Konig R, Pillai S, Chiles K, Kearney M, Palmer S, Richman D, Coffin JM, Landau NR. 2004.
Single-strand specificity of APOBEC3G accounts for minus-strand deamination of the HIV
genome. Nature structural & molecular biology 11:435-442.

149. Ganser-Pornillos BK, Chandrasekaran V, Pornillos O, Sodroski JG, Sundquist WI, Yeager M.
2011. Hexagonal assembly of a restricting TRIM5alpha protein. Proceedings of the National
Academy of Sciences of the United States of America 108:534-539.

150. Diaz-Griffero F, Qin XR, Hayashi F, Kigawa T, Finzi A, Sarnak Z, Lienlaf M, Yokoyama S, Sodroski
J. 2009. A B-box 2 surface patch important for TRIM5alpha self-association, capsid binding
avidity, and retrovirus restriction. Journal of virology 83:10737-10751.
134

151. Sayah DM, Sokolskaja E, Berthoux L, Luban J. 2004. Cyclophilin A retrotransposition into TRIM5
explains owl monkey resistance to HIV-1. Nature 430:569-573.

152. Liao CH, Kuang YQ, Liu HL, Zheng YT, Su B. 2007. A novel fusion gene, TRIM5-Cyclophilin A in
the pig-tailed macaque determines its susceptibility to HIV-1 infection. Aids 21 Suppl 8:S19-26.

153. Brennan G, Kozyrev Y, Hu SL. 2008. TRIMCyp expression in Old World primates Macaca
nemestrina and Macaca fascicularis. Proceedings of the National Academy of Sciences of the
United States of America 105:3569-3574.

154. Newman RM, Hall L, Kirmaier A, Pozzi LA, Pery E, Farzan M, O'Neil SP, Johnson W. 2008.
Evolution of a TRIM5-CypA splice isoform in old world monkeys. PLoS pathogens 4:e1000003.

155. Virgen CA, Kratovac Z, Bieniasz PD, Hatziioannou T. 2008. Independent genesis of chimeric
TRIM5-cyclophilin proteins in two primate species. Proceedings of the National Academy of
Sciences of the United States of America 105:3563-3568.

156. Wilson SJ, Webb BL, Maplanka C, Newman RM, Verschoor EJ, Heeney JL, Towers GJ. 2008.
Rhesus macaque TRIM5 alleles have divergent antiretroviral specificities. Journal of virology
82:7243-7247.

157. Price AJ, Marzetta F, Lammers M, Ylinen LM, Schaller T, Wilson SJ, Towers GJ, James LC. 2009.
Active site remodeling switches HIV specificity of antiretroviral TRIMCyp. Nature structural &
molecular biology 16:1036-1042.

158. Hrecka K, Hao C, Gierszewska M, Swanson SK, Kesik-Brodacka M, Srivastava S, Florens L,
Washburn MP, Skowronski J. 2011. Vpx relieves inhibition of HIV-1 infection of macrophages
mediated by the SAMHD1 protein. Nature 474:658-661.

159. Laguette N, Sobhian B, Casartelli N, Ringeard M, Chable-Bessia C, Segeral E, Yatim A, Emiliani
S, Schwartz O, Benkirane M. 2011. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1
restriction factor counteracted by Vpx. Nature 474:654-657.

160. Goldstone DC, Ennis-Adeniran V, Hedden JJ, Groom HC, Rice GI, Christodoulou E, Walker PA,
Kelly G, Haire LF, Yap MW, de Carvalho LP, Stoye JP, Crow YJ, Taylor IA, Webb M. 2011. HIV-1
restriction factor SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase. Nature
480:379-382.

161. Lahouassa H, Daddacha W, Hofmann H, Ayinde D, Logue EC, Dragin L, Bloch N, Maudet C,
Bertrand M, Gramberg T, Pancino G, Priet S, Canard B, Laguette N, Benkirane M, Transy C,
Landau NR, Kim B, Margottin-Goguet F. 2012. SAMHD1 restricts the replication of human
immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside
triphosphates. Nature immunology 13:223-228.

