University of Southern California Dissertations and Theses

Virus customization of host protein machinery for efficient propagation

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Virus customization of host protein machinery for efficient propagation

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Content VIRUS CUSTOMIZATION OF HOST PROTEIN

MACHINERY FOR EFFICIENT PROPAGATION

By
Sara Pirooz
______________________________________________________________________

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)

August 2016
Copyright 2016 Sara Pirooz
Dedication
To Mahdi
This is for you.
I couldn’t have done this without you.

ii
Acknowledgements
The work described in this thesis would not have been possible without guidance from
many people.
I would like to, first and foremost, acknowledge the support of my advisor, Dr. Chengyu
Liang. None of this work would have been possible without her mentorship and
inspiration through countless discussions. Dr. Liang always brought in boundless energy
and brilliant thinking to stimulate fresh and insightful views on research problems. She
has always made herself available for discussion and I am grateful for her patience
during all the times I hassled her with requests or for advice. Her enthusiasm and
dedication to science has been a true inspiration to me.
I want to show my great appreciation to my committee members, Dr. Omid Akbari, Dr.
Pinghui Feng, Dr. Keigo Machida, and Dr. Weiming Yuan for their invaluable
suggestions, criticism, and spiritual influence as passionate scientists. It has been an
honor to work with such a distinguished group of scientists
I would also like to thank all my past and present colleagues in the Liang Lab for
providing me with amiable environment and support during these years.
Most importantly, I would like to express my appreciation of my loved ones who are
always a constant source of support: my exceptional parents who taught me the value
of unconditional love and support, my love for you has no boundaries; my brother who
always had trust in me; and my husband who has always been there for me, sharing my
happiness and success, cheering me up when science treated me unfriendly, my
daughter Ava for letting me enjoy this totally incredible feeling of being a mom, making
my world that much sweeter. Without my family’s endless support, I know I cannot be
who I am today or accomplish what I have accomplished.
iii
Table of Contents
Dedication ii
Acknowledgements iii
Table of Contents iv
List of Tables x
List of Figures xi
Abstract xiii
Introduction xv
Thesis Statement xxi
Chapter 1:
UVRAG is required for virus entry through combinatorial
interaction with the class C-Vps complex and SNAREs 1
1.1 Introduction 2
1.2 Classification and general properties of enveloped RNA viruses 5
1.2.1 Enveloped RNA virus structure 5
1.2.2 Enveloped RNA virus life cycle 8
1.3 Mechanism of enveloped RNA virus entry 10
1.3.1 Endocytic Trafficking 10
1.3.2 Virus interaction with Autophagy 14
iv
1.4 UV radiation resistance-associated (UVRAG)
as a universal connector 17
1.5 Materials and methods
1.5.1 Cell culture 20
1.5.2 Plasmid Constructs 21
1.5.3 Gene Knockdown by RNAi 22
1.5.4 Antibodies, Fluorscent Dyes, and Reagents 22
1.5.5 Flow Cytometry Analysis 23
1.5.6 Immunofluorescence and
Confocal Laser Scanning Microscopy 23
1.5.7 Virus Purification, Infection, and viral growth 24
1.5.8 Virus Labeling 25
1.5.9 VSV Internalization and VSV-G Protein
Expression Assay 26
1.5.10 MLV-GFP Pseudotyped Virus Preparation
and Entry Assay 26
1.5.11 Virus-Matrix protein release assay 27
1.5.12 Western Blot and Immunoprecipitation 27
1.5.13 Quantitative RT-PCR 28
1.5.14 Statistical Analysis 29
v
1.6 Results 30
1.6.1 VSV replication is suppressed in
UVRAG-deficient cells 30
1.6.2 Effect of UVRAG on viral infection
is not IFN-dependent 31
1.6.3 Autophagy-independent role of
UVRAG in viral infection 32
1.6.4 UVRAG is required for efficient viral entry 33
1.6.5 UVRAG promotes endocytic transport of virions 35
1.6.6 Domains of UVRAG required for virus entry 37
1.6.7 Class C vacuolar protein sorting (C-Vps), but not
Beclin 1, is required for UVRAG-mediated virus entry 38
1.6.8 UVRAG interacts with SNAREs 39
1.6.9 UVRAG enhances C-Vps interaction with
SNAREs and trans-SNARE assembly 40
1.6.10 Vamp8, but not Vamp7 is required for virus entry 42
1.6.11 Virus entry stimulates a specific complex assembly
of UVRAG, C-Vps, and SNAREs 43
1.6.12 Vamp8 recruitment to the virus-containing vesicles 44
1.7 Discussion 46
vi
1.8 Summary 50
1.9 Supplementary Data 54
1.9.1 UVRAG promotes VSV replication 54
1.9.2 Effect of IFN on UVRAG-mediated VSV infection 55
1.9.3 Autophagy-independent effect of UVRAG in
viral infection 56
1.9.4 UVRAG is essential for virus entry 57
1.9.5 Domain of UVRAG in viral infection 59
1.9.6 Role of Beclin 1 and C-Vps in
UVRAG-mediated viral infection 60
1.9.7 UVRAG mediates efficient interaction of the
C-Vps complex with SNAREs 62
1.9.8 Effect of SNARE proteins in
UVRAG-mediated virus entry 63
1.9.9 UVRAG facilitates the recruitment of VAMP8,
but not VAMP7, to VSV-containing vesicles 64
1.10 Conclusions 65
vii
Chapter 2:
Kaposi’s sarcoma-associated herpesvirus-Bcl-2-mediated
regulation of NM23-H2 is required for KSHV infection 67
2.1 Introduction 68
2.2 Materials and methods 76
2.2.1 Cell culture and viruses 76
2.2.2 Plasmid Constructs 77
2.2.3 Western Blot and Immunoprecipitation 77
2.2.4 Antibodies, Fluorscent Dyes, and Reagents 78
2.2.5 Gene Knockdown by shRNA 78
2.2.6 Immunofluorescence and
Confocal Laser Scanning Microscopy 78
2.2.7 Flow Cytometry Analysis 79
2.2.8 RNA extraction and qRT-PCR 79
2.2.9 Gel filtration chromatography 79
2.2.10 NDPK Assay 80
2.2.11 Statistical Analysis 80
2.3 Results 81
2.3.1 NM23-H2, but not NM23-H1,
is a new target of KSHV-viral Bcl-2 81
viii
2.3.2 KSHV-viral Bcl-2 stabilizes NM23-H2
at the post translational level 84
2.3.3 KSHV-Bcl-2-E
14
and NM23-H2-N
69

are the functional residues required 85
2.3.4 KSHV-viral Bcl-2 co-localizes with
and stabilizes NM23-H2 87
2.3.5 NM23-H2, similar to KSHV-Bcl-2 is required
for KSHV lytic replication 89
2.3.6 vBcl-2 doesn’t regulate NM23-H2 or
NM23-H2 target genes transcriptional activity 91
2.3.7 vBcl-2 influences the disruption of
NM23-H2 oligomerization 93
2.3.8 vBcl-2 positively regulates
NDPK activity of NM23-H2 96
2.4 Discussion 98
2.5 Future Perspectives 103
Bibliography 105

ix
List of Tables
Table 1.1 RNA virus classification

x
List of Figures
Figure 1.1 Enveloped RNA virus life cycle
Figure 1.2.1 VSV virion structure
Figure 1.2.2 VSV life cycle
Figure 1.3.1 VSV clathrin-mediated endocytosis
Figure 1.3.2 Schematic representation of Autophagy pathway
Figure 1.4 UVRAG as the interlocutor between autophagy and homotypic endosome
fusion
Figure 1.6.1 UVRAG is required for VSV replication
Figure 1.6.4 UVRAG is required for efficient virus entry
Figure 1.6.6 Domains of UVRAG required for virus entry
Figure 1.6.8 UVRAG functions together with C-Vps and SNAREs
Figure 1.6.10 Distinct roles of the SNARE proteins in virus entry
Figure S1.9.1 UVRAG promotes VSV replication
Figure S1.9.2 Effect of IFN on UVRAG-mediated VSV infection
Figure S1.9.3 Autophagy-independent effect of UVRAG in viral infection
Figure S1.9.4 UVRAG is essential for virus entry
Figure S1.9.5 Domain of UVRAG in viral infection
Figure S1.9.6 Role of Beclin 1 and C-Vps in UVRAG-mediated viral infection
Figure S1.9.7 UVRAG mediates efficient interaction of the C-Vps complex
with SNAREs
Figure S1.9.8 Effect of SNARE proteins in UVRAG-mediated virus entry.
Figure S1.9.9 UVRAG facilitates the recruitment of VAMP8, but not VAMP7,
to VSV-containing vesicles
xi
Figure 2.1 KSHV life cycle
Figure 2.3.1 NM23-H2, but not NM23-H1, is a new target of KSHV-viral Bcl-2
Figure 2.3.2 KSHV-viral Bcl-2 stabilizes NM23-H2 at the post translational level
Figure 2.3.3 KSHV-Bcl-2-E
14
and NM23-H2-N
69
are the functional residues required
Figure 2.3.4 KSHV-viral Bcl-2 co-localizes with and stabilizes NM23-H2
Figure 2.3.5 NM23-H2, similar to KSHV-Bcl-2 is required for KSHV lytic replication
Figure 2.3.6 vBcl-2 doesn’t regulate NM23-H2 or NM23-H2 target genes transcriptional
activity
Figure 2.3.7 vBcl-2 influences the disruption of NM23-H2 oligomerization
Figure 2.3.8 vBcl-2 positively regulates NDPK activity of NM23-H2

xii
Abstract
The ability of a virus to initiate a complex journey into the host cell leading to efficient
replication and assembly ultimately dictates the fate of an incoming virus. Many
enveloped viruses begin their infection process by exploiting the endomembrane
system to enter the host cell. Then the virus hijacks the host cell's machinery, to reach
the appropriate replication site. The new viruses are assembled and released via
budding at the host cell membrane, thereby maintaining the integrity of the host cell
membrane and allowing initiation of a new infection. Therefore, for virus egress, again
viruses need to exploit the endomembrane system to traffic and eventually reach an
open door for exit. Every step of the viral life cycle, from entry to budding, is
orchestrated through interactions with cellular proteins. Accordingly, viruses hijack and
use these proteins utilizing any achievable mechanism to their benefit, making it
challenging to trace and target viruses. Although virology studies have been fruitful, how
viruses commandeer so many diverse pathways, yet still escape, remains elusive.
Our results show a vision of different the viral strategies in modulating both cellular
signaling and its own life cycle. In chapter 1, I focus on RNA viruses and show that upon
entry, the RNA enveloped viruses represented by VSV and Influenza hitchhike and
remodel the endomembrane system and traffic within, eventually escaping endosomal
organelles for their genome release. Here I revealed that the UV-radiation resistance-
associated gene (UVRAG), an autophagic tumor suppressor well known for regulating
autophagy and intracellular trafficking, is a critical factor for RNA enveloped virus entry.
xiii
In chapter 2, I shift my focus to DNA viruses, and by exploring the role of Kaposi’s
sarcoma-associated herpesvirus (KSHV)-Bcl-2 (B-cell lymphoma 2), an autophagy-
regulating gene, I show that the KSHV-Bcl-2 interaction with NM23-H2, an abundant
NDP kinase is required for virus lytic replication. This interaction of KSHV-Bcl-2 is
genetically separable from its antiapoptotic and antiautophagic functions. Surprisingly,
the role of NM23-H2 in virus lytic replication is independent of its catalytic activity. My
results will probably reveal a novel function of the KSHV-Bcl-2/NM23-H2 complex in
viral lytic replication, which is genetically separate from its previous known functions.
New aspects of host regulation by viruses offer potential therapeutic targets, echoing
the importance of earlier viral-based discoveries.

xiv
Introduction
Viruses are masters of deception, manipulating their host’s cells environment for a
favorable home to grow and spread. Disrupting host cells normally tight signaling
mechanisms is advantageous for viruses. As obligate parasites, viruses are unable to
reproduce on their own, therefore rely on co-opting their host’s cellular machinery, like
an occupying army taking over a local factory. They are especially good at overriding or
bypassing built-in control mechanisms. A viruses ability to hijack cellular processes
relies on the ability to mimic the structure or function of cellular proteins. Viruses are
well known to encode proteins that share magnificently similar activities to cellular
proteins, but still different enough that they’re beneficial to the virus.
Humans have been battling viruses since the beginning of their existence. Ranging from
the common cold to cancer, viral diseases persistently remain a burden in todays
society. 15-20% of human cancers [1] [2] as well as many other chronic disorders are
initiated by viral infections. Finding cures for ongoing viral infections and the need to
prevent viral diseases has fueled decades of scientific research by studying these
organisms and their interactions with the host. Studies of virus-hosts interactions have
not only led to the development of vaccines and antiviral treatments, but also revealed
fundamental principles of cell and structural biology, immunology and biochemistry. For
example, cellular RNA splicing mechanisms were elucidated by studying adenoviruses
[3] [4] .Crystallization of tobacco mosaic virus [5] was a huge leap for structural biology.
In the field of immunology, the discovery of interferon [6] and major histocompatibility
(MHC) locus restriction [7]. In the field of cellular biochemistry, there are landmark
xv
discoveries showing that single-stranded RNA can be transcribed into double-stranded
DNA by the enzyme reverse transcriptase [8] [9]. Cell cycle regulatory mechanisms
have also been revealed through the study of virus interactions with host cells [10].
Viruses target different cell cycle regulators and studying how viruses manipulate host
cells will continue to reveal cell cycle regulatory mechanisms [10]. Lastly, the
understanding of signal transduction pathways and their contribution to diseases has
also been heavily influenced by the study of viruses [11] [12].
The complex set of interactions between a virus and a host organism starts off with the
viruses gaining entry into cells. Viruses must then deliver their genome into the host
cells to initiate replication Finally, viral proteins and genomic material are assembled into
mature virus particles and the virus exits the cell by budding through the plasma
membrane.
In Chapter 1, I begin to tackle the first complex interaction between a virus and its host
leading to an efficient virus infection: entry. In this study, I have focused on using the
Vesicular stomatitis virus (VSV) and influenza virus. VSV is a well-characterized, acid-
activated, enveloped, prototypic RNA virus that belongs to the family of Rhabdoviridae.
VSV due to its ease of handling, high titers, and broad cell tropism; the virus and its
membrane glycoprotein G (VSVG), has long been used as a model to study
endocytosis [13] [14] [15] [16], secretory traffic [17] [18] [19], pseudotyping of retroviral
vectors for gene delivery [20] [21] [22], viral entry mechanisms, intracellular trafficking,
and overall host endosome biology [23]. Therefore, I use this prototypic virus as a
model to push forward my studies. Influenza viruses are enveloped RNA viruses,
belonging to the family Orthomyxoviridae. Influenza is a serious disease that can lead to
xvi
hospitalization and sometimes even death. There have been four major flu pandemics
in the last century in 1918, 1957, 1968 and 2009. All four pandemics were caused by
different types of influenza viruses that had been introduced to humans from animals.
Current therapeutics against influenza include those that target the virus itself, like the
uncoating process after entry as well as those that prevent viral budding. While there
are therapeutics in development that target entry, currently there are none clinically
available. Studying flu virus entry may lead to efficient discovery of novel, broad-
spectrum viral entry inhibitors, therefore, bypassing influenza virus pandemic potential.
Some viruses penetrate cells directly through the plasma membrane, however, most
take advantage of existing portals of entry evolved for nutrient uptake and receptor
signaling while moving within the endosomal apparatus of the cell to reach the site for
replication [23]. Although it is established that transport to the acidic endosome is
required for release of VSV and other negative-strand RNA viruses, such as influenza
virus [23] [14], the molecular machinery that ferries these viruses through the
endomembrane remains poorly understood. Moreover, the ride is not free for the virus
because the lysosome station on the endocytic pathway is a potentially hazardous
environment that degrades viral components and reports infection. How the virus traffics
within, and eventually escapes from, specific endosomal organelles before lysosome
degradation is an important question in virus entry.
Evidence shows that endocytic transport is not a random priming event hitchhiked by
the virus, but an important strategy the virus exploits to route itself to a specific
compartment for fusion and genome release [23]. This process is achieved by SNARE-
regulated sequential fusion between virion-containing vesicles and intracellular
xvii
organelles, including early endosomes, late endosomes (LEs), and lysosomes [24].
Fusogenic trans-SNARE complexes are assembled to form a four-helix bundle
consisting of glutamine Qa-, Qb-, and Qc-SNAREs embedded in one membrane and
arginine (R)-SNAREs embedded in the other [24]. Specifically, syntaxin 7 (STX7; Qa),
Vti1b (Qb), and STX8 (Qc) on the LE, when paired with VAMP7 (R), mediate the LE
fusion with the lysosome, but when paired with VAMP8 (R), regulate homotypic fusion of
the LEs [25]. The upstream process regulating LE-associated SNARE pairing relies on
the class C Vps (hereafter referred to as C-Vps) complex, composed of Vps11, Vps16,
Vps18, and Vps33 as core subunits [26] [27].
Here, I found that UVRAG, an autophagic tumor suppressor, is required for the entry of
the prototypic negative-strand RNA virus, including influenza A virus and VSV, by a
mechanism independent of IFN and autophagy. UVRAG mediates viral endocytic
transport and membrane penetration through interactions with the C-Vps tethering
complex and endosomal glutamine-containing SNAREs [syntaxin 7 (STX7), STX8, and
vesicle transport through t-SNARE homolog 1B (Vti1b)], leading to the assembly of a
fusogenic trans-SNARE complex involving vesicle-associated membrane protein
(VAMP8), but not VAMP7. Indeed, UVRAG stimulates VAMP8 translocation to virus-
bearing endosomes. Inhibition of VAMP8, but not VAMP7, significantly reduces viral
entry. My data indicates that UVRAG, in concert with C-Vps, regulates viral entry by
assembling a specific fusogenic SNARE complex. Thus, UVRAG governs downstream
viral entry, highlighting an important pathway capable of potential antiviral therapeutics
In Chapter 2 I focus on tackling the second step for an efficient virus infection:
replication. KSHV evades host defenses through tight suppression of autophagy by
xviii
targeting its signal transduction: by viral Bcl-2. vBcl-2 family proteins in addition to being
negative regulators of apoptosis, have emerged as critical negative regulators of
autophagy. vBcl-2 is known to be required for KSHV lytic gene expression, viral DNA
replication, and progeny virus production. More importantly, the antiapoptotic and
antiautophagic functions of vBcl-2 are not required for KSHV lytic replication [28]. The
identified glutamic acid 14 (E
14
) of vBcl-2 is critical for KSHV lytic replication, indicating
a novel function of vBcl-2 in the virus life cycle. However the why and how of this novel
function remains elusive. My results show the formation of a distinct complex between
vBcl-2 with NM23-H2, an abundant NDP Kinase. Surprisingly. this interaction does not
require the catalytic activity of NM23-H2. In general, NDP kinases have been
acknowledged as a large family of highly conserved proteins that participate in
nucleotide metabolism, yet the actual biological significance of their enzymatic activity
has remained elusive. In my studies, I show that Nm23-H2 is also required for KSHV
replication similar to vBcl-2. I further dissect how vBcl-2 may target and regulate this
important host factor. vBcl-2 stabilizes NM23-H2 protein levels at the post-translational
level, without touching the transcriptional expression. NM23-H2 exists as stable
hexamers in solution, however vBcl-2 disrupts the oligomerization of NM23-H2. And
finally, although based on the crystal structure of NM23-H2, the active site of NM23
doesn't seem to be affected in monomeric and hexameric state, vBcl-2 increases the
NDPK activity of NM23-H2. This may indicate a novel function of NM23-H2 in virus
replication and eventually propagation.
Elucidating viral-cell entry mechanisms and their interaction with the host trafficking
network, their replication process, and their egress will continue to open new paths in
xix
understanding the gaps in disease mechanisms as well as basic cellular biology.
Discoveries from basic research on the cell biology of viral life cycle are actively being
translated into the development of host-targeted therapies for treatment purposes.

xx
Thesis Statement
In my dissertation research I aim to elucidate how viruses customize host protein
machinery for efficient entry and infection by dissecting two distinct virus-host
interactions steps: entry and replication.
xxi
Chapter 1
UVRAG IS REQUIRED FOR VIRUS ENTRY THROUGH COMBINATORIAL
INTERACTION WITH THE CLASS C-Vps COMPLEX AND SNAREs
1
1.1 Introduction
RNA viruses display diverse evolutionary patterns, which makes it difficult to predict
future viral disease emergences. Viruses with RNA as their genetic material can quickly
adapt to and exploit varying conditions because of the high error rates of the virus
enzymes (polymerases) that replicate their genomes. It comes as no surprise, then, that
recent prominent examples of emerging or re-emerging diseases are caused by RNA
viruses. Currently approved therapies for infections with these pathogens are limited,
and the development of specific viral enzyme-targeted inhibitors is frequently
complicated by the inherently high mutation rate of viral RNA polymerases and the rapid
development of resistance [29]. New approaches are being advocated for the
development of novel antivirals by the targeting of host processes, versus the virus itself
[30]. However, since the cell-centric approach to antiviral development requires
substantial understanding of the host-pathogen interactions that control virus infection,
there is still a long way to go in bridging the remaining gaps in our current knowledge .
Enveloped RNA viruses include pathogens with exceptional histories of morbidity, such
as HIV, and influenza, Ebola, as well as important emerging pathogens such as the Zika
virus (Table 1). The medical importance and fascinating biology of these viruses has
made them the subject of ongoing research, which has revealed shared themes that
underlie many viral infection strategies. First, the limited coding capacity of RNA viruses
forces them to use host cell factors to extend their capabilities. Second, viral proteins
often achieve this by mimicking the structures and functions of cellular proteins. Third,
different viruses, as well as cells themselves, frequently use similar mechanisms to
accomplish difficult molecular transformations. All steps of the RNA virus life cycle
2
3
Virus Family Virus Capsid Nucleic
Acid
Disease Organs
Affected
Transmission
Togaviridae Rubella virus,
alphavirus
Enveloped ss Rubella,
encephalitis
Skin,
brain
droplets,
contact,
mosquito
Flaviviridae Dengue virus,
hepatitis C
virus, yellow
fever virus,
arbovirus
Enveloped ss Dengue
Fever,
Yellow
fever,
arboviral
encephalitis
, NANB
hepatitis
Blood,
muscles,
liver,
brain
Mosquito,
contact with
body fluids
Arenaviridae Lymphocytic
choriomeningitis
virus
Enveloped ss(-) Lymphocyti
c
Choriomeni
ngitis (LCM)
Blood Droplets,
contact with
bodily fluids,
vertical
transmission
Orthomyxoviridae Influenzavirus
A,
influenzavirus
B,
influenzavirus
C, isavirus,
thogotovirus
Enveloped ss(-) Influenza Respirato
ry tract
Droplets
Paramyxoviridae Measles virus,
mumps virus,
respiratory
syncytial virus,
Rinderpest
virus, canine
distemper virus
Enveloped ss(-) Respiratory
Syncytial,
measles,
mumps
Respirato
ry tract,
skin,
salivary
glands,
blood
Droplets,
contact
Bunyaviridae California
encephalitis
virus, hantavirus
Enveloped ss(-) Encephalitis
, Hantavirus
Pulmonary
Syndrome
Brain,
blood,
lungs
Arthropod,
Droplets
Rhabdoviridae Rabies virus,
Vesicular
stomatitis virus
Enveloped ss(-) Rabies Brain,
spinal
cord
Contact with
bodily fluids
Filoviridae Ebola virus,
Marburg virus
Enveloped ss(-) Ebola Whole
body
Bodily fluids
Bornaviridae Borna disease
virus
Enveloped ss(-) Borna
disease
Blood,
brain
Droplets
Retroviridae HIV Enveloped ss AIDS T-
lymphocy
tes
Contact with
body fluids
Table 1. Enveloped RNA virus classification
Schematic modified from endmemo.com.
require the participation of host factors (Fig. 1.1). The molecular interplay between host
factors and invading viruses is a continuous process that governs virus host range,
tissue specificity, and viral pathogenesis and is a driving force in viral evolution [31].

The
study of host factors in the viral life cycle provides insights into their normal cellular
functions and helps identify attractive targets for developing new, effective, antiviral
drugs.
4
Figure 1.1. Enveloped RNA virus life cycle
Taken from Stapleford and Miller et al.
1.2 Classification and general properties of enveloped RNA viruses
1.2.1 Enveloped RNA virus structure
In recent years, remarkable progress has been made in determining the structures of
key viral proteins and of the virion capsids of RNA viruses, highlighting the significance
in the structure of many of these viruses. Viruses have a very simple structure, yet are
so small that a microscope is necessary to visualize them. A virus particle alone
consists of a viral genome or genetic material (typically several kilo basepairs (kb) and
usually containing several genes), surrounded by a protein shell called a capsid.
Enveloped RNA virus protein shell is enclosed in a membrane called an envelope. All
enveloped RNA viruses carry an RNA genome contained by an envelope made of virus-
modified host membrane. Viruses are then further categorized into separate families
based on the different modes of entry into host cells, how they strategize replication and
transcription, their assembly process and the structure of their virions. VSV, is a well-
characterized, enveloped, prototypic RNA virus that belongs to the family of
Rhabdoviridae. VSV has been long used to study the endomembrane system. The
genome this enveloped RNA virus is always encapsidated by proteins generating the
nucleocapsid (NC). These proteins play essential roles in virus replication, maintain the
structure of the NC, and may help the virus avoid the host immune system. To initiate a
productive infection, the virus must translocate their genome across the cell membrane.
For enveloped viruses, this step is mediated by virally encoded glycoproteins that
promote both receptor recognition and membrane fusion.
5
Glycoproteins on the surface of the envelope serve to identify and bind to receptor sites
on the host's membrane. The viral envelope then fuses with the host's membrane,
allowing the capsid and viral genome to enter and infect the host. Therefore, the
envelope mediates interaction with the surfaces of host cells so that the NC can infect
these cells and replicate. Envelopes are lipid bilayers into which virus glycoproteins are
inserted, giving each virus unique antigenic determinants. The lipid composition of a
virus envelope usually reflects that of the host cell membranes [32] [33].
VSV virions are bullet-shaped particles 180 nm in length and 80 nm in diameter [34].
VSV has one of the the simplest RNA genome consisting of five VSV genes encoding
nucleocapsid (N), phospho- (P), matrix (M), glyco- (G), and large (L) proteins (Fig.
1.2.1). VSV genome is encapsidated with the N protein to form a nuclease-resistant
6
Figure 1.2.1. VSV virion structure
Taken from Li and Zhang et al.
helical N-RNA complex that is the functional template for mRNA synthesis as well as
genomic RNA replication. The N-RNA complex is tightly associated with the viral RNA-
dependent RNA polymerase (RdRp), which is comprised of the 241-kDa L protein
catalytic subunit and the 29-kDa essential P phospho-protein cofactor, and results in the
assembly of a viral ribonucleoprotein (RNP) complex [35] [36]. This structure contains
the minimum virus encoded components of the VSV RNA synthesis machinery [37]. The
RNP complex is further surrounded by the M protein which plays a crucial role in virus
assembly, budding, and maintenance of the structural integrity of the virus particle [38].
The outer membrane of virion is the envelope composed of a cellular lipid bilayer. The
transmembrane G protein is anchored in the viral envelope, which is essential for
receptor binding and cell entry [39]. G protein forms the spikes that protrude from the
viral surface. G protein plays a critical role during the initial steps of virus infection since
it is responsible for virus attachment to specific receptors.
Furthermore, Influenza virus has also long been used as a model system to understand
virus entry. However, despite the results, much is still unknown about the viruses
infection pathway. Influenza is an enveloped virus with a lipid-bilayer membrane and
three types of integral membrane proteins, Hemagglutinin (HA), Neuraminidase (NA)
and M2. Beneath the lipid membrane is a protein matrix made of M1. A segmented
genome, comprised of eight single-stranded RNAs packed into ribonucleoprotein
(vRNP) complexes, resides inside the virus. The protein components of the vRNPs are
the nucleoproteins (NP) and RNA-polymerases (PA, PB1 and PB2). Other proteins
encoded by the influenza genes include NS1 and NS2 [40].
7
1.2.2 Enveloped RNA virus life cycle
Most viruses with RNA genomes complete their life cycle in the cytoplasm of infected
cells. Upon attaching to an unknown cell receptor(s), VSV enters host cells via receptor
mediated endocytosis [41]. Following low pH triggered fusion and uncoating, the RNP
complex is delivered into the cytoplasm where RNA synthesis and viral replication
occurs (Fig. 1.2.2) [42]. During primary transcription, the input RdRp recognizes the
specific signals in the N-RNA template to transcribe six discrete RNAs: a 47-nucleotide
leader RNA, which is neither capped nor polyadenylated, and 5 mRNAs that are capped
and methylated at the 5’ end and polyadenylated at the 3’end. These mature mRNAs
are then translated by host ribosomes to yield functional viral proteins which are
8
Figure 1.2.2. VSV life cycle
Taken from Li and Zhang et al.
required for viral genome replication. During replication, the RdRP initiates at the 3’ end
of the genome and synthesizes a full-length complementary antigenome, which
subsequently serves as template for synthesis of full-length progeny genomes. These
progeny genomes can then be utilized as templates for secondary transcription, or
assembled into infectious particles. Finally, viral proteins and genomic RNA are
assembled into complete virus particles and the virus exits the cell by budding through
the plasma membrane.
Influenza viruses infect cells in a multi-step process: (i) viruses are internalization via
receptor-mediated endocytosis; (ii) internalized viruses are trafficked along the
endocytic pathway to acidic late endosomes; (iii) exposure to low pH triggers HA
catalyzed fusion between the viral and endosomal membranes, releasing vRNPs; (iv)
vRNPs are imported into the nucleus for viral gene expression and replication [40].

