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Modulation of host antigen presentation by herpes simplex virus 1

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Modulation of host antigen presentation by herpes simplex virus 1

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Content MODULATION OF HOST ANTIGEN PRESENTATION BY
HERPES SIMPLEX VIRUS 1

by
Arpita Kulkarni

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)

December 2010

Copyright 2010 Arpita Kulkarni

ii
ACKNOWLEDGEMENTS

This dissertation would not have been possible without the support of several individuals
who in one way or the other contributed their valuable assistance in the preparation of
this study.

First and foremost, I would like to record my gratitude to my supervisor, Dr. Weiming
Yuan, for his guidance, supervision and encouragement at every stage of this work. I am
extremely thankful to Dr. Ping Rao and Dr. Hong Thanh Pham for their constant
guidance and day-to-day interaction, without which, this thesis would not be completed.

Secondly, I am very grateful to my committee members Dr. Axel Schönthal and Dr.
Keigo Machida for their valuable contribution, ideas and constructive comments.

I would also like to extend a very warm thanks to all my lab mates and friends who have
been there as a source of constant encouragement and support.

Last but not the least; I would like to thank my family for their blessings and for
supporting me in any respect during the completion of this thesis.

iii
TABLE OF CONTENTS

ACKNOWLEDGEMENTS ii
LIST OF FIGURES iv
ABBREVIATIONS v
ABSTRACT vii
CHAPTER 1: INTRODUCTION
1.1. Immune responses against HSV-1 infection 1
1.2. Role of inhibition of antigen presentation during HSV-1 infection 3
1.3. HSV-1 proteins U
S
3, U
S
3.5, U
S
3K220M and Glycoprotein B 5
CHAPTER 2: MATERIALS AND METHODS
2.1. Plasmids 8
2.2. Cell Lines 9
2.3. Transfection 9
2.4. Antibodies and Reagents 10
2.4.1. Primary Antibodies 10
2.4.2. Secondary and Conjugated Antibodies 10
2.5. Immunoprecipitation 11
2.6. Immunoblot 11
2.7. Flow Cytometry 12

CHAPTER 3: RESULTS
3.1. MHC-I levels in HeLa.CD1d.U
S
3.myc cells are lower than those in 13
HeLa.CD1d cells
3.2. Interferon-gamma partially restores MHC-I levels in U
S
3 15
expressing clones
3.3. Cells transiently transfected with U
S
3 also show reduced MHC-I levels 17
3.4. U
S
3K220M, a kinase dead mutant, fails to down-regulate MHC-I 18
3.5. U
S
3.5, a shorter transcript of U
S
3, also down-regulates MHC-I 20
3.6. MHC-I is retained in the ER in U
S
3 expressing cells 21
3.7. gB binds to CD1d through its extracellular domain 25
3.8. CD1d binds to gB through its extracellular domain 27

CHAPTER 4: DISCUSSION 29

BIBLIOGRAPHY 33

iv
LIST OF FIGURES

Figure 1 CD8 T-cell monitoring of HSV-1 latency in sensory ganglia 4

Figure 2 MHC-I cell-surface levels in HeLa.CD1d.U
S
3.myc pool cells 13

Figure 3 MHC-I total and cell-surface levels in U
S
3 expressing cells 14

Figure 4 Interferon-gamma treatment of U
S
3 expressing cells 16

Figure 5 Schematic diagram of transient transfection 17

Figure 6 Western blot analysis after U
S
3-pTracer plasmid transfection 17

Figure 7 Western blot analysis of cells transfected with U
S
3K220M, 18
U
S
3, U
S
3 (1/5) and U
S
3 (1/10)

Figure 8 U
S
3K220M fails to down-regulate cell-surface MHC-I 20

Figure 9 MHC-I down-regulation of U
S
3 is independent of its 21
anti-apoptotic function

Figure 10 Drug inhibition and localization of MHC-I in the U
S
3 23
expressing cells

Figure 11 Confirmation of drug inhibition and localization of MHC-I 24
in two other experimental models

Figure 12 Developing gB mutants 26

Figure 13 Co-IP 1: gB interacts with CD1d at its extracellular domain 27

Figure 14 Co-IP 2: CD1d interacts with gB at its extracellular domain 28

v
ABBREVIATIONS

APC Allophycocyanin
BSA Bovine Serum Albumin
CD Cluster of Differentiation
CD1d Cluster of Differentiation 1 (class d)
Co-IP Co-Immunoprecipitation
CTL Cytotoxic T Lymphocyte
DMSO Dimethyl Sulfoxide
EF1 Elongation factor 1-alpha
ER Endoplasmic Reticulum
ERAD ER-Associated Degradation
FACS Fluorescence Activated Cell Sorting
FITC Fluorescein Isothiocyanate

gBGPI Glycoprotein B-GPI anchored
gBTD Glycoprotein B-Tail Deleted
gBWT Glycoprotein B Wild Type
GPI Glycosylphosphatidylinositol
GRP94 Glucose-Regulated Protein 94
HRP Horseradish Peroxidase
HSV-1 Herpes simplex virus 1
IAA Iodoacetic Acid
ICP47 Infected Cell Protein 47

vi
LAT Latency Associated Transcript
LLnL N-acetyl-l-leucinyl-l-leucinyl-norleucinal
MHC-I Major Histocompatibility Complex - I
NaF Sodium Fluoride
NaPP Sodium Pyrophosphate
NKT Natural Killer T
ORF Open Reading Frame
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PMN Poly Morpho Nuclear
PMSF Phenylmethanesulfonylfluoride
PVDF Polyvinylidene Fluoride
RPM Rotations Per Minute
SCID Severe Combined Immunodeficiency
SDS PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
T cell Thymus cell
TAP Transporter associated with Antigen Processing
TBST Tris-Buffered Saline Tween-20
TCR T Cell Receptor
Th 1 T helper cell 1
U
L
Unique long region
U
S
Unique short region
-GalCer Alpha-Galactosylceramide

vii
ABSTRACT

Herpes simplex virus 1 is a contagious human pathogen, which affects about 70% of the
world population. After a productive infection phase on the skin/mucous membrane, a
latency phase follows in the sensory ganglia, which is life-long. CD8 T cells and NKT
cells play vital roles in both acute and latent herpes simplex virus 1 infections. Both these
components of cell-mediated immunity are activated by antigen presentation through
molecules like MHC-I and CD1d. HSV-1 has evolved to evade immune surveillance
through mechanisms like latency and establishment of infection in the neurons. By
transient and stable expression in mammalian cells, we found that U
S
3 protein kinase and
U
S
3.5, its shorter transcript, significantly down-regulate MHC-I from the cell-surface and
cause its degradation in the proteasome. Also, U
S
3K220M is a loss of function mutant of
this kinase and is not able to down-regulate MHC-I. These results reveal a new role for
viral protein kinases in modulation of host antigen presentation. Further, through co-IP
and Western blot analysis, we mapped the interaction of CD1d and HSV-1 glycoprotein
B to their extracellular domains.