162. Accola MA, Bukovsky AA, Jones MS, Gottlinger HG. 1999. A conserved dileucine-containing
motif in p6(gag) governs the particle association of Vpx and Vpr of simian immunodeficiency
viruses SIV(mac) and SIV(agm). Journal of virology 73:9992-9999.
135

163. Selig L, Pages JC, Tanchou V, Preveral S, Berlioz-Torrent C, Liu LX, Erdtmann L, Darlix J,
Benarous R, Benichou S. 1999. Interaction with the p6 domain of the gag precursor mediates
incorporation into virions of Vpr and Vpx proteins from primate lentiviruses. Journal of virology
73:592-600.

164. Baldauf HM, Pan X, Erikson E, Schmidt S, Daddacha W, Burggraf M, Schenkova K, Ambiel I,
Wabnitz G, Gramberg T, Panitz S, Flory E, Landau NR, Sertel S, Rutsch F, Lasitschka F, Kim B,
Konig R, Fackler OT, Keppler OT. 2012. SAMHD1 restricts HIV-1 infection in resting CD4(+) T
cells. Nature medicine 18:1682-1687.

165. Blake JA, Richardson JE, Bult CJ, Kadin JA, Eppig JT, Mouse Genome Database G. 2002. The
Mouse Genome Database (MGD): the model organism database for the laboratory mouse.
Nucleic acids research 30:113-115.

166. Renz JF, Lin Z, de Roos M, Dalal AA, Ascher NL. 1996. SCID mouse as a model for transplantation
studies. The Journal of surgical research 65:34-41.

167. Mosier DE, Gulizia RJ, Baird SM, Wilson DB, Spector DH, Spector SA. 1991. Human
immunodeficiency virus infection of human-PBL-SCID mice. Science 251:791-794.

168. Tary-Lehmann M, Lehmann PV, Schols D, Roncarolo MG, Saxon A. 1994. Anti-SCID mouse
reactivity shapes the human CD4+ T cell repertoire in hu-PBL-SCID chimeras. The Journal of
experimental medicine 180:1817-1827.

169. Hoffmann-Fezer G, Gall C, Zengerle U, Kranz B, Thierfelder S. 1993. Immunohistology and
immunocytology of human T-cell chimerism and graft-versus-host disease in SCID mice. Blood
81:3440-3448.

170. Traggiai E, Chicha L, Mazzucchelli L, Bronz L, Piffaretti JC, Lanzavecchia A, Manz MG. 2004.
Development of a human adaptive immune system in cord blood cell-transplanted mice. Science
304:104-107.

171. van der Loo JC, Hanenberg H, Cooper RJ, Luo FY, Lazaridis EN, Williams DA. 1998. Nonobese
diabetic/severe combined immunodeficiency (NOD/SCID) mouse as a model system to study the
engraftment and mobilization of human peripheral blood stem cells. Blood 92:2556-2570.

172. Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, Kotb M, Gillies SD, King M,
Mangada J, Greiner DL, Handgretinger R. 2005. Human lymphoid and myeloid cell development
in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.
Journal of immunology 174:6477-6489.

173. Brehm MA, Cuthbert A, Yang C, Miller DM, DiIorio P, Laning J, Burzenski L, Gott B, Foreman O,
Kavirayani A, Herlihy M, Rossini AA, Shultz LD, Greiner DL. 2010. Parameters for establishing
humanized mouse models to study human immunity: analysis of human hematopoietic stem cell
engraftment in three immunodeficient strains of mice bearing the IL2rgamma(null) mutation.
Clinical immunology 135:84-98.

136

174. Berges BK, Wheat WH, Palmer BE, Connick E, Akkina R. 2006. HIV-1 infection and CD4 T cell
depletion in the humanized Rag2-/-gamma c-/- (RAG-hu) mouse model. Retrovirology 3:76.

175. Watanabe S, Ohta S, Yajima M, Terashima K, Ito M, Mugishima H, Fujiwara S, Shimizu K,
Honda M, Shimizu N, Yamamoto N. 2007. Humanized NOD/SCID/IL2Rgamma(null) mice
transplanted with hematopoietic stem cells under nonmyeloablative conditions show prolonged
life spans and allow detailed analysis of human immunodeficiency virus type 1 pathogenesis.
Journal of virology 81:13259-13264.