9
1.3 Mechanism of enveloped RNA virus entry
1.3.1 Endocytic Trafficking
The majority of viruses enter cells via endocytosis [40], similar to the transport of
extracellular cargo through the endocytic route. The best characterized of these is the
clathrin-mediated pathway (Fig. 1.3.1) that is essential for productive infection by many
viruses [43]. Clathrin-mediated endocytosis occurs by the concentration of receptors
and bound ligands into clathrin-coated pits (CCPs), which pinch off from the plasma
membrane to form clathrin-coated vesicles (CCVs) [40] [44] [45] [46] [47] [48]. These
CCVs then uncoat and fuse with other endocytic vesicles or endosomes [40].
10
Figure 1.3.1. VSV clathrin-mediated endocytosis
Taken from ViralZone Swiss Institute of Bioinformatics
Endocytosis provides a pathway through which viruses can pass through by exploiting
intrinsic properties of endocytic vesicles to migrate, enabling the virus to hijack the cell’s
own machinery to be transported past the plasma membrane [40]. As with virtually all
endocytic cargoes, regardless of the mechanism of uptake, most viruses internalized by
endocytosis are delivered into intracellular compartments, endosomes, and then
penetrate the endosome to release their genome [40]. For enveloped viruses, this
process occurs via a protein-catalyzed membrane fusion process between viruses and
endosomes. Many viruses will use endosomal environmental cues, usually low pH, to
trigger the membrane fusion/penetration reactions that deliver the viral genetic material
to the cytoplasm [49] [50]. Fusion or penetration from endosomes offers several
potential advantages to a virus: it ensures that there is no cortical actin barrier to
contend with, and limits the display of viral components on the surface of the cell where
they may be targets for the immune system [43]. Therefore, taking advantage of existing
portals of entry evolved for nutrient uptake and receptor signaling while moving within
the endosomal apparatus of the cell to reach the site for replication, is a smart strategy
on the viruses end . Although it is established that transport to the acidic endosome is
required for release of VSV and other negative-strand RNA viruses, such as influenza
virus [23] [14], the molecular machinery that ferries these viruses through the
endomembrane remains poorly understood. Moreover, the ride is not free for the virus
because the lysosome station on the endocytic pathway is a potentially hazardous
environment that degrades viral components and reports infection. How the virus traffics
within, and eventually escapes from, specific endosomal organelles before lysosome
degradation is another important question in virus entry.
11
RNA viruses are highly pathogenic and cause many severe diseases in humans and
animals. These viruses generally use existing cellular pathways to enter cells, which
involves intensive interaction with the endomembrane network, offering the endocytic
pathway as an attractive scheme for therapeutic intervention. Molecular mechanisms
governing virus entry remain incompletely understood. I found that UVRAG, well known
for regulating intracellular trafficking by accelerating late endocytic transport through
pairing with the C-Vps complex [51], is a critical factor for virus entry. Understanding the
mechanism that allows the virus to interact with late endocytic organelles could identify
the specific set of proteins that have a role in virus entry, which will help us to design
specific therapeutic agents against endocytic virus entries.
Virus exploits endocytic transportation to route itself to a specific compartment for fusion
and genome release [23]. This process is achieved by SNARE-regulated sequential
fusion between virion-containing vesicles and intracellular organelles, including early
endosomes, late endosomes (LEs), and lysosomes [24]. Fusogenic trans-SNARE
complexes are assembled to form a four-helix bundle consisting of glutamine (Q) a-,
Qb-, and Qc-SNAREs embedded in one membrane and arginine (R)-SNAREs
embedded in the other [24]. Specifically, syntaxin 7 (STX7; Qa), Vti1b (Qb), and STX8
(Qc) on the LE, when paired with VAMP7 (R), mediate the LE fusion with the lysosome,
but when paired with VAMP8 (R), regulate homotypic fusion of the LEs [25]. The
upstream process regulating LE-associated SNARE pairing relies on the C-Vps,
composed of Vps11, Vps16, Vps18, and Vps33 as core subunits [26] [27]. A recent
study indicated that C-Vps interaction with endosomal Q-SNAREs allows the assembly
12
of fusogenic trans-SNAREs leading to vesicle fusion [26]. The C-Vps complex was also
found to mediate the entry of Ebola virus [52], but the mechanism of action remains
unknown.
13
1.3.2 Virus Interaction with Autophagy
As obligate intracellular parasites, viruses need to exploit existing host cell processes
for successful survival. Viruses use diverse strategies and mechanisms to govern host
defenses in order to co-exist with their hosts. One such mechanism is “Autophagy”
meaning“self-eating” in Greek, which has been the subject of research for the last 60
years [53]. Autophagy is one of the most basic and primitive processes of eukaryotic
cells that is essential for survival and immune defense. It mediates the removal and
recycling of cytoplasmic constituents via lysosomal degenerative pathways [54], thereby
prolonging cell survival and maintaining celllular homeostasis by balancing metabolism
in cells. Autophagy takes place in all eukaryotic cells and involves the generation of
double-membrane structures that engulf and sequester portions of the cytoplasm into
enclosed autophagosome vesicles. This phagophore then expands to form double-
membrane vesicles, termed autophagosomes. Autophagosomes mature by fusing with
endosomes and/or lysosomes to form autolysosomes, where degradation of the
internal contents occurs by resident lysosomal hydrolases [54].
Two central proteins in the regulation of autophagy are mTORC1 and Beclin 1 [55]. In
mammals, mTORC1 negatively regulates autophagy under normal conditions through
the binding, phosphorylation and consequent inactivation of the proautophagy ULK1/2
kinase complex [54]. However, upon inactivation of mTORC1, the autophagy cascade is
triggered by the assembly of the ULK complex [56]. Downstream of mTORC1, Beclin 1
is at the heart of the class III PI-3-Kinase (hVps34) regulatory complex, which is
essential for autophagosome formation [57]. The Vps34-Beclin 1 complex is a central
regulator of autophagosome biogenesis [54]. Vps34 is a class III phosphatidylinositol-3-
14
kinase (PI3K) that generates phosphatidylinostiol-3-phosphate (PI3P), which recruits
many Autophagy related genes (ATGs) and autophagy regulators to the membrane
nucleation site (phagophore). Autophagy was originally identified in mammalian cells,
however genetic screens in yeast have provided us tools to identify autophagy-related
genes (ATGs) [58]. The activity of Vps34 is regulated by its interaction with Beclin 1,
normally sequestered away from Vps34 by Bcl-2, and a variety of Beclin 1 binding
partners [53]. Furthermore, Vps34-Beclin 1 binding proteins also regulate the fusion of
the autophagosome with endocytic compartments and the lysosome [53] (Fig. 1.3.2).
Therefore, Beclin 1, by itself, does not carry any enzymatic activity but functions as a
scaffold to recruit autophagy activating cofactors including Atg14, UVRAG, endophilin
15
Figure 1.3.2. Schematic Representation of Autophagy Pathway.
Taken from http://dx.doi.org/10.3389/fncel.2014.00450
B1 (Bif-1), and Ambra-1, or autophagy counteracting cofactors such as Bcl-2, NAF-1,
and Rubicon [56][59][60][61]. Numerous other cellular factors either have been shown
or hypothesized to regulate autophagy, many of which are significant in viral infections
[62] [63] [64]. From the nucleation to maturation of autophagosomes, ATGs play
fundamental roles. LC3, the mammalian ortholog of Atg8, is a common marker of the
autophagosome, and has significant roles in the expansion and closure of the
autophagosome, as well as the selective recruitment of cargo to the autophagosomal
membrane [54]. LC3-II associates exclusively with the autophagosome, defining it as a
classical marker of autophagosome maturation [54].
Cellular autophagy is an intracellular membrane trafficking pathway which delivers
cytoplasmic material to lysosomes where the material is degraded thereby removing
unwanted material. How cells dispose of waste is of critical importance to all living
organisms. Depending on the circumstances, autophagy can be pro-survival or pro-
death, can enhance viral infection or aid in generation of an anti-viral response.
Autophagy is clearly a double-edged sword that can cut both ways [65] [57].
UVRAG is well known for its crucial role in both pathways: activating autophagy by
pairing with Beclin 1 and accelerating late endocytic transport by pairing with the C-Vps
complex [51] [66]. Yet, despite its importance, the functionality of UVRAG in viral
infection has not been addressed.
16
1.4 UV radiation resistance-associated (UVRAG) as a universal connector
Endocytic and autophagic trafficking are 2 tightly regulated processes evolved for the
transport and clearance of extracellular and intracellular cargoes, respectively, and
which are both implicated in viral infection and pathogenesis. Most enveloped viruses
exploit the endosomal machinery to route themselves to specific compartments for
membrane fusion and delivery of their genetic material, while autophagy acts as an
essential part of the host antiviral defense mechanism. UVRAG is well known for its
crucial role in both pathways [66] [51]. Yet, despite its importance, until recently, the
functionality of UVRAG in viral infection had not been addressed. For the first time, our
studies investigated the cellular outcome of UVRAG in viral infection.
Autophagy, an intracellular membrane trafficking pathway, is induced under conditions
of cellular stress such as nutrient withdrawal, DNA damage, or viral infections. During
this process, isolation membranes (the phagosome) derived from the ER are formed
and expanded into double-membrane autophagosomes that engulf the cellular cargo for
[67]. Autophagosomes then fuse either with late endosomes or lysosomes forming the
autolysosome where the cargo is degraded by lysosomal enzymes. In contrast to
intracellular cargo, extracellular cargo that is internalized by endocytosis, endocytosed
macromolecules are delivered to lysosomes via endosomes, bypassing the autophagy
pathway. This pathway involves a “kiss and run” as well as a complete fusion event, the
latter dependent on the presence of a target membrane (t-) SNARE complex and a
vesicle membrane (v-) SNARE complex which form a tetrameric trans-SNARE complex.
Late endosomes can either fuse with other late endosomes (“homotypic fusion”) or
other lysosomes (“heterotypic fusion”), the former requiring Syntaxin-8, VAMP-8, and
17
Vti-1b and the latter Syntaxin -11, VAMP-7 as well as Vti-1b. Both homotypic and
heterotypic fusion are also dependent on Rab5GTPase, Rab7GTPase,
N-ethylmaleimide (NEM) sensitive factor (NSF) and its soluble associated proteins
(SNAPs) [67].
Both the autophagy and the endocytic pathway are not completely separated but united
by a common interlocutor, UVRAG. UVRAG activates autophagy by associating with
Beclin-1 (thus extending the phagophore) as well as inducing the maturation of the
autophagosome in later stages via binding to C-Vps, and accelerating endocytic
transport by activating Rab7 (thus leading to heterotypic fusion of the late endosome
with lysosomes). In addition to the role in autophagy and fusion of endosomes with
18
Figure 1.4. UVRAG as the interlocutor between
autophagy and homotypic endosome fusion
Taken from virologytidbits.blogspot.com
lysosomes, UVRAG also plays a role in the integrity of the ER and the Golgi as well in
the DNA damage response [67].
Although the role of various autophagy related proteins in viral infections is well
established, however, the role of UVRAG in particular in mediating viral entry remained
elusive. A potential role for UVRAG in mediating viral entry can be postulated from the
observation that in UVRAG deficient cells cell surface receptor degradation is
downregulated. In conclusion, UVRAG might be an universal connector between viral
entry, autophagy, and endocytic trafficking [67].
19
1.5 Materials and methods
1.5.1 Cell culture
NIH 3T3, HeLa, HCT116, African green monkey kidney epithelial Vero cells, BHK-21,
immortalized mouse embryonic fibroblasts, and HEK 293T cells were cultured in DMEM
supplemented with 10% (vol/vol) FBS, 2 mM L-glutamine, and 1% penicillin/
streptomycin (Gibco–BRL). Transient transfection was performed with Fugene 6
(Roche), Lipofectanine 2000 (In- vitrogen), or calcium phosphate (Clontech). NIH 3T3,
HeLa, and HCT116 stable cell lines were established using a standard pro- tocol of
selection with 2 µg/mL puromycin (Sigma–Aldrich), as described previously [23] [14].
UV-radiation resistance-associated gene (UVRAG)
+ /+
(E14TG2a.4) and UVRAG
+/−
(AC0571) feeder-free mouse ES cells were obtained from the Mutant Mouse Regional
Resource Center (MMRRC) and maintained at a comparable passage in Glasgow
Minimum Essential Medium (Sigma) with 15% (vol/vol) FBS (Invitrogen), following the
MMRRC’s cell culture protocol (www/mmrrc.org/strains/E14/ ctr_protocol.pdf). UVRAG
gene trapping was confirmed by sequencing and Western blotting. UVRAG
+/−
ES cells
stably expressing UVRAG were generated using a puromycin selection method [24]
after nucleofection with a mouse ES cell kit (VPH 1001; Lonza). Beclin 1 KO ES cells
were cultured as previously described [25] [26].
20
1.5.2 Plasmid Constructs
The Flag-, HA-, or GST-tagged WT UVRAG and the UVRAG
C2
, UVRAG
CCD
(CCD,
coiled-coil domain), UVRAG
270-CT
(270-CT, 270-C-terminal), UVRAG
ΔC2
, and
UVRAG
ΔCCD
mutants and the HA-tagged vacuolar protein sorting 16 (Vps16) and
Vps18 plasmids used in this study have been described in our previous work [14]. All
constructs were confirmed by sequencing, using an ABI PRISM 377 automatic DNA
sequencer (Applied Biosystems).
1.5.3 Gene Knockdown by RNAi
All siRNA and shRNA constructs were purchased from Open Biosystems. The siRNA
targeting sequences (5′ to 3′) are as follows:
siRNA 5’-sequence-3’
Vps16 AGCCACATCCTCATCTGAGACATCCTT
Vps18 AGGCTCATGCACAGCTGATTGCTGG
Beclin1 AAGATCCTGGACCGTGTCACC
siRNA duplex sense antisense
STX7 CAGAGGAUCUCUUCUAACAtt UGUUAGAAGAGAUCCUCUGtt
GAGAGAGAAUCUUCUAUCAtt UGAUAGAAGAUUCUCUCUCtt
CAAGGGCAGCAGAUUAUCAtt UGAUAAUCUGCUGCCCUUGtt
STX8 CCUCUUGGAUGAUCUUGUAtt UACAAGAUCAUCCAAGAGGtt
CCUUUCCUCUAUCAUAAGUtt ACUUAUGAUAGAGGAAAGGtt
Vti1b
GUAGAGAAUGAGCAUAUGAtt UCAUAUGCUCAUUCUCUACtt
CCAAGAGUAGACUGGUAAAtt UUUACCAGUCUACUCUUGGtt
siRNA duplex
21
All siRNAs were transfected using FuGENE reagent (Roche) according to the
manufacturer’s protocol.
1.5.4 Antibodies, Fluorscent Dyes, and Reagents
The following antibodies were used in this study: polyclonal rabbit anti-UVRAG (U7058;
Sigma–Aldrich) at 1:1,000, monoclonal mouse anti-UVRAG (SAB4200005; Sigma–
Aldrich) at 1:200, polyclonal goat anti–VSV-glycoprotein G (VSV-G; sc-138076; Santa
Cruz Biotechnology) at 1:500, anti-VSVM (8G5F11; KeraFAST), polyclonal rabbit Atg5
(2630; Cell Signaling) at 1:1,000, monoclonal rabbit Atg7 (NBP1-95872; Novus
Biologicals) at 1:1,000, polyclonal rabbit Atg16 (NBP1-54386; Novus Biologicals) at
1:1,000, monoclonal mouse anti-lysobisphosphatidic acid (Z-SLBPA; Echelon) at 1:200,
rabbit anti–Beclin 1 (3738; Cell Signaling) at 1:1,000, polyclonal rabbit anti-Vps18
(NBP1-70366; Novus Biologicals) at 1:200, polyclonal goat anti-Vps16 (sc-86939; Santa
Cruz Biotechnology) at 1:200, anti-Vps11 (SAB2700359; Sigma), anti-syntaxin 7 (STX7;
GGAAGAUUCUCCGUUCAAUtt AUUGAACGGAGAAUCUUCCtt
VAMP8 CCACAUCUGAGCACUUCAAtt UUGAAGUGCUCAGAUGUGGtt
CACUGGUGCCUUCUCUUAAtt UUAAGAGAAGGCACCAGUGtt
GCAUUUCUUGGGUCCUUAGtt CUAAGGACCCAAGAAAUGCtt
VAMP7 CCAGACUACUUACGGUUCAtt UGAACCGUAAGUAGUCUGGtt
CAAGGAUAUGAGAGAACAAtt UUGUUCUCUCAUAUCCUUGtt
CCAUUUAACUGCAGUGUAAtt UUACACUGCAGUUAAAUGGtt
sense antisense siRNA duplex
shRNA 5’-sequence-3’
UVRAG
ACGGAACATTGTTAATAGAAAT
22
110-072; Synaptic Systems), anti-STX8 (110-083; Synaptic Systems), anti-Vti1b
(164-002; Synaptic Systems), anti-VAMP7 (ab36195; Abcam), anti-VAMP8 (ab89158;
Abcam), anti-actin (SC-47778; Santa Cruz Biotechnology) at 1:2,000; anti-Flag (F1804;
Sigma), anti-GST (2624; Cell Signaling), and anti-HA (MMS-101P; Covance).
HRP-labeled or fluorescently labeled secondary antibody conjugates were purchased
from Molecular Probes (Invitrogen). Purified rabbit IgG was purchased from Pierce. All
other chemical and reagents were obtained from Sigma–Aldrich unless otherwise noted.
1.5.5 Flow Cytometry Analysis
For flow cytometry preparation, cells were treated with cell dissociation buffer (Sigma),
washed twice with PBS, and then fixed in 4% (wt/vol) paraformaldehyde/PBS. Cells
were assayed for phycoerythrin fluorescence gated on cells that were positive for GFP
fluorescence. At least 10,000 cells were analyzed for each sample in triplicate.
1.5.6 Immunofluorescence and Confocal Laser Scanning Microscopy
Immunofluorescence microscopy was carried out as described previously [66]. Briefly,
cells were fixed with 4% (wt/vol) paraformaldehyde (20 min at room temperature). After
fixation, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with
10% (vol/vol) goat serum (Gibco) for 1 h. Primary antibody staining was carried out
using antiserum or purified antibody in 1% goat serum for 2 h at room temperature or
overnight in 4 °C. Cells were then extensively washed with PBS and incubated with
Alexa 488-, Alexa 594-, and/or Alexa 633-conjugated secondary antibodies for 1 h,
followed by DAPI staining. Cells were mounted using Vectashield (Vector Laboratories,
Inc.). Confocal images were acquired using a Nikon Eclipse C1 laser scanning
23
microscope fitted with a Nikon objective (plan apochromat, 1.4 N.A.) with a
magnification of 60× and Nikon image software.
1.5.7 Virus Purification, Infection, and viral growth curve
Vesicular stomatitis virus (VSV; Indiana serotype) and recombinant VSV containing a
GFP reporter were propagated in BHK-21 cells, and viral titers were determined by
plaque assay on BHK-21 cells as described previously [27]. Briefly, the supernatant of
infected culture was harvested when 75% of the cells showed a cytopathic effect and
was clarified by centrifugation at 1,000 × g for 30 min at 4 °C. Virus in the supernatant
was concentrated by centrifugation at 30,000 × g for 2 h at 4 °C. The pellet was
resuspended in 5 mM Hepes, 150 mM NaCl, and 0.1 mM EDTA (HNE buffer, pH 7.4)
and purified further on a 30% (wt/vol) sucrose cushion by ultracentrifugation in a
Beckman SW41Ti rotor at 80,000 × g for 15 h at 4 °C. Purified virus was resuspended in
HNE buffer and stored in 200-µL aliquots at −80 °C. A limiting dilution plaque assay was
performed on BHK-21 cells to determine the viral titer. For the viral infection, virus
stocks were diluted to the indicated multiplicity of infection (MOI) and were added to
cells for 1 h at 37 °C. Viral inoculum was removed thereafter, and cells were washed
twice with PBS and cultured with fresh culture media for the indicated time. Infected
cells were analyzed by GFP fluorescence or quantified by counting the number of GFP
+
cells using flow cytometry 8–16 h after infection, as described previously [52]. When
indicated, microtubules were predepolymerized with 10 µM nocodazole for 2 h, with the
drug remaining present during infection, or cells were treated with 2 µM
vacuolar-type H
+
-ATPase (V-ATPase) inhibitor bafilomycin A1. For the viral growth
24
curve, the supernatants of infected cultures were harvested at various time points
postinfection, subjected to three freeze/thaw cycles, and then titered by plaque assay in
triplicate as previously described [51]. Briefly, 10-fold serial dilutions of virus were added
to BHK-21 or Vero cells in 24-well plates and incubated for 1 h at 37 °C, with gentle
rocking every 15 min. The medium was then aspirated and replaced with 0.7%
methylcellulose in maintenance medium [RPMI-1640 and 2% (vol/vol) FBS,
supplemented with 1% penicillin/streptomycin]. After 4 d at 37 °C, the cells were fixed
with 25% (wt/vol) formaldehyde and stained with 2% (wt/vol) crystal violet in 20% (vol/
vol) ethanol. The plates were washed and dried, and the number of plaque-forming
units per milliliter was calculated.
1.5.8 Virus Labeling
DiI (Molecular Probes, Invitrogen) labeling of VSV was performed as described
previously [68] Briefly, ∼3 × 10
7
pfu/mL of VSV diluted in sodium bicarbonate buffer, pH
8.3, was incubated with DiI at a final concentration of 50 µM dye, while stirring gently.
The dye and virus mixture was incubated at room temperature for 1 h with gentle
inversions every 15 min. The labeling reaction was stopped by adding freshly prepared
1.5 M hydroxylamine, pH 8.5 (Sigma–Aldrich) and incubated at room temperature for
1 h with gentle inversions every 15 min. Labeled VSV was purified using Sephadex
G-25 columns (Amersham, GE Healthcare) to remove the unbound dye, and the labeled
virus was retitrated and stored in 100-µL aliquots at −80 °C and tested for fluorescence
before use.
25
1.5.9 VSV Internalization and VSV-G Protein Expression Assay
For viral internalization, cells were inoculated with VSV with a MOI of 1 at 4 °C for 30
min to allow binding of virus to the cell surface, but not internalization. The
virus-containing medium was replaced with fresh medium and shifted to 37 °C. At
various time points following the 37 °C shift, cells were treated with citric acid buffer [40
mM citric acid, 10 mM KCl, 135 mM NaCl (pH 3.0)] for 1 min to inactivate any particles
that remained on the surface. The cells were washed two times with medium to remove
the acidic buffer, and fresh medium was added. Cells were lysed immediately for
endosome fractionation. Endocytosed viral RNA was quantified by RT-PCR. RNA levels
when there was no acid wash were included as a control. For VSV-G protein
expression, infected cells were lysed with 1% Nonidet P-40 buffer, followed by
immunoblotting using anti–VSV-G antibody.
1.5.10 MLV-GFP Pseudotyped Virus Preparation and Entry Assay
Pseudotyped MLV-GFP bearing different viral entry proteins was generated as
described [69] [70]. The entry proteins include IAV HA proteins from A/PR/8/34 [H1N1;
H1(PR)], A/Udorn/72 [H3N2; H3(Ud)], and A/Thailand/2(SP-33)/2004(H5N1) [H5(Thai)],
as well as glycoproteins from VSV, LASV, and LCMV. At 48h postinfection with
pseudotyped viruses, the cells were subjected to fluorescence microscopy and flow
cytometry analysis to determine the GFP
+
cell population.
26
1.5.11 Virus-Matrix protein release assay
Cells grown on eight-well chamber slides were inoculated with VSV at a MOI of
200–500 in the presence of 10 µM nocodazole and 2 µM bafilomycin, a V-ATPase
inhibitor. After the indicated time points, cells were washed once with PBS and fixed
with 4% (wt/vol) paraformaldehyde in PBS for 20 min at room temperature. To detect
VSV-Matrix (M) protein, fixed cells were incubated with 0.2% Triton X-100 for 5 min at
room temperature and a 1:700 dilution of monoclonal antibody 23H12 (anti-VSVM;
KeraFAST) in PBS containing 1% goat serum overnight at 4 °C. Cells were washed
three times with PBS, and the anti-M antibodies were detected using a 1:500 dilution of
Alexa 568-conjugated goat anti-mouse secondary antibodies. Cells were counterstained
with DAPI to visualize nuclei. Cells were washed three times and mounted onto glass
slides, after which M localization images were acquired using a Nikon Eclipse C1 laser-
scanning microscope fitted with a Nikon objective (plan apochromat, 1.4 N.A.) with a
magnification of 60× and Nikon image software.
1.5.12 Western Blot and Immunoprecipitation
For immunoblotting, polypeptides were resolved by SDS/PAGE and transferred to a
PVDF membrane (Bio-Rad). Membranes were blocked with 5% (wt/vol) nonfat milk and
probed with the indicated antibodies. HRP-conjugated goat secondary antibodies
(1:10,000; Invitrogen) were used. Immunodetection was achieved with
Hyglo chemiluminescence reagent (Denville Scientific) and detected by a Fuji ECL
machine (LAS-3000). For immunoprecipitation, cells were harvested and then lysed in
1% Nonidet P-40 lysis buffer supplemented with complete protease inhibitor mixture
27
(Roche). After preclearing with protein A/G agarose beads for 1 h at 4 °C, whole-cell
lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1–4
µg of commercial antibody was added to 1 mL of cell lysate, which was incubated at 4
°C for 8–12 h. After addition of protein A/G agarose beads, incubation was continued for
another 2 h. Immunoprecipitates were extensively washed with Nonidet P-40 lysis buffer
and eluted with SDS/ PAGE loading buffer by boiling for 5 min.
1.5.13 Quantitative RT-PCR
For quantification of viral RNA replication, total RNA from the infected cells was
extracted using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions.
Purified total RNA (0.5 µg) was reverse-transcribed with the SuperScript III RT kit, using
the following oligonucleotide: 5′-TTACCATTATTGGCCCGTCAAGCT-3′. One microliter
of RT template was incubated with the VSV-G protein–specific primers (forward primer,
5′-AGGCACAGCCATACAAGTCAAA-3′; reverse primer, 5′-
TTTGGAAGCATGACACATCCA-3′) and with 2× master mix (Biorad iQ SYBR Green
Supermix) according to the supplier’s instructions. The PCR assay was performed at 95
°C for 15 min and with 35 cycles of 95°C for 30s, 60°C for 30s, and 72°C for 30s,
followed by melting curve analyses. DNA (100–500 ng) was analyzed in duplicate for
each sample and compared with a standard curve of actin as a control serially diluted
with uninfected cellular DNAs and amplified in parallel. Amplification and detection were
performed using a C1000 ThermalCycler (Bio-Rad). The specificity of the amplified
products was confirmed by agarose gel electrophoresis. Quantitative analyses of IFN
transcripts in polyinosinic:polycytidylic acid-treated cells were performed using the
primer sets for IFN-α (forward, 5′-CCTTCCACAGGATCACTGTGTACCT-3′; reverse,
28
5′ -TTCTGCTCTGACCACCTCCC-3′ ); IFN-β (forward, 5′ -
CACAGCCCTCTCCATCAACT-3′; reverse, 5′-TCCCACGTCAATCTTTCCTC-3′); and
β-actin (forward, 5′-CGAGGCCCAGAGCAAGAGAG-3′; reverse,
5′-CGGTTGGCCTTAGGGTTCAG-3′). PCR reactions were optimized to measure the
exponential phase on the amplification curve, and the size of the amplified products was
confirmed by agarose gel electrophoresis.
1.5.14 Statistical Analysis
All experiments were independently repeated at least three times. Data are presented
as mean ± SD. Statistical significance was calculated using the Student t test or one-
way ANOVA test, unless otherwise stated. A P value of ≤0.05 was considered
statistically significant.
29
1.6 RESULTS
1.6.1 VSV replication is suppressed in UVRAG-deficient cells
To investigate whether UVRAG is involved in the cell’s response to RNA virus infection, I
depleted endogenous UVRAG from HeLa cells by shRNA and infected them with
30
UVRAG has a distinct role in mediating virus entry, working in
concert with C-Vps and the endosomal SNAREs during late endo-
cytic membrane fusion. We have alsoidentifiedspecificSNAREs
andtheirinteractionsthatarereprogrammedbythevirustoassistin
their entry and infection.
Results
VSVReplicationIsSuppressedinUVRAG-DeficientCells.Toinvestigate
whether UVRAG is involved in the cell’s response to RNA virus
infection, we depleted endogenous UVRAG from HeLa cells by
shRNAandinfectedthemwithrecombinantVSVexpressingGFP
(rVSV-GFP) as a reporter. Knockdown of UVRAG resulted in
asubstantialreduction inVSVreplication,compared with control
shRNA-treated cells (Fig. 1A,B,andD). Both the number of cells
expressingGFP(Fig.1B)andtheabundanceofviralRNAencoding
glycoprotein G (Fig. 1C)weremarkedlyreducedinUVRAG-de-
pleted cells. To verify this, we further assessed VSV infectivity in
mouseEScellswithalleliclossofUVRAGgene(UVRAG
+/−
)(Fig.1
E–G). Compared with the WT control (UVRAG
+/+
), UVRAG
+/−
cells were significantly less susceptible after infection with different
doses [multiplicity of infection (MOI)] of VSV, with the virus titers
dropping precipitously (Fig. 1F). The replication restriction in
UVRAG
+/−
cellswasrevertedwhenWTUVRAGwasreintroduced,
suggesting that this is not an off-target, but a UVRAG-specific, ef-
fect (Fig. S1 A and B). Consistent with the resistance to viral in-
fectioninUVRAG-deficientcells,ectopicexpressionofUVRAGin
UVRAG-deficient HCT116 cells markedly increased infection
compared with control, as shown by the proportionality in GFP
signals at varying MOIs (Fig. S1 C and D). Taken together, these
data indicate that UVRAG is required for efficient VSV infection.
Effect of UVRAG on Viral Infection Is Not IFN-Dependent. How does
UVRAGpromoteviralinfection? Asimpleinterpretation would
be an altered type I IFN response, the first line of defense against
virus infection (13). To test this, we compared the levels of type I
IFN production between control and UVRAG knockdown cells
after stimulation of polyinosinic:polycytidylic acid [poly(I:C)], a po-
tent inducer of type I IFN. We found that untreated cells had
marginal expression of Ifnα and Ifnβ mRNA, and that treatment
with poly(I:C) stimulated similarly high Ifnα and Ifnβ mRNA ex-
pression in WT and UVRAG knockdown cells (Fig. S2 A and B).
To verify this, we collected culture medium from control and
UVRAG knockdown cells after poly(I:C) treatment and examined
itsprotectionofnaiveHeLacellsagainstVSVinfection(Fig.S2C).
Although the virus titers were drastically reduced in the poly(I:C)-
treated cultures by >90%, no discernible difference was observed
between control and UVRAG knockdown cultures (Fig. S2 D and
E).TheseresultsindicatethatUVRAGdoesnotaffectoveralltype
IIFNproductionandUVRAG-inducedVSVinfectionisnotdue
to an altered IFN response.
Autophagy-Independent Role of UVRAG in Viral Infection. We next
assessed whether the virus-resistant phenotype of UVRAG-
deficient cells was due to inhibition of autophagy. This seemed
unlikely, because repression of autophagy was found to increase
VSV replication in cultured cells (14, 15). Consistent with this
view, we detected a robust increase of viral GFP in autophagy-
deficient cells, including Atg5
−/−
, Atg7
−/−
, and Atg16
+/−
immor-
talizedmouseembryonicfibroblastsafterinfectionwithincreasing
doses of VSV (Fig. S3 A–C), suggesting that the autophagy
pathwayislargelyantiviral.Nonetheless,toruleoutthepossibility
of autophagy participation, we examined the effect of UVRAG
knockdownonVSVinfectionintheautophagy-deficientAtg5KO
cells. We found that whereas loss of Atg5 increased viral repli-
cationasnotedbefore(14,15),depletionofUVRAGsignificantly
reduced virus titers (50- to 100-fold) regardless of Atg5 activity
(Fig. S3 D–F). These data indicate that UVRAG is necessary for
viral infection through a nonautophagic mechanism.
UVRAG Is Required for Efficient Virus Entry. To determine which
step in the replication cycle was affected by UVRAG, we forced
fusion of the viral envelope with the plasma membrane by adding
medium with a low pH (pH = 5.0) to cells with bound virus. By
doing this, the normal route of entry of VSV via endocytic trans-
port was bypassed, leading to the direct release of viral genomes
into the cytosol for replication (16). Under this condition, VSV
replication was insensitive to UVRAG and no discernible differ-
ence wasdetected between controland UVRAGknockdowncells
(Fig. S4A). Notably, the low-pH–induced membrane fusion was
sufficient because the virus exhibited resistance to bafilomycin,
which blocks the acidification of endosomes, and therefore of
mature VSV entry (Fig. S4A). We also observed that UVRAG
knockdowndidnotaffectthenumbersofvirionsbeinginternalized
into cells at 5 minand 15 minafter infection,suggesting nodefect
in the early virus uptake (Fig. S4B). These data suggest that VSV
replication was impaired at an early stage of infection in cells
lackingUVRAG,somewherebetweenviraluptakeandtherelease
of viral nucleocapsid.
WenextinvestigatedwhetherUVRAGregulatedtheendocytic
virusentry.Tothisend,weconductedasingle-cycleentryassayin
UVRAG-depleted cells. This assay uses defective pseudo-retro-
virusescarryingdifferentviralenvelopesasentryfactorsandGFP
as an indicator (17, 18). As such, infection of these pseudoviruses
differs only in the entry step mediated by their respective envelope
0.01 MOI 0.1 MOI 1 MOI 10 MOI
UVRAG +/+ ES
UVRAG +/- ES
E F
!"
!#"
!##"
!$%&" !#$%&"
0
1
2
3
4
+/+ +/-
G
100
ES cells
UVRAG
Actin
UVRAG+/+
UVRAG+/-
Virus titers [Log
10
(PFU/ml)]
**
**
A
rVSV-GFP
UVRAG sh Control sh
BC
D
!" # $ % "&'()' *+, -.'()'
ND ND
*
VSV G RNA (AU)
0
20
40
60
80
100
120
VSV
- + - +
Control sh
UVRAG sh
100
35
shRNA:
Control UVRAG
UVRAG
Actin
0
20
40
60
80
100
120
!"
***
Relative Infectivity
1 MOI 10 MOI
Control sh
UVRAG sh
Fig. 1. UVRAG is required for VSV replication. (A and B) Impaired VSV
replication in UVRAG knockdown cells. (A) HeLa cells were pretreated with
control- or UVRAG-specific shRNA for 72 h and then infected (MOI of 10)
with rVSV-GFP and processed for immunofluorescence microscopy. sh, short
hairpin. (Scale bar, 50 μm.) (B) Viral infectivity is expressed as mean GFP
fluorescence relative to control cells, as determined by flow cytometry.
Valuesrepresentmean±SD(n=3independentexperiments).***P<0.001.
(C)QuantitativeRT-PCRanalysisofRNAencodingtheVSVGproteinin
controlsh-transducedandUVRAGsh-transducedHeLacellsmock-infectedor
infected for 8 h with rVSV-GFP. ND, not detectable. *P< 0.05. AU, artificial
unit. (D)WesternblotshowstheexpressionofendogenousUVRAG,and
actin serves as a loading control. (E–G) Fluorescence microscopy analysis of
UVRAG
+/+
and UVRAG
+/−
ES cells infected for 8 h with rVSV-GFP at the in-
dicated MOIs. Viral infectivity in E was determined by plaque assay (F), and
theWesternblotshowsthelevelsofUVRAGinthesecells(G).Datarepresent
mean± SD (n= 4). **P< 0.01. (Scale bar, 50 μm.)
Pirooz et al. PNAS | February 18, 2014 | vol. 111 | no. 7 | 2717
MICROBIOLOGY
Figure 1.6.1 UVRAG is required for VSV replication. (A and B) Impaired VSV replication in
UVRAG knockdown cells. (A) HeLa cells were pretreated with control- or UVRAG-specific
shRNA for 72 h and then infected (MOI of 10) with rVSV-GFP and processed for
immunofluorescence microscopy. sh, short hairpin. (Scale bar, 50 µm.) (B) Viral infectivity is
expressed as mean GFP fluorescence relative to control cells, as determined by flow cytometry.
Values represent mean ± SD (n = 3 independent experiments). ***P < 0.001. (C) Quantitative
RT-PCR analysis of RNA encoding the VSV G protein in control sh-transduced and UVRAG sh-
transduced HeLa cells mock-infected or infected for 8 h with rVSV-GFP. ND, not detectable. *P <
0.05. AU, artificial unit. (D) Western blot shows the expression of endogenous UVRAG, and
actin serves as a loading control. (E–G) Fluorescence microscopy analysis of UVRAG
+/+
and
UVRAG
+/−
ES cells infected for 8 h with rVSV-GFP at the in- dicated MOIs. Viral infectivity in E
was determined by plaque assay (F), and the Western blot shows the levels of UVRAG in these
cells (G). Data represent mean ± SD (n = 4). **P < 0.01. (Scale bar, 50 µm.)
recombinant VSV expressing GFP (rVSV-GFP) as a reporter. Knockdown of UVRAG
resulted in a substantial reduction in VSV replication, compared with control shRNA-
treated cells (Fig. 1.6.1 A, B, and D). Both the number of cells expressing GFP (Fig.
1.6.1 B) and the abundance of viral RNA encoding glycoprotein G (Fig. 1.6.1 C) were
markedly reduced in UVRAG-depleted cells. To verify this, I further assessed VSV
infectivity in mouse ES cells with allelic loss of UVRAG gene (UVRAG
+/−
) (Fig. 1.6.1 E–
G). Compared with the WT control (UVRAG
+/+
), UVRAG
+/−
cells were significantly less
susceptible after infection with different doses [multiplicity of infection (MOI)] of VSV,
with the virus titers dropping precipitously (Fig. 1.6.1 F). The replication restriction in
UVRAG
+/−
cells was reverted when WT UVRAG was reintroduced, suggesting that this
is not an off-target, but a UVRAG-specific, effect (Fig. S1.9.1 A and B). Consistent with
the resistance to viral infection in UVRAG-deficient cells, ectopic expression of UVRAG
in UVRAG-deficient HCT116 cells markedly increased infection compared with control,
as shown by the proportionality in GFP signals at varying MOIs (Fig. S1.9.1 C and D).
Taken together, these data indicate that UVRAG is required for efficient VSV infection.
1.6.2 Effect of UVRAG on viral infection is not IFN-dependent
How does UVRAG promote viral infection? A simple interpretation would be an altered
type I IFN response, the first line of defense against virus infection [71]. To test this, I
compared the levels of type I IFN production between control and UVRAG knockdown
cells after stimulation of polyinosinic:polycytidylic acid [poly(I:C)], a potent inducer of
type I IFN. I found that untreated cells had marginal expression of Ifnα and Ifnβ mRNA,
31
and that treatment with poly(I:C) stimulated similarly high Ifnα and Ifnβ mRNA
expression in WT and UVRAG knockdown cells (Fig. S1.9.2 A and B). To verify this, I
collected culture medium from control and UVRAG knockdown cells after poly(I:C)
treatment and examined its protection of naive HeLa cells against VSV infection (Fig.
S1.9.2 C). Although the virus titers were drastically reduced in the poly(I:C)- treated
cultures by >90%, no discernible difference was observed between control and UVRAG
knockdown cultures (Fig. S1.9.2 D and E). These results indicate that UVRAG does not
affect overall type I IFN production and UVRAG-induced VSV infection is not due to an
altered IFN response.
1.6.3 Autophagy-independent role of UVRAG in viral infection
I next assessed whether the virus-resistant phenotype of UVRAG- deficient cells was
due to inhibition of autophagy. This seemed unlikely, because repression of autophagy
was found to increase VSV replication in cultured cells [72] [73] . Consistent with this
view, I detected a robust increase of viral GFP in autophagy-deficient cells, including
Atg5
−/−
, Atg7
−/−
, and Atg16
+/−
immortalized mouse embryonic fibroblasts after
infection with increasing doses of VSV (Fig. S1.9.3 A–C), suggesting that the autophagy
pathway is largely antiviral. Nonetheless, to rule out the possibility of autophagy
participation, I examined the effect of UVRAG knockdown on VSV infection in the
autophagy-deficient Atg5 KO cells. I found that whereas loss of Atg5 increased viral
replication as noted before [72] [73] , depletion of UVRAG significantly reduced virus
titers (50- to 100-fold) regardless of Atg5 activity (Fig. S1.9.3 D–F). These data indicate
that UVRAG is necessary for viral infection through a nonautophagic mechanism.
32
1.6.4 UVRAG is required for efficient viral entry
To determine which step in the replication cycle was affected by UVRAG, I forced fusion
of the viral envelope with the plasma membrane by adding medium with a low pH (pH =
33
proteins (18). We found that down-regulation of UVRAG con-
siderably inhibited VSV-glycoprotein G (VSV-G)–pseudotyped
virus infection (Fig. 2A), whereas no inhibition was observed with
Lassa virus (LASV) and lymphocytic choriomeningitis virus
(LCMV),whichuseadifferentpathwaytoentercells(19,20).
There was a fourfold increase in VSV-G–mediated pseudovirus
infection in HCT116 and HeLa cells stably expressing UVRAG,
whereasLASVinfectionremainedunaffectedbyUVRAG(Fig.S4
C and D). To determine whether the entry block in UVRAG-de-
ficient cells was unique for VSV, we infected cells with the pseu-
doviruses carrying the entry proteins of IAV PR8 (H1N1), Udorn
(H3N1), and Thai (H5N1) (Fig. 2A andB). Similar to VSV, none
of the three IAVs replicated efficiently in UVRAG-depleted cells,
andtheirinfectivitywas20%ofthatobservedinWTcells(Fig.2A
and B). These results indicate that UVRAG is essential for the
entry of VSV, IAV, and likely other endocytic RNA viruses.
UVRAG Promotes Endocytic Transport of Virions. To assess the mech-
anism of action of UVRAG in virus entry, we used a viral fusion
assay that tracks the real-time entry of virions in living cells (2, 21).
We labeled VSV with the self-quenching dye DiI (Molecular
Probes,Invitrogen), which isincorporatedinto thevirion envelopes
without affecting their infectivity (2, 21). Upon binding onto the
cellsurfaceat4°C,DiI-labeledVSVwasnotfluorescent(Fig.S4E;
t=0).Whencellswereincubatedat37°Ctotriggerviralentry,the
endocytic transport of DiI-labeled VSV and its exposure to acidic
endosomes were visualized as red particles due to acid-induced
dye dequenching and fusion (Fig. S4E; t =30). Knockdown of
UVRAG resulted in a substantial reduction in intracellular DiI-
labeled VSV (Fig. S4E–G). The signals were specific because they
wereabolishedwhentreatedwithbafilomycinA1,whichneutralizes
endosomal pH (Fig. S4G). We also examined the intracellular
distribution of the Matrix (M) protein of native VSV to the cyto-
plasm as a marker for membrane fusion (7). Control cells had
diffused distribution of M protein (Fig. S4H). By contrast, only
punctate,perinuclearstainingofMproteinwasdetectedininfected
cells with UVRAG knockdown, which was further blocked by bafi-
lomycin A1 or nocodazole (Fig. S4 H and I). These data suggest
that viral fusion is inhibited upon UVRAG deficiency.
To examine whether UVRAG routes virions to acidic endo-
somes for membrane fusion, we examined the distribution of
endocytosed VSV (as indicated by the staining of VSV-G) within
1hafterinfectionrelativetolysobisphosphatidic acid (LBPA),
which accounts for the vast majority of LE membranes (22) and
promotes viral fusion (23). In agreement with reports that pro-
ductive VSV fusion takes place in LEs (2), we found that most
VSV-G colocalized with the juxtanuclear LBPA
+
LEs in control
cells at 45 min after infection. By contrast, there was a significant
reduction in the costaining of VSV-G and LBPA upon UVRAG
depletion (Fig. 2C andD). A similar reduction was also observed
when cells were treated with nocodazole, which depolymerizes
microtubulesinterruptingendosomaltransport(Fig.2CandD).Of
note, the LBPA-containing membranes remained unchanged upon
UVRAG knockdown, and equivalent numbers of virions were
endocytosedintocells(Fig.2CandFig.S4B).Tofurtherverifythat
this is entry-related, we infected cells with the VSV-G–coated
pseudovirus. Again, knockdown of UVRAG and/or treatment of
cellswithnocodazoledrasticallyinhibitedtheacquisitionofLBPA
+
membrane to the pseudovirion-containing vesicles (Fig. S4J). To-
gether, these results indicate that UVRAG is required for the late
endocytic transport of the virus,leadingtoefficientmembrane
fusion and productive penetration.
Domains of UVRAG Required for Virus Entry. Next, we performed
astructure/functionanalysis to map the domain of UVRAG re-
sponsible for virus entry. We expressed Flag-tagged WT and mu-
tant UVRAG proteins at equivalent levels in HCT116 cells
and assessed the ability of these mutants to promote the entry of
pseudovirusesbearingtheentryproteinsofVSV,IAV,andLASV.
UVRAG contains an N-terminal C2 domain (residues 42–147),
followed by a central coiled-coil domain (CCD; residues 200–269)
and a C-terminal region (residues 270–699) (Fig. S5A). We found
that unlike WT UVRAG, which promotes the entry of VSV and
IAV, but not that of LASV, UVRAG lacking the C2 (ΔC2) or
coiled-coildomain(CCD)(ΔCCD)domainbothfailedtopromote
cells’ susceptibility to VSV and IAV infection (Fig. 3 A and B).
Moreover,expressionoftheΔC2mutanthadadominant-negative
phenotype, drastically inhibiting the entry of the viruses by more
than 90% compared with the vector (Fig. 3 A and B). We also
detectedmuchlowerabundanceofVSVGproteinsinΔCCDand
ΔC2 cells after native VSV infection (Fig. S5B). Consistently, in-
tracellular transport of VSV to LBPA
+
vesicles was markedly re-
duced to 50% and 5% of WT levels by the expression of ΔCCD
andΔC2,respectively(Fig.S5CandD).Theseresultsindicatethat
both the C2 and CCD domains are required for UVRAG to
promote virus entry.
VSV-G LBPA Overlay
Control sh UVRAG sh
%"
A
- Noc + Noc
shRNA:
Noc
+
Ctrl UVRAGCtrl UVRAG
+ - -
-
%cells with VSV-G/LBPA fusion
Control sh
UVRAG sh
B
Control UVRAG
shRNA:
C
D
Control sh UVRAG sh
0
10
20
30
40
50
60
**
0
UVRAG
Actin
100
VSV H5(Thai) LASV
LASV
**
*
* **
Relative Infectivity
0.2
0.4
0.6
1.0
1.2
0.8
0
VSV
Control sh
UVRAG sh
H1
(PR)
LCMV H3
(Udorn)
H5
(Thai)
Fig. 2. UVRAG is required for efficient virus entry. (A and B)UVRAG
knockdown impairs virus entry of VSV and IAV. HeLa cells were transfected
with control- or UVRAG-specific shRNA and then infected with MLV-GFP
pseudotyped withthe indicated envelopeprotein [VSV, IAVH1N1 (PR8), IAV
H3N1 (Udom), IAV H5N1 (Thai), LASV, or LCMV]. (A)Representativeimages
of viral infection (green) are shown. (Scale bar, 50 μm.) (B) Viral entry is
expressed as mean GFP fluorescence relative to control cells, as determined
by flow cytometry. Values represent mean ± SD (n = 5 independent ex-
periments). (Lower Right) Western blot shows the expression of UVRAG in
cells. *P<0.05;**P<0.01. (Cand D)UVRAG isrequiredforviralaccesstoLE
compartments. HeLa cells were transfected with control- or UVRAG-specific
shRNA, preincubated with 10 μMnocadazole(+Noc) or without (−Noc) for
2 h, and then infected with VSV (MOI of 0.5) for 45 min. Infected cells were
fixed and immunostained with antibody against VSV-G (green) and LBPA
(red). (C) Representative images from three independent experiments are
shown. Note that colocalization between VSV-G and LBPA was observed in
control cells and highlighted (Insets)butwasinhibitedbyUVRAGknock-
down or by Noc treatment. (Scale bar, 20 μm.) (D) Percentage of infected
cells with VSV-G staining colocalized with LBPA was quantified. Data rep-
resentmean±SD(n=100)fromthreeindependentexperiments.**P<0.01.
Ctrl, control.
2718 | www.pnas.org/cgi/doi/10.1073/pnas.1320629111 Pirooz et al.
Figure 1.6.4 UVRAG is required for efficient virus entry. (A and B) UVRAG knockdown impairs
virus entry of VSV and IAV. HeLa cells were transfected with control- or UVRAG-specific shRNA
and then infected with MLV-GFP pseudotyped with the indicated envelope protein [VSV, IAV H1N1
(PR8), IAV H3N1 (Udorn), IAV H5N1 (Thai), LASV, or LCMV]. (A) Representative images of viral
infection (green) are shown. (Scale bar, 50 µm.) (B) Viral entry is expressed as mean GFP
fluorescence relative to control cells, as determined by flow cytometry. Values represent mean ±
SD (n = 5 independent experiments). (Lower Right) Western blot shows the expression of UVRAG
in cells. *P < 0.05; **P < 0.01. (C and D) UVRAG is required for viral access to LE compartments.
HeLa cells were transfected with control- or UVRAG-specific shRNA, preincubated with 10 µM
nocadazole (+Noc) or without (−Noc) for 2 h, and then infected with VSV (MOI of 0.5) for 45 min.
Infected cells were fixed and immunostained with antibody against VSV-G (green) and LBPA (red).
(C) Representative images from three independent experiments are shown. Note that
colocalization between VSV-G and LBPA was observed in control cells and highlighted (Insets) but
was inhibited by UVRAG knock- down or by Noc treatment. (Scale bar, 20 µm.) (D) Percentage of
infected cells with VSV-G staining colocalized with LBPA was quantified. Data rep- resent mean ±
SD (n = 100) from three independent experiments. **P < 0.01. Ctrl, control.
5.0) to cells with bound virus. By doing this, the normal route of entry of VSV via
endocytic transport was bypassed, leading to the direct release of viral genomes into
the cytosol for replication [74]. Under this condition, VSV replication was insensitive to
UVRAG and no discernible difference was detected between control and UVRAG
knockdown cells (Fig. S1.9.4 A). Notably, the low-pH–induced membrane fusion was
sufficient because the virus exhibited resistance to bafilomycin, which blocks the
acidification of endosomes, and therefore of mature VSV entry (Fig. S1.9.4 A). I also
observed that UVRAG knockdown did not affect the numbers of virions being
internalized into cells at 5 min and 15 min after infection, suggesting no defect in the
early virus uptake (Fig. S1.9.4 B). These data suggest that VSV replication was
impaired at an early stage of infection in cells lacking UVRAG, somewhere between
viral uptake and the release of viral nucleocapsid.
I next investigated whether UVRAG regulated the endocytic virus entry. To this end, I
conducted a single-cycle entry assay in UVRAG-depleted cells. This assay uses
defective pseudo-retroviruses carrying different viral envelopes as entry factors and
GFP as an indicator [75] [69] [70] [76]. As such, infection of these pseudoviruses differs
only in the entry step mediated by their respective envelope proteins [69]. We found that
downregulation of UVRAG considerably inhibited VSV-glycoprotein G (VSV-G)-
pseudotyped virus infection (Fig. 1.6.4 A), whereas no inhibition was observed with
Lassa virus (LASV) and lymphocytic choriomeningitis virus (LCMV), which use a
different pathway to enter cells [70] [76]. There was a fourfold increase in VSV-G-
mediated pseudovirus infection in HCT116 and HeLa cells stably expressing UVRAG,
whereas LASV infection remained unaffected by UVRAG (Fig. S1.9.4 C and D). To
34
determine whether the entry block in UVRAG-deficient cells was unique for VSV, I
infected cells with the pseudoviruses carrying the entry proteins of IAV PR8 (H1N1),
Udorn (H3N1), and Thai (H5N1) (Fig. 1.6.4 A and B). Similar to VSV, none of the three
IAVs replicated efficiently in UVRAG-depleted cells, and their infectivity was 20% of that
observed in WT cells (Fig. 1.6.4 A and B). These results indicate that UVRAG is
essential for the entry of VSV, IAV, and likely other endocytic RNA viruses.
1.6.5 UVRAG promotes endocytic transport of virions
To assess the mechanism of action of UVRAG in virus entry, I used a viral fusion assay
that tracks the real-time entry of virions in living cells [14] [77]. I labeled VSV with the
self-quenching dye DiI (Molecular Probes, Invitrogen), which is incorporated into the
virion envelopes without affecting their infectivity [14] [77]. Upon binding onto the cell
surface at 4 °C, DiI-labeled VSV was not fluorescent (Fig. S1.9.4 E; t = 0). When cells
were incubated at 37 °C to trigger viral entry, the endocytic transport of DiI-labeled VSV
and its exposure to acidic endosomes were visualized as red particles due to acid-
induced dye dequenching and fusion (Fig. S1.9.4 E; t =30). Knockdown of UVRAG
resulted in a substantial reduction in intracellular DiI-labeled VSV (Fig. S1.9.4 E–G).
The signals were specific because they were abolished when treated with bafilomycin
A1, which neutralizes endosomal pH (Fig. S1.9.4 G). I also examined the intracellular
distribution of the Matrix (M) protein of native VSV to the cytoplasm as a marker for
membrane fusion [52]. Control cells had diffused distribution of M protein (Fig. S1.9.4
H). By contrast, only punctate, perinuclear staining of M protein was detected in infected
cells with UVRAG knockdown, which was further blocked by bafilomycin A1 or
35
nocodazole (Fig. S1.9.4 H and I). These data suggest that viral fusion is inhibited upon
UVRAG deficiency.
To examine whether UVRAG routes virions to acidic endosomes for membrane fusion, I
examined the distribution of endocytosed VSV (as indicated by the staining of VSV-G)
within 1 h after infection relative to lysobisphosphatidic acid (LBPA), which accounts for
the vast majority of LE membranes [78] and promotes viral fusion [79]. In agreement
with reports that productive VSV fusion takes place in LEs [14], I found that most VSV-G
colocalized with the juxtanuclear LBPA
+
LEs in control cells at 45 min after infection. By
contrast, there was a significant reduction in the costaining of VSV-G and LBPA upon
UVRAG depletion (Fig. 1.6.4 C and D). A similar reduction was also observed when
cells were treated with nocodazole, which depolymerizes microtubules interrupting
endosomal transport (Fig. 1.6.4 C and D). Of note, the LBPA-containing membranes
remained unchanged upon UVRAG knockdown, and equivalent numbers of virions were
endocytosed into cells (Fig. 1.6.4C and Fig. S1.9.4 B). To further verify that this is entry-
related, I infected cells with the VSV-G-coated pseudovirus. Again, knockdown of
UVRAG and/or treatment of cells with nocodazole drastically inhibited the acquisition of
LBPA
+
membrane to the pseudovirion-containing vesicles (Fig. S1.9.4 J). Together,
these results indicate that UVRAG is required for the late endocytic transport of the
virus, leading to efficient membrane fusion and productive penetration.
36
1.6.6 Domains of UVRAG required for virus entry
Next, I performed a structure/function analysis to map the domain of UVRAG
responsible for virus entry. I expressed Flag-tagged WT and mutant UVRAG proteins at
equivalent levels in HCT116 cells and assessed the ability of these mutants to promote
the entry of pseudoviruses bearing the entry proteins of VSV, IAV, and LASV.
UVRAG contains an N-terminal C2 domain (residues 42–147), followed by a central
coiled-coil domain (CCD; residues 200–269) and a C-terminal region (residues 270–
699) (Fig. S1.9.5 A). I found that unlike WT UVRAG, which promotes the entry of VSV
and IAV, but not that of LASV, UVRAG lacking the C2 (ΔC2) or coiled-coil domain (CCD)
(ΔCCD) domain both failed to promote cells’ susceptibility to VSV and IAV infection (Fig.
37
C-Vps, but Not Beclin1, Is Required for UVRAG-Mediated Virus Entry.
Our previous studies showed that the C2 of UVRAG associates
with C-Vps to enhance endocytic protein degradation, whereas
the CCD binds Beclin1 and activates Beclin1-mediated auto-
phagy (8, 9). We next determined the importance of C-Vps and
Beclin1 in UVRAG-mediated viral infection. As observed with
UVRAG knockdown, treatment of cells with Vps16- or Vps18-
siRNA, but not with scrambled siRNA, significantly suppressed
VSVreplicationatdifferentMOIs(Fig.S6AandB).Toconfirm
the significance of UVRAG–C-Vps interaction in viral infection,
we depletedVps18fromcellsexpressing WTormutant UVRAG
and found that removal of Vps18 diminished the capability of
UVRAG to promote VSV infection (Fig. S6 C–E). Our results
demonstrate that UVRAG and its interaction with C-Vps are
required for VSV infection.
Unlike depletion of C-Vps, knockdown of Beclin1 showed
minimal effect on VSV replication (Fig. S6F). Likewise, no re-
ductioninviralinfectionwasobservedinWTormutantUVRAG-
expressing cells by inhibition of Beclin1 (Fig. S6 G–I). Although
UVRAG ΔCCD inhibited viral entry, Beclin1 is clearly not re-
quiredinthisactivity;otherfactor(s)maybeinvolvedwithrespect
to CCD function. Collectively, these results demonstrate that
UVRAG interactions with C-Vps, but not Beclin1, are important
for efficient viral infection.
UVRAG Interacts with SNAREs. One essential role of C-Vps in
endosomal transport is to facilitate assembly of the fusogenic
SNARE complex, including STX7, Vti1b, and STX8, pairing
withVAMP8orVAMP7inthehomotypicorheterotypicfusion
ofLEs,respectively(4,24).WeaskedwhetherUVRAG,likeits
interactorC-Vps,alsobindsSNAREsonLEs/lysosomes,whereby
much acid-induced virus fusion takes place (25). Our immuno-
precipitation analyses demonstrated that both endogenous and
Flag-tagged UVRAG efficiently coprecipitated with endogenous
Q-SNAREs(i.e.,STX7,Vti1b,STX8)(Fig.4AandFig.S7A).No
discernibleinteractionwasdetectedwithVAMP7andVAMP8or
with STX6, which is the trans-Golgi–related SNARE, suggesting
a compartment-specific Q-SNARE interaction of UVRAG (Fig.
4A). Moreover, the CCD is sufficient and necessary for the in-
teractionofUVRAGwithQ-SNAREs(Fig.S7AandB).Notably,
the ΔC2 mutant of UVRAG, defective in C-Vps binding (8),
preserved efficient interaction with Q-SNAREs (Fig. S7A), sug-
gesting that UVRAG association with Q-SNAREs is not de-
pendent on its interaction with C-Vps. Because the CCD of
UVRAG directly binds Beclin1 (9), we asked whether Beclin1 is
involved in the UVRAG–SNARE interactions. As shown in Fig.
S7C, depletion of Beclin1 did not alter the binding efficiency of
UVRAG with Q-SNAREs. These results indicate that UVRAG
forms a complex with endosomal Q-SNAREs through its CCD in
a Beclin1-independent manner (Fig. S7G).
UVRAG Enhances C-Vps Interaction with SNAREs and trans-SNARE
Assembly. The dual interactions of UVRAG with C-Vps and
SNAREsthroughdistinctdomains(Fig.S7G)suggestthatUVRAG
may coordinate the complex assembly of C-Vps and SNAREs.
More Vps16 or Vps18 coimmunoprecipitated with the Q-
SNAREswhenUVRAGwasectopicallyexpressed(Fig.S7D).By
contrast,deletionoftheC2orCCD,whichabrogatesC-VpsorQ-
SNARE binding of UVRAG, respectively, failed to promote the
C-Vps–SNAREinteractions(Fig.S7D).Inaccord,knockdownof
UVRAGseverelyhinderedendogenousinteractionoftheC-Vps
proteins with the SNAREs (Fig. 4B). These data indicate that
UVRAGmediatestheinteractionofC-VpswithQ-SNAREsand
mayparticipateintheSNARE-mediated membranefusion.
ToinvestigatefurtherwhetherUVRAGisalsorequiredinthe
assembly of cognate SNAREs into core complexes (Qa-Qb-Qc-
R),adecisivestepindrivingmembranefusion(5,24),weevaluated
the cis-and trans-SNARE pairing in WT and UVRAG-depleted
cells. Knockdown of UVRAG clearly reduced the interaction be-
tween Q-SNAREs without affecting their steady-level expression
(Fig. S7E). Furthermore, the trans-SNARE assembly of the Q-
SNARE with its cognate R-SNAREs VAMP8 and VAMP7 was
also reduced when UVRAG was deficient (Fig. S7E). The marked
effect of UVRAG on trans-SNAREs may influence their relative
distribution at endosomal membranes. To explore this, we treated
controlandUVRAG-expressingHeLacellswithN-ethylmaleimide,
whichinhibitsN-ethylmaleimide-sensitivefusionprotein(NSF)and
disassembly of SNARE complexes, and assessed the formation of
thetrans-SNAREcomplexbyconfocalmicroscopy.BothSTX8and
the R-SNARE proteins VAMP7 and VAMP8 displayed punctate
staining (Fig. S7F). Nearly complete colocalization was observed
between the SNAREs in UVRAG-expressing cells, whereas only
partialcolocalizationwasdetectedincontrolcells(Fig.S7F).These
results suggest that UVRAG promotes fusogenic SNARE complex
formation during late endocytic membrane fusion in normal
conditions.
VAMP8, but Not VAMP7, Is Required for Virus Entry. To determine
the SNAREs involved in UVRAG-mediated virus entry, we
depleted cells of individual SNAREs, including STX7, Vti1b,
Fig. 3. Domains of UVRAG required for virus entry. HCT116 cells stably
expressing vector and Flag-tagged UVRAG or itsΔC2 orΔCCD mutant were
infected with MLV-GFP virus pseudotyped with the indicated entry protein
for 48 h. Pseudovirus infectivity (green) was visualized by fluorescence mi-
croscopy (A), and viral entry is expressed as mean GFP fluorescence relative
to the vector (Vec) cells, as examined by flow cytometry (B). Data represent
mean ± SD (n = 4 independent experiments). *P < 0.05; **P < 0.01. (Scale
bar, 50 μm.)
A
B
Input IgG
UVRAG
Q-SNAREs R-SNARE
IgG
UVRAG sh
Ctrl sh
IgG
UVRAG sh
Ctrl sh
IgG
UVRAG sh
Ctrl sh
15
25
35-
25-
100-
-
-
35-
35-
25-
100-
35-
25-
100-
35-
25-
25-
100-
100-
100-
IB: STX7
IB: STX7
IB: STX7
IB: STX7
IB: Vti1b
IB: Vti1b
IB: Vti1b
IB: Vti1b
IB: STX8
IB: VAMP7
IB: VAMP8
IB: UVRAG IB: UVRAG
IB: STX6
IB: Vps16
IB: Vps16
IB: Vps18
IB: Vps18
WCL:
IP: Vps18
IP: Vps16 IP:
Fig. 4. UVRAG functions together with C-Vps and SNAREs. (A)Endogenous
interaction of UVRAG with endosomal SNAREs. Whole-cell lysates (WCLs) of
293T cells were used for immunoprecipitation (IP) with control serum (IgG)
or anti-UVRAG, followed by immunoblotting (IB) with the indicated anti-
bodies. Ten percent of the WCLs were used as input. (B)UVRAGdeficiency
impairs C-Vps interaction with Q-SNAREs. 293T cells were transfected with
control shRNA or UVRAG shRNA. WCLs were used for IP with anti-Vps16
(Upper) or anti-Vps18 (Lower), followed by IB with the indicated antibodies.
(Right)Endogenousproteinexpressionsareshown.
Pirooz et al. PNAS | February 18, 2014 | vol. 111 | no. 7 | 2719
MICROBIOLOGY
Figure 1.6.6 Domains of UVRAG required for virus entry. HCT116
cells stably expressing vector and Flag-tagged UVRAG or its ΔC2 or
ΔCCD mutant were infected with MLV-GFP virus pseudotyped with
the indicated entry protein for 48 h. Pseudovirus infectivity (green)
was visualized by fluorescence mi- croscopy (A), and viral entry is
expressed as mean GFP fluorescence relative to the vector (Vec)
cells, as examined by flow cytometry (B). Data represent mean ± SD
(n = 4 independent experiments). *P < 0.05; **P < 0.01. (Scale bar, 50
µm.)
1.6.6 A and B). Moreover, expression of the ΔC2 mutant had a dominant-negative
phenotype, drastically inhibiting the entry of the viruses by more than 90% compared
with the vector (Fig. 1.6.6 A and B). I also detected much lower abundance of VSVG
proteins in ΔCCD and ΔC2 cells after native VSV infection (Fig. S1.9.5 B). Consistently,
intracellular transport of VSV to LBPA
+
vesicles was markedly reduced to 50% and 5%
of WT levels by the expression of ΔCCD and ΔC2, respectively (Fig. S1.9.5 C and D).
These results indicate that both the C2 and CCD domains are required for UVRAG to
promote virus entry.
1.6.7 C-Vps, but not Beclin 1, is required for UVRAG-mediated virus entry
Previous studies showed that the C2 of UVRAG associates with C-Vps to enhance
endocytic protein degradation, whereas the CCD binds Beclin 1 and activates Beclin
1-mediated autophagy [51] [66]. I next determined the importance of C-Vps and Beclin 1
in UVRAG-mediated viral infection. As observed with UVRAG knockdown, treatment of
cells with Vps16- or Vps18- siRNA, but not with scrambled siRNA, significantly
suppressed VSV replication at different MOIs (Fig. S1.9.6 A and B). To confirm the
significance of UVRAG-C-Vps interaction in viral infection, I depleted Vps18 from cells
expressing WT or mutant UVRAG and found that removal of Vps18 diminished the
capability of UVRAG to promote VSV infection (Fig. S1.9.6 C–E). My results
demonstrate that UVRAG and its interaction with C-Vps are required for VSV infection.
Unlike depletion of C-Vps, knockdown of Beclin 1 showed minimal effect on VSV
replication (Fig. S1.9.6 F). Likewise, no reduction in viral infection was observed in WT
or mutant UVRAG-expressing cells by inhibition of Beclin 1 (Fig. S1.9.6 G–I). Although
38
UVRAG ΔCCD inhibited viral entry, Beclin 1 is clearly not required in this activity; other
factor(s) may be involved with respect to CCD function. Collectively, these results
demonstrate that UVRAG interactions with C-Vps, but not Beclin 1, are important for
efficient viral infection.
1.6.8 UVRAG interacts with SNAREs
One essential role of C-Vps in endosomal transport is to facilitate assembly of the
fusogenic SNARE complex, including STX7, Vti1b, and STX8, pairing with VAMP8 or
VAMP7 in the homotypic or heterotypic fusion of LEs, respectively [25] [80]. I asked
whether UVRAG, like its interactor C-Vps, also binds SNAREs on LEs/lysosomes,
whereby much acid-induced virus fusion takes place [81]. My immunoprecipitation
analyses demonstrated that both endogenous and Flag-tagged UVRAG efficiently
coprecipitated with endogenous Q-SNAREs (i.e., STX7, Vti1b, STX8) (Fig. 1.6.8 A and
Fig. S1.9.7 A). No discernible interaction was detected with VAMP7 and VAMP8 or with
STX6, which is the trans-Golgi–related SNARE, suggesting a compartment-specific
Q-SNARE interaction of UVRAG (Fig. 1.6.8 A). Moreover, the CCD is sufficient and
necessary for the interaction of UVRAG with Q-SNAREs (Fig. S1.9.7 A and B). Notably,
the ΔC2 mutant of UVRAG, defective in C-Vps binding [51], preserved efficient
interaction with Q-SNAREs (Fig. S1.9.7 A), suggesting that UVRAG association with
Q-SNAREs is not dependent on its interaction with C-Vps. Because the CCD of UVRAG
directly binds Beclin 1 [66], I asked whether Beclin 1 is involved in the UVRAG-SNARE
interactions. As shown in Fig. S1.9.7 C, depletion of Beclin 1 did not alter the binding
efficiency of UVRAG with Q-SNAREs. These results indicate that UVRAG forms a
39
complex with endosomal Q-SNAREs through its CCD in a Beclin 1-independent manner
(Fig. S1.9.7 G).
1.6.9 UVRAG enhances C-Vps interaction with SNAREs and trans-SNARE
assembly
The dual interactions of UVRAG with C-Vps and SNAREs through distinct domains
(Fig. S1.9.7 G) suggest that UVRAG may coordinate the complex assembly of C-Vps
and SNAREs. More Vps16 or Vps18 coimmunoprecipitated with the Q- SNAREs when
UVRAG was ectopically expressed (Fig. S1.9.7 D). By contrast, deletion of the C2
orCCD, which abrogates C-Vps or Q- SNARE binding of UVRAG, respectively, failed to
40
C-Vps, but Not Beclin1, Is Required for UVRAG-Mediated Virus Entry.
Our previous studies showed that the C2 of UVRAG associates
with C-Vps to enhance endocytic protein degradation, whereas
the CCD binds Beclin1 and activates Beclin1-mediated auto-
phagy (8, 9). We next determined the importance of C-Vps and
Beclin1 in UVRAG-mediated viral infection. As observed with
UVRAG knockdown, treatment of cells with Vps16- or Vps18-
siRNA, but not with scrambled siRNA, significantly suppressed
VSVreplicationatdifferentMOIs(Fig.S6AandB).Toconfirm
the significance of UVRAG–C-Vps interaction in viral infection,
we depletedVps18fromcellsexpressing WTormutant UVRAG
and found that removal of Vps18 diminished the capability of
UVRAG to promote VSV infection (Fig. S6 C–E). Our results
demonstrate that UVRAG and its interaction with C-Vps are
required for VSV infection.
Unlike depletion of C-Vps, knockdown of Beclin1 showed
minimal effect on VSV replication (Fig. S6F). Likewise, no re-
ductioninviralinfectionwasobservedinWTormutantUVRAG-
expressing cells by inhibition of Beclin1 (Fig. S6 G–I). Although
UVRAG ΔCCD inhibited viral entry, Beclin1 is clearly not re-
quiredinthisactivity;otherfactor(s)maybeinvolvedwithrespect
to CCD function. Collectively, these results demonstrate that
UVRAG interactions with C-Vps, but not Beclin1, are important
for efficient viral infection.
UVRAG Interacts with SNAREs. One essential role of C-Vps in
endosomal transport is to facilitate assembly of the fusogenic
SNARE complex, including STX7, Vti1b, and STX8, pairing
withVAMP8orVAMP7inthehomotypicorheterotypicfusion
ofLEs,respectively(4,24).WeaskedwhetherUVRAG,likeits
interactorC-Vps,alsobindsSNAREsonLEs/lysosomes,whereby
much acid-induced virus fusion takes place (25). Our immuno-
precipitation analyses demonstrated that both endogenous and
Flag-tagged UVRAG efficiently coprecipitated with endogenous
Q-SNAREs(i.e.,STX7,Vti1b,STX8)(Fig.4AandFig.S7A).No
discernibleinteractionwasdetectedwithVAMP7andVAMP8or
with STX6, which is the trans-Golgi–related SNARE, suggesting
a compartment-specific Q-SNARE interaction of UVRAG (Fig.
4A). Moreover, the CCD is sufficient and necessary for the in-
teractionofUVRAGwithQ-SNAREs(Fig.S7AandB).Notably,
the ΔC2 mutant of UVRAG, defective in C-Vps binding (8),
preserved efficient interaction with Q-SNAREs (Fig. S7A), sug-
gesting that UVRAG association with Q-SNAREs is not de-
pendent on its interaction with C-Vps. Because the CCD of
UVRAG directly binds Beclin1 (9), we asked whether Beclin1 is
involved in the UVRAG–SNARE interactions. As shown in Fig.
S7C, depletion of Beclin1 did not alter the binding efficiency of
UVRAG with Q-SNAREs. These results indicate that UVRAG
forms a complex with endosomal Q-SNAREs through its CCD in
a Beclin1-independent manner (Fig. S7G).
UVRAG Enhances C-Vps Interaction with SNAREs and trans-SNARE
Assembly. The dual interactions of UVRAG with C-Vps and
SNAREsthroughdistinctdomains(Fig.S7G)suggestthatUVRAG
may coordinate the complex assembly of C-Vps and SNAREs.
More Vps16 or Vps18 coimmunoprecipitated with the Q-
SNAREswhenUVRAGwasectopicallyexpressed(Fig.S7D).By
contrast,deletionoftheC2orCCD,whichabrogatesC-VpsorQ-
SNARE binding of UVRAG, respectively, failed to promote the
C-Vps–SNAREinteractions(Fig.S7D).Inaccord,knockdownof
UVRAGseverelyhinderedendogenousinteractionoftheC-Vps
proteins with the SNAREs (Fig. 4B). These data indicate that
UVRAGmediatestheinteractionofC-VpswithQ-SNAREsand
mayparticipateintheSNARE-mediated membranefusion.
ToinvestigatefurtherwhetherUVRAGisalsorequiredinthe
assembly of cognate SNAREs into core complexes (Qa-Qb-Qc-
R),adecisivestepindrivingmembranefusion(5,24),weevaluated
the cis-and trans-SNARE pairing in WT and UVRAG-depleted
cells. Knockdown of UVRAG clearly reduced the interaction be-
tween Q-SNAREs without affecting their steady-level expression
(Fig. S7E). Furthermore, the trans-SNARE assembly of the Q-
SNARE with its cognate R-SNAREs VAMP8 and VAMP7 was
also reduced when UVRAG was deficient (Fig. S7E). The marked
effect of UVRAG on trans-SNAREs may influence their relative
distribution at endosomal membranes. To explore this, we treated
controlandUVRAG-expressingHeLacellswithN-ethylmaleimide,
whichinhibitsN-ethylmaleimide-sensitivefusionprotein(NSF)and
disassembly of SNARE complexes, and assessed the formation of
thetrans-SNAREcomplexbyconfocalmicroscopy.BothSTX8and
the R-SNARE proteins VAMP7 and VAMP8 displayed punctate
staining (Fig. S7F). Nearly complete colocalization was observed
between the SNAREs in UVRAG-expressing cells, whereas only
partialcolocalizationwasdetectedincontrolcells(Fig.S7F).These
results suggest that UVRAG promotes fusogenic SNARE complex
formation during late endocytic membrane fusion in normal
conditions.
VAMP8, but Not VAMP7, Is Required for Virus Entry. To determine
the SNAREs involved in UVRAG-mediated virus entry, we
depleted cells of individual SNAREs, including STX7, Vti1b,
Fig. 3. Domains of UVRAG required for virus entry. HCT116 cells stably
expressing vector and Flag-tagged UVRAG or itsΔC2 orΔCCD mutant were
infected with MLV-GFP virus pseudotyped with the indicated entry protein
for 48 h. Pseudovirus infectivity (green) was visualized by fluorescence mi-
croscopy (A), and viral entry is expressed as mean GFP fluorescence relative
to the vector (Vec) cells, as examined by flow cytometry (B). Data represent
mean ± SD (n = 4 independent experiments). *P < 0.05; **P < 0.01. (Scale
bar, 50 μm.)
A
B
Input IgG
UVRAG
Q-SNAREs R-SNARE
IgG
UVRAG sh
Ctrl sh
IgG
UVRAG sh
Ctrl sh
IgG
UVRAG sh
Ctrl sh
15
25
35-
25-
100-
-
-
35-
35-
25-
100-
35-
25-
100-
35-
25-
25-
100-
100-
100-
IB: STX7
IB: STX7
IB: STX7
IB: STX7
IB: Vti1b
IB: Vti1b
IB: Vti1b
IB: Vti1b
IB: STX8
IB: VAMP7
IB: VAMP8
IB: UVRAG IB: UVRAG
IB: STX6
IB: Vps16
IB: Vps16
IB: Vps18
IB: Vps18
WCL:
IP: Vps18
IP: Vps16 IP:
Fig. 4. UVRAG functions together with C-Vps and SNAREs. (A)Endogenous
interaction of UVRAG with endosomal SNAREs. Whole-cell lysates (WCLs) of
293T cells were used for immunoprecipitation (IP) with control serum (IgG)
or anti-UVRAG, followed by immunoblotting (IB) with the indicated anti-
bodies. Ten percent of the WCLs were used as input. (B)UVRAGdeficiency
impairs C-Vps interaction with Q-SNAREs. 293T cells were transfected with
control shRNA or UVRAG shRNA. WCLs were used for IP with anti-Vps16
(Upper) or anti-Vps18 (Lower), followed by IB with the indicated antibodies.
(Right)Endogenousproteinexpressionsareshown.
Pirooz et al. PNAS | February 18, 2014 | vol. 111 | no. 7 | 2719
MICROBIOLOGY
Figure 1.6.8 UVRAG functions together with C-Vps and SNAREs. (A)
Endogenous interaction of UVRAG with endosomal SNAREs. Whole-cell lysates
(WCLs) of 293T cells were used for immunoprecipitation (IP) with control serum
(IgG) or anti-UVRAG, followed by immunoblotting (IB) with the indicated anti-
bodies. Ten percent of the WCLs were used as input. (B) UVRAG deficiency
impairs C-Vps interaction with Q-SNAREs. 293T cells were transfected with
control shRNA or UVRAG shRNA. WCLs were used for IP with anti-Vps16
(Upper) or anti-Vps18 (Lower), followed by IB with the indicated antibodies.
(Right) Endogenous protein expressions are shown.
promote the C-Vps-SNARE interactions (Fig. S1.9.7 D). In accord, knockdown of
UVRAG severely hindered endogenous interaction of the C-Vps proteins with the
SNAREs (Fig. 1.6.8 B). These data indicate that UVRAG mediates the interaction of
C-Vps with Q-SNAREs and may participate in the SNARE-mediated membrane fusion.
To investigate further whether UVRAG is also required in the assembly of cognate
SNAREs into core complexes (Qa-Qb-Qc- R), a decisive step in driving membrane
fusion [26] [80], I evaluated the cis- and trans-SNARE pairing in WT and UVRAG-
depleted cells. Knockdown of UVRAG clearly reduced the interaction between
Q-SNAREs without affecting their steady-level expression (Fig. S1.9.7 E). Furthermore,
the trans-SNARE assembly of the Q- SNARE with its cognate R-SNAREs VAMP8 and
VAMP7 was also reduced when UVRAG was deficient (Fig. S1.9.7 E). The marked
effect of UVRAG on trans-SNAREs may influence their relative distribution at
endosomal membranes. To explore this, I treated control and UVRAG-expressing HeLa
cells with N-ethylmaleimide, which inhibits N-ethylmaleimide-sensitive fusion protein
(NSF) and disassembly of SNARE complexes, and assessed the formation of the trans-
SNARE complex by confocal microscopy. Both STX8 and the R-SNARE proteins
VAMP7 and VAMP8 displayed punctate staining (Fig. S1.9.7 F). Nearly complete
colocalization was observed between the SNAREs in UVRAG-expressing cells,
whereas only partial colocalization was detected in control cells (Fig. S1.9.7 F). These
results suggest that UVRAG promotes fusogenic SNARE complex formation during late
endocytic membrane fusion in normal conditions.
41
1.6.10 Vamp8, but not Vamp7 is required for virus entry
To determine the SNAREs involved in UVRAG-mediated virus entry, I depleted cells of
individual SNAREs, including STX7, Vti1b, STX8, VAMP7, and VAMP8, and conducted
one-step virus entry. As seen with inhibition of UVRAG, depletion of the Q- SNAREs or
VAMP8 significantly blocked VSV-G–mediated virus entry (Fig. 1.6.10 A). Intriguingly, no
reduction was detected in VAMP7-deficient cells; instead, a slight increase resulted (Fig.
1.6.10 A). As seen with VSV, suppression of VAMP8, but not VAMP7, rendered cells
42
STX8, VAMP7, and VAMP8, and conducted one-step virus
entry. As seen with inhibition of UVRAG, depletion of the Q-
SNAREs or VAMP8 significantly blocked VSV-G–mediated
virus entry (Fig. 5A). Intriguingly, no reduction was detected in
VAMP7-deficient cells; instead, a slight increase resulted (Fig.
5A). As seen with VSV, suppression of VAMP8, but not
VAMP7, rendered cells resistant to the pseudovirus infection of
IAV, and it also drastically disabled the ability of UVRAG to
promote virus entry, suggesting that UVRAG works in concert
with specific SNAREs to regulate virus entry (Fig. S8 A and B).
Given that UVRAG forms a complex with SNAREs through its
CCD, these data explain the antiviral restriction ofΔCCD (Fig.
3A). Consistently, when cells were infected with live rVSV-GFP,
only VAMP7 knockdown cells remained susceptible, whereas
siRNA depletion of all other SNAREs was refractory to VSV
infection (Fig. S8C). These results indicate that LE-associated
Q-SNAREs and VAMP8 are specifically required for virus entry
and infection.
Virus Entry Stimulates a Specific Complex Assembly of UVRAG, C-Vps,
and SNAREs. The data above imply that UVRAG action with
C-Vps and SNAREs is a key regulator during virus entry. This
raises the question of whether this reflects a random event
hitchhiked by the virus or, instead, a strategy of the virus to in-
duce a specific fusogenic complex for efficient entry. To test this,
weinfectedHeLacellsstablyexpressingemptyvectororUVRAG
with VSV and assessed the complex-forming ability of UVRAG
with endogenous C-Vps and SNAREs within 2 h after infection
(Fig. S8D). Compared with the complex formation in mock-
infected cells, VSV infection triggered a robust increase ofC-Vps
and SNAREs coimmunoprecipitated with UVRAG (Fig. S8D).
Theincreasedbindingwasnotduetodifferingexpressionbecause
no evident alteration of the protein levels was detected in mock-
and virus-infected cells (Fig. S8D). However, bafilomycin treat-
mentsignificantlyreducedthecomplexassemblyofUVRAGwith
C-VpsandSNAREs(Fig.S8D),suggestingthatthisentry-induced
complex assembly of UVRAG lies downstream of the low-pH
trigger.Asacontrol,theUVRAG–Beclin1interactionwasneither
affected by VSV nor changed by bafilomycin (Fig. S8D), further
suggesting that Beclin1 is not involved in this process. Analogous
results were obtained when we infected cells with the pseudo-
particles. As expected, VSV-G– and IAV-H5–mediated entry pro-
cessesaresufficienttopromotethecomplexformationofUVRAG,
C-Vps, and Q-SNAREs (Fig. S8 F and G).
We next examined UVRAG-mediated cis- and trans-SNARE
assembly upon viral infection. As shown in Fig. S8E, UVRAG
expressionenhancedinteractionsofSTX7withSTX8andVti1b,
as well as its pairing with VAMP8, which was further induced by
VSV infection. In contrast, minimal amounts of VAMP7 coim-
munoprecipitated with STX7, suggesting that VAMP7 is largely
excluded from UVRAG-promoted SNARE complex formation
upon viral infection (Fig. S8E). These findings demonstrate that
virus entry triggers a specific supercomplex formation involving
UVRAG, C-Vps, Q-SNARES, and VAMP8, but not VAMP7.
VAMP8 Recruitment to the Virus-Containing Vesicles. To examine
whether VAMP7 and VAMP8 are differentially required for the
virus entry-induced membrane remodeling, we examined endog-
enous redistribution of VAMP8 and VAMP7 in control and
UVRAG-expressing cells after 1 h of infection with VSV (Fig.
S9). Both VAMP8 and VAMP7 revealed vesicular staining with
a perinuclear concentration in mock-infected cells (Fig. S9).
UponVSVinfection,however,VAMP8wasprominentlyrecruited
to the VSV-G–containing vesicles, whereas VAMP7 was largely
excluded (Fig. S9). Furthermore, expression of UVRAG resulted
in a twofold increase in the acquisition of VAMP8-containing
membranestotheVSV-G
+
vesiclescomparedwiththecontrolbut
had a minimal effect on VAMP7 accumulation in the virus-con-
taining vesicles (Fig. S9). Although UVRAG is required for trans-
SNARE complex assembly (Fig. S7E), only VAMP8 was selected
to function with UVRAG in facilitating virus entry, whereas
VAMP7 is largely absent from the majority of virus-containing
compartments, consistent with an earlier finding that VAMP7 is
not required for the entry of VSV and IAV.
Discussion
Initially discovered in a screen for UV resistance, UVRAG has
been identified in our studies, as well as those of others, to
possess various functions, including intracellular trafficking, chro-
mosomal stability, autophagy activation, and tumor suppression
(8–10, 12, 26, 27). However, the cellular outcome of UVRAG in
viral infection has not been investigated. We show here that
UVRAG-deficient cells are largely refractory to infection by VSV
and the different types of IAV. Despite the fact that reduced
autophagy makes a cellular milieu favorable for viral growth (14),
suppression of UVRAG seems to override host autophagy ma-
chinery and to inhibit virus infection at a step upstream of viral
replication. Indeed, the viral-resistant phenotype in UVRAG-
depletedcellscorrelateswiththeimpairedentryofVSVandIAV,
both of which require acidic compartments to trigger their entry.
WefoundthatUVRAGoperateslateintheendocyticpathwayby
facilitatingvirusaccesstotheLEsformembranefusioninthecell.
Furthermore, UVRAG regulation of viral entry requires C2 and
CCDregions,whichengageC-VpsandBeclin1,respectively(8,9).
Deletion of the C2 that disrupts the interaction with C-Vps tem-
pered UVRAG-mediated viral entry. Unlike C-Vps, depletion of
Beclin1hasaminimaleffectontheactionofUVRAGtopromote
viral infection, suggesting that other factors are involved in this
processthroughtheCCD.Indeed,UVRAGCCDissufficientand
necessary to bind LE Q-SNAREs in a manner independent of its
association with C-Vps, serving as a regulator or a scaffold-like
siRNA:
35
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VSV LASV
Control si
STX7 si
STX8 si
Vti1b si
VAMP8 si
VAMP7 si
0
20
40
60
80
100
120
140
Relative Infectivity
n.s.
***
*
25 --
25
--
15
--
25
--
Ctrl STX7 Ctrl STX8
Ctrl VAMP7
Ctrl VAMP8
Ctrl Vti1b
Actin
STX7
Actin
STX8
Actin
Vti1b
Actin
VAMP8
Actin
VAMP7
A
B
Fig. 5. Distinct roles of the SNARE proteins in virus entry. (A and B) HeLa
cellsweretransfectedwithcontrol-orSNARE-specificsiRNAasindicatedand
then infected with pseudotyped viruses with the envelope protein of VSV
and LASV for 48 h. (A)ViralentryisexpressedasmeanGFPfluorescence
relative to control sh-treated cells. Values represent mean ± SD (n = 3 in-
dependent experiments). (B) Western blot shows endogenous protein ex-
pression in cells. n.s., not significant; *P< 0.05; ***P< 0.001.
2720 | www.pnas.org/cgi/doi/10.1073/pnas.1320629111 Pirooz et al.
Figure 1.6.10 Distinct roles of the SNARE proteins in virus entry. (A and B)
HeLa cells were transfected with control- or SNARE-specific siRNA as
indicated and then infected with pseudotyped viruses with the envelope
protein of VSV and LASV for 48 h. (A) Viral entry is expressed as mean
GFP fluorescence relative to control sh-treated cells. Values represent mean
± SD (n = 3 in- dependent experiments). (B) Western blot shows
endogenous protein expression in cells. n.s., not significant; *P < 0.05; ***P
< 0.001.
resistant to the pseudovirus infection of IAV, and it also drastically disabled the ability of
UVRAG to promote virus entry, suggesting that UVRAG works in concert with specific
SNAREs to regulate virus entry (Fig. S1.9.8 A and B). Given that UVRAG forms a
complex with SNAREs through its CCD, these data explain the antiviral restriction of
ΔCCD (Fig. 1.6.6 A). Consistently, when cells were infected with live rVSV-GFP, only
VAMP7 knockdown cells remained susceptible, whereas siRNA depletion of all other
SNAREs was refractory to VSV infection (Fig. S1.9.8 C). These results indicate that LE-
associated Q-SNAREs and VAMP8 are specifically required for virus entry and infection.
1.6.11 Virus entry stimulates a specific complex assembly of UVRAG, C-Vps,
and SNAREs
The data above imply that UVRAG action with C-Vps and SNAREs is a key regulator
during virus entry. This raises the question of whether this reflects a random event
hitchhiked by the virus or, instead, a strategy of the virus to induce a specific fusogenic
complex for efficient entry. To test this, I infected HeLa cells stably expressing empty
vector or UVRAG with VSV and assessed the complex-forming ability of UVRAG with
endogenous C-Vps and SNAREs within 2 h after infection (Fig. S1.9.8 D). Compared
with the complex formation in mock-infected cells, VSV infection triggered a robust
increase of C-Vps and SNAREs coimmunoprecipitated with UVRAG (Fig. S1.9.8 D).
The increased binding was not due to differing expression because no evident alteration
of the protein levels was detected in mock-and virus-infected cells (Fig. S1.9.8 D).
However, bafilomycin treatment significantly reduced the complex assembly of UVRAG
with C-Vps and SNAREs (Fig. S1.9.8 D), suggesting that this entry-induced complex
assembly of UVRAG lies downstream of the low-pH trigger. As a control, the UVRAG-
43
Beclin 1 interaction was neither affected by VSV nor changed by bafilomycin (Fig.
S1.9.8 D), further suggesting that Beclin 1 is not involved in this process. Analogous
results were obtained when I infected cells with the pseudo-particles. As expected, VSV-
G– and IAV-H5–mediated entry processes are sufficient to promote the complex
formation of UVRAG, C-Vps, and Q-SNAREs (Fig. S1.9.8 F and G).
I next examined UVRAG-mediated cis- and trans-SNARE assembly upon viral infection.
As shown in Fig. S1.9.8 E, UVRAG expression enhanced interactions of STX7 with
STX8 and Vti1b, as well as its pairing with VAMP8, which was further induced by VSV
infection. In contrast, minimal amounts of VAMP7 co-immunoprecipitated with STX7,
suggesting that VAMP7 is largely excluded from UVRAG-promoted SNARE complex
formation upon viral infection (Fig. S1.9.8 E). These findings demonstrate that virus
entry triggers a specific supercomplex formation involving UVRAG, C-Vps, Q-SNARES,
and VAMP8, but not VAMP7.
1.6.12 Vamp8 is recruited to virus-containing vesicles
To examine whether VAMP7 and VAMP8 are differentially required for the virus
entry-induced membrane remodeling, I examined endogenous redistribution of VAMP8
and VAMP7 in control and UVRAG-expressing cells after 1 h of infection with VSV (Fig.
S1.9.9). Both VAMP8 and VAMP7 revealed vesicular staining with a perinuclear
concentration in mock-infected cells (Fig. S1.9.9). Upon VSV infection, however, VAMP8
was prominently recruited to the VSV-G–containing vesicles, whereas VAMP7 was
largely excluded (Fig. S1.9.9). Furthermore, expression of UVRAG resulted in a twofold
increase in the acquisition of VAMP8-containing membranes to the VSV-G
+
vesicles
44
compared with the control but had a minimal effect on VAMP7 accumulation in the virus-
containing vesicles (Fig. S1.9.9). Although UVRAG is required for trans-SNARE
complex assembly (Fig. S1.9.7 E), only VAMP8 was selected to function with UVRAG in
facilitating virus entry, whereas VAMP7 is largely absent from the majority of virus-
containing compartments, consistent with an earlier finding that VAMP7 is not required
for the entry of VSV and IAV.
45
1.7 Discussion
Initially discovered in a screen for UV resistance, UVRAG has been identified in our
studies, as well as those of others, to possess various functions, including intracellular
trafficking, chromosomal stability, autophagy activation, and tumor suppression [51] [66]
[68] [82] [83] [84]. However, the cellular outcome of UVRAG in viral infection has not
been investigated. We show here that UVRAG-deficient cells are largely refractory to
infection by VSV and the different types of IAV. Despite the fact that reduced autophagy
makes a cellular milieu favorable for viral growth [72], suppression of UVRAG seems to
override host autophagy machinery and to inhibit virus infection at a step upstream of
viral replication. Indeed, the viral-resistant phenotype in UVRAG- depleted cells
correlates with the impaired entry of VSV and Influenza, both of which require acidic
compartments to trigger their entry. We found that UVRAG operates late in the
endocytic pathway by facilitating virus access to the LEs for membrane fusion in the
cell. Furthermore, UVRAG regulation of viral entry requires C2 and CCD regions, which
engage C-Vps and Beclin 1, respectively [51] [66]. Deletion of the C2 that disrupts the
interaction with C-Vps tempered UVRAG-mediated viral entry. Unlike C-Vps, depletion
of Beclin 1 has a minimal effect on the action of UVRAG to promote viral infection,
suggesting that other factors are involved in this process through the CCD. Indeed,
UVRAG CCD is sufficient and necessary to bind LE Q-SNAREs in a manner
independent of its association with C-Vps, serving as a regulator or a scaffold-like
protein in the assembly of the C-Vps–SNARE complex on the endosomes. Although
C-Vps and SNAREs are central regulators of late endocytic organelles, their roles in
modifying infection are less established. We demonstrated that abrogation of the
46
subunit of C-Vps or Q-SNAREs drastically reduced entry processes of VSV and IAV.
Unlike our findings, a recent study showed that C-Vps, albeit necessary for Ebola virus
infection, is not needed in VSV infection [52]. The discrepancy between published data
and ours may be due to differences in experimental design and/or to the different cell
lines used in the study. Nonetheless, our data suggests that for productive virus entry,
both VSV and Influenza cell entry must occur through the late endocytic pathway that is
regulated by a functional UVRAG–C-Vps–Q-SNARE complex.
We found that a trans-SNARE complex consisting of the Q- SNAREs STX7, Vti1b, and
STX8 and the R-SNARE VAMP8 is critical for virus entry and that the interaction of
these SNAREs is enhanced shortly after VSV and IAV infection. In contrast, the Q-
SNARE pairing with VAMP7 was drastically reduced. Previous studies have shown that
VAMP7 is enriched on lysosomes, where it mediates heterotypic endosome–lysosome
fusion, but that VAMP8 is present on LEs, where it controls homotypic fusion between
these organelles [25]. Conceivably, suppression of VAMP7, and thereby VAMP7-
mediated lysosome fusion, may allow exclusion of endocytosed virions from the
lysosome delivery designed for pathogen destruction and antigen presentation, whereas
VAMP8 is needed for virus-induced membrane remodeling. In support of this, we found
that VAMP8 is recruited to the virus-carrying vesicles, whereas VAMP7 is excluded.
Moreover, unlike VAMP8, the depletion of which decreased virus entry, the knockdown
of VAMP7 led to a slight but consistent increase of viral entry. Although further validation
to elucidate the impact of SNAREs during in vivo infection is warranted, it is tempting to
speculate that discrepant binding and SNARE assembly induced by viral infection could
47
potentially reflect a strategy of the virus to evade lysosome degradation and immune
recognition.
Although it is known that UVRAG overexpression is known to target viral proteins to
autophagosomes [85] [86] [87] [58] (and thus lead to potential degradation of viral
components) and cell surface receptors to lysosomal degradation, our findings that
suggest that UVRAG overexpression leads to increased viral replication of two negative
strand RNA viruses -Influenza A and Vesicular Stomatitis Virus (VSV) - may seem
counterintuitive at first. Both viruses however encode proteins that inhibit the autophagy
pathway (VSV-G and Influenza Virus M2 protein) thus counteracting the antiviral
autophagy response, either by inhibiting activation of mTOR (VSV-G) or the degradation
of the autophagosome (Influenza Virus M2). On the other hand, UVRAG seems to be
required for the replication of VSV and Influenza Virus, suggesting that UVRAG may
target internalized viral particles to structures, that prevent them from being degraded
and/or recognized by Pattern Recognition Receptors (PRRs) which are an essential part
of the cellular antiviral response. In addition, since UVRAG is required for viral particle
localization to (acidic) late endosomes and endosomes are involved in recognizing viral
RNA via Toll-like receptors, it might be interesting to determine if these endosomes are
positive for TLRs or if viral proteins inhibit the antiviral signaling [67].
This is the first time the cellular outcome of UVRAG in viral infection has been
investigated, which also poses interesting questions. First, what is the molecular
mechanism that allows the virus to interact with and remodel host cell fusion
machinery? Are there any viral entry factors also involved in this process? Furthermore,
are different endocytic cargoes (viral and nonviral) differentially regulated by UVRAG?
48
How does UVRAG coordinate its distinct membrane-associated (autophagosome and
endosome) trafficking activities in the context of viral infection? It remains to be tested
whether this regulation of virus entry we have discovered with VSV and IAV might also
be seen with other viruses. For example, African Swine Fever Virus (AFSV) particles
localize to the late endosome, suggesting that UVRAG might be important as well.
Nonetheless, the existence of intensive interactions between viral particles and the host
endomembrane network suggests that remodeling host cell fusion machinery may be a
shared strategy for productive entry, and that technologies that interfere with the viral
entry step and/or their interaction with the host endomembrane system could have
considerable promise in treating some virally associated diseases.
In summary, our study has identified a previously unknown function of UVRAG in the
regulation of virus entry through multiple interactions with the membrane fusion
machinery of cells, independent of IFN and autophagy activation. we have defined
SNAREs required for endocytic transport and fusion of the virus, which, to our
knowledge, has not been directly linked to virus entry before. Finally, the recruitment of
specific cognate SNARE partners onto the target membrane reflects a virus-induced,
highly programmed cascade of membrane fusion. Further understanding of the
mechanism by which this cascade is regulated will have implications not only for the
understanding of pathogenic mechanisms of virus entry but for development of
attractive new targets for antiviral therapy.
49
1.8 Summary
Most enveloped viruses exploit the endosomal machinery to route themselves to
specific compartments for membrane fusion and delivery of their genetic material, while
autophagy acts as an essential part of the host antiviral defense mechanism. UVRAG is
well known for its crucial role in both pathways: activating autophagy by pairing with
Beclin 1 and accelerating late endocytic transport by pairing with the class C-Vps
complex. Yet, despite its importance, the functionality of UVRAG in viral infection has
not been addressed. Our findings suggest that overexpression of UVRAG confers
efficient infection of vesicular stomatitis virus (VSV) and influenza A virus (IAV), both
negative-strand RNA viruses; in contrast, silencing of UVRAG results in a substantial
reduction of viral replication. This proviral activity of UVRAG is counterintuitive and hard
to reconcile with the role of UVRAG in autophagy activation that is largely antiviral. In
fact, even in autophagy-deficient Atg5 null cells, removal of UVRAG renders cells less
susceptible to VSV infection, suggesting that an alternative mechanism beyond
autophagy may be associated with UVRAG. In line with this, expression of UVRAG
does not affect overall type I-interferon (IFN) production, and UVRAG-induced viral
infection is not due to an altered IFN response––the first line of defense against
infection. On the basis of these data, we argue that UVRAG may directly regulate the
viral life cycle.
To determine the step(s) in the replication cycle that are regulated by UVRAG, we
tracked the movement of VSV labeled with self-quenching dye in living cells, and found
that UVRAG does not affect the initial uptake of the virus into cells, nor does it alter the
route of delivery of the endocytosed viral particles to early endosomes.
50
However,knockdown of UVRAG results in a significant delay in viral access to late
endosomes and in virus-endosome fusion for cytoplasmic delivery of nucleocapsids.
Encouraged by these results, we conducted a single- cycle entry assay using a Moloney
murine leukemia virus (MLV)-based pseudo-retroviral system carrying different viral
envelopes as entry factors, and found that knockdown of UVRAG considerably inhibited
infection mediated by the glycoprotein of VSV; it also blocked the entry of multiple highly
pathogenic strains of Influenza. In contrast, suppression of UVRAG did not prevent
arenavirus infection that enters cells via a different endocytic pathway. Our data suggest
a functional specificity and essential role of UVRAG during virus entry.
How might UVRAG regulate the virus entry process? UVRAG appears to have close
association with the endosome machinery. In addition to C-Vps, interactions between
UVRAG and late endosomal SNARE proteins have been detected in our study. The
region of UVRAG that is responsible for this interaction is mapped to the CCD domain in
UVRAG,the region that had previously been shown to confer Beclin 1-binding and
autophagy activation. Although Beclin 1 has been implicated in the antiviral response
and mutations in Beclin 1 are associated with pathogenesis, its function in virus entry is
unclear. In fact, our findings suggest that Beclin 1 is a minor participant in UVRAG-
mediated viral entry. Genetic deletion or siRNA-mediated depletion of Beclin 1 has
minimal effect on viral infection, and it does not affect UVRAG-SNAREs interactions.
Instead, our observations strongly support an important role for the UVRAG-C-Vps-
SNAREs complex in promoting virus entry. First, deletion of the C2 or CCD domain of
UVRAG that disrupts the interaction of UVRAG with C-Vps and SNAREs, respectively,
severely impairs the ability of UVRAG to promote cells’ susceptibility to VSV and
51
Inflienza infection. Second, the siRNA-mediated depletion of the individual C-Vps
complex subunits and SNARE proteins potently suppresses virus infection. Finally, the
overexpression of wild-type UVRAG, but not the C-Vps- or SNAREs-binding defective
UVRAG mutant, promotes the assembly of fusogenic trans-SNAREs at endosomes, a
decisive step in driving membrane fusion.
The formation of fusogenic trans- SNARE complexes confers specificity to the
membrane fusion process in cells. We observed that the UVRAG-associated C-Vps is
able to mediate the pairing of Q-SNAREs, including STX7 (syntaxin 7), STX8 (syntaxin
8), and Vti1B, with both the late endosome-related R-SNARE VAMP8 and the
lysosome-related R-SNARE VAMP7 in normal conditions. However, an altered
interaction pattern of endosomal SNAREs is observed shortly after viral infection.
Concomitant with a robust increase in the pairing of Q-SNAREs with VAMP8, we
observed a significant decrease in their pairing with VAMP7. In support of this,
suppression of VAMP7 that is required for lysosome-related membrane fusion, does not
inhibit virus entry; rather, it results in a slight increase in viral infection. Moreover, unlike
VAMP8 that is recruited to the virus-carrying vacuoles, VAMP7 is largely excluded, and
this process requires UVRAG and its interaction with C-Vps. These findings suggest
that UVRAG- mediated SNARE assembly is not a random event hitchhiked by the virus;
instead, it may represent a mechanism of viral evasion of lysosome fusion that is
designed for pathogen destruction and immune detection. In summary, our study clearly
reveals a previously unknown function of UVRAG in regulating virus entry through
multiple interactions with the membrane fusion machinery of cells, independent of
52
autophagy. To the best of our knowledge, this is the first time the cellular outcome of
UVRAG in viral infection has been investigated.
53
1.9 Supplementary Data
Figure 1.9.1 UVRAG promotes VSV replication
54
1. Liang C, et al. (2006) Autophagic and tumour suppressor activity of a novel Beclin1-
binding protein UVRAG. Nat Cell Biol 8(7):688–699.
2. Liang C, et al. (2008) Beclin1-binding UVRAG targets the class C Vps complex to
coordinateautophagosome maturation and endocytic trafficking. Nat Cell Biol 10(7):
776–787.
3. Joubert PE, et al. (2012) Chikungunya-induced cell death is limited by ER and
oxidative stress-induced autophagy. Autophagy 8(8):1261–1263.
4. YueZ,JinS,YangC,LevineAJ,HeintzN(2003)Beclin1,anautophagygeneessential
for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl
Acad Sci USA 100(25):15077–15082.
5. Zhao Z, et al. (2012) A dual role for UVRAG in maintaining chromosomal stability
independent of autophagy. Dev Cell 22(5):1001–1016.
6. Whelan SP, Ball LA, Barr JN, Wertz GT (1995) Efficient recovery of infectious vesicular
stomatitis virus entirely from cDNA clones. Proc Natl Acad Sci USA 92(18):8388–8392.
7. Yoshida T, et al. (2010) Clinical omics analysis of colorectal cancer incorporating copy
number aberrations and gene expression data. Cancer Inform 9:147–161.
8. Gangappa S, van Dyk LF, Jewett TJ, Speck SH, Virgin HW, 4th (2002) Identification of
the in vivo role of a viral bcl-2.JExpMed 195(7):931–940.
9. Sumpter R, Jr., Levine B (2011) Selective autophagy and viruses. Autophagy 7(3):
260–265.
10. Le Blanc I, et al. (2005) Endosome-to-cytosol transport of viral nucleocapsids. Nat Cell
Biol 7(7):653–664.
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Vector UVRAG
Vec UVRAG
UVRAG
Actin
Vector UVRAG
UVRAG+/- ES
-
/ +
ES
1MOI 1MOI 10MOI
r o t c e V G A R V U
UVRAG r o t c e V . 6 1 1 T C H G A R V U . 6 1 1 T C H
1 MOI
10 MOI
Vec
UVRAG
UVRAG+/- ES
Fig. S1. UVRAGpromotesVSVreplication.UVRAG
+/−
EScellsreconstitutedwithemptyvector(rows1–2),orFlag-taggedUVRAG(rows3–4)wasinfectedwith
recombinant VSV expressing GFP (rVSV-GFP) at the indicated MOI for 18 h. The infected cells (green) were processed for immunofluorescence microscopy (A),
and viral titers were determined by plaque assay (B). Data represent mean± SD (n= 4). (B, Lower Right) Reconstituted UVRAG expression was confirmed by
immunoblotting. Vec, vector. (C and D) Expression of UVRAG in HCT116 cells enhances viral replication. HCT116 cells stably expressing empty vector (HCT116.
Vector) or Flag-UVRAG (HCT116.UVRAG) were infected with rVSV-GFP at the indicated MOI for 12 h. Infected cells (green) were visualized by fluorescence
microscopy (C), and viral infectivity at 1 MOI was measured by plaque assay (D). Values represent mean ± SD (n = 3). (D, Inset) Flag-UVRAG expression in
HCT116 cells with actin served as a loading control. **P< 0.01. (Scale bars, 50 μm.)
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 3 of 11
Figure 1.9.1 UVRAG promotes VSV replication. UVRAG
+/−
ES cells reconstituted with empty vector
(rows 1–2), or Flag-tagged UVRAG (rows 3–4) was infected with recombinant VSV expressing GFP
(rVSV-GFP) at the indicated MOI for 18 h. The infected cells (green) were processed for
immunofluorescence microscopy (A), and viral titers were determined by plaque assay (B). Data
represent mean ± SD (n = 4). (B, Lower Right) Reconstituted UVRAG expression was confirmed by
immunoblotting. Vec, vector. (C and D) Expression of UVRAG in HCT116 cells enhances viral
replication. HCT116 cells stably expressing empty vector (HCT116. Vector) or Flag-UVRAG
(HCT116.UVRAG) were infected with rVSV-GFP at the indicated MOI for 12 h. Infected cells (green)
were visualized by fluorescence microscopy (C), and viral infectivity at 1 MOI was measured by
plaque assay (D). Values represent mean ± SD (n = 3). (D, Inset) Flag-UVRAG expression in HCT116
cells with actin served as a loading control. **P < 0.01. (Scale bars, 50 µm.)
Figure 1.9.2 Effect of IFN on UVRAG-mediated VSV infection
55
Fig. S2. Effect of IFN on UVRAG-mediated VSV infection. (A and B)QuantitativeRT-PCRanalysisofRNAsencodingtheIFN-α, IFN-β,andUVRAGproteins.
Control and UVRAG knockdown HeLa cells were treated with polyinosinic:polycytidylic acid [poly(I:C); 0.1 μg/mL] for 6 h, and the induction of ifnα and ifnβ
gene expression was quantified by real-time RT-PCR with the gene actin as a reference to normalize data. Data shown represent mean ± SD of three in-
dependent experiments. n.s., not significant. (A)RepresentativegelimagesofthePCRproductsofIFN-α, IFN-β, UVRAG [confirming UVRAG knockdown (KD)],
andactinareshown.Theimageisinvertedforclarity.(C)SchematicrepresentationoftheexperimentalprocedurefortypeIIFNproductioninUVRAGKDcells.
HeLa cells were transfected with control- or UVRAG-specific shRNA; they were then treated with poly(I:C) for 6 h, and medium was collected. Naive HeLa cells
were incubated overnight (O/N) with the collected medium from controland UVRAG KD cells and then infected with rVSV-GFP; the results are shown inD and
E. (Scale bar, 20 μm.) Infected cells (green) in C were visualized by fluorescence microscopy 8 h after infection (D), and viral titers were determined by plaque
assay (E). Data were normalized to values in untreated cells, and error bars indicate SD from three independent experiments.
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 4 of 11
Figure 1.9.2 Effect of IFN on UVRAG-mediated VSV infection. (A and B) Quantitative RT-PCR analysis
of RNAs encoding the IFN-α, IFN-β, and UVRAG proteins. Control and UVRAG knockdown HeLa cells
were treated with polyinosinic:polycytidylic acid [poly(I:C); 0.1 µg/mL] for 6 h, and the induction of ifnα
and ifnβ gene expression was quantified by real-time RT-PCR with the gene actin as a reference to
normalize data. Data shown represent mean ± SD of three in- dependent experiments. n.s., not
significant. (A) Representative gel images of the PCR products of IFN-α, IFN-β, UVRAG [confirming
UVRAG knockdown (KD)], and actin are shown. The image is inverted for clarity. (C) Schematic
representation of the experimental procedure for type I IFN production in UVRAG KD cells. HeLa cells
were transfected with control- or UVRAG-specific shRNA; they were then treated with poly(I:C) for 6 h,
and medium was collected. Naive HeLa cells were incubated overnight (O/N) with the collected medium
from control and UVRAG KD cells and then infected with rVSV-GFP; the results are shown in D and E.
(Scale bar, 20 µm.) Infected cells (green) in C were visualized by fluorescence microscopy 8 h after
infection (D), and viral titers were determined by plaque assay (E). Data were normalized to values in
untreated cells, and error bars indicate SD from three independent experiments.
Figure 1.9.3 Autophagy-independent effect of UVRAG in viral infection
56
Control shRNA: UVRAG Control UVRAG
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2
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Atg5+/+ Atg5-/-
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10
(PFU/ml)]
Atg5+/+ Atg5-/-
***
F
UVRAG
Actin
100
37
Control sh
Control sh
UVRAG sh
UVRAG
sh
Atg5+/+ Atg5-/-
Atg5
A
0.1 MOI 1 MOI 10 MOI
Atg7
+/+
Atg7
-/-
0.1 MOI 1 MOI 10 MOI
B
0.1 MOI 1 MOI 10 MOI
Atg5
+/+
Atg5
-/-
0.1 MOI 1 MOI 10 MOI
0.1 MOI 1 MOI 10 MOI
Atg16
+/+
Atg16
+/-
0.1 MOI 1 MOI 10 MOI
C
Atg5
Actin
Atg7
Actin
Atg16
Actin
+/+ +/-
+/+ -/-
+/+ -/-
70
55
70
iMEF
iMEF
iMEF
Fig. S3. Autophagy-independent effect of UVRAG in viral infection. Role of autophagy in viral infection. Atg7
−/−
(A), Atg5
−/−
(B), and Atg16
+/−
(C) immor-
talized mouse embryonic fibroblast (iMEF) cells with their relevant control cells were infected with rVSV-GFP at the indicated MOI (0.1–10) for 12 h, and viral
replication (green) was visualized by fluorescence microscopy. (Scale bars, 50 μm.) (Right) Endogenous protein expression in these cells is shown. (D–F)Atg5
+/+
andAtg5
−/−
iMEFcellsweretreatedwithcontrolshRNAorUVRAG-specificshRNAfor72handinfectedwithrVSV-GFPfor12h.Theinfectedcells(green)were
processed for immunofluorescence microscopy (D), and viral titers were determined by plaque assay (E). ND, not detectable; sh, short hairpin. ***P< 0.001.
(Scale bar, 20 μm.) (F) Western blots show the levels of Atg5 and UVRAG. Data represent mean ± SD (n = 3).
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 5 of 11
Figure 1.9.3 Autophagy-independent effect of UVRAG in viral infection. Role of autophagy in viral
infection. Atg7
−/−
(A), Atg5
−/−
(B), and Atg16
+/−
(C) immor- talized mouse embryonic fibroblast
(iMEF) cells with their relevant control cells were infected with rVSV-GFP at the indicated MOI
(0.1–10) for 12 h, and viral replication (green) was visualized by fluorescence microscopy. (Scale
bars, 50 µm.) (Right) Endogenous protein expression in these cells is shown. (D–F) Atg5
+/+
and
Atg5
−/−
iMEF cells were treated with control shRNA or UVRAG-specific shRNA for 72 h and
infected with rVSV-GFP for 12 h. The infected cells (green) were processed for
immunofluorescence microscopy (D), and viral titers were determined by plaque assay (E). ND,
not detectable; sh, short hairpin. ***P < 0.001. (Scale bar, 20 µm.) (F) Western blots show the
levels of Atg5 and UVRAG. Data represent mean ± SD (n = 3).
Figure 1.9.4 UVRAG is essential for virus entry