1
CHAPTER 1: INTRODUCTION

Herpes simplex virus 1 (HSV-1), also known as human herpes virus 1 (HHV-1), is a
member of the Alphaherpesvirinae subfamily. It is a contagious human pathogen, which
affects 50%-70% of the world population and spreads through secretions, tears and
saliva. HSV-1 causes infection in the primary skin or the orofacial mucosal surfaces (in
the form of lesions) followed by a life-long latency, which involves intra-axonal
transmission to the sensory ganglion (Nash, 2000; Taylor et al., 2002). A recurrent
infection can occur in the dermatosome following the reactivation of the latent virus.
HSV-1 commonly causes cold sores but in severe cases, can cause keratitis (infection of
the cornea) or fatal encephalitis (infection of the central nervous system). The disease is
very severe in infants and immunocompromised patients and can cause death (Whitley
and Roizman, 2001). Common antiviral drugs include Acyclovir, which inhibits viral
replication. There have been no efficient vaccines available yet.

1.1. Immune responses against HSV-1 infection

The life cycle of HSV-1 involves a primary infection for one to two weeks followed by
transmission of the virus to the sensory ganglion through the axon. A study showed that
an HSV-1 infection in the cornea of mice led to a corneal epithelial lesion for two to four
days (Liu et al., 1996). Th 1 cytokines like interleukin-2 and interferon- regulate this
infiltration, which is dominated by the CD4 T cell population. However, after the virus

2
was transported to the trigeminal ganglion, the inflammatory infiltrate was drastically
different form that of the cornea. There was a massive infiltration of CD8 T cells in the
ganglion about seven days post-infection (Liu et al., 1996). Before this infiltration, there
is viral replication and glycoprotein expression on the neuronal cell surface, which is
under surveillance of T cells (that don’t need MHC-I antigen presentation and can
recognize viral antigens by itself). Three to five days after ganglionic infection, CD8 T
cell population in the ganglion increases in large numbers. This supported by other
studies showing how SCID mice (deficient in B and T cell population) had increased
levels of HSV replication in their sensory ganglia (Liu et al., 1996). MHC-I molecules
are heterodimers of (heavy) chain and a 2 (light) microglobulin chain. They present
peptides, generated by proteases in the cytosol and transported to the ER via the TAP, to
CD8 T lymphocytes. MHC-I, complexed with the peptide, undergoes maturation and is
transported to the cell surface from the ER through the Golgi (Lehner and Trowsdale,
1998).

CD1d, similar to MHC-I, is a heterodimer of a heavy chain and a 2 microglobulin light
chain. It is expressed on the cell-surface of antigen presenting cells (Porcelli, 1995). They
present glycolipid antigens and lipids to NKT cells (Bendelac et al., 2002). NKT cells are
a special subset of T cells that carry receptors of both T cells and NK cells. Studies show
that during early stages of HSV-1 infection, a lack of CD1-restricted subset of NKT cells
leads to delayed clearance of virus and a more severe disease state. Moreover, the CD1d
ligand -GalCer activates CD8 T and NK cells. An inhibition in CD1d antigen

3
presentation results in delayed innate and CTL immune responses (Carnaud et al.,
1999)(Grubor-Bauk et al., 2003). NKT response is considered to be very crucial in HSV-
1 infection because it promotes innate and adaptive immune responses rapidly after
primary infection. The first couple of days after infection are determinant of whether the
disease will be a severe one because there is a constant battle between the immune
system and viral amplification. If NKT cells fail to activate these responses, the virus is
more likely to have access to the peripheral nervous system and the central nervous
system, in more severe conditions (Grubor-Bauk et al., 2008) (Van Kaer, 2004).

1.2. Role of inhibition of antigen presentation during HSV-1 infection

Immediately after infecting the mucous membrane, an HSV-1 immediate early protein
ICP47 plays an important role in inhibiting MHC-I antigen presentation. TAP protein,
present on the ER membrane, transports viral peptides processed in the proteasome and
helps load it onto the MHC-I molecule, which eventually reaches the cell surface for
TCR recognition and CTL action. ICP47 inhibits TAP, prevents peptide loading and
prevents MHC-I cell-surface expression (York et al., 1994).

HSV-1 infection of the dendritic cells causes rapid down-regulation of CD1d molecules
from the cell-surface. Pulse-chase and immunofluorescence experiments proved that
CD1d recycling was blocked by HSV-1 infection and CD1d was accumulated in large
intracellular vescicles resembling lysosomes. Rapid endocytosis of CD1d was observed
partly responsible for this phenomenon (Yuan et al., 2006).

4
Lastly, the virus uses latency as a strategy to evade the immune response by being
‘invisible’ to any humoral or cell-mediated response, where it expresses only LAT genes
(Nash, 2000). However, recent studies show that even during latency, there is a low yet
detectable viral gene expression. HSV-specific CD8 T cells surround latently infected
ganglion cells and control the reactivation of HSV-1. Studies show that viral inhibition of
MHC-I in the latently infected cells, by expressing viral antigens other than LAT genes,
results in an efficient reactivation of the virus.
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Figure 1: (Khanna et al., 2004) CD8 T-cell monitoring of HSV-1 latency in sensory ganglia. (a) During
a primary infection of mice (affecting the cornea of the eye) HSV-1 invades the termini of sensory
neurons, the nucleo-capsid travels by retrograde axonal transport to the neuron cell bodies within the
trigeminal ganglion (TG), viral DNA is inserted into the nucleus, and a brief period of virus replication
ensues. (b) An initial infiltration of macrophages and TCR T cells gives rise to an infiltrate
dominated by CD4 and CD8 T cells, and macrophages that persists for the life of the animal. The CD8
T cells associate closely with the neuron cell bodies, whereas the CD4 T cells localize mainly to the
axonal areas. CD8 T cells directly monitor viral gene expression in neurons by detecting epitopes of
viral proteins that are produced early in a reactivation event and presented on the surface of the neuron
with MHC class I. (c) The CD8 T cells force the viral genome into a quiescent state through IFN-g
production (early in reactivation) or through the release of lytic granules (at later stages of reactivation).
If CD8 T-cell function is compromised (e.g. by stress-related hormones), viral glycoproteins and
nucleocapsids are formed, transported by anterograde axonal transport, virions are assembled at nerve
termini, and infectious virus is released, potentially leading to recurrent disease. immediate early
genes; , early genes; 1 and 2, late genes.

5

Figure 1 (Khanna et al., 2004) elucidates the CD8 T cell mediated control of latently
infected cells. Thus, HSV-1 inhibition of MHC-I is important for prevention of viral
reactivation and its spread to other individuals through shedding and other secretions (Orr
et al., 2007).