176. Sun Z, Denton PW, Estes JD, Othieno FA, Wei BL, Wege AK, Melkus MW, Padgett-Thomas A,
Zupancic M, Haase AT, Garcia JV. 2007. Intrarectal transmission, systemic infection, and CD4+ T
cell depletion in humanized mice infected with HIV-1. The Journal of experimental medicine
204:705-714.

177. Joseph A, Zheng JH, Chen K, Dutta M, Chen C, Stiegler G, Kunert R, Follenzi A, Goldstein H.
2010. Inhibition of in vivo HIV infection in humanized mice by gene therapy of human
hematopoietic stem cells with a lentiviral vector encoding a broadly neutralizing anti-HIV
antibody. Journal of virology 84:6645-6653.

178. Holt N, Wang J, Kim K, Friedman G, Wang X, Taupin V, Crooks GM, Kohn DB, Gregory PD,
Holmes MC, Cannon PM. 2010. Human hematopoietic stem/progenitor cells modified by zinc-
finger nucleases targeted to CCR5 control HIV-1 in vivo. Nature biotechnology 28:839-847.

179. Terwilliger EF, Cohen EA, Lu YC, Sodroski JG, Haseltine WA. 1989. Functional role of human
immunodeficiency virus type 1 vpu. Proceedings of the National Academy of Sciences of the
United States of America 86:5163-5167.

180. Ewart GD, Sutherland T, Gage PW, Cox GB. 1996. The Vpu protein of human immunodeficiency
virus type 1 forms cation-selective ion channels. Journal of virology 70:7108-7115.

181. Neil SJ, Eastman SW, Jouvenet N, Bieniasz PD. 2006. HIV-1 Vpu promotes release and prevents
endocytosis of nascent retrovirus particles from the plasma membrane. PLoS pathogens 2:e39.

182. Lopez LA, Yang SJ, Hauser H, Exline CM, Haworth KG, Oldenburg J, Cannon PM. 2010. Ebola
virus glycoprotein counteracts BST-2/Tetherin restriction in a sequence-independent manner
that does not require tetherin surface removal. Journal of virology 84:7243-7255.

183. Miyagi E, Andrew AJ, Kao S, Strebel K. 2009. Vpu enhances HIV-1 virus release in the absence of
Bst-2 cell surface down-modulation and intracellular depletion. Proceedings of the National
Academy of Sciences of the United States of America 106:2868-2873.

184. Varthakavi V, Smith RM, Martin KL, Derdowski A, Lapierre LA, Goldenring JR, Spearman P.
2006. The pericentriolar recycling endosome plays a key role in Vpu-mediated enhancement of
HIV-1 particle release. Traffic 7:298-307.

185. Yang SJ, Lopez LA, Exline CM, Haworth KG, Cannon PM. 2011. Lack of adaptation to human
tetherin in HIV-1 group O and P. Retrovirology 8:78.
137

186. Vigan R, Neil SJ. 2010. Determinants of tetherin antagonism in the transmembrane domain of
the human immunodeficiency virus type 1 Vpu protein. Journal of virology 84:12958-12970.

187. Hout DR, Gomez LM, Pacyniak E, Miller JM, Hill MS, Stephens EB. 2006. A single amino acid
substitution within the transmembrane domain of the human immunodeficiency virus type 1
Vpu protein renders simian-human immunodeficiency virus (SHIV(KU-1bMC33)) susceptible to
rimantadine. Virology 348:449-461.

188. Shah AH, Sowrirajan B, Davis ZB, Ward JP, Campbell EM, Planelles V, Barker E. 2010.
Degranulation of natural killer cells following interaction with HIV-1-infected cells is hindered by
downmodulation of NTB-A by Vpu. Cell host & microbe 8:397-409.

189. Doehle BP, Chang K, Fleming L, McNevin J, Hladik F, McElrath MJ, Gale M, Jr. 2012. Vpu-
deficient HIV strains stimulate innate immune signaling responses in target cells. Journal of
virology 86:8499-8506.