57
Fig. S4. UVRAG is essential for virus entry. (A) Effect of UVRAG on VSV infection upon acid-mediated bypass of endocytosis. HeLa cells were transfected with
control shRNA or UVRAG-specific shRNA for 72 h and then incubated with VSV (MOI of 1) at pH 5.0 (to induce fusion of the viral envelope and the plasma
membrane) as indicated. Cells were then incubated at 37 °C for 2 h without or with 2 μM bafilomycin A1 (Baf A1). Viral RNA replication was quantified by RT-
PCR and expressed as a percentage of the control condition. Data represent mean± SD from three independent experiments. n.s., not significant; pm, plasma
membrane. (B) UVRAG does not affect VSV uptake. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA for 72 h and infected with VSV
(MOIof1)onice for30 min.Cells werethenincubatedfor theindicatedtimeat37°C toallowVSVinternalization. Viral RNAsinternalizedwerequantified by
real-timeRT-PCR,andnostatisticalsignificancewasdetected.RNAlevelswhentherewasnoacidwashwereincludedasacontrol.Valuesrepresentmean±SD
(n= 3). (C and D)GainofUVRAGexpressionpromotesvirusentry.HCT116(C)andHeLa(D) cells stably expressing vector or Flag-UVRAG were infected with
MLV-GFP pseudotyped with the entry proteins of VSV or Lassa virus (LASV). Forty-eight hours after infection, pseudovirus infectivity (green) was visualized by
fluorescence microscopy (C, Left and D) and expressed as mean EGFP fluorescence relative to vector control cells, as measured by flow cytometry (C, Right).
Data represent mean± SD from five independent experiments. **P< 0.01. (Scale bars, 50 μm.) (E–G) UVRAG depletion inhibits late endosomal transport of
VSV.HeLacellsweretransfectedwithcontrol-orUVRAG-specificshRNAfor72handinfectedwithDiI-labeledVSVat4°C.(E)Temperaturewasthenshiftedto
37 °C to allow endocytosis; cells at the indicated time frames were analyzed by fluorescence microscopy, and representative images from three independent
experimentsweretaken.ArrowsdenotedequenchedDiIfluorescentspotsthatrepresentviralexposuretoacidiccompartments.(Scalebar,50μm.)(F)Number
of cells containing DiI-dequenching signals was counted at the indicated time and is expressed as a percentage of the total cell numbers. Cells in E were also
treated with 2 μMBafA1.(G) Number of cells containing DiI-spots was counted 45 min postinfection and expressed as the percentage of control. Data
representmean±SD(n=60)fromthreeindependentexperiments.**P<0.01;***P<0.001.(HandI)Viralnucleocapsidreleaseintothecytoplasmisarrested
by UVRAG knockdown. (H) HeLa cells were transfected with control- or UVRAG-specific shRNA for 72 h and then infected with VSV for 2 h in the presence or
absence of nocodazole (Noc) or Baf A1 and processed for VSV M staining (red) by confocal microscopy; representative images are shown. Nuclei were stained
with DAPI (blue). (Scale bars, 20 μm.) (I) Number of infected cells with diffused M staining in the cytoplasm was quantified and expressed as a percentage of
that of control. Data represent mean ± SD (n = 100) from three independent experiments. **P < 0.01. (J) UVRAG deficiency inhibits virus transport to late
endosomalcompartments.HeLacellsweretransfectedwithcontrol-orUVRAG-specificshRNAfor72h,preincubatedwithoutorwith10μMNoc(+Noc)for2h,
and then infected with MLV-GFP pseudotyped with VSV-G. Infected cells were fixed and immunostained with antibody against VSV-G (green) and lysobi-
sphosphatidicacid(LBPA;red).NucleiwerestainedwithDAPI(blue).(Insets)RelativedistributionofVSV-GwithLBPAishighlightedintheindicatedcells.(Scale
bars, 20 μm.)
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58
Figure 1.9.4 UVRAG is essential for virus entry. (A) Effect of UVRAG on VSV infection upon acid-mediated
bypass of endocytosis. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA for 72 h and
then incubated with VSV (MOI of 1) at pH 5.0 (to induce fusion of the viral envelope and the plasma membrane)
as indicated. Cells were then incubated at 37 °C for 2 h without or with 2 µM bafilomycin A1 (Baf A1). Viral RNA
replication was quantified by RT- PCR and expressed as a percentage of the control condition. Data represent
mean ± SD from three independent experiments. n.s., not significant; pm, plasma membrane. (B) UVRAG does
not affect VSV uptake. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA for 72 h and
infected with VSV (MOI of 1) on ice for 30 min. Cells were then incubated for the indicated time at 37 °C to allow
VSV internalization. Viral RNAs internalized were quantified by real-time RT-PCR, and no statistical significance
was detected. RNA levels when there was no acid wash were included as a control. Values represent mean ±
SD (n = 3). (C and D) Gain of UVRAG expression promotes virus entry. HCT116 (C) and HeLa (D) cells stably
expressing vector or Flag-UVRAG were infected with MLV-GFP pseudotyped with the entry proteins of VSV or
Lassa virus (LASV). Forty-eight hours after infection, pseudovirus infectivity (green) was visualized by
fluorescence microscopy (C, Left and D) and expressed as mean EGFP fluorescence relative to vector control
cells, as measured by flow cytometry (C, Right). Data represent mean ± SD from five independent experiments.
**P < 0.01. (Scale bars, 50 µm.) (E–G) UVRAG depletion inhibits late endosomal transport of VSV. HeLa cells
were transfected with control- or UVRAG-specific shRNA for 72 h and infected with DiI-labeled VSV at 4 °C. (E)
Temperature was then shifted to 37 °C to allow endocytosis; cells at the indicated time frames were analyzed by
fluorescence microscopy, and representative images from three independent experiments were taken. Arrows
denote dequenched DiI fluorescent spots that represent viral exposure to acidic compartments. (Scale bar, 50
µm.) (F) Number of cells containing DiI-dequenching signals was counted at the indicated time and is expressed
as a percentage of the total cell numbers. Cells in E were also treated with 2 µM Baf A1. (G) Number of cells
containing DiI-spots was counted 45 min postinfection and expressed as the percentage of control. Data
represent mean ± SD (n = 60) from three independent experiments. **P < 0.01; ***P < 0.001. (H and I) Viral
nucleocapsid release into the cytoplasm is arrested by UVRAG knockdown. (H) HeLa cells were transfected with
control- or UVRAG-specific shRNA for 72 h and then infected with VSV for 2 h in the presence or absence of
nocodazole (Noc) or Baf A1 and processed for VSV M staining (red) by confocal microscopy; representative
images are shown. Nuclei were stained with DAPI (blue). (Scale bars, 20 µm.) (I) Number of infected cells with
diffused M staining in the cytoplasm was quantified and expressed as a percentage of that of control. Data
represent mean ± SD (n = 100) from three independent experiments. **P < 0.01. (J) UVRAG deficiency inhibits
virus transport to late endosomal compartments. HeLa cells were transfected with control- or UVRAG-specific
shRNA for 72 h, preincubated without or with 10 µM Noc (+Noc) for 2 h, and then infected with MLV-GFP
pseudotyped with VSV-G. Infected cells were fixed and immunostained with antibody against VSV-G (green) and
lysobisphosphatidic acid (LBPA; red). Nuclei were stained with DAPI (blue). (Insets) Relative distribution of VSV-
G with LBPA is highlighted in the indicated cells. (Scale bars, 20 µm.)
Figure 1.9.5 Domain of UVRAG in viral infection
59
Fig. S5. Domain of UVRAG in viral infection. (A) Domain organization of UVRAG. The C2, CCD, and C-terminal regions of UVRAG are indicated. Amino acid
positionsareshown abovethefigure.(B)Immunoblotting(IB)analysisofVSVGinWT-andmutantUVRAG-expressingHCT116cells infectedfor12hwithVSV
(MOI of 1). Actin serves as a loading control throughout. (C and D) HCT116 cells stably expressing WT or mutant UVRAG were infected with VSV for 1 h and
processedforconfocalmicroscopyusingantibodiesagainstVSV-G(green)andLBPA(red).NucleiwerestainedwithDAPI(blue).(Insets)Relativedistributionof
VSV-G with LBPA in cells is highlighted. (Scale bars, 10 μm.) (D)PercentageofVSV-G–containing vesicles colocalized with LBPA was quantified. Data represent
mean± SD (n = 3). **P< 0.01; ***P< 0.001.
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 7 of 11
Figure 1.9.5 Domain of UVRAG in viral infection. (A) Domain organization of UVRAG. The
C2, CCD, and C-terminal regions of UVRAG are indicated. Amino acid positions are shown
above the figure. (B) Immunoblotting (IB) analysis of VSV G in WT- and mutant UVRAG-
expressing HCT116 cells infected for 12 h with VSV (MOI of 1). Actin serves as a loading
control throughout. (C and D) HCT116 cells stably expressing WT or mutant UVRAG were
infected with VSV for 1 h and processed for confocal microscopy using antibodies against
VSV-G (green) and LBPA (red). Nuclei were stained with DAPI (blue). (Insets) Relative
distribution of VSV-G with LBPA in cells is highlighted. (Scale bars, 10 µm.) (D) Percentage
of VSV-G–containing vesicles colocalized with LBPA was quantified. Data represent mean
± SD (n = 3). **P < 0.01; ***P < 0.001.
Figure 1.9.6 Role of Beclin 1 and class C vacuolar protein sorting (C-Vps) in
UVRAG-mediated viral infection