1.3. HSV-1 proteins U
S
3, U
S
3.5, U
S
3K220M and Glycoprotein B

HSV-1 U
S
3 (a 481 amino acid serine/threonine protein kinase) (Kato et al., 2005) (Poon
and Roizman, 2005) has diverse functions such as activating protein kinase A and egress
of viral nucleocapsids from nuclei (Reynolds et al., 2002). U
S
3 also plays a role in
blocking apoptosis induced by mutated viral genes, exogenous agents such as sorbitol
(Poon and Roizman, 2007) or activated proapoptotic genes (Benetti et al., 2003; Benetti
and Roizman, 2004; Munger et al., 2001). A homologue of U
S
3, ORF66 protein kinase
from Varicella Zoster Virus, is one example of a protein kinase that down-regulates
MHC-I cell surface expression. Here, MHC-I maturation from the ER to the Golgi
complex is delayed (Eisfeld et al., 2007).

HSV-1 proteins are multifunctional. HSV-1 controls the timing and abundance of protein
expression, compartmentalizes the viral products by expressing full length and truncated
versions of proteins like U
L
26 and U
L
26.5, 22 and U
S
1.5, etc (Poon et al., 2006a). Two
transcripts are encoded by the U
S
3 transcriptional unit. A shorter, less dominant truncated
transcript, which initiates at methionine 77 and lacks 76 amino-terminal amino acids,

6
is designated as U
S
3.5. U
S
3 and U
S
3.5 both post-translationally modify histone
deacetylase 1 and 2 and activate protein kinase A. However, only U
S
3 (and not U
S
3.5) is
able to block apoptosis and affect the modification of nuclear membrane during viral
egress more efficiently (Poon et al., 2006a).

HSV-1 glycoprotein B is a viral envelope protein that helps the fusion of the virus to the
host cell membrane during viral entry. gB is a potent immunogen and activates a special
subset of T cells known as gB-CD8 T cells, which are 50% of the population of the CD8
T cells near the latency site (Khanna et al., 2003). While these gB-CD8 T cells are able to
block virus reactivation, their number decreases during latency because if gB, a late
protein, was to be the sole immunogen during latency, there would be more reactivations
(Ramachandran et al., 2010). gB cytoplasmic tail is phosphorylated by U
S
3 protein
kinase, which could result in its rapid endocytosis, delay in recycling or similar such
functions which are yet to be studied (Kato et al., 2009). These reports have suggested
that the presence of gB on the cell surface is crucial in determining immune response to
acutely or latently infected cells. Also, when HSV-1 infects a CD1d expressing cell line,
CD1d interacts with gB (WY-Unpublished results).

The purpose of this report is to study if HSV-1 U
S
3 protein kinase regulates MHC-I cell
surface expression based on two major findings, CD8 T cells play a major role in
controlling viral replication and reactivation (Liu et al., 2000) and VZV ORF 66, a
homologue of US3, down-regulates MHC-I(Eisfeld et al., 2007). We test this hypothesis
through flow cytometry and Western blot analyses on stable cell line and transient

7
transfection models. The second purpose of this report is to map the interaction of HSV-1
glycoprotein B and CD1d (Yuan et al., 2006) and to study its significance with respect to
immune evasion. We found that U
S
3, when expressed in mammalian cells, does in fact
down-regulate MHC-I from the cell surface. U
S
3.5, its shorter, truncated transcript, which
is similar to U
S
3 in most of its functions except one major function of blocking apoptosis,
also down-regulates MHC-I. U
S
3K220M, a point mutant of U
S
3, fails to perform this
function. Through experiments involving proteasome/lysosome inhibition followed by
Western blot, we found that U
S
3 uses a mechanism to accumulate MHC-I in the ER,
prevent its biosynthesis and maturation to the cell surface. MHC-I is ultimately targeted
to the ERAD pathway. Lastly, through co-IP and Western blot experiments on domain-
deletion mutants, we found that glycoprotein B and CD1d interact with each other
through their extracellular domains. This result, when combined with a previously
published result, U
S
3 protein kinase down-regulates gB from the cell surface by
phosphorylating its cytoplasmic tail (Kato et al., 2009), could be one possible mechanism
that HSV-1 uses to down-regulate CD1d from the cell surface in order to escape the NKT
cell response.

8
CHAPTER 2: MATERIALS AND METHODS

2.1. Plasmids

Plasmid expressing HSV-1 glycoprotein B wild type (gBWT) (904 amino acids) in the
vector pcDNA3.1 was generated from HSV-1 KOS strain by Dr. Weiming Yuan. A
cytoplasmic tail-deleted glycoprotein B mutant (gBTD) (802 amino acids), was cloned by
amplifying the N terminal extracellular and transmembrane domains of gB.
Glycosylphosphatidylinositol (GPI)-anchored gB mutant (gBGPI), which expresses only
the gB extracellular domain attached to a GPI anchor, was constructed by amplifying the
gB extracellular and the GPI coding sequence from the CD1dGPI.pLPCX plasmid and
combining the purified PCR product to amplify the final gBGPI (814 amino acids)
construct by using the most 5’ and 3’ primers.

Plasmid expressing HSV-1 U
S
3 protein kinase ORF (481 amino acids) with a myc-tag
was amplified from HSV-1 Strain 17 and ligated to pTracer-EF/V5-HisA and pLPCX
vectors. An alternate transcription product U
S
3.5 protein kinase (405 amino acids), which
initiated from methionine 77 on the Us3 gene, was also cloned with a myc-tag into
pTracer-EF/V5-HisA vector. A kinase-inactivated mutant of U
S
3 (U
S
3K220M), where the
Lysine at amino acid 220 is mutated to a methionine, was amplified by PCR and ligated
in vectors pTracer-EF/V5-HisA. All plasmids were generated by PCR using primers from
Integrated DNA Technologies and Phusion DNA polymerase (NEB), digested and ligated

9
using different restriction enzymes (NEB) and Quick Ligation Kit (NEB). Plasmids were
amplified by maxiprep (Qiagen)

2.2. Cell Lines

HeLa.CD1d cell line and 293T.CD1d cell line (cells constitutively and stably expressing
human CD1d protein) were provided by Dr. Yuan. A stable U
S
3 expressing subline of
HeLa.CD1d cells (HeLa.CD1d.Us3.myc) was generated by co-expressing the packaging
plasmid pVSV.g and U
S
3myc.pLPCX plasmid into GP2 293 packaging cells. Another
cell line Hela.CD1d.GPI, which stably expresses CD1d extracellular domain attached to a
GPI anchor was provided by Dr. Yuan. All cells were maintained in Dulbecco’s Modified
Eagle Medium (Invitrogen) containing 10% fetal bovine serum (FBS) (Gibco) and 1%
penicillin/streptomycin antibiotics.