190. Doehle BP, Chang K, Rustagi A, McNevin J, McElrath MJ, Gale M, Jr. 2012. Vpu mediates
depletion of interferon regulatory factor 3 during HIV infection by a lysosome-dependent
mechanism. Journal of virology 86:8367-8374.

191. Hotter D, Kirchhoff F, Sauter D. 2013. HIV-1 Vpu does not degrade the interferon regulatory
factor 3. Journal of virology.

192. Cao W, Bover L, Cho M, Wen X, Hanabuchi S, Bao M, Rosen DB, Wang YH, Shaw JL, Du Q, Li C,
Arai N, Yao Z, Lanier LL, Liu YJ. 2009. Regulation of TLR7/9 responses in plasmacytoid dendritic
cells by BST2 and ILT7 receptor interaction. The Journal of experimental medicine 206:1603-
1614.

193. Matsuda A, Suzuki Y, Honda G, Muramatsu S, Matsuzaki O, Nagano Y, Doi T, Shimotohno K,
Harada T, Nishida E, Hayashi H, Sugano S. 2003. Large-scale identification and characterization
of human genes that activate NF-kappaB and MAPK signaling pathways. Oncogene 22:3307-
3318.

194. Cocka LJ, Bates P. 2012. Identification of alternatively translated Tetherin isoforms with differing
antiviral and signaling activities. PLoS pathogens 8:e1002931.

195. Galao RP, Le Tortorec A, Pickering S, Kueck T, Neil SJ. 2012. Innate sensing of HIV-1 assembly by
Tetherin induces NFkappaB-dependent proinflammatory responses. Cell host & microbe 12:633-
644.

196. Du B, Wolf A, Lee S, Terwilliger E. 1993. Changes in the host range and growth potential of an
HIV-1 clone are conferred by the vpu gene. Virology 195:260-264.

197. Deora A, Ratner L. 2001. Viral protein U (Vpu)-mediated enhancement of human
immunodeficiency virus type 1 particle release depends on the rate of cellular proliferation.
Journal of virology 75:6714-6718.
138

198. Gibbs JS, Regier DA, Desrosiers RC. 1994. Construction and in vitro properties of SIVmac
mutants with deletions in "nonessential" genes. AIDS research and human retroviruses 10:333-
342.

199. Sato K, Misawa N, Fukuhara M, Iwami S, An DS, Ito M, Koyanagi Y. 2012. Vpu augments the
initial burst phase of HIV-1 propagation and downregulates BST2 and CD4 in humanized mice.
Journal of virology 86:5000-5013.

200. Schubert U, Bour S, Willey RL, Strebel K. 1999. Regulation of virus release by the macrophage-
tropic human immunodeficiency virus type 1 AD8 isolate is redundant and can be controlled by
either Vpu or Env. Journal of virology 73:887-896.

201. Kutsch O, Levy DN, Bates PJ, Decker J, Kosloff BR, Shaw GM, Priebe W, Benveniste EN. 2004.
Bis-anthracycline antibiotics inhibit human immunodeficiency virus type 1 transcription.
Antimicrobial agents and chemotherapy 48:1652-1663.

202. Ochsenbauer-Jambor C, Jones J, Heil M, Zammit KP, Kutsch O. 2006. T-cell line for HIV drug
screening using EGFP as a quantitative marker of HIV-1 replication. BioTechniques 40:91-100.

203. Abada P, Noble B, Cannon PM. 2005. Functional domains within the human immunodeficiency
virus type 2 envelope protein required to enhance virus production. Journal of virology 79:3627-
3638.

204. Adachi A, Gendelman HE, Koenig S, Folks T, Willey R, Rabson A, Martin MA. 1986. Production
of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells
transfected with an infectious molecular clone. Journal of virology 59:284-291.

205. Strebel K, Klimkait T, Martin MA. 1988. A novel gene of HIV-1, vpu, and its 16-kilodalton
product. Science 241:1221-1223.

206. Rouet F, Ekouevi DK, Chaix ML, Burgard M, Inwoley A, Tony TD, Danel C, Anglaret X, Leroy V,
Msellati P, Dabis F, Rouzioux C. 2005. Transfer and evaluation of an automated, low-cost real-
time reverse transcription-PCR test for diagnosis and monitoring of human immunodeficiency
virus type 1 infection in a West African resource-limited setting. Journal of clinical microbiology
43:2709-2717.