60
Fig. S6. Role of Beclin1 and class C vacuolar protein sorting (C-Vps) in UVRAG-mediated viral infection. (A and B) HeLa cells were transfected with control-,
Vps16-, or Vps18-specific siRNA as indicated and then infected with rVSV-GFP at the indicated MOI for 12 h. Infected cells (green) were detected by fluo-
rescencemicroscopy(A),andviralinfectivitywasquantifiedbythepercentageofGFP
+
cellsdetectedbyflowcytometry(B).si,smallinterfering.(Scalebars,50
μm.) (B, Right) Western blots show endogenous protein expression. Ctrl, control. (C–E) HCT116 cells stably expressing WT or mutant UVRAG were transfected
with control- or Vps18-specific siRNA for 72 h and then infected with rVSV-GFP. (C) Infected cells (green) were processed for fluorescence microscopy. (Scale
bar,50μm.)(D)PercentageofGFP
+
-positivecellsincontrol-orVps18-siRNA–treatedcellswasquantifiedbyflowcytometry.*P<0.05;**P<0.01;***P<0.001.
Legend continued on following page
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61
Figure 1.9.6 Role of Beclin 1 and C-Vps in UVRAG-mediated viral infection. (A and B) HeLa cells
were transfected with control-, Vps16-, or Vps18-specific siRNA as indicated and then infected with
rVSV-GFP at the indicated MOI for 12 h. Infected cells (green) were detected by fluorescence
microscopy (A), and viral infectivity was quantified by the percentage of GFP
+
cells detected by flow
cytometry (B). si, small interfering. (Scale bars, 50 µm.) (B, Right) Western blots show endogenous
protein expression. Ctrl, control. (C–E) HCT116 cells stably expressing WT or mutant UVRAG were
transfected with control- or Vps18-specific siRNA for 72 h and then infected with rVSV-GFP. (C)
Infected cells (green) were processed for fluorescence microscopy. (Scale bar, 50 µm.) (D)
Percentage of GFP
+
-positive cells in control- or Vps18-siRNA–treated cells was quantified by flow
cytometry. *P < 0.05; **P < 0.01; ***P < 0.001. (E) Western blot shows the expression levels of
Vps18 in cells with actin serving as a loading control. (F) Fluorescence microscopy of WT and Beclin
1 knockdown HeLa cells infected for 12 h with rVSV-GFP. Viral infectivity was expressed as the
percentage of GFP
+
cells by flow cytometry (Center), and Western blots show endogenous protein
expression (Right). (Scale bar, 50 µm.) (G–I) HCT116 cells stably expressing vector, WT UVRAG,
ΔC2, or ΔCCD were transfected with control- or Beclin 1-specific shRNA for 72 h and then infected
with rVSV-GFP. (G) Infected cells (green) were visualized by fluorescence microscopy. (Scale bar, 50
µm.) (H) Percentage of GFP
+
-positive cells in control- or Beclin 1-shRNA–treated cells was
Figure 1.9.7 UVRAG mediates efficient interaction of the C-Vps complex
with SNAREs
62
(E)WesternblotshowstheexpressionlevelsofVps18incellswithactinservingasaloadingcontrol.(F)FluorescencemicroscopyofWTandBeclin1knockdown
HeLa cells infected for 12 h with rVSV-GFP. Viral infectivity was expressed asthe percentage of GFP
+
cells by flow cytometry (Center), and Western blots show
endogenousproteinexpression(Right).(Scalebar,50μm.)(G–I)HCT116cellsstablyexpressingvector,WTUVRAG,ΔC2,orΔCCDweretransfectedwithcontrol-
orBeclin1-specificshRNAfor72handtheninfectedwithrVSV-GFP.(G)Infectedcells(green)werevisualizedbyfluorescencemicroscopy.(Scalebar,50μm.)(H)
Percentage of GFP
+
-positive cells in control- or Beclin1-shRNA–treated cells was determined by flow cytometry. (I) Western blot shows the expression levels of
Beclin1 in these cells, with actin serving as a loading control. Bec, Beclin1.
Fig. S7. UVRAG mediates efficient interaction of the C-Vps complex with SNAREs. (A) CCD region of UVRAG interacts with SNAREs. Forty-eight hours
posttransfection with Flag-tagged WT and mutant UVRAG proteins, 293T whole-cell lysates (WCLs) were immunoprecipitated with anti-Flag, followed by IB
with the indicated antibodies. (Right) WCL protein expressions are shown. (B) UVRAG CCD region is sufficient for SNARE binding. 293T cells transfected with
GST-tagged UVRAG deletion mutants as indicated were immunoprecipitated with anti-GST, followed by IB with the indicated antibodies. (Right) GST-tagged
and endogenous protein expression is shown. (C) Effect of Beclin1 on UVRAG interaction with SNAREs. 293T cells were transfected with control shRNA or
Beclin1-specific shRNA for 72 h, followed by immunoprecipitation (IP) with a UVRAG antibody and IB for UVRAG and the SNARE complex subunits. (Right)
Endogenous protein expression is shown. Input represents 10% WCLs. Note that SNARE interaction was unaffected by Beclin1 knockdown. (D)UVRAGpro-
motes the complex assembly of C-Vps and SNAREs. 293T cells were transfected with Flag-tagged WT and mutant UVRAG proteins, followed by IP with anti-
Vps16 (Left)oranti-Vps18(Right) and then IB with the indicated antibodies. (Right) Transfected and endogenous protein expression is shown. (E)UVRAGis
required for the SNARE assembly. 293T cells were transfected with control shRNA or UVRAG shRNA for 72 h. WCLs were used for IP with anti-STX8 (Left)or
anti-Vti1b (Center), followed by IB with the indicated antibodies. (Right)Endogenousproteinexpression.(F) SNARE distribution in UVRAG-expressing cells.
HeLa.Vector and HeLa.UVRAG cells were incubated with 1 mM N-ethylmaleimide for 15 min and processed for confocal microscopy analysis using the anti-
VAMP7 (green), anti-VAMP8 (green), and anti-STX8 (red) antibodies. (Insets) Relative colocalization of SNARE proteins is highlighted. (Scale bars, 10μm.) (G)
Schematic representation of the UVRAG/C-Vps/SNARE supercomplex.
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 9 of 11
Figure 1.9.7 UVRAG mediates efficient interaction of the C-Vps complex with SNAREs. (A) CCD region of
UVRAG interacts with SNAREs. Forty-eight hours posttransfection with Flag-tagged WT and mutant UVRAG
proteins, 293T whole-cell lysates (WCLs) were immunoprecipitated with anti-Flag, followed by IB with the
indicated antibodies. (B) UVRAG CCD region is sufficient for SNARE binding. 293T cells transfected with
GST-tagged UVRAG deletion mutants as indicated were immunoprecipitated with anti-GST. (C) Effect of
Beclin 1 on UVRAG interaction with SNAREs. 293T cells were transfected with control shRNA or Beclin 1-
specific shRNA for 72 h, followed by immunoprecipitation (IP) with a UVRAG antibody and IB for UVRAG and
the SNARE complex subunits. Input represents 10% WCLs. (D) UVRAG promotes the complex assembly of
C-Vps and SNAREs. 293T cells were transfected with Flag-tagged WT and mutant UVRAG proteins, followed
by IP with anti- Vps16 (Left) or anti-Vps18 (Right) and then IB with the indicated antibodies. (E) UVRAG is
required for the SNARE assembly. 293T cells were transfected with control shRNA or UVRAG shRNA for 72
h. (F) SNARE distribution in UVRAG-expressing cells. HeLa.Vector and HeLa.UVRAG cells were incubated
with 1 mM N-ethylmaleimide for 15 min and processed for confocal microscopy analysis using the anti-
VAMP7 (green), anti-VAMP8 (green), and anti-STX8 (red) antibodies. (Insets) Relative colocalization of
SNARE proteins is highlighted. (Scale bars, 10 µm.) (G) Schematic representation of the UVRAG/C-Vps/
SNARE supercomplex.
Figure 1.9.8 Effect of SNARE proteins in UVRAG-mediated virus entry.
63
A
B
F
vec 0 2
VSV-Pseudotyped
virus entry (h)
Baf A1
__ _
+
2
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Flag
IB: Beclin1
IB: STX7
IB: STX8
IP: Flag
Flag-UVRAG
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Beclin1
IB: STX7
IB: STX8
vec 0 2
Baf A1
__
+
2
Flag-UVRAG
_
C-Vps SNAREs
WCLs
1234
VSV-Pseudovirus (h)
G
Control
siRNA:
VAMP8
UVRAG Vector
VAMP7 STX8 Vti1b STX7
Pseudovirus [IAV (H5)]
vec 0 2
IAV(H5)-Pseudotyped
virus entry (h)
Baf A1
__ _
+
2
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Flag
IB: Beclin1
IB: STX7
IB: STX8
IP: Flag
Flag-UVRAG
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Beclin1
IB: STX7
IB: STX8
vec 0 2
Baf A1
__
+
2
Flag-UVRAG
_
C-Vps SNAREs
WCLs
1234
Pseudovirus (h)
0
20
40
60
80
100
HeLa-vector HeLa-UVRAG
control sh
STX7 sh
Vti 1b sh
STX8 sh
vamp8 sh
vamp7 sh
HeLa. Vector HeLa. UVRAG
% GFP
**
**
** *
Control si
STX7si
Vti1b si
STX8si
VAMP8 si
VAMP7 si
**
**
***
**
100 -
100 -
100 -
35 -
25 -
25 -
60 -
100 -
100 -
100 -
35-
25-
25-
60-
rVSV-GFP
Ctrl STX7 Vti1b STX8 VAMP8 VAMP7 siRNA:
0
100
Relative Infectivity
50
Control si
STX7 si
STX8 si
Vti1b si
VAMP8 si
VAMP7
si
***
*
C
E D
vec 0 2 viral entry (h)
Baf A1
__ _
+
2
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Flag
IB: Beclin1
IB: STX7
IB: STX8
IP: Flag
IB: UVRAG
Flag-UVRAG
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Beclin1
IB: STX7
IB: STX8
vec 0 2 viral entry (h)
Baf A1 +
2
Flag-UVRAG
__ _
C-Vps SNAREs
WCLs
35-
100-
25-
100-
25-
60-
100-
100-
1 2 3 4
WCLs
02
VSV (h)
IP: STX7
IgG
IB: STX8
IB: Vti1b
IB: VAMP7
IB: VAMP8
vec
UVRAG
UVRAG
0 0
15
25-
25-
15-
-
-
25-
-
25
IB: STX7
IB: VAMP7
IB: VAMP8
- 35
1 2 3 4
Fig. S8. Effect of SNARE proteins in UVRAG-mediated virus entry. (A and B) HCT116 cells stably expressing empty vector or UVRAG were transfected with
control- or SNARE-specific siRNA as indicated for 72 h and then infected with MLV-GFP pseudotyped with the entry proteins of influenza A virus (IAV; H5). (A)
Infected cells (green) were visualized by fluorescence microscopy. (Scale bar, 50 μm.) (B)PercentageofGFP
+
cells was determined by flow cytometry and
expressedas thepercentage relativetothecontrol.*P<0.05; **P<0.01. (C)Fluorescencemicroscopy(Left)andflowcytometry(Right)analysis ofcontroland
SNARE knockdown cells infected for 12 h with rVSV-GFP. Viral infectivity is expressed as mean GFP fluorescence relative to control sh-treated cells. Data
represent mean± SD from three independent experiments. ***P< 0.001. (Scale bar, 20 μm.) (D) VSV infection induces the complex assembly of UVRAG with
Legend continued on following page
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 10 of 11
A
B
F
vec 0 2
VSV-Pseudotyped
virus entry (h)
Baf A1
__ _
+
2
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Flag
IB: Beclin1
IB: STX7
IB: STX8
IP: Flag
Flag-UVRAG
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Beclin1
IB: STX7
IB: STX8
vec 0 2
Baf A1
__
+
2
Flag-UVRAG
_
C-Vps SNAREs
WCLs
1234
VSV-Pseudovirus (h)
G
Control
siRNA:
VAMP8
UVRAG Vector
VAMP7 STX8 Vti1b STX7
Pseudovirus [IAV (H5)]
vec 0 2
IAV(H5)-Pseudotyped
virus entry (h)
Baf A1
__ _
+
2
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Flag
IB: Beclin1
IB: STX7
IB: STX8
IP: Flag
Flag-UVRAG
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Beclin1
IB: STX7
IB: STX8
vec 0 2
Baf A1
__
+
2
Flag-UVRAG
_
C-Vps SNAREs
WCLs
1234
Pseudovirus (h)
0
20
40
60
80
100
HeLa-vector HeLa-UVRAG
control sh
STX7 sh
Vti 1b sh
STX8 sh
vamp8 sh
vamp7 sh
HeLa. Vector HeLa. UVRAG
% GFP
**
**
** *
Control si
STX7si
Vti1b si
STX8si
VAMP8 si
VAMP7 si
**
**
***
**
100 -
100 -
100 -
35 -
25 -
25 -
60 -
100 -
100 -
100 -
35-
25-
25-
60-
rVSV-GFP
Ctrl STX7 Vti1b STX8 VAMP8 VAMP7 siRNA:
0
100
Relative Infectivity
50
Control si
STX7 si
STX8 si
Vti1b si
VAMP8 si
VAMP7
si
***
*
C
E D
vec 0 2 viral entry (h)
Baf A1
__ _
+
2
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Flag
IB: Beclin1
IB: STX7
IB: STX8
IP: Flag
IB: UVRAG
Flag-UVRAG
IB: Vps16
IB: Vps18
IB: Vps11
IB: Vti1b
IB: Beclin1
IB: STX7
IB: STX8
vec 0 2 viral entry (h)
Baf A1 +
2
Flag-UVRAG
__ _
C-Vps SNAREs
WCLs
35-
100-
25-
100-
25-
60-
100-
100-
1 2 3 4
WCLs
02
VSV (h)
IP: STX7
IgG
IB: STX8
IB: Vti1b
IB: VAMP7
IB: VAMP8
vec
UVRAG
UVRAG
0 0
15
25 -
25-
15-
-
-
25 -
-
25
IB: STX7
IB: VAMP7
IB: VAMP8
- 35
1 2 3 4
Fig. S8. Effect of SNARE proteins in UVRAG-mediated virus entry. (A and B) HCT116 cells stably expressing empty vector or UVRAG were transfected with
control- or SNARE-specific siRNA as indicated for 72 h and then infected with MLV-GFP pseudotyped with the entry proteins of influenza A virus (IAV; H5). (A)
Infected cells (green) were visualized by fluorescence microscopy. (Scale bar, 50 μm.) (B)PercentageofGFP
+
cells was determined by flow cytometry and
expressedas thepercentage relativetothecontrol.*P<0.05; **P<0.01. (C)Fluorescencemicroscopy(Left)andflowcytometry(Right)analysis ofcontroland
SNARE knockdown cells infected for 12 h with rVSV-GFP. Viral infectivity is expressed as mean GFP fluorescence relative to control sh-treated cells. Data
represent mean± SD from three independent experiments. ***P< 0.001. (Scale bar, 20 μm.) (D) VSV infection induces the complex assembly of UVRAG with
Legend continued on following page
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 10 of 11
Figure 1.9.8 Effect of SNARE proteins in UVRAG-mediated virus entry. (A and B) HCT116 cells stably expressing
empty vector or UVRAG were transfected with control- or SNARE-specific siRNA as indicated for 72 h and then
infected with MLV-GFP pseudotyped with the entry proteins of influenza A virus (IAV; H5). (A) Infected cells (green)
were visualized by fluorescence microscopy. (Scale bar, 50 µm.) (B) Percentage of GFP
+
cells was determined by flow
cytometry and expressed as the percentage relative to the control. *P < 0.05; **P < 0.01. (C) Fluorescence microscopy
(Left) and flow cytometry (Right) analysis of control and SNARE knockdown cells infected for 12 h with rVSV-GFP. Viral
infectivity is expressed as mean GFP fluorescence relative to control sh-treated cells.(D) VSV infection induces the
complex assembly of UVRAG with C-Vps and SNAREs. 293T cells were transfected with empty vector (lane 1) or Flag-
UVRAG (lanes 2–4) for 48 h and then mock-infected (lane 2) or infected with VSV (lanes 3–4) for 2 h in the presence or
absence of Baf A1 (2 µM). WCLs were immunoprecipitated with anti-Flag, followed by IB with the indicated antibodies
against C-Vps, glutamine (Q)-SNAREs, and Beclin 1. (E) Arginine (R)-SNARE VAMP8, but not VAMP7, is involved in
UVRAG-mediated SNARE complex assembly upon virus entry. 293T cells were transfected with empty vector (lane 2)
or Flag-UVRAG (lanes 3–4) for 48 h and then mock-infected (lanes 1–3) or infected with VSV (lane 4) for 2 h. The 293T
cells were transfected with empty vector or Flag-UVRAG for 48 h and then infected for 2 h with pseudovirus of VSV (F)
and IAV (G) in the presence of absence of Baf A1 (2 µM). WCLs were immunoprecipitated with anti-UVRAG, followed
by IB with the indicated antibodies against C-Vps subunits and SNARE proteins.
Figure 1.9.9 UVRAG facilitates the recruitment of VAMP8, but not VAMP7,
to VSV-containing vesicles
64
C-VpsandSNAREs.293Tcellswere transfected with empty vector(lane 1)orFlag-UVRAG(lanes 2–4)for 48handthen mock-infected(lane2) orinfectedwith
VSV(lanes3–4)for2hinthepresenceorabsenceofBafA1(2μM).WCLswereimmunoprecipitatedwithanti-Flag,followedbyIBwiththeindicatedantibodies
against C-Vps, glutamine (Q)-SNAREs, and Beclin1. (Right) Endogenous protein expressions are shown. (E) Arginine (R)-SNARE VAMP8, but not VAMP7, is
involved in UVRAG-mediated SNARE complex assembly uponvirus entry. 293T cells were transfectedwith empty vector (lane 2) or Flag-UVRAG (lanes 3–4)for
48handthenmock-infected(lanes1–3)orinfectedwithVSV(lane4)for2h.WCLswereimmunoprecipitatedwithcontrolIgG(lane1)oranti-STX7(lanes2–4),
followed by IB with the indicated antibodies. Note that the VAMP8–STX7 interaction was drastically induced by VSV infection, whereas the VAMP7–STX7
interactionwasinhibited.The293TcellsweretransfectedwithemptyvectororFlag-UVRAGfor48handtheninfectedfor2hwithpseudovirusofVSV(F)and
IAV (G) in the presence of absence of Baf A1 (2 μM). WCLs were immunoprecipitated with anti-UVRAG, followed by IB with the indicated antibodies against
C-Vps subunits and SNARE proteins. (Right) Endogenous protein expressions in cells are shown.
0
20
40
60
80
VAMP8 VAMP7
Vector
UVRAG
Vector Vector UVRAG
VSV Mock
VAMP8 acquisition
of VSV-G
+
-vesicles (%)
UVRAG
*** ***
VSV-G VAMP8 VAMP7 Merge
Fig. S9. UVRAG facilitates the recruitment of VAMP8, but not VAMP7, to VSV-containing vesicles. Confocal microscopy analysis of HeLa.Vector and HeLa.
UVRAG cells mock-infected or infected for 2 h with VSV, using anti–VSV-G (green), anti-VAMP8 (red), and anti-VAMP7 (purple) antibodies. (Insets) VAMP8
acquisition of virion-containing vesicles is highlighted. (Scale bars, 10 μm.) (Right)PercentageofVSV-G
+
vesicles colocalized with VAMP8 was quantified. Data
represent mean± SD (n = 60). ***P< 0.001.
Pirooz et al. www.pnas.org/cgi/content/short/1320629111 11 of 11
Figure 1.9.9 UVRAG facilitates the recruitment of VAMP8, but not VAMP7, to
VSV-containing vesicles. Confocal microscopy analysis of HeLa.Vector and
HeLa. UVRAG cells mock-infected or infected for 2 h with VSV, using anti–VSV-
G (green), anti-VAMP8 (red), and anti-VAMP7 (purple) antibodies. (Insets)
VAMP8 acquisition of virion-containing vesicles is highlighted. (Scale bars, 10
µm.) (Right) Percentage of VSV-G
+
vesicles colocalized with VAMP8 was
quantified. Data represent mean ± SD (n = 60). ***P < 0.001.
1.10 Conclusions
Although the cell imposes multiple barriers to virus entry, enveloped viruses persistently
hitchhike and remodel the endomembrane system of their hosts to traffic within, and
eventually escape from, endosomal organelles for their genome release. Through a
cascade of membrane-trafficking events, virus-bearing vesicles fuse with acidic
endosomes and/or lysosomes mediated by SNAREs triggering viral fusion. However,
the molecular mechanisms underlying this process remain elusive. My findings reveal
that UVRAG, an autophagic tumor suppressor, is required for the entry of the prototypic
negative- strand RNA virus, including influenza A virus and vesicular stomatitis virus, by
a mechanism independent of IFN and autophagy. UVRAG mediates viral endocytic
transport and membrane penetration through interactions with the C-Vps tethering
complex and endosomal glutamine-containing SNAREs [syntaxin 7 (STX7), STX8, and
vesicle transport through t-SNARE homolog 1B (Vti1b)], leading to the assembly of a
fusogenic trans-SNARE complex involving vesicle-associated membrane protein
(VAMP8), but not VAMP7. Indeed, UVRAG stimulates VAMP8 translocation to virus-
bearing endosomes. Inhibition of VAMP8, but not VAMP7, significantly reduces viral
entry. My data indicates that UVRAG, in concert with C-Vps, regulates viral entry by
assembling a specific fusogenic SNARE complex. Thus, UVRAG governs downstream
viral entry, highlighting an important pathway capable of potential antiviral therapeutics.
Studies of virus-host interactions will certainly continue to open new paths in
understanding disease mechanisms as well as basic cellular biology. The complex set
of interactions between a virus and a host organism starts off with the virus breaching
65
physical cellular barriers and gaining entry into cells. This virus-cell entry process is the
primary focus of the first chapter of my dissertation research. Elucidating viral entry
mechanisms and their interaction with the host trafficking network is necessary for
antiviral therapy. In this project, I focused on the use of host autophagy molecular
factors during the entry of prototypic negative-stranded RNA viruses, and highlight
recent progress in our understanding of the role of one such factor, UVRAG, in both
viral and cellular endocytic membrane trafficking and fusion events.
Collectively, my results indicate that multiple factors, operate to facilitate prototypic
negative-strand RNA virus entry into cells. These results obtained using vesicular
stomatitis virus and influenza A virus as models likely apply generally to prototypic
negative-strand RNA viruses and may serve as the basis for a general appreciation of
viral entry mechanisms. These results also lay the groundwork for future evaluation of
viral entry determinants.
66
Chapter 2
KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS-BCL-2-MEDIATED
REGULATION OF NM23-H2 IS REQUIRED FOR KSHV INFECTION
67
2.1 Introduction
Viruses have been linked to the cause of cancer in humans, accounting for 10-20% of
cancer worldwide [88]. Kaposi’s sarcoma-associated herpesvirus (KSHV), also referred
to as human herpesvirus-8 (HHV-8) is a double stranded DNA gamma-herpesviruses,
known to infect humans. Gamma-herpesvirus family, also includes Epstein-Barr virus
(EBV), herpesvirus saimiri (HSV), and murine gammaherpesvirus 68 (MHV-68) [90].
This virus is of particular interest because it has the ability to establish life-long infection
in its host. More importantly, infection triggers Kaposi’s sarcoma (KS), and two
lymphoproliferative disorders: primary effusion lymphoma (PEL) and multicentric
castleman’s disease (MCD). Kaposi’s sarcoma herpesvirus (KSHV)-induced tumors
remain the most common cause of malignancy among HIV-infected patients. Even with
effective HAART and well-controlled HIV infection, some AIDS patients develop
progressive KS causing profoundly increased viral load and mortality. The ability of
KSHV infection to allude host detection and drive tumor progression towards more
invasive stages, has become a concerning issue, especially when considering the
immune compromised population, who are extremely vulnerable. Unfortunately, the
development of new chemotherapeutic agents has, so far, only incrementally improved
patient survival.
Among human viruses, KSHV is most closely related to the Epstein-Barr virus (EBV), a
tumorigenic gamma-1 herpesvirus, known to be associated with lymphomas and
nasopharyngeal carcinoma. Like all other herpesviruses, the infectious cycle of KSHV
includes two alternative infection phases, latent and lytic [91]. Latent infection allows the
virus to establish long-term persistent infection, and involves the presence of viral
68
episomes and expression of a small set of viral genes. Lytic infection is needed for the
maintenance of viral reservoirs and for virus spread, and involves a temporally
regulated cascade of viral gene expression and DNA replication, leading to the
production and release of new virions (Fig. 2.1) [92]. Lytic virus reactivation can be
induced in these cells by treatment with a variety of soluble cytokines, co-infection by
another viral agent, or treatment with chemical reagents such as 12-O
tetradecanoylphorbol-13-acetate (TPA) and sodium butyrate. Initiation of the lytic
replication proceeds mainly through the activities of the viral replication and transcription
activator (RTA) protein, which activates the expression of certain KSHV genes,
eventually leading to the upregulation of all the lytic cycle KSHV genes [91] [93] [94] [95]
[96].
In KSHV oncogenesis, based on previous studies, one critical virulence factor for
γ-herpesviruses persistence and oncogenicity is the homologue of the cellular Bcl-2,
vBcl-2 protein encoded by all γ-herpesviruses (γ-HVs), including EBV, KSHV, and
murine γ-herpesvirus 68 (γ-HV68) [97]. Cellular and viral Bcl-2 exhibit a 3-dimensional
structure similarity, raising the possibility that these members may share some common
biochemical function [98]. The finding that vBcl-2 is encoded by all γ-herpesviruses
implies how critical vBcl-2 is in the virus life cycle [97]. Bcl-2 was originally discovered
as an oncogenic protein in B-cell lymphomas [99] [100] [101]. The Bcl-2 family consists
of both the anti-apoptotic (e.g., Bcl-2, Bcl-XL, and Bcl-w) and pro-apoptotic (e.g, Bax,
Bid, Bak, and Bad) members, which all share the Bcl-2 homology domain (BH). Bcl-2
mediated cell death evasion is one of the hallmarks of most cancers. The biological
activities of Bcl-2 have been largely attributed to its effects on the apoptotic pathway.
69
However, more recent studies have shown that Bcl-2 is also intimately involved in
regulating autophagy. Recent studies have indicated the apoptotic and autophagic
machinery converge, narrowing their direct crosstalk to be mediated by the interaction
of Bcl-2, an apoptosis inhibitor, and Beclin 1, an essential autophagy activator.
Structural analyses further implicated that the two mentioned functions of vBcl-2, anti-
apoptosis and anti-autophagy, engage similar structural cassettes, the hydrophobic
BH3-binding groove on the surface of vBcl-2 [102] [103]. vBcl-2 counteracts the host’s
apoptotic response (‘self-killing’) by sequestering normal functioning pro-apoptotic Bcl-2
molecules (e.g. Bax and Bak), allowing for the survival of infected cells and spread of
progeny virus, resulting in virus induced oncogenesis. In addition to preventing
apoptosis, vBcl-2 concomitantly couples with the pro-autophagic Beclin 1 protein,
averting autophagy (‘self-eating’) and allowing persistent infection within the host’s cells.
Since persistent infection is critical for γ-herpesvirus-associated malignancy [104], the
induction of vBcl-2 antagonism of cellular pathways, disrupting homeostasis, may
contribute to the oncogenic potential of γ-herpesviruses. Moreover, complex
interrelationships are likely to exist between these pathways, since they share common
regulators. Given the importance of Bcl-2 in cell survival, many viruses have developed
strategies to prevent the death of infected cells from sustained viral replication and
associated disease, through Bcl-2 mediation[105]. Thus, inhibition of apoptosis and
autophagy by KSHV virus Bcl-2 may provide an attractive mechanism for prolonging the
life-span of KSHV-infected cells, which in-turn enables increased virus production,
establishment of latency and/or efficient reactivation. This only further highlights the
importance of Bcl-2 coordinated regulation.
70