2.3. Transfection

gBWT, gBTD or gBGPI plasmids were transiently transfected in HeLa.CD1d by calcium
phosphate transfection method. U
S
3myc.pTracer and U
S
3K220Mmyc.pTracer plasmids
were transiently transfected in 293T-CD1d cells using the BioT reagent (Bioland). Cells
were seeded in either 6-well plates or 10 cm plates and transfected at 60-75% confluency.
24 hours post-transfection, the media was changed and 48 hours post-transfection cells
were harvested for flow cytometry, co-IP or Western blot analysis.

10
2.4. Antibodies and Reagents

2.4.1. Primary Antibodies: R69 anti-gB Rabbit monoclonal antibody was provided by
Roselyn Eisenberg (University of Pennsylvania) and used in dilution 1.5ug/IP for
immunoprecipitation or 1:500 for Western blot. 10B7 anti-gB mouse antibody (IgG1)
from ‘Virusys’ was used in dilution 1.5ug/IP for immunoprecipitation and 1:1000 for
Western blot. HC10 anti-MHC-I Ascites antibody (IgG2a) (Dr. Peter Cresswell) was
used in 1:2000 dilution for Western blot. Anti- U
S
3 rabbit antibody was provided by Dr.
Bernard Roizman (University of Chicago) and used in a dilution of 1:1000 for Western
blot. Biotin anti-CD1d D5 antibody (Dr. Yuan) used in 1:200 dilution. Anti-GRP94 Rat
antibody (Stressgen) was used as a loading control in a 1:500 dilution. All primary
antibodies were diluted in 2% Milk in TBST (Tris-Buffered Saline Tween-20)

2.4.2. Secondary and Conjugated Antibodies: Goat anti mouse-HRP, Goat anti rat-HRP
and Goat anti rabbit-HRP antibodies (Jackson ImmunoResearch Laboratories, Inc) were
used in dilutions 1:10,000, 1:5000 and 1:10,000 respectively in TBST. Streptavidin-HRP
(Molecular Probes) against Biotin anti-CD1d antibody was used in 1:5000 dilutions.
APC-conjugated MHC-I antibody (BD Pharmingen) was used in a 1:10 dilution for Flow
cytometry cell-surface staining.

11
2.5. Immunoprecipitation

48 hr post-transfection of HeLa.CD1d or HeLa.CD1d.GPI cells with gBWT, gBTD or
gBGPI plasmids, cells were harvested, washed twice with ice-cold PBS and lysed in 500
ul to 900 ul of 1% Brij-98 in PBS with protease inhibitors IAA and PMSF for 30 minutes
on ice. Lysates were spun at 13,000 RPM for 7 minutes to get post-nuclear supernatant
500ug to 900 ug of protein was rotated overnight with 1.5 ug of antibody followed by 2
hour incubation with 30 ul of pre-washed protein-A sepharose beads. Beads are then
washed 3 times with PBS and further treated for SDS-PAGE and immunoblot analyses

2.6. Immunoblot

After IP, beads were boiled at 95 degree Celsius in 2X SDS loading buffer for 5 minutes.
For other immunoblots, cells were harvested, washed 2 times with ice cold PBS and lysed
in 1% Triton-X-100 in Tris-buffered Saline with freshly added protease inhibitors PMSF,
Leupeptin, Pepstatin A and phosphatase inhibitors NaF, NaPP, -glycerophosphate,
Sodium Vanadate for 30 min on ice, post-nuclear supernatant was obtained by spinning
lysates at 600xg for 5 minutes. Protein concentration was determined by standard BSA
assay (Biorad). 25-40 ug of protein were boiled in 2X SDS loading buffer for 5 minutes
at 95 degree Celsius. Samples were loaded on 10% SDS Polyacrylamide gels (Biorad)
and run at 20 mAmp/gel. Gels were transferred to polyvinylidene fluoride (PVDF)
membranes (Millipore) at 10 Volts for 90 minutes. PVDF membranes were blocked with

12
5% Milk in TBST for 1 hour with gentle shaking, followed by primary antibody
incubation overnight on a rotator in 4 degree Celsius. Membranes were washed and
incubated with secondary antibody for 45 minutes to 1 hour followed by washing 3 times
with TBST. Membranes were coated evenly with HyGLO Quick spray
Chemiluminescent HRP antigen detection reagent (Denville Scientific, Inc) and exposed
to 4 – 30 seconds on Fujifilm Las 3000 detector.

2.7. Flow Cytometry

Cell surface MHC-I was detected by APC conjugated MHC-I antibody and fluorescence
was measured on the FACS machine (Becton Dickinson). 25 ul of conjugated antibody
diluted in PBS+1% BSA was used for staining cells for 30 minutes on ice. Cells were
washed extensively and fixed with 4 % paraformaldehyde solution for 5 minutes.

13
CHAPTER 3: RESULTS

3.1. MHC-I levels in HeLa.CD1d.U
S
3.myc cells are lower than those in HeLa.CD1d
cells

To test whether U
S
3 protein kinase down-regulates MHC-I antigen presenting molecule,
a molecule that presents viral peptides to CD8 T cells (Peaper and Cresswell, 2008), we
generated a sub-line (HeLa.CD1d.Us3.myc)
that stably and constitutively expresses U
S
3
protein kinase. The advantage of using a
stable cell line is that protein is expressed at
a relatively lower level as compared to
other over-expression methods and
represents its expression level during actual
viral infection. Cells were grown on
DMEM medium (Sigma) with 10% fetal
bovine serum and 1%
penicillin/streptomycin antibiotic mixture.
HeLa.CD1d.U
S
3.myc pool cells (cells that contained a mixture of parental and stably
transfected cells) also needed 1 ug/ml puromycin selection antibiotic to reduce the
number of untransfected cells. Figure 2 shows staining of cell-surface MHC-I with FITC
labeled anti-MHC-I antibody of HeLa.CD1d.U
S
3.myc pool cells versus HeLa.CD1d

Figure 2: MHC-I cell-surface levels in
HeLa.CD1d.U
S
3.myc pool cells. Pool cell
MHC-I level represented by the blue peak is
lower than those of parental HeLa.CD1d cells
in red. The green peak is the basal signal from
the unstained cells.