207. Ishikawa F, Yasukawa M, Lyons B, Yoshida S, Miyamoto T, Yoshimoto G, Watanabe T, Akashi
K, Shultz LD, Harada M. 2005. Development of functional human blood and immune systems in
NOD/SCID/IL2 receptor {gamma} chain(null) mice. Blood 106:1565-1573.

208. Kumar P, Ban HS, Kim SS, Wu H, Pearson T, Greiner DL, Laouar A, Yao J, Haridas V, Habiro K,
Yang YG, Jeong JH, Lee KY, Kim YH, Kim SW, Peipp M, Fey GH, Manjunath N, Shultz LD, Lee SK,
Shankar P. 2008. T cell-specific siRNA delivery suppresses HIV-1 infection in humanized mice.
Cell 134:577-586.

209. Kuhl BD, Sloan RD, Donahue DA, Bar-Magen T, Liang C, Wainberg MA. 2010. Tetherin restricts
direct cell-to-cell infection of HIV-1. Retrovirology 7:115.
139

210. Jolly C, Booth NJ, Neil SJ. 2010. Cell-cell spread of human immunodeficiency virus type 1
overcomes tetherin/BST-2-mediated restriction in T cells. Journal of virology 84:12185-12199.

211. Zhong P, Agosto LM, Ilinskaya A, Dorjbal B, Truong R, Derse D, Uchil PD, Heidecker G, Mothes
W. 2013. Cell-to-cell transmission can overcome multiple donor and target cell barriers imposed
on cell-free HIV. PloS one 8:e53138.

212. Zhang Q, Liu Z, Mi Z, Li X, Jia P, Zhou J, Yin X, You X, Yu L, Guo F, Ma J, Liang C, Cen S. 2011.
High-throughput assay to identify inhibitors of Vpu-mediated down-regulation of cell surface
BST-2. Antiviral research 91:321-329.

213. Pang XJ, Hu SQ, Zhang Y, Cen S, Jin Q, Guo F. 2012. [Establishment of a high-throughput
screening assay for interaction inhibitor between BST-2 and Vpu]. Bing du xue bao = Chinese
journal of virology / [bian ji, Bing du xue bao bian ji wei yuan hui] 28:633-638.

214. Tavano B, Galao RP, Graham DR, Neil SJ, Aquino VN, Fuchs D, Boasso A. 2013. Ig-like transcript
7, but not bone marrow stromal cell antigen 2 (also known as HM1.24, tetherin, or CD317),
modulates plasmacytoid dendritic cell function in primary human blood leukocytes. Journal of
immunology 190:2622-2630.

215. Bego MG, Mercier J, Cohen EA. 2012. Virus-activated interferon regulatory factor 7 upregulates
expression of the interferon-regulated BST2 gene independently of interferon signaling. Journal
of virology 86:3513-3527.

216. Boasso A, Shearer GM. 2008. Chronic innate immune activation as a cause of HIV-1
immunopathogenesis. Clinical immunology 126:235-242.

217. d'Ettorre G, Paiardini M, Ceccarelli G, Silvestri G, Vullo V. 2011. HIV-associated immune
activation: from bench to bedside. AIDS research and human retroviruses 27:355-364.

218. Benecke A, Gale M, Jr., Katze MG. 2012. Dynamics of innate immunity are key to chronic
immune activation in AIDS. Current opinion in HIV and AIDS 7:79-85.

219. Wilson EB, Yamada DH, Elsaesser H, Herskovitz J, Deng J, Cheng G, Aronow BJ, Karp CL, Brooks
DG. 2013. Blockade of chronic type I interferon signaling to control persistent LCMV infection.
Science 340:202-207.

220. Grandvaux N, tenOever BR, Servant MJ, Hiscott J. 2002. The interferon antiviral response: from
viral invasion to evasion. Current opinion in infectious diseases 15:259-267.

221. Rollason R, Korolchuk V, Hamilton C, Schu P, Banting G. 2007. Clathrin-mediated endocytosis of
a lipid-raft-associated protein is mediated through a dual tyrosine motif. Journal of cell science
120:3850-3858.