KS-Bcl-2 is transcribed during lytic virus infection [98] [106] [107]. However, the function
of KS-Bcl-2 during virus infection only recently was found to be essential for KSHV lytic
replication [28] [108]. It was shown that knockout of vBcl-2 dramatically reduces KSHV
lytic gene expression, viral DNA replication, and progeny virus production. Most
importantly, it was found that this novel function of vBcl-2 for lytic replication does not
depend on its previously known antiapoptotic and antiautophagic functions [28] [108].
These results further suggest that KSHV Bcl-2 carries a novel function in viral lytic
replication specific for KSHV infection.
71
RNAi screening of the human kinome
High-throughput RNAi screening has been successfully
applied to study the contribution of protein kinases on
HCMV infection and KSHV reactivation [42,81]. The same
strategy can be applied to study the function of host protein
kinases in other herpesvirus infections. The disadvantages
of RNAi screening are that the strategy is expensive and
labor-intensive. Further, effective protein kinase knock-
down usually requires more than 24 h of transfection with
the appropriate siRNAs, a factor that can result in second-
ary downregulation of other protein kinases and compli-
cate interpretation of the results.
Protein kinase inhibitor screening
With the commercial availability of protein kinase inhibi-
tor libraries (containing around 200 protein kinase inhi-
bitors), it is also feasible to perform protein kinase
inhibitor screens to identify those with anti-herpesvirus
potential. However, such screens are complicated by the
choice of infection system because different cell back-
grounds may influence the observed effectiveness, treat-
ment concentration, or toxicity. In addition, off-target
effects should also be kept in mind because protein kinase
inhibitors frequently affect the activity of multiple kinases
when used at the concentrations required for potent inhi-
bition of their primary targets.
Quantitative mass spectrometry analysis
To identify the key cellular protein kinases critical for the
viral life cycle, it will be necessary to globally understand
the phosphorylation events that occur upon viral infection,
upon lytic reactivation, or during the establishment of
latency. Stable isotope labeling by amino acids in cell
culture (SILAC) coupled with liquid chromatography–
mass spectrometry/mass spectrometry (LC–MS/MS) anal-
ysis has been widely used to dissect cellular signaling
pathways [82]. This technology can be applied to investi-
gate the host signaling pathways manipulated by herpes-
viruses. Multiplex SILAC-based strategies will further
provide the temporal analysis of host protein phosphory-
lation during the course of viral infection, which will
facilitate the identification of key protein kinases critical
at different stages. Target inhibition by RNAi knockdown
or specific protein kinase inhibitors will allow the function
of these potentially key protein kinases in the viral
life cycle to be elucidated and will provide a rationale for
anti-viral drug development.
Preclinical animal models
Laboratory mice are good models for HSV-1/2 replication
and pathogenesis [83]. Human dorsal root ganglia (DRGs)
xenografts in immunodeficient mice provide a good model
for evaluating HSV-1 and VZV neuropathogenesis [84,85].
Cytoplasm
Nucleus
mTOR
AMPK
CAMKK
Btk
MEK
PI3K
IKK
EphA2
Entry and
uncoa!ng
DNA
replica!on
DNA
encapsida!on
Nuclear
egress
ER
Golgi complex
Virus
release
Final
envelopment
A"achment
PDGFR
ATM
CHK2
CDKs
Aurora A
VEGFR
CHPKs
US3/ORF66
CHPKs
US3/ORF66
1
2
3
12
11
4
7
8
9
10
b
c
d
Capsid
proteins
a
Early
proteins
5
6
mRNA
mRNA
Envelope proteins
CK2
CDKs
JNK
RSKs
PI3K
CDKs
TRENDS in Microbiology
Figure 1. Protein kinases involved in the herpesvirus life cycle. (1) Viral glycoproteins bind to host cell receptors on the cell membrane (tyrosine kinases PDGFR and EphA2
are the receptors for HCMV and KSHV, respectively). (2) Viral envelope fuses with the cell membrane and releases the nucleocapsid into the cytoplasm. (3) The nucleocapsid
associates with cytoskeletal elements and translocates to the nucleus where the nucleocapsid interacts with nuclear pores and releases the viral genome into the nucleus.
(4) The viral DNA is transcribed into early mRNAs which are then transported to the cytoplasm for early protein synthesis. (5) These early proteins are imported into the
nucleus and promote viral DNA replication. (6) The viral DNA is then transcribed into the late mRNAs which are responsible for late viral protein synthesis (capsid and
envelope proteins). (a–d) The viral envelope proteins (pink, green, and blue circles) are processed in the endoplasmic reticulum (ER) and Golgi complex. (7) The capsid
proteins are imported into the nucleus and then encapsidate the newly replicated genomes. (8–10) These capsids egress from the nucleus and bud into the exocytotic
vesicles. (11) The virion containing vesicles migrate to and fuse with the cell membrane. (12) Infectious viruses are then released from the cell [1]. Viral (black circle) and
cellular (red circle) kinases discussed in this review are shown in the cell compartment most likely to be relevant to their impact on the viral life cycle (steps 2–9). The viral
and cellular kinases involved in steps a–d and 10–12 have yet to be identified.
Review
Trends in Microbiology June 2013, Vol. 21, No. 6
292
Episome
Fig. 2.1 KSHV life cycle
Furthermore, I have collected preliminary data of the formation of a distinct complex
between vBcl-2 and NM23-H2/NDP kinase B, a key NDP Kinase. The human NM23
family, which is characterized enzymatically as nucleoside diphosphate kinase (NDPK),
consists of eight related genes that encode widely expressed proteins known as NM23-
H1 through NM23-H10. NDPKs were originally identified as essential housekeeping
enzymes that play a role in maintaining the intracellular nucleotide concentration by
catalyzing the transfer of phosphate groups between nucleoside diphosphates. NDPK
expression upon interacting with other regulatory proteins has been reported to be
involved in many cellular processes, including oncogenesis [109], cellular proliferation
[110], differentiation [111] [112], motility [113], development [114], DNA repair [115]
[116] , apoptosis [117], and metastasis. Although the NM23 family of genes has been
studied extensively for more than a decade, the exact mechanism of how NM23-H2 is
regulated has yet to be fully defined. The non-metastasis protein NM23 was originally
identified as a tumor marker associated with reduced metastatic activity in melanoma
[118]. NM23 was the first and most extensively described metastasis suppressor gene
[119] [120], the expression of which could abrogate spontaneous and experimental
metastasis without affecting tumor growth [120]. Among ten human isoforms (NM23-H1
to H10) being identified, NM23-H1 and NM23-H2 represent the two most widely
expressed isoforms, sharing 88% sequence identity but having distinct functions in
development, metastasis, and transcriptional regulation [119]. NM23-H1 activity is
reverted by EBV latent antigen EBNA-3C, resulting in a relief of NM23-H1-dependent
anti-migration and metastasis [121] [122]. KSHV LANA was also found to interact with
NM23-H1 to induce cellular pathogenesis [123]. Furthermore, NM23-H2 activity was
72
shown to significantly increase by TPA and UV radiation, both of which are triggers of
herpesviral reactivation, in vivo and in vitro [124]. NM23-H2 is also known to directly
mediate the neoplastic transformation of epidermal cells in the early stages of skin
carcinogenesis [124]. Lastly, NM23-H2 has been shown to function as a transcriptional
activator of c-myc, which activates expression of both H1, and H2 genes [125] [126]
[115]. Yet, no viral interactions have been reported to target NM23-H2 and the biological
functions of NM23-H2 in virus-induced oncogenesis are still unclear.
The structure of NM23 includes α-helical regions, β sheets, the K-pn loop and a Head
region involved in nucleotide interactions [121]. NM23-H1/2 is rather simple with six α-
helices partially covering a four-stranded antiparallel β-sheet. Formation of hexamers
would allow NM23-H1/2 to create new surface areas for protein-protein interaction, and
this might explain the diverse range of associated proteins [127]. The question
remaining is whether the biological functions of NM23-H2 involves hexamer formation
as an absolute requirement. The hexameric structure of NM23-H2 is typically viewed as
a trimer of dimeric subunits. Early mutagenesis studies indicate that point mutations
may not be sufficient to effectively prevent hexamer formation [127]. It is interesting that
NM23-H1 and NM23-H2 differ by only 18 amino acids and, excluding the two residues
that form part of the multimer contact sites, they are all located on exposed surfaces of
the hexamer. As a result, the surface nature of NM23-H2 is basic in contrast to the
acidic character of NM23-H1, which may also explain the differences of their interacting
partners and functions [127]. Both NM23-H1 and NM23-H2 are mainly present in the
cytoplasm, but they can also be found, at least transiently, associated to membranes
and in nuclei. Although these two isoforms share highly identical sequences and similar
73
secondary structures, the 12 % structural difference is likely to constitute molecular
determinants for interaction with specific protein partners.
The unifying feature of this family is their evolutionarily conserved NDPK protein
domain, an archain peptide sequence also found in many prokaryotes. Surprisingly, only
isoforms H1-H4, which function as hexamers, and H6 actually exhibit NDPK activity
[128]. Experimental characterization of H2 and H1 in particular has elucidated in vitro
biochemical activities in addition to NDPK, including a 3’-5’ exonuclease activity and a
histidine-dependent protein kinase function. The contribution of each activity to H1/2
biological functions at this point is unclear.
My recent studies have further expanded the functions of vBcl-2, in targeting and
altering the role of the NM23-H2, a key human metastasis suppressor. The theory of
vBcl-2 regulating NM23-H2 may be intriguing because, as popular as Bcl-2 may be, so
far mainly apoptotic and autophagic factor have been found to pair with Bcl-2. Moreover,
a specific interaction was found between vBcl-2 and NM23-H2, indicating the
identification of a new partner. However, no interaction was detected for vBcl-2 with
NM23-H1, indicating that this is an isoform-specific interaction. Moreover, a limited
number of published studies suggest a role for NM23-H1, a member of the NM23 family
in the suppression of cell invasion and tumor progression induced by viruses [122] [129]
[121] [130]. And although NM23H1/H2 proteins share high sequence identity, however,
they share more distinct interacting partners than common ones. Therefore it was not
surprising that our studies show the formation of a distinct complex between vBcl-2 and
NM23-H2 only, excluding NM23-H1 from this interaction. This highlights a specific role
74
for KSHV-Bcl-2-NM23-H2 complex in KSHV infection. Since metastasis suppressors
regulate multiple steps in the metastatic cascade, the role of vBcl-2 has the potential to
be of fundamental importance in the therapy of KSHV associated cancer. My results
pave the way for the discovery of a novel function of vBcl-2 targeting and altering, a
critical host factor, NM23-H2.
Thus, an innovative approach to understand KSHV-host interactions regulating infection
and/or pathogenesis is of enduring importance. In an effort to increase our
understanding of the detailed function of KS-Bcl-2 in the context of viral infection, I
aimed to dissect the mechanism of how KS-Bcl-2-NM23-H2 complex regulates viral
infection. The successful outcome of my studies may significantly impact our
understanding of viral oncology and provide valuable insight into identifying novel host-
factor determinants. The mechanism of KSHV induced oncogenesis can be used to
expand upon known antiviral mechanisms of host machinery, and ultimately be applied
for developing new treatment for progressive KSHV malignancies, and KSHV-
associated tumors for AIDS patients.