14
cells. The MHC-I cell-surface levels are reduced in the U
S
3 expressing pool cells as
compared to those in parental cells. Next, the pool cells were individually screened for
U
S
3 expression and cloned to get a homogenous population of cells expressing this
protein. Finally, ‘Clone #3’ and ‘Clone #11’ were selected as two homogenous
populations of cells expressing U
S
3 protein kinase in HeLa.CD1d cells, which were
grown without any puromycin selection in the medium. Cultured Clone #3, Clone #11,
HeLa.CD1d.U
S
3myc pool cells and HeLa.CD1d cells were harvested and cell-surface
MHC-I was stained with anti MHC-I antibody labeled with FITC dye. Figure 3A shows
dramatic reduction in cell surface MHC-I levels in Clone #3 and Clone #11 when
compared to pool cells. This indicates that the number of cells in the pool cells positively
transfected with U
S
3; lose U
S
3 expression due to its foreign and potentially toxic nature,
when cells are passaged multiple times in culture, despite puromycin selection. This
makes using cloned cells (Clone #3 and Clone #11) more advantageous since they are

A B
Figure 3: MHC-I total and cell surface levels in U
S
3 expressing cells
A) FACS analysis, MHC-I cell surface levels stained with FITC labeled antibody and B) Western blot
analysis of MHC-I shows dramatic down-regulation in clone#3 and clone#11, when compared to
parental HeLa.CD1d (HC) cells. Pool cells no longer show U
S
3 stable expression.

15
homogenous. Further, cells were lysed in a non-ionic detergent like Triton-X-100, protein
lysates were separated on SDS-PAGE and MHC-I total protein levels were detected by
immunoblot. Figure 3B shows comparison of MHC-I levels between Clone #3 and Clone
#11 when compared to pool and HeLa.CD1d cells. Total MHC-I levels are dramatically
reduced in cells stably expressing U
S
3 protein kinase. Further investigation needs to be
done to understand if this regulation is at the transcriptional or a post-transcriptional
level.

3.2. Interferon-gamma partially restores MHC-I levels in U
S
3 expressing clones

Interferon gamma is a cytokine that up-regulates the transcription of MHC-I in order to
overcome any MHC-I modulation by a pathogen. It is observed in increased levels on
clinical lesions. As a first step to elucidate the mechanism that U
S
3 uses to down-
regulate MHC-I, we induced MHC-I over-expression by adding interferon-gamma and
detected MHC-I levels by FACS and Western blot. Similar to other immunoevasins like
human cytomegalovirus U
S
3, U
S
2 and U
S
11 (Jones et al., 1996), if U
S
3 down-regulates
MHC-I expression by inhibiting one single step during MHC-I synthesis and biogenesis,
treatment with interferon-gamma will result in increased MHC-I expression.

Three sets of HeLa.CD1d, Clone #3 and Clone #11 cells cultured in complete medium
were treated with interferon-gamma for time points 0, 36 and 72 hours. Cells were
harvested and stained for cell-surface MHC-I with FITC labeled anti-MHC-I antibody or
lysed with 1% Triton-X-100 buffer. Lysates were analyzed by Western blot.

16
Figure 4A shows the increase in MHC-I cell surface levels in parental HeLa.CD1d cells
from 0 hour (orange) to 36 hour (green) and 72 hour (pink). In Figure 4B and 4C, U
S
3
expressing Clone #3 and Clone #11 also show increase from 0 (red) to 36 (green) and 72
hour (brown). However, the increase is not significant. U
S
3 is a potent kinase and might
inhibit multiple steps in MHC-I biogenesis, which are yet to be determined. As for this
report, we focus on any post-transcriptional regulation of U
S
3 on MHC-I expression.
A B
C D
Figure 4: Interferon-gamma treatment of Us3 expressing cells
A) Cell surface MHC-I levels of HeLa.CD1d cells increase profoundly after 36 hour (blue) interferon-
gamma treatment and slightly more after 72 hour (pink) as compared to untreated (0 hour) (orange)
samples. Plots B) and C) cell surface MHC-I levels in US3 expressing clone#3 and
clone#11respectively also show an increase when treated with interferon gamma after 36 hour (green)
and 72 hour (brown) but are not significant or completely restored, suggesting that while US3 may not
transcriptionally inhibit MHC-I expression, it is very potent and may take multiple paths to inhibit
MHC-I post-transcriptionally. D) Total MHC-I heavy chain levels from Western blot analysis show
similar trends.

17
3.3. Cells transiently transfected with U
S
3 also show reduced MHC-I levels

Transient transfection
is a method used to
transiently express a
gene in mammalian
cells. To eliminate any
possibility of stable
transfection of U
S
3
resulting in artifacts
due to adverse effects
caused by its constant presence in the cells, we used the transient method to study U
S
3-
MHC-I downregulation. U
S
3
gene was cloned into pTracer-EF/V5-HisA
(Invitrogen) vector, where its high-level
expression is driven by a human EF1
promoter (Invitrogen manual). This vector also
expresses a GFP gene, which can be detected
by fluorescence microscopy or FACS
(Invitrogen manual). Figure 5 shows a
schematic diagram of transient transfection.
Figure 5: Schematic diagram of transient transfection: the gene of interest
(Us3, Us3K220M etc) into pTracer vector which contains the GFP gene.
The plasmid is transfected into 293T cells using BioT reagent for 48
hours. Positively transfected cells express your gene and the GFP gene,
which can the used as a marker for cell sorting for FACS experiments
pTracer-EF/V5-HisA

Figure 6: Western blot analysis after
Us3-pTracer plasmid transfection. when
expressed transiently in 293T cells cause
the MHC-I heavy chain levels to reduce
shown in the third lane

18
U
S
3-pTracer plasmid
was transfected into
293T-CD1d cells using
BioT, a lipid based
reagent (Bioland). Two
controls were included, a
vector-only transfection
and an un-transfected
control. Cells were lysed
after 48 hours and MHC-
I levels were detected by
Western blot analysis.
Total MHC-I heavy chain levels, detected by anti-MHC-I HC10 antibody, were reduced
as compared to vector-only and untransfected control cells seen in figure 6. Thus, MHC-I
total levels in the cell are reduced when U
S
3 is stably or transiently expressed in cells.