222. Mani M, Kandavelou K, Dy FJ, Durai S, Chandrasegaran S. 2005. Design, engineering, and
characterization of zinc finger nucleases. Biochemical and biophysical research communications
335:447-457.
140

223. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable
dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816-821.

224. Voit RA, McMahon MA, Sawyer SL, Porteus MH. 2013. Generation of an HIV resistant T-cell line
by targeted "stacking" of restriction factors. Molecular therapy : the journal of the American
Society of Gene Therapy 21:786-795.

225. Waheed AA, Ablan SD, Soheilian F, Nagashima K, Ono A, Schaffner CP, Freed EO. 2008.
Inhibition of human immunodeficiency virus type 1 assembly and release by the cholesterol-
binding compound amphotericin B methyl ester: evidence for Vpu dependence. Journal of
virology 82:9776-9781.

Asset Metadata

Creator Haworth, Kevin G. (author)

Core Title Molecular characterization of the HIV-1 Vpu protein and its role in antagonizing the cellular restriction factor BST-2/tetherin both in vitro and in vivo

Contributor Electronically uploaded by the author (provenance)

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 07/23/2013

Defense Date 06/03/2013

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag BST2,HIV,humanized mice,OAI-PMH Harvest,tetherin,Vpu

Format application/pdf (imt)

Language English

Advisor Cannon, Paula M. (committee chair), Camarero, Julio A. (committee member), Neamati, Nouri (committee member), Yuan, Weiming (committee member)

Creator Email kevin.haworth@gmail.com,kevin.haworth@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-295396

Unique identifier UC11294021

Identifier etd-HaworthKev-1815.pdf (filename),usctheses-c3-295396 (legacy record id)

Legacy Identifier etd-HaworthKev-1815-0.pdf

Dmrecord 295396

Document Type Dissertation

Format application/pdf (imt)

Rights Haworth, Kevin G.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA

Abstract (if available)

Abstract The cellular restriction factor BST-2/tetherin exerts a late stage anti-viral activity against enveloped viruses, retaining newly formed virions at the cell surface and effectively lowering virus output from infected cells. It is also a key player in the regulation of interferon production through binding to ILT7 on plasmacytoid dendritic cells, which also impacts the virus-host interaction. A number of pathogenic viruses have been found to express counter strategies to tetherin, with the human immunodeficiency viruses using Vpu. This suggests that a strong selective pressure exists for HIV to block tetherin expression. ❧ This potent antagonism of tetherin by Vpu could pose as a novel target for development of additional therapeutic compounds to combat viral infection. Currently, the accessory proteins of HIV are not the main targets for current HAART therapy of infected individuals. New compounds targeting these proteins could dramatically aid in the fight against HIV infection, especially considering these proteins are responsible for mediating cellular conditions, permitting efficient viral replication and dissemination. ❧ Current systems to investigate tetherin countermeasures are limited since in vitro cell cultures do not adequately recapitulate certain aspects of viral replication or innate immune activation. Additionally, in vivo testing using SIV or SHIV derivatives in monkey models of infection can be misrepresentative of HIV infections in humans. We are using humanized mice as a small animal model to study HIV-1 infections in vivo. The mice are created by engraftment of NOD/SCID/IL2Rγ-/- mice with human hematopoietic stem cells, which results in the development of mature human CD4+ T cells that support infection by HIV-1. ❧ We have created a series of Vpu deficient viruses in the NL4-3 backbone, using either a null mutant or a specific point mutation (A₁₈H) that blocks tetherin antagonism without affecting other functions of Vpu, such as CD4 degradation. Interestingly, neither of these Vpu mutations had any effect on virus replication that was apparent in the Jurkat-based JLTRG reporter cell line, but cause decreased replicative fitness in PBMC cultures. By infecting humanized mice with these viruses which vary in their ability to counteract tetherin, we were able to gain a better understanding on the role of tetherin restriction during the course of an in vivo infection. Together, these studies were meant to better characterize the interaction between tetherin and its viral antagonist Vpu by both determining the significance of restriction during infection, and discovering novel ways to disrupt this interaction through therapeutic intervention.