75
2.2 Materials and methods
2.2.1 Cell culture and viruses
iSLK cells were cultured in the presence of 1 g/ml puromycin and 250 g/ml G418.
BAC16 and its derivatives were introduced into iSLK cells via transfection with the
Fugene HD reagent (Roche), and transfected iSLK cells were selected with 200 g/ml
hygromycin (Invitrogen) as explained [28]. Wild-type (WT) KSHV and its mutant
derivatives were all produced from iSLK-BAC16 or iSLK-BAC16 mutant cell lines upon
doxycycline (1 g/ml) induction for 3 to 4 days. SLK stable cell lines were established
using lentivirus vector infection with selection with 200 g/ml hygromycin. iSLK.219
iSLK.219 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum and 100 U penicillin-streptomycin, G418 (250 µg/ml),
hygromycin (400 µg/ml) and puromycin (10 µg/ml). To get lytic reactivation phase,
iSLK.219 cell line is induced with doxycycline (1ug/ml) and Sodium Butyrate (1mM)
HEK-293T, SLK cells, NIH 3T3, and HeLa were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and
1% penicillin-streptomycin (Gibco-BRL)., were cultured in DMEM supplemented with.
Transient transfection was performed with Fugene 6 (Roche), Lipofectanine 2000 (In-
vitrogen), or calcium phosphate (Clontech). NIH 3T3 stable cell lines were established
using a standard protocol of selection with 2 µg/mL puromycin (Sigma–Aldrich), as
described previously.
76
2.2.2 Plasmid Constructs
The vBcl-2-coding sequence was amplified from KSHV BAC16 genomic DNA and
cloned into the pCDH-CMV- MSC-ef1-hygromycin vector (System Biosciences) carrying
an N-terminal hemagglutinin (HA) tag (pCDH-HA-vBcl-2). Deletions and mutations in
the vBcl-2 gene were generated using a QuikChange site- directed mutagenesis kit
(Stratagene) as explained [28]. The following NM23-H2 constructs were cloned into
pcDNA5 containing Flag at the N terminus, wild-type, D94-114, K12A, R34A, H47A,
N69H, P96S, C109A, H118C, S120G and C145A. All plasmids containing KSHV-Bcl2
constructs were cloned into pcDNA5 containing HA at the N Terminus. This includes
wild-type and mutants, AAA and E14A.
2.2.3 Western Blot and Immunoprecipitation
For immunoblotting, polypeptides were resolved by SDS/PAGE and transferred to a
PVDF membrane (Bio-Rad). Membranes were blocked with 5% (wt/vol) nonfat milk and
probed with the indicated antibodies. Immunodetection was achieved with
Hyglo chemiluminescence reagent (Denville Scientific) and detected by a Fuji ECL
machine (LAS-3000). For immunoprecipitation, cells were harvested and then lysed in
1% Nonidet P-40 lysis buffer supplemented with complete protease inhibitor mixture
(Roche). After preclearing with protein A/G agarose beads for 1 h at 4 °C, whole-cell
lysates were used for immunoprecipitation with the indicated antibodies. Generally, 1–4
µg of commercial antibody was added to 1 mL of cell lysate, which was incubated at 4
°C for 8–12 h. After addition of protein A/G agarose beads, incubation was continued for
another 2 h. Immunoprecipitates were extensively washed with Nonidet P-40 lysis buffer
and eluted with SDS/ PAGE loading buffer by boiling for 5 min. Primary antibodies
77
included mouse Flag (Sigma), mouse HA (Covance), rabbit HA (Covance), mouse cmyc
(Covance), rabbit GST, mouse GST, rat KSHV latency-associated nuclear antigen
(LANA; Advanced Biotechnologies), mouse NM23H2 (ab606020; abcam), rabbit
NM23H2 (ab131329; abcam), rabbit NM23H1 (GTX108325; Genetex), rabbit NM23H2
for IP (H00004831-D01; Abnova), and actin (Santa Cruz). Appropriate horseradish
peroxidase- conjugated secondary antibodies were incubated on membranes in 5%
milk.
2.2.4 Antibodies, Fluorscent Dyes, and Reagents
HRP-labeled or fluorescently labeled secondary antibody conjugates were purchased
from Molecular Probes (Invitrogen). Purified mouse and rabbit IgG was purchased from
Pierce. All other chemical and reagents were obtained from Sigma–Aldrich unless
otherwise noted.
2.2.5 Gene Knockdown by shRNA
NM23 shRNA constructs were purchased from Open Biosystems. All shRNAs were
transfected using using lentivirus vector infection according to the published protocol.
2.2.6 Immunofluorescence and Confocal Laser Scanning Microscopy
Immunofluorescence microscopy was carried out as described previously. Briefly, cells
were fixed with 4% (wt/vol) paraformaldehyde (20 min at room temperature). After
fixation, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with
10% (vol/vol) goat serum (Gibco) for 1 h. Primary antibody staining was carried out
using antiserum or purified antibody in 1% goat serum for 2 h at room temperature or
overnight in 4 °C. Cells were then extensively washed with PBS and incubated with
78
Alexa 488-, and Alexa 594-conjugated secondary antibodies for 1 h, followed by DAPI
staining. Cells were mounted using Vectashield (Vector Laboratories, Inc.). Confocal
images were acquired using a Nikon Eclipse C1 laser- scanning microscope fitted with a
Nikon objective (plan apochromat, 1.4 N.A.) with a magnification of 60× and Nikon
image software.
2.2.7 Flow Cytometry Analysis
For flow cytometry preparation, cells were treated with cell dissociation buffer (Sigma),
washed twice with PBS, and then fixed in 4% (wt/vol) paraformaldehyde/PBS. Cells
were assayed for phycoerythrin fluorescence gated on cells that were positive for GFP
fluorescence and/or RFP. At least 10,000 cells were analyzed for each sample in
triplicate.
2.2.8 RNA extraction and qRT-PCR
Total RNA was isolated from cells with an RNeasy minikit (Qiagen) and treated with
RNase-free DNase per the manufacturer’s protocol. cDNA was reverse transcribed from
2 g of total RNA using an iScript cDNA synthesis kit (Bio-Rad), and quantitative reverse
transcription-PCR (qRT-PCR) was performed with the iQ SYBR green Supermix (Bio-
Rad).
2.2.9 Gel filtration chromatography
iSLK-BAC16 or iSLK-BAC16 mutant cell lines were induced with doxycycline (1 g/ml),
collected and lysed in cold 1% Tritonx 100 buffer (buffer A) supplemented with a
complete protease inhibitor cocktail (Roche). After pre-clearing with protein A/G agarose
beads, whole-cell lysates were incubated immunoprecipitation with the indicated
79
antibodies. Agarose was extensively washed with buffer A. Gel filtration chromatography
was performed as described previously [131]. Briefly, lysates (200 µl) were loaded onto
a Superpose-6 column (Sigma-Aldrich) and subjected to gel filtration analysis with
buffer A. Elutions were collected in 0.5 ml fractions and aliquots of fractions were
analysed by immunoblotting. The gel filtration standards (Sigma-Aldrich) were bovine
thyroglobulin (relative molecular mass 669,000 (M
r
669K)), horse spleen apoferritin (M
r
443K), sweet potato β- amylase (M
r
200K), bovine serum albumin (M
r
66K) and bovine
erythrocyte carbonic anhydrase (M
r
29K). The elution profile of each protein was
quantified by densitometry analysis and normalized. Relative densitometry units were
plotted against fraction number.
2.2.10 NDPK Assay
A well-known procedure to assay NDP kinase activities was used [132] [133]. In brief,
10 ul of diluted enzyme (lysate) was added to a 990 ul reaction mixture containing 100
mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM KCl, 0.4 mM NADH, 6 mM ATP, 0.7 mM
TDP, 4 mM phosphoenolpyruvate (PEP), and 10 U of pyruvate kinase and lactate
dehydrogenase each. The absorbance of NADH at 340 nm was the recorded. A unit of
activity is defined as the amount required to convert 1umol of NADH to NAD+.
2.2.11 Statistical Analysis
All experiments were independently repeated at least three times. Data are presented
as mean ± SD. Statistical significance was calculated using the Student t test or one-
way ANOVA test, unless otherwise stated. A P value of ≤0.05 was considered
statistically significant.
80
2.3 Results
2.3.1 NM23-H2, but not NM23-H1, is a new target of KSHV-viral Bcl-2
Liang et al [28] utilized the new KSHV BAC16 genome modification system and the
iSLK virus production cell line to study the role of viral autophagy-modulating genes on
KSHV lytic replication. They provide evidence that KSHV Bcl-2 is essential for KSHV
lytic replication. By a series of mutagenesis analyses, they identified the crucial amino
acid of vBcl-2 (E
14
) for KSHV lytic replication and found that it is genetically separable
from its antiapoptotic and antiautophagic functions. The E
14
residue of KSHV Bcl-2 is
located in the first helix (the 1 helix) [134], which has a low degree of sequence
conservation with the 1 helix sequence of MHV-68 M11 [105]. The 1 helix forms a BH4
domain but is not involved in the formation of the central hydrophobic BH3-peptide
binding groove, which structurally explains why the vBcl-2 E14A mutant still showed
antiapoptotic and antiautophagic activities similar to those of wild-type vBcl-2. Their
study indicated for the first time that vBcl-2 contains an additional function besides
antiapoptotic and antiautophagic functions which is directly essential for the KSHV lytic
cycle. On the other hand, the vBcl-2 AAA mutant can still support KSHV lytic replication.
Although the detailed mechanism by which vBcl-2 supports KSHV lytic replication is still
elusive, they propose that vBcl-2 might interact with some novel cellular or viral binding
partners through its 1 helix peptide, and this interaction might be essential for the tight
regulation of KSHV lytic gene expression, viral DNA replication, or viral particle
assembly. In summary, they have identified a critical additional function of vBcl-2 which
81
is required for KSHV lytic replication. However the detailed mechanism remains unclear
and requires further investigation.
Surprisingly, my preliminary data of the formation of a distinct complex between vBcl-2
with exogenous and endogenous NM23-H2 in 293T cells (Fig. 2.3.1 A-D), uncovers an
additional novel function of this small viral protein. To exclude the possibility of tagging, I
determined interaction between GST-vBcl-2 and Flag-NM23-H2 (Fig. 2.3.1 C). The
formation of a distinct complex was specific between vBcl-2 and NM23-H2 only,
excluding NM23-H1 from this interaction. Negative NM23-H1 interaction may be due to
false negativity, therefore, I confirmed the binding of NM3-H1 with LANA based on
previous publications [123] (Fig. 2.3.1 E), further highlighting a specific role for
KSHV-Bcl-2-NM23-H2 complex in KSHV infection.
82
A
IB: Actin
IP: HA
IB: Flag
Input
HA-mBcl-2
Flag-NM23-H2
-
+ + +
15-
IP: HA
IB: HA
25-
IB: HA
25-
15-
1 2 3
IB: Flag
HA-kBcl-2
HA-cBcl-2
+
4
+
+
+
42-
WCLs
IB: Actin
IP: HA
IB: Myc
Input
HA-mBcl-2
Myc-NM23-H1
-
+ + +
21-
IP: HA
IB: HA
25-
IB: HA 25-
21-
1 2 3
IB: Myc
HA-kBcl-2
HA-cBcl-2
+
4
+
+
+
42-
WCLs
83
Figure 2.3.1. NM23-H2, but not NM23-H1, is a new target of viral Bcl-2 of KSHV
B
C
D
E
IP: Flag
IB: HA
Input
IB: HA
IB: Flag
IP: Flag
IB: Flag
WCLs
15-
15-
IB: Actin
42-
25-
25-
Flag-NM23-H2
HA-kBcl-2
+
+
+
+
1 2 3
IP: Flag
IB: HA
Input
IB: HA
IB: Flag
IP: Flag
IB: Flag
WCLs
15-
15-
IB: Actin 42-
25-
25-
Flag-NM23-H2
HA-mBcl-2
+
+
+
+
1 2 3
IP: GST
IB: GST
Input
IB: GST
IB: Flag
IP: GST
IB: Flag
WCLs
15-
15-
IB: Actin 42-
25-
Flag-NM23-H2
GST-kBcl-2
+
+
+
+
1 2 3
IP: GST
IB: GST
Input
IB: GST
IB: Flag
IP: GST
IB: Flag
WCLs
15-
15-
IB: Actin
42-
25-
1 2 3
Flag-NM23-H2
GST-mBcl-2
+
+
+
+
25-
25-
WCLs
Input
Vec
mBcl-2
IP:HA
15- IB: NM23-H2
IB: NM23-H2
IB: NM23-H1
IB: NM23-H1
IB: HA
IB: Actin
21-
42-
kBcl-2
21-
15-
25-
IB: Actin
IP: Myc
IB: Flag
Flag-Lana
Myc-NM23-H1
+
150-
IP: Myc
IB: Myc
21-
IB: Myc 21-
150-
1 2 3
IB: Flag
+
+
42-
WCLs
+
Input
2.3.2 KSHV-viral Bcl-2 stabilizes NM23-H2 at the post translational level
The binding experiments clearly showed vBcl-2 may stabilize NM23-H2. To further
investigate this, I treated 293T cells dose dependently with vBcl-2 and confirmed the
vBcl-2-mediated stabilization of NM23-H2 (Fig. 2.3.2 A). Furthermore, I used
cycloheximide as inhibitor of protein synthesis, treated the cells to block the protein
synthesis and followed the kinetics of protein stability the protein half-life in 3T3 cells
stably expressing vBcl-2 and confirmed vBcl-2 indeed stabilizes NM23-H2 protein levels
(Fig. 2.3.2 B). I claim that KSHV-Bcl-2 stabilizes NM23-H2 at the post-translational level
since NM23-H2 mRNA levels were not affected upon introducing KSHV-Bcl-2
exogenously in 293T cells, or in islk-Bac16-Bcl-2 WT and KO after the induction of lytic
reactivation.
84
A
30 60 90 120 180 0
NM23-H2
Actin
NIH3T3.Vector
30 60 90 120 180 0
NIH3T3.kBcl-2
NM23-H2
Actin
CHX(min)
3T3 Vector
3T3 kBcl-2
B
Density Relative to Actin
Flag-NM23-H2
0 2 4 8
HA-kBcl-2
IB: HA
IB: NM23-H2
25-
15-
IB: Actin
42-
Flag-NM23-H2
0 2 4 8
HA-mBcl-2
IB: HA
IB: NM23-H2
25-
15-
IB: Actin
42-
Fig. 2.3.2 KSHV-viral Bcl-2 stabilizes NM23-H2
2.3.3 KSHV-Bcl-2-E
14
and NM23-H2-N
69
are the functional residues required
Next, I performed a structure/function analysis to map the domain responsible for vBcl-2
interaction with NM23-H2 (Fig 2.3.3 A). I expressed HA-tagged WT and mutant Bcl-2
proteins at equivalent levels in 293T cells and assessed the ability of these mutants to
interact with NM23-H2. As shown in (Fig. 2.3.3 B ), a change of glutamic acid 14 (E
14
) to
alanine in vBcl-2 completely abolished KSHV-Bcl-2 interaction, whereas other mutations
and deletions showed little or no effect. In parallel I expressed Flag-tagged WT and
mutant NM23-H2 proteins at equivalent levels in 293T cells and assessed the ability of
these mutants to interact with vBcl-2. As shown in (Fig. 2.3.3 C ), a change of
asparagine 69 (N
69
) to histidine in NM23-H2 decreased NM23-H2 interaction, whereas
other mutations and deletions showed little or no effect.These results suggest that the
E
14
residue of KSHV Bcl-2, is critical for KSHV lytic replication and progeny infectious
virus production, and the N
69
is required for NM23-H2 interaction.
85
Figure 1. NM23-H2, but not NM23-H1, is a new target of viral Bcl-2 of KSHV
A
B
C
IP: Flag
IB: HA
Input
IB: HA
IB: Flag
IP: Flag
IB: Flag
WCLs
15-
15-
IB: Actin
42-
25-
25-
Flag-NM23-H2
HA-kBcl-2
+
+
+
+
1 2 3
IP: Flag
IB: HA
Input
IB: HA
IB: Flag
IP: Flag
IB: Flag
WCLs
15-
15-
IB: Actin 42-
25-
25-
Flag-NM23-H2
HA-mBcl-2
+
+
+
+
1 2 3
D
Actin
IP: HA
IB: Flag
Input
HA-mBcl2
Flag-NM23-H2
-
+ + +
15-
IP: HA
IB: HA
25-
IB: HA
25-
15-
1 2 3
IB: Flag
HA-kBcl2
HA-cBcl2
+
4
+
+
+
42-
WCLs
Actin
IP: HA
IB: Myc
Input
HA-mBcl2
Myc-NM23-H1
-
+ + +
21-
IP: HA
IB: HA
25-
IB: HA 25-
21-
1 2 3
IB: Myc
HA-kBcl2
HA-cBcl2
+
4
+
+
+
42-
E
WCLs
KSHV Bcl-2-NM23-H2 Interaction MHV68 vBcl-2-NM23-H2 Interaction No interaction with NM23-H1
F
IP: GST
IB: GST
Input
IB: GST
IB: Flag
IP: GST
IB: Flag
WCLs
15-
15-
IB: Actin
42-
25-
Flag-NM23-H2
GST-kBcl-2
+
+
+
+
1 2 3
IP: GST
IB: GST
Input
IB: GST
IB: Flag
IP: GST
IB: Flag
WCLs
15-
15-
IB: Actin
42-
25-
1 2 3
Flag-NM23-H2
GST-mBcl-2
+
+
+
+
Exclude the efffect of tagging
All 3 Bcl-2 aligned
H
G
Endogenous Binding
WCLs
Input
Vec
mBcl2
IP:HA
15- IB: NM23-H2
IB: NM23H2
IB: NM23-H1
IB: NM23H1
IB: HA
IB: Actin
21-
42-
kBcl2
C.Elegans_NDK-1 -MSNTERTFIAIKPDGVHRGLVGKIIARFEERGYKLVALKQMTASKAHLEVHYQDLKDKPFFPSLIEYMSSGPVVAMVWQGLDVVKQGRSMLGATNPLASAPGTIRGDFCIQTGRNICHG
D.Melanogaster MAANKERTFIMVKPDGVQRGLVGKIIERFEQKGFKLVALKFTWASKELLEKHYADLSARPFFPGLVNYMNSGPVVPMVWEGLNVVKTGRQMLGATNPADSLPGTIRGDFCIQVGRNIIHG
Daniorerio_NDK-B MAGKTERTFIAVKPDGVQRGIMGEIIKRFEGKGFRLVAAKFVQASEDLAKGHYIDLKDQPFYAGLVKYTSSGPLLAMVWEGLNVIKTGRVMLGETDPFASKPGTIRGDFCIEVGRNLIHG
HU_NM23H2 -MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHG
Mu_NM23 -MANSERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFLQASEDLLKEHYTDLKDRPFFTGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHG
Ra_NM23 -MANSERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFIQASEDLLKEHYIDLKDRPFFSGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHG
.: ***** :*****:**::*:** *** :*::**. * **: : ** **. :**: .*::* ***:: ***:**:*:* ** *** *:* * **********:.***: **