3.4. U
S
3K220M, kinase dead mutant, fails to down-regulate MHC-I

The lysine 220 in U
S
3 is mutated to a methionine by quick-change PCR mutagenesis
because other eukaryotic protein kinases have been known to have a lysine in this
position and a mutation results in loss of function (Kato et al., 2005). This U
S
3K220M
gene is inserted into pTracer-EF/V5-HisA vector for transient transfection experiments.
U
S
3 protein kinase is known to block histone deacetylation by inhibiting HDAC1
Figure 7: Western blot analysis of cells transfected with
Us3K220M, Us3, Us3 (1/5) and Us3 (1/10). It clearly shows that
even when US3 levels (lanes 4 and 5) are almost equal to
US3K220M (lane 2), MHC-I total levels are lowered. This
suggests that MHC-I down-regulation in Us3 transfected cells are
not a result of artifact by mere over-expression.
1/5 1/10

19
and HDAC2 to enable gene expression at high levels (Poon et al., 2006b). The
U
S
3K220M kinase mutant possibly fails to do so and its expression in transfected cells is
much lower than that of U
S
3. To over-compensate this observation, we transfected 5
times and 10 times lower U
S
3 plasmid DNA as controls and compared its effect on
MHC-I levels by Western blot. Figure 7, middle panel shows U
S
3/U
S
3K220M expression
after transfecting 293T-CD1d cells with U
S
3K220M, U
S
3, (1/5) and U
S
3 (1/10) DNA in
lanes 2, 3, 4 and 5 respectively. U
S
3K220M is much lower than U
S
3, however, it is higher
than U
S
3 (1/10). When we compare the total MHC-I down-regulation between these two
samples, we clearly see that U
S
3K220M still fails to do so. Thus, failure of U
S
3K220M to
reduce levels of MHC-I is not merely due to lower expression of U
S
3K220M, since U
S
3
(1/10) is much lower than U
S
3K220M and still reduces MHC-I as seen in figure 7.

Figure 8 shows APC labeled MHC-I cell-surface staining of 293T-CD1d cells transfected
with either U
S
3-pTracer or U
S
3K220M-pTracer plasmid for 48 hours using BioT reagent
(Bioland). pTracer-EF/V5HisA vector encodes for a GFP protein, which expresses itself
in the cells which are positively transfected. The regular FITC channel used in the FACS
machine uses a filter set that excites GFP protein and detects its fluorescence. Thus, FITC
channel is used to sort the positively transfected cells from the remaining untransfected
cells. GFP fluorescence (FITC-H) is used to sort the FITC positive and FITC negative
populations within each sample of transfected cells and APC signals are measured in
each subset and compared to each other. The APC excitation and emission peaks are far
different from those of GFP, hence it is unlikely that the signals are mixed or leaked.
Transfection efficiency is shown by upper panel and MHC-I levels in the lower

20
panel. U
S
3 significantly
down-regulates MHC-I
cell-surface levels
(Figure 8A) where as
U
S
3K220M fails to do
so (Figure 8B)

3.5. U
S
3.5, a shorter
transcript of Us3,
also down-
regulates MHC-I

U
S
3.5 is initiated at
methionine 77 (encodes 77 to 481) and is a less dominant, shorter U
S
3 transcript, which
is similar to U
S
3 is most functions except its anti-apoptotic role (Poon et al., 2006b). By
studying effect of U
S
3.5 expression on MHC-I down-regulation, we will be able to map
down this function to a part of its gene, which is independent of its anti-apoptotic
domain. Also, whether U
S
3 acts as a monomeric or heteromeric kinase is still a matter of
investigation. U
S
3.5 was cloned into pTracer-EF/V5-HisA vector. Figure 9 lower panel
clearly shows how U
S
3.5 transfected cells have lower cell-surface MHC-I levels
(represented by pink peaks versus untransfected blue peaks). The top panel shows GFP
levels, which represent the transfection efficiency.
A) U
S
3 B) U
S
3K220M

Figure 8: U
S
3K220M fails to down-regulate cell-surface MHC-I. A)
U
S
3 transfection of 293T-CD1d cells causes MHC-I down-
regulation. Top panel shows transfection efficiency and lower panel
shows MHC-I levels in transfected (pink) vs untransfected (blue)
cells. B) U
S
3K220M fails to down-regulate MHC-I (pink transfected
vs blue untransfected cells)

21
.
These results clearly show that in the presence of U
S
3
or U
S
3.5 expression, the MHC-I is down-regulated
form the cell surface and this function is lost when the
lysine 220 of U
S
3 is mutated to Methionine. It is yet to
be investigated whether this function of U
S
3 protein
kinase is a result of direct phosphorylation of MHC-I
or involves phosphorylation of another protein kinase
substrate of viral or cellular origin. One possible
mechanism that could explain this phenomenon is the
fact that U
S
3 activates and phosphorylates protein
kinase A, which is known to phosphorylates MHC-I
cytoplasmic tail and other such substrates (Guild and
Strominger, 1984).

3.6. MHC-I is retained in the ER in U
S
3 expressing
cells

To understand the molecular mechanism underlying the down-regulation of MHC-I by
U
S
3 protein kinase is the next step. To elucidate this process more clearly, we carried out
drug inhibition treatment to two important organelles involving in protein turnover, the
lysosome and the proteasome. Previously researchers have used similar techniques to
study mechanisms used by viral proteins to carry out retention of antigen presenting
APC-H
U
S
3.5

Figure 9: MHC-I down-
regulation by U
S
3 is
independent of its anti-
apoptotic function. Top panel
shows the transfection
efficiency of U
S
3.5-pTracer
transfection in 293T-CD1d cells
represented by GFP (FITC-H)
levels. Bottom panel shows
MHC-I down-regulation in
transfected cells (pink) vs
untransfected cells (blue)

22
molecules in the cytoplasm, protease inhibition treatment of HCMV U
S
11+ cells by
LLnL drug revealed that breakdown intermediates of MHC-I accumulated in the
ER/Golgi compartment and its maturation was delayed in the presence of U
S
11 (Wiertz et
al., 1996). In our study, we used bafilomycin A1 that specifically inhibits vacuolar types
H (+) ATPase (predominantly present on lysosomes) and inhibits protein degradation on
the lysosomes (Yoshimori et al., 1991). By adding this drug to cultured cells, if MHC-I
heavy chain levels in the cell lysate increases, we can possibly conclude that lysosome
(or late endosomes) may have trapped MHC-I heavy chain molecules in the presence of
U
S
3. Similarly, we used MG132 or lactacystin to inhibit the proteasome (Cole, 2001). ER
misfolded proteins are retro-translocated to the cytosol by an elaborate ubiquitinylation
process and degraded in the proteasome (Hampton, 2002). Proteasome inhibition will
cause accumulation of immature (non-modified/ unglycosylated/ misfolded) ER MHC-I
heavy chain molecules, which can be detected by Western blot owing to its faster
migration (lower molecular weight) on SDS PAGE as compared to mature MHC-I heavy
chains.

100 nM bafilomycin A1 was added to HeLa.CD1d and U
S
3 expressing stable cell lines
Clone #3 and Clone #11 for two time periods; 2 hours and 12 hours, an untreated control
was included. At the same time, HeLa.CD1d cells and Clone #3 were treated with 10 M
MG132, another set was treated with 10 M Lactacystin. Since these drugs are prepared
in a DMSO base, a DMSO control was included, along with an untreated control. A
titration of drug concentrations and time duration was conducted with the cells to check
toxicity and concentrations at which no significant cell toxicity was seen was used

23
(data not shown). Cells were harvested, lysed in 1% Triton-X-100 with freshly added
cocktail of phosphatase and protease inhibitors. Lysates were analysed by Western blot.
MHC-I was detected by anti-MHC-I HC10 antibody, which recognizes an epitope on
heavy chain. Figure 10 shows A) Bafilomycin A1 treated samples where there is no
obvious increase in MHC-I total heavy chain levels and B) shows MG132 and lactacystin
treated Clone #3 (lanes 7 and 8) where a lower band to MHC-I has appeared, which is the
unglycosylated/ non-modified ER retained MHC-I heavy chain. Thus, in U
S
3 expressing
Figure 10: Drug inhibition and localization of MHC-I in the Us3 expressing cells
A) Bafilomycin A1 treated HeLa.CD1d (lanes 1-3), Clone #3 (4-6) and Clone #11(lanes 7-9) for
0, 12 hours and 2 hours. As compared to untreated samples, no obvious accumulation of MHC-I
can be observed. B) HeLa.CD1d (lanes1-4) and Clone#3 (Lanes 5-8) are treated with proteasome
inhibitors MG132 or lactacystin including DMSO and untreated controls. A lower molecular
weight MHC-I unglycosylated breakdown intermediate that is an ER resident is accumulated.
However, MHC-I levels are still pretty low as compared to HeLa.CD1d samples and complete
recovery is not obtained through proteasome inhibition, suggesting that one possible mechanism
of down-regulation MHC-I by Us3 is ER retention but is not the only one.

24
stable cell lines MHC-I is accumulated in the ER, which is possibly and is led to
proteasomal degradation through the ERAD pathway. There was no accumulation found
after lysosomal inhibition suggesting that MHC-I may not be down-regulated by rapid
endocytosis followed by lysosomal degradation by U
S
3.

To confirm this observation, we repeated the same experiment in two other experimental
conditions, a U
S
3 expressing inducible cell line and U
S
3 transfected cells. The inducible
cell line HeLa.CD1d.Tet-On.U
S
3 is a selective cell line in which expression of U
S
3 is
B) Us3 Transfection
A) Us3 inducible cell line
Figure 11: Confirmation of drug
inhibition and localization of
MHC-I in two other
experimental models
A) HeLa.CD1d.Tet-On.Us3
inducible cell line clone#1 and
clone#5 are induced (using
doxycyclin) to express US3 in
the cells for 48 hours, then
treated with 10 micromolar
MG132 for 12 hours, as
compared to DMSO and
untreated controls (lanes 1 and
2 in both panels) there is a
significant accumulation of the
lower molecular weight
unglycosylated MHC-I heavy
chain intermediate (lanes 3 in
both panels). B) Us3.ptracer
was transfected in 293T-CD1d
cells and treated with
lactacystin for 12 hours.
Comparing to DMSO and
untreated controls, this
experiment also shows lower
molecular weight MHC-I
accumulation

25
induced by adding doxycyclin. Then, two such U
S
3 inducible cell lines Clone #1 and
Clone #5 were treated with 10 M MG132 along with DMSO and untreated controls
(figure 11A) there is an accumulation of unglycosylated MHC-I in the drug treated
samples. Next, U
S
3.pTracer was transiently transfected in 293T-CD1d cells and treated
with lactacystin, a DMSO and untreated control was included (figure 11B) also shows
accumulation. Thus, it is most likely that in U
S
3 expressing cells, there is a delay in
maturation of MHC-I form the ER to the Golgi complex and to the cell surface, which
results in rapid degradation of MHC-I by processes like ubiquitination.

3.7. gB binds to CD1d through its extracellular domain

Plasmids expressing the open reading frames of HSV- 1 wild type glycoprotein B
(gBWT), cytoplasmic tail-deleted gB (gBTD) and GPI anchored gB (gBGPI) were
generated by amplifying the full length, the tail-deleted and the ectodomain attached to a
GPI anchor respectively, in the mammalian expression vector pcDNA3.1 (figure 12). The
plasmids were transfected in HeLa.CD1d cells by calcium phosphate transfection
method. Cells were lysed using a non-ionic detergent like 1% Brij 98. Lysates were used
for two co-IP experiments. Co-IP 1 involved pull down of gB using anti-gB antibody and
protein A beads followed by Western blot to detect CD1d. Co-IP 2 involved pull down of
CD1d using anti-CD1d antibody and protein A beads followed by Western blot to detect
gB. Pre IP samples (lysate only) were loaded onto the gel to confirm CD1d and gB
expression in the cell. Anti GRP 94 antibody was used as a loading control antibody.

26

Immunoblot results (figure 13A) show that CD1d is pulled down by all three variants of
gB (top panel, lanes 4, 5 and 6) suggesting that only the extracellular domain of gB binds
to CD1d. By including a reciprocal IP experiment, we eliminate the possibility of any
non-specific binding of either of the antibodies used in the IP or non-specific binding of
protein A beads to the proteins. Figure 13B shows a pull down of CD1d using anti-CD1d
antibody followed by detection of gB through Western blot. The top panel shows the
interaction in all three gB versions. However, the protein expression of the gBTD and
gBGPI mutants is much lower as compared to the gBWT. This is because of the structure
of the domain-deletion mutants is probably not as stable as the wild type. These two co-
IP experiments showed that only the extracellular domain of gB is participating in the
interaction between CD1d and gB. Next, we test to see what CD1d domain is involved in
this interaction.
Figure 12: Developing gB mutants. gBWT is a 904 amino acid long protein with all three
domains, gBTD is an 802 amino acid protein with ectodomain and transmembrane domain.
gBGPI is gB ectodomain only attached to GPI anchor which tethers it to the lipid membrane
gBWT gBTD gBGPI

27

3.7. CD1d binds to gB through its extracellular domain

Hela.CD1d.GPI cells (provided by Dr. Yuan), which express CD1d ectodomain attached
to a GPI anchor were transfected with gBGPI-pcDNA3.1 in mammalian expression
vector pcDNA3.1 by Lipofectamine reagent (Invitrogen). Cells were lysed using a non-
ionic detergent like 1% Brij 98 and cleared to get protein lyates. gB was
immunoprecipitated by R69 anti-gB antibody and protein A beads. Complexes were
boiled in SDS buffer and separated on SDS-PAGE. CD1d was detected by immunoblot
using anti-CD1d antibody. Figure 14 shows Western blot results after IP, CD1d
ectodomain was pulled down when gB ectodomain is precipitated. Thus, CD1d binds gB

Figure 13: CoIP-1: gB interacts with CD1d at its extracellular domain. A) gBWT, gBTD and
gBGPI were expressed in HeLa.CD1d cells. First, gB antibody + protein A beads were used to
pull down gB, then the protein complex was washed and separated on SDS PAGE and CD1d was
detected by anti-CD1d antibody. All three gB versions can pull down CD1d (top panel), which
means that gB ectodomain interacts with CD1d. B) The same experiment is conducted except the
antibodies are switched. Results show that all three gB versions are pulled down by CD1d (top
panel). The purpose of this co-IP was to eliminate the possibility of either antibody non-
specifically binding with the proteins. For example, gB antibody non-specifically binding with
CD1d will result into a false positive but since the reciprocal IP shows similar results, the chances
of non-specificity has reduced.