C.Elegans_NDK-1 SDAVDSANREIAHWFKQEEINDYASPFINSWVYE
D.Melanogaster SDAVESAEKEIALWFNEKELVTWTP-AAKDWIYE
Daniorerio_NDK-B SDSEKSAATEVSLWFKPEELVSYRS-CAQEWIYE
HU_NM23H2 SDSVKSAEKEISLWFKPEELVDYKS-CAHDWVYE
Mu_NM23 SDSVKSAEKEISLWFQPEELVEYKS-CAQNWIYE
Ra_NM23 SDSVESAEKEISLWFQPEELVDYKS-CAQNWIYE
**: .** *:: **: :*: : ..*:**
HU_NM23H1 MANCERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVGLKFMQASEDLLKEHYVDLKDRPFFAGLVKYMHSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGS
HU_NM23H2 MANLERTFIAIKPDGVQRGLVGEIIKRFEQKGFRLVAMKFLRASEEHLKQHYIDLKDRPFFPGLVKYMNSGPVVAMVWEGLNVVKTGRVMLGETNPADSKPGTIRGDFCIQVGRNIIHGS
*** ********************************.:**::***: **:**:******** ******.***************************************************
HU_NM23H1 DSVESAEKEIGLWFHPEELVDYTSCAQNWIYE
HU_NM23H2 DSVKSAEKEISLWFKPEELVDYKSCAHDWVYE
***:******.***:*******.***::*:**
Actin
IP: Myc
IB: Flag
Flag-Lana
Myc-NM23-H1
+
150-
IP: Myc
IB: Myc
21-
IB: Myc 21-
150-
1 2 3
IB: Flag
+
+
42-
WCLs

β1 α0 α1 β2 αΑ α2 β3 α3 α4 β4

α5
Kpn Loop
+
Input
IP: HA
IB: H2
15-
IP: HA
IB: H1
21-
IB: H1 21-
25-
IB: HA
42-
WCLs
Input
BAC16-vBcl2-HA
IP: HA
IB: HA
IB: H2
IB: Actin
25-
15-
IP:HA
IgG
IP: HA
IB: H2
15-
IP: HA
IB: H1
21-
IB: H1 21-
25-
IB: HA
42-
WCLs
Input
HA-KBcl2
BAC16-vBcl2-HA
IP: HA
IB: HA
IB: H2
IB: Actin
25-
15-
IP:HA
Dox +
+ + Dox
IP:HA
vBcl2 KO
IP: HA
IB: H2
15-
IP: HA
IB: H1
21-
IB: H1 21-
25-
IB: HA
42-
WCLs
Input
vBcl2 KO
BAC16-vBcl2-HA
IP: HA
IB: HA
IB: H2
IB: Actin
25-
15-
+ + Dox
vBcl2-HA
21-
15-
25-
25-
25-
A
HU_NM23H1
HU_NM23H2
WT +
AAA
Δ1-18
Δ43-74
NM23-H2
binding
E14A
Δ19-42
Δ75-95
Δ96-125
Δ126-143
Δ144-175
MDEDVLPGEVLAIEGIFMACGLNEPEYLYHPLLSPIKLYITGLMRDKESLFEAMLANVRFHSTTGINGLGLSMLQVSGDGNMNWGRALAILTFGSFVAQKLSNEPHLRDFALAVLPVYAYEAIGPQWFRARGGWRGLKAYCTQVLTRRRGRRMTALLGSIALLATILAAVAMSRR*

α1 α2 α3 α4 α5 α6 α6’ α7 ΤΜ
ΒΗ4 ΒΗ3 ΒΗ1 ΒΗ2
+
+
+
+
+
+
+
-
-
KSHV-Bcl-2

86
Fig. 2.3.3 KSHV-Bcl-2-E
14
and NM23-H2-N
69
are the functional residues required
IB: Flag
IB: HA
IP: HA
IP: HA
IB: HA
IB: Flag
Input
WCLs
HA-KSHV-Bcl-2
Vec
Δ96-125
WT
Δ1-18
Δ19-42
Δ43-74
Δ75-95
15-
25-
25-
IB: Actin 42-
15-
Δ126-143
Δ144-175
AAA
E14A
IB: Flag
IB: HA
IP: HA
IP: HA
IB: HA
IB: Flag
Input
WCLs
HA-KSHV-Bcl-2
Vec
15-
25-
25-
IB: Actin
42-
15-
AAA
E14A
WT
B
15-
25-
15-
42-
25-
IB: Flag
IB: HA
IP: Flag
IP: Flag
IB: Flag
IB: HA
Input
WCLs
Flag-NM23-H2
H47A
Vec
Δ94-114
WT
K12A
R34A
25-
25-
15-
IB: Actin
42-
15-
N69H
P96S
C109A
H118C
IB: HA
IB: Flag
IP: Flag
IP: Flag
IB: Flag
IB: HA
Input
WCLs
Flag-NM23-H2
Vec
IB: Actin
WT
N69H
S120G
C145A
NM23-H2
C
2.3.4 KSHV-viral Bcl-2 co-localizes with and stabilizes NM23-H2
To further explore the interaction of vBcl-2 with Nm23-H2, I assessed the formation of
the complex by confocal microscopy in different cell lines (Fig. 2.3.4). Complete
co-localization and stabilization was observed between KSHV-Bcl-2 WT and NM23-H2
upon introducing KSHV-Bcl-2 WT exogenously in HeLa, and primary slk cells.
Surprisingly the KSHV-Bcl-2-AAA mutant to also showed a similar phenotype as Bcl-2
WT, whereas no co-localization or stabilization was detected between KSHV-Bcl-2-E
14
A
with NM23-H2. This further confirms the E
14
residue of KSHV Bcl-2, is critical for
87
a L e H
NM23-H2 kBcl-2 Overlay
NM23-H2 kBcl-2 Overlay
SLK
kBcl-2
kBcl-2-E14A
kBcl-2-AAA
NM23-H2
NM23-H2
NM23-H2
Overlay
Overlay
Overlay
KSHV-Bcl-2-NM23-H2 interaction, co-localization, and stabilization. It would be
interesting to determine where in the cell KSHV-Bcl-2 and NM23-H2 colocalize? ER,
Golgi, nucleus membrane? Succeeding lytic replication, is egress, where viruses pass
through ER for assembly and Golgi for maturation. Therefore using ER and Golgi
markers to confirm their relative distribution would reveal interesting results.
88
NM23-H1
kBcl-2 Overlay
* * *
* * *
* * *
Colocalization
&Stabilization
No colocalization
& no Stabilization
Slight colocalization
& no Stabilize
colocalization
& Slight Stabilization
3 T 3
NM23-H2 kBcl-2 Overlay
NM23-H2 mBcl-2 Overlay
NM23-H1
mBcl-2 Overlay
Fig. 2.3.4 KSHV-viral-Bcl2 co-localizes and stabilizes NM23-H2
2.3.5 NM23-H2, similar to KSHV-Bcl-2 is required for KSHV lytic replication
To pinpoint the function of vBcl-2 targeting NM23-H2, I examined the lytic cycle in the
iSLK.219 cell line, which carries green fluorescent protein (GFP) as an infection marker
(latency) and red fluorescent protein (RFP; under the control of the PAN promoter) as a
lytic replication marker [135]. Post induction, FACs analysis was used to sort the cells.
Upon knockdown of NM23-H2 (Fig. 2.3.5 A), cells yielded lower RFP intensity and
amount, likely due to decreased viral genome replication (Fig. 2.3.5 B and C). In support
of this, knockdown of NM23-H1 (Fig. 2.3.5 A) showed similar elevated RFP+ cells well,
suggesting more robust lytic gene expression similar to the control (Fig. 2.3.5 B and C).
This is consistent with our hypothesis that the KSHV-Bcl-2 is required for virus lytic
replication through NM23-H2. However, the mechanism of how vBcl-2 regulates
NM23-H2 for efficient replication and probably egress remains unanswered.
89
C
+ Dox + Dox
shcontrol
shcontrol
shcontrol
shcontrol
shNM23-H2
shNM23-H2
shNM23-H1
shNM23-H1
IB: NM23-H2
IB: NM23-H1
IB: Actin
IB: NM23-H1
IB: NM23-H2
IB: Actin
1 2 3 4 1 2 3 4
15-
25-
42- 42-
25-
15-
A
If our hypothesis is correct and KSHV-Bcl-2 interaction with NM23-H2 is essential for
viral egress and thereof infectivity mutants on both sides lose this function. For the virus
side, KSHV-Bcl-2-E
14
A, it will be reduced replication and for the host side, NM23-H2-
N
69
H, it is resistant to infection and/or replication.
90
Fig. 2.3.5 NM23-H2, similar to KSHV-Bcl-2 is required for KSHV lytic replication
-Dox +Dox islk.219-48hr
shNM23-H1 shNM23-H2 shcon
% of GFP
% of RFP
shcon -Dox
shcon +Dox
shNM23-H2 -Dox
shNM23-H1 -Dox
shNM23-H2 +Dox
shNM23-H1 +Dox
shcon -Dox
shcon +Dox
shNM23-H2 -Dox
shNM23-H1 -Dox
shNM23-H2 +Dox
shNM23-H1 +Dox
shcon -Dox
shcon +Dox
shNM23-H2 -Dox
shNM23-H1 -Dox
shNM23-H2 +Dox
shNM23-H1 +Dox
shcon -Dox
shcon +Dox
shNM23-H2 -Dox
shNM23-H1 -Dox
shNM23-H2 +Dox
shNM23-H1 +Dox
NS
ND ND ND
NS
**
C
B
2.3.6 vBcl-2 doesn’t regulate NM23-H2 or NM23-H2 target genes transcriptional
activity
NM23-H2 has been identified as a sequence-specific DNA-binding protein with affinity
for a nuclease-hypersensitive element of the c-MYC gene promoter [125]. In a DNA
sequence-dependent manner, Nm23-H2 recognizes additional target genes for
activation, including myeloperoxidase, CD11b, and CCR5, all involved in myeloid-
specific differentiation [115]. Moreover, Nm23-H2 binds to nuclease hypersensitive
elements in the platelet-derived growth factor PDGF-A gene promoter sequence-
specifically, correlating with either positive or negative transcriptional regulation [115].
Mutational analyses have identified Arg34, Asn69 and Lys135 as critical for DNA
binding, but not required for the NDP kinase reaction [115]. However, the catalytically
important His118 residue is dispensable for sequence-specific DNA binding, suggesting
that sequence-specific DNA recognition and phosphoryl transfer are independent
91
Fig. 2.3.6 vBcl-2 is not required for the regulation of NM23-H2 or
NM23-H2 target gene transcription
cMyc
NM23-H1
KSHV-Bcl-2
NM23-H2
WT KO WT KO
Vinculin
PDGF-A
+Dox
Actin
properties [115]. These data support a model in which NM23/NDP kinase modulates
gene expression through DNA binding and subsequent structural transactions.
To assess the impact of vBcl-2 knockout on NM23-H2 target gene expression at the
mRNA level, I analyzed the transcription of several NM23-H2 target genes post
induction by reverse transcription-PCR (RT-PCR) (Fig. 2.3.6). The results of RT-PCR
indicated that the mRNA levels of the NM23-H2, as well as its target genes, were not
affected in iSLK-BAC16-vBcl-2-KO cells. This indicated KSHV-Bcl-2 targeting and
regulating NM23-H2 may require some post-translational modifications.
92
2.3.7 vBcl-2 influences the disruption of NM23-H2 oligomerization
Structural analysis has crystalized NM23-H2 as a hexamer. Crystal packing and
quaternary structure are the primary determinants of variation in the backbone
conformations of NDP kinases. It is rather surprising that NM23-H2, which is a small
17-kDa protein, is capable of forming homohexamers as well as heterohexamers.
Formation of hexamers would allow Nm23-H2 to create new surface areas for protein-
protein interaction. In order to identify whether KSHV-Bcl-2 influences NM23-H2
dimeric oligomerization, I co-transfected HA-NM23-H2 and Flag-NM23-H2, along with
increased transfection of GST-k-Bcl-2 (Fig. 2.3.7 A). The correlative data suggest that
k-Bcl-2 de-stablizes Nm23-H2 dimeric structure.
To further detail how k-Bcl-2 regulates NM23-H2 oligomerization, I also performed gel
filtration analysis of NM23-H2 complex (Fig. 2.3.7 B) and found that k-Bcl2 and
NM23-H2 co-elute as a complex (∼M
r
40K), however, k-Bcl2, seems to disrupt the
elution profile of NM23-H2. The induction of virus lytic reactivation in iSLK-BAC16-
93
IB: HA
IB: Flag
IP: HA
IP: HA
IB: HA
IB: Flag
Input
WCLs
HA-NM23-H2+Flag-NM23-H2
IB: Actin
0 2
4
GST-kBcl-2
IB: GST
15-
15-
25-
25-
25-
42-
A
vBcl-2 cells, also caused a clear shift of NM23-H2 peak elution further confirming that
k-Bcl2 influences the disruption of NM23-H2 oligomerization. One unresolved issue
pertaining to the biological functions of Nm23-H2 is whether hexamer formation is an
absolute requirement.
94
NM23-H2
Fraction Number
Relative Densitometry Units
100
50
0
Vec
K-Bcl-2
E14A
19 20 21 22 23 24 25 26 27 28 29 30 31
B
IB: HA
IB:Flag
IB: HA
IB:Flag
20 21 22 23 24 25 26 27 28 29 30 31

293T
+Vec
293T
+KBcl-2
293T
+k-E14A
IB: HA
IB:Flag
220 70
20 21 22 23 24 25 26 27 28 29 30 31
20 21 22 23 24 25 26 27 28 29 30 31

95
Fig. 2.3.7 vBcl-2 influences the disruption of NM23-H2 oligomerization
NM23-H2
Fraction Number
Relative Densitometry Units
100
50
0
islk WT -Dox
islk WT +Dox
islk Bcl-2-KO +Dox
iSLK WT
-Dox
iSLK WT
+ Dox
iSLK KO
+ Dox

IB: HA
IB: NM23-H2
IB: HA
IB: NM23-H2
IB: HA
IB: NM23-H2
20 21 22 23 24 25 26 27 28 29 30 31
20 21 22 23 24 25 26 27 28 29 30 31
20 21 22 23 24 25 26 27 28 29 30 31
2.3.8 vBcl-2 positively regulates NDPK activity of NM23-H2
NM23-H2 is a member of the NDK family, whose biochemical role is to use ATP to
generate a different nucleoside triphosphate [136]. Although kinase activity doesn’t
seem to be required for NM23-H2 interaction with vBcl-2, I would like to further
determine if vBcl-2 regulates NM23-H2 kinase activity. Previous studies have shown
that members of the NM23/NDPK family interact directly and specifically with members
of the dynamin superfamily both in-vitro and in mammalian cells and as GTPase-
activating proteins, are positioned to maintain high local GTP concentrations and
promote dynamin-dependent membrane remodeling. Small GTPase families have been
reportedly implicated in regulated exocytosis and might play key roles in certain events
of membrane trafficking [137] [138]. On the other hand, several GTPases have been
involved in the morphogenesis of viruses, specifically herpesviruses [139] [140] [141]
[142]. Recent studies suggest a role for GTPases in herpesvirus infection. In order to
determine whether KSHV-Bcl-2 regulates NDPK activity of NM23-H2 for the benefit of
viral morphogenesis and egress, I determined NDPK activity upon introduction of
KSHV-Bcl-2 wild type, mutants, as well as NM23-H2 mutants, by a well-known
procedure to assay NDP kinase activities (Fig. 2.3.8) [132] [133]. My results indicated
that KSHV-Bcl-2 strikingly increased the NDPK activity of NM23-H2, as did the KSHV-
AAA mutant. However, KSHV-E
14
A mutant shared similar NDPK activity as endogenous
NM23-H2. The NM23-H2-N
69
H mutant apparently lost the ability to be regulated by
KSHV-Bcl-2. My data suggests KSHV may utilize the GTPase-activating protein function
of NM23-H2, as a source of GTP, to further pursue their agenda.
96

97
Fig. 2.3.8 vBcl-2 positively regulates NDPK activity of NM23-H2
0 1 2 3 4 5
0.0
0.5
1.0
1.5
Time(min)
OD 340/OD 340 at t=0
no enzyme
enzyme+PBS
Vec
NM23H2
NM23H2-N69H
NM23H2-H118C
kBcl2
NM23H2+kBcl2
NM23H2+kBcl2-AAA
NM23H2-H118C+kBcl2
NM23H2+kBcl2-E14A
NM23H2-N69H+kBcl2
OD 340
no lysate
lysate+PBS
Flag-NM23-H2
Flag-H118C
Flag-N69H
HA-kBcl-2
NM23-H2+kBcl-2
H118C+kBcl-2
Vec
N69H+kBcl-2
NM23-H2+AAA
NM23-H2+E14A
NS
NS
*
NS
**
* *
*
2.4 Discussion
For successful infection and propagation, viruses use and modulate cellular signaling
machineries. Herpesviruses, are not exceptions, and have evolved multiple strategies to
overcome or modulate cellular signaling pathways, ultimately leading to the
establishment of persistent infection and tumorigenesis [56]. KSHV dedicates a large
portion of its genome to functions that sabotage almost every aspect of host immunity,
including autophagy [56] [143] [105] [144]. Bcl-2 of KSHV targets Beclin 1 to
downregulate the nucleation step of autophagy [105], FLIP of KSHV blocks the
elongation step of autophagy by preventing the interaction between Atg3 and LC3 [144],
and K7 of KSHV is associated with Rubicon to impair the autophagosome maturation
step [143].
During the virus lytic replication, KSHV expresses a full panel of viral genes and
successful completion of this lytic replication leads to the release of progeny viruses
and, ultimately, cell death. A critical virulence factor involved in KSHV persistence and
oncogenicity is the homologue of the cellular Bcl-2, vBcl-2, encoded by KSHV.
Bcl-2 homologs in other herpesviruses exhibit similar antiautophagic activity. For
instance, Epstein-Barr virus (EBV) encodes two Bcl-2 homologs, BHRF1 and BALF1,
which demonstrate antiapoptotic activity and are required for transforming primary
resting B lymphocytes [145]. MHV-68 encodes one Bcl-2 homolog, M11, which
demonstrates antiapoptotic as well as antiautophagic activity [105] [146] [147] [148]
[102]. In vivo studies suggested that MHV-68 lacking M11 exhibits an impaired ability to
establish latency and compromised reactivation from latency; however, M11 is not
required for MHV-68 lytic replication in vitro [105] [146] [147] [148] [102]. Although Bcl-2
98
of KSHV and M11 of MHV-68 have similar crystal structures, they have a low degree of
sequence identity [105] [134]. KSHV Bcl-2 and MHV-68 M11 bind to Bak and Beclin 1
with different binding affinities [105] [134], suggesting that KSHV Bcl-2 may have
additional cellular or viral interaction partners compared to those of MHV-68 M11. The
different positions with respect to the viral genome may cause the differences in function
between KSHV Bcl-2 and MHV-68 M11. The gene for KSHV Bcl-2 is a lytic gene and is
located right after the origin of lytic replication [149]; however, the gene for MHV-68 M11
is located within the latency locus [150]. The positional difference between KSHV Bcl-2
and MHV-68 M11 suggests that during evolution KSHV Bcl-2 may have tried to gain
some additional functions for effective lytic replication.
vBcl-2 is essential for KSHV lytic replication and probably for its subsequent
propagation [28] [108]. Knocking out vBcl-2 from the KSHV genome results in
decreased lytic gene expression at the mRNA and protein levels, impaired viral DNA
replication, and consequently, a dramatic reduction in infectious virus progeny.
Interestingly, this novel function of vBcl-2 for lytic replication does not depend on its
known anti-apoptotic and anti-autophagic functions [28] [108]. Many viral proteins
perform multiple functions. Therefore, it is not surprising that KSHV Bcl-2 harbors
several functions which are not only required for modulating cellular cell death signaling
but also essential for viral lytic replication.
Our preliminary data has shown the formation of a distinct complex between vBcl-2 and
NM23-H2, an abundant NDP Kinase. The formation of a distinct complex between
vBcl-2 with NM23-H2 is exclusive. KSHV-Bcl-2 further stabilizes NM23-H2 at the post-
translational level and co-localizes together mainly in the cytoplasm. The functional
99
mutants involved in this interaction are the E
14
residue on the virus side (previously
shown to be critical for KSHV lytic replication and progeny infectious virus production)
and the N
69
residue on the host side. Interestingly, complete co-localization and
stabilization was observed between KSHV-Bcl-2 WT and NM23-H2, and KSHV-Bcl2-
AAA mutant and NM23-H2, whereas no co-localization or stabilization was detected
between KSHV-Bcl-2-E14A with NM23-H2. This further confirms the E
14
residue of
KSHV Bcl-2, is critical for KSHV-Bcl-2-NM23-H2 interaction, co-localization, and
stabilization. It would be interesting to determine where in the cell KSHV-Bcl-2
regulates NM23-H2? ER, Golgi, nucleus membrane? Succeeding lytic replication, is
egress, where viruses pass through ER for assembly and Golgi for maturation.
Therefore the ER may be the site of initial regulation post-replication.
To distinguish the function of vBcl-2 targeting NM23-H2, we examined the lytic cycle in
the iSLK.219 cells and found that KSHV-Bcl-2 is required for virus lytic replication
through NM23-H2. However, the mechanism of how vBcl-2 regulates NM23-H2 for
efficient replication and probably egress remains unanswered. If our hypothesis is
correct and KSHV-Bcl-2 interaction with NM23-H2 is essential for viral egress and
thereof infectivity, mutants on both sides will lose this function. For the virus side,
KSHV-Bcl-2-E
14
A, it will be reduced replication and for the host side, NM23-H2-N
69
H, it
is resistant to infection and/or replication.
To assess the mechanistic interaction of KSHV-Bcl-2 and NM23-H2, we pursued to
determine whether NM23-H2 transcriptional activity is required? Whether the
oligomerization and hexamer formation of NM23-H2 is targeted? Or whether KSHV-
100
Bcl-2 regulates NM23-H2 NDPK activity? Neither the transcription of NM23-H2 itself,
nor NM23-H2 target gene expression at the mRNA level was affected upon knockout of
KSHV-Bcl-2. This may mean KSHV-Bcl-2 targeting and regulating NM23-H2 may
require some post-translational modifications. We next sought to determine the elution
profile of KSHV-Bcl-2 and NM23-H2. We found that KSHV-Bcl-2 and NM23-H2 co-elute
as a complex, however, KSHV-Bcl-2, seems to disrupt the elution profile of NM23-H2.
However, one unresolved issue pertaining to the biological functions of NM23-H2 is
whether hexamer formation is an absolute requirement. Finally in order to determine
whether KSHV-Bcl-2 regulates the NDPK activity of NM23-H2 for the benefit of viral
morphogenesis and egress, we determined KSHV-Bcl-2 strikingly increased the NDPK
activity of NM23-H2, as did the KSHV-AAA mutant. However, KSHV-E
14
A mutant shared
similar NDPK activity as endogenous NM23-H2. The NM23-H2-N
69
H mutant apparently
lost the ability to be regulated by KSHV-Bcl-2. Our data suggests KSHV may utilize the
GTPase-activating protein function of NM23-H2, as a source of GTP, to further pursue
their agenda.
Previous studies have shown that members of the NM23/NDPK family interact directly
and specifically with members of the dynamin superfamily (Dynamin guanosine
triphosphatase (GTPase)) both in-vitro and in mammalian cells [151] and thus are
positioned to maintain high local GTP concentrations and promote dynamin-dependent
membrane remodeling. Therefore, a the mechanism by which different NDPKs maintain
high GTP concentration to high-turnover GTPase dynamins for efficient work in different
membrane compartments has been revealed. Furthermore, it has been shown that
knockdown of NDPKs NM23-H1/H2, which produce GTP through ATP-driven
101
conversion of GDP, inhibited dynamin-mediated endocytosis [152]. NM23-H1/H2
localized at clathrin-coated pits and interacted with the proline-rich domain of dynamin
[152]. In vitro, NM23-H1/H2 are recruited to dynamin-induced tubules, stimulated GTP-
loading on dynamin, and triggered fission in the presence of ATP and GDP [152]. Thus,
NDPKs interact with and provide GTP to dynamins, allowing these motor proteins to
work with high thermodynamic efficiency. Based on our current data, we speculate the
mechanism of KSHV-Bcl-2-NM23-H2 complex on viral replication and egress to be as:
KSHV-Bcl-2 may interact with NM23-H2 at the ER, and through some ER-related
dynamin-like protein, somehow regulates ER-mediated vesicle formation. This will
facilitate vesicle egress, helping the virus to escape and generate a new infection.
The theory of vBcl-2 targeting and regulating NM23-H2 may be intriguing, paving the
way for a detailed mechanism for a novel functional of vBcl-2. Thus, an innovative
approach to understand KSHV-host interactions regulating virus replication and egress
is of enduring importance. The mechanism of KSHV induced oncogenesis can be used
to expand upon known antiviral mechanisms of host machinery, and ultimately be
applied for developing new treatment for progressive KSHV malignancies, and KSHV-
associated tumors for AIDS patients.
102
2.5 Future Perspectives
Previous studies have implicated the role of kinases in regulating transport between the
ER and Golgi compartments [153] [154] [155]. Moreover, the role of GTP as a source of
energy has been the topic of focus in regulating virus exocytosis, and it seems to play a
key role in certain membrane trafficking events. But how do viruses escape this tightly
regulated secretory pathway? What are the components involved?
Components comprising the cytosolic coat complex II (COPII)

are now recognized to be
involved in cargo selection and export from the ER [156] [157]. Export from the ER is
initiated by the activation of a small GTPase. This activation step leads to the
recruitment of the COPII subunits Sec23/24 from the cytosol to the membrane to form a
complex that interacts with cargo and cargo receptors, before export. Subsequent
recruitment of the Sec13/31 complex allows the selected cargo to be exported from the
ER by budding vesicles. Following COPII-mediated sorting from the ER, cargo-
containing COPII vesicles are believed to fuse to form pre-Golgi intermediates
containing tubular elements [158]. Pre-Golgi intermediates are the first step in the
exocytic pathway involved in the retrieval of recycling components to the ER using the
coat complex I (COPI) components [159], thus separating forward moving cargo from
the membrane-bound components of the COPII budding and fusion machinery [157]
[160] [161] [162]. The integration of these two sorting steps enables the forward moving
cargo to be selectively delivered to the Golgi complex for transport to the cell surface
[160]. NM23-H2, was identified as a factor that promotes COPII assembly and ER
export in mammalian cells [163]. The identification of NM23-H2, a GTPase activating
protein, as a positive factor for COPII assembly and ER export [163], raises the
103
possibility that members of the NM23-H2 may act at multiple points in the secretory
pathway to regulate vesicle formation.
Although a detailed understanding of the how viruses utilize the ER export machinery
has been achieved, key unanswered questions suggest the existence of unidentified
factors and steps in virus egress. Unraveling the mechanism by which vBcl-2 supports
KSHV lytic replication is still elusive, we propose that vBcl-2 might interact with
NM23-H2 through its 1 helix peptide. This interaction might be essential for the tight
regulation of KSHV lytic gene expression, viral DNA replication, as a source of GTP
facilitate vesicle formation at egress sites, and finally through COPII assembly push
forward ER export and infectivity.
104
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Abstract (if available)

Abstract The ability of a virus to initiate a complex journey into the host cell leading to efficient replication and assembly ultimately dictates the fate of an incoming virus. Many enveloped viruses begin their infection process by exploiting the endomembrane system to enter the host cell. Then the virus hijacks the host cell's machinery, to reach the appropriate replication site. The new viruses are assembled and released via budding at the host cell membrane, thereby maintaining the integrity of the host cell membrane and allowing initiation of a new infection. Therefore, for virus egress, again viruses need to exploit the endomembrane system to traffic and eventually reach an open door for exit. Every step of the viral life cycle, from entry to budding, is orchestrated through interactions with cellular proteins. Accordingly, viruses hijack and use these proteins utilizing any achievable mechanism to their benefit, making it challenging to trace and target viruses. Although virology studies have been fruitful, how viruses commandeer so many diverse pathways, yet still escape, remains elusive. ❧ Our results show a vision of different the viral strategies in modulating both cellular signaling and its own life cycle. In chapter 1, I focus on RNA viruses and show that upon entry, the RNA enveloped viruses represented by VSV and Influenza hitchhike and remodel the endomembrane system and traffic within, eventually escaping endosomal organelles for their genome release. Here I revealed that the UV-radiation resistance-associated gene (UVRAG), an autophagic tumor suppressor well known for regulating autophagy and intracellular trafficking, is a critical factor for RNA enveloped virus entry. ❧ In chapter 2, I shift my focus to DNA viruses, and by exploring the role of Kaposi’s sarcoma-associated herpesvirus (KSHV)-Bcl-2 (B-cell lymphoma 2), an autophagy-regulating gene, I show that the KSHV-Bcl-2 interaction with NM23-H2, an abundant NDP kinase is required for virus lytic replication. This interaction of KSHV-Bcl-2 is genetically separable from its antiapoptotic and antiautophagic functions. Surprisingly, the role of NM23-H2 in virus lytic replication is independent of its catalytic activity. My results will probably reveal a novel function of the KSHV-Bcl-2/NM23-H2 complex in viral lytic replication, which is genetically separate from its previous known functions. New aspects of host regulation by viruses offer potential therapeutic targets, echoing the importance of earlier viral-based discoveries.

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Asset Metadata

Creator Pirooz, Sara (author)

Core Title Virus customization of host protein machinery for efficient propagation

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 07/25/2016

Defense Date 05/02/2016

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

Tag OAI-PMH Harvest,virus,virus entry,virus infection

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Feng, Pinghui (committee chair), Akbari, Omid (committee member), Machida, Keigo (committee member), Yuan, Weiming (committee member)

Creator Email spirooz@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-278586

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Legacy Identifier etd-PiroozSara-4608.pdf

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Document Type Dissertation

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Rights Pirooz, Sara

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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...

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