28
at its extracellular domain and the transmembrane and cytoplasmic domains are not
required for this interaction.

Figure 14: Co-IP 2. CD1d interacts with gB at its extracellular domain.
HeLa.CD1d.GPI cells that express CD1d ectodomain were transfected with gBGPI.
First, gBGPI was pulled down by anti-gB antibody and the complex was washed and
separated on SDS PAGE followed by CD1d immunoblot. CD1d was detected (lowest
panel), this suggests that CD1d ectodomain interacts with gB ectodomain)

29
CHAPTER 4: DISCUSSION

Herpes simplex virus (type 1 or 2) infection of mice shows virus-induced or host-induced
apoptosis with characteristic condensed nuclear chromatin. This phenomenon could result
in a severe disease state but is not ideal for the virus to survive and persist in the host.
Thus, the organism is known to delay apoptosis (Irie et al., 2004). Several studies have
elucidated this apoptosis-blocking function of U
S
3 protein kinase (Benetti et al., 2003;
Benetti and Roizman, 2004; Munger et al., 2001). An example shows how an infection of
the trigeminal ganglion of mice with a U
S
3-deficient mutant of HSV-2, induced apoptosis
in the ganglionic cells (Asano et al., 2000). It is very clear that CD8 T cells play a pivotal
role in HSV latency and reactivation and could induce apoptosis in virus-infected cells.
Very few reports have delineated the mechanism by which U
S
3 carries out this anti-
apoptotic function. Only one report suggested that U
S
3 blocks the cytotoxic effects of
CD8 T cells without modifying MHC-I antigen presentation (Cartier et al., 2003).
However, the evidence they showed to prove that U
S
3 does not modify MHC-I was not
strong enough. The notion of inhibition of MHC-I antigen presentation playing a role in
HSV infection is highlighted in several cases. For example, when a U
S
3 deficient HSV-2
is infected intravaginally, there is an enhanced infiltration of T cells and antigen
presenting cells and the levels of interferon- and interleukin-12 increase (Inagaki-Ohara
et al., 2001). These cytokines are stimulated in the presence of activated CTLs, Th1 and
NK cells in the presence of pathogens. Consistent with our results, if MHC-I antigen
presentation is not inhibited in the presence of a U
S
3 deficient virus, an enhanced T cell
response will be observed.

30
From our stable and transient transfection data, we found that U
S
3 causes down-
regulation of MHC-I. U
S
3 and U
S
3.5 exemplify those proteins of the HSV genome those
are full and truncated versions of the same protein in overlapping transcriptional units
(Poon et al., 2006a). Our results show that both these proteins down-regulate MHC-I. It is
beneficial for the virus to use two forms of a protein in order to control their timing and
expression as well as their distribution in the cell (Poon et al., 2006a). The presence of
HSV-1 protein ICP22 in infected cells increases the ratio of U
S
3 to U
S
3.5, which are two
kinases, translated from independently regulated mRNAs (Poon and Roizman, 2005).
They have common as well as distinct functions. Although their full range of functions
still remain to be discovered, it is clear that this strategy provides several advantages to
the virus such as directing them to different sub-cellular compartments through distinct
post-translational modifications (Poon and Roizman, 2005).

By inhibiting the proteasome in the U
S
3 expressing cells, we found that the ER-form of
MHC-I gets accumulated in the cell. This suggests that U
S
3 uses some mechanism to
delay or halt the trafficking of MHC-I from the ER to the cell surface, ultimately causing
ER-associated degradation. Several viral proteins utilize this mechanism to evade antigen
presentation. HCMV glycoproteins U
S
2 and U
S
11 destabilize MHC-I molecules and
cause rapid degradation while HCMV U
S
3 delays the egress of MHC-I to the cell surface.
All three proteins are expressed at different time points thus altering MHC-I biosynthesis
throughout the infection (Jones et al., 1996). An HSV-1 U
S
3 homologue, VZV ORF66
protein kinase, does not co-precipitate with MHC-I but delays its maturation through the
ER/Golgi complex. The mechanism is unknown but the speculation is that it either

31
hyper-phosphorylates MHC-I chaperone proteins or its cytoplasmic tail to alter its
trafficking (Eisfeld et al., 2007). These studies provide a lot of insight into the possible
mechanism of down-regulation of MHC-I by HSV-1 U
S
3. It is yet to be determined
whether U
S
3 directly phosphorylates MHC-I or not. But, protein kinase A (PKA) is
known to phosphorylate the cytoplasmic tail of MHC-I (Guild and Strominger, 1984) and
it is known that U
S
3 substrate specificity is similar to PKA and that U
S
3 activates PKA.
Thus, it is believed that U
S
3 mimics the functions of activated PKA (Benetti and
Roizman, 2004). Lastly, MHC-I cytoplasmic tail phosphorylation in the ER/Golgi is
aberrant and can cause deviations in normal trafficking (Eisfeld et al., 2007; Eisfeld et al.,
2007). These findings are consistent with our current results when put together as a
proposed model for U
S
3 -MHC-I down-regulation.

Immediately after infection, NKT cells promote both innate and adaptive immunity by
recognizing lipids on CD1d. Failure of NKT cells to activate these responses causes the
virus to have more access to the peripheral nervous system and central nervous system in
more severe conditions (Grubor-Bauk et al., 2008) (Van Kaer, 2004). A special subset of
T cells, the gB-CD8 T cells, activated by gB, do play a role in controlling the virus but
eventually decrease in number (Sheridan et al., 2009). Thus, CD1d antigen presentation is
crucial in eliciting immune responses in the early stages of infection. We found that
CD1d and gB interact with each other through their ectodomains. The significance of this
interaction can be explained using a phenomenon where U
S
3 regulates gB cell surface
expression by phosphorylating it on its cytoplasmic tail (Kato et al., 2009). This
phenomenon could result in endocytosis of the gB-CD1d complex, or could prevent

32
the recycling of the gB-CD1d complex back to the cell surface, thus evading NKT cell
recognition. Also, recently we found that U
S
3 has also been able to down-regulate CD1d
when expressed in HeLa.CD1d cells (WY-Unpublished results). This could result in
down-regulation of gB in the gB-CD1d complex to evade the gB-CD8 T cell recognition.
By further generating serial deletion mutants, this interaction could be mapped down
further to a specific region or a linear peptide on each molecule. Antibodies targeted
against this region could help us block this interaction, study its effect on viral evasion
and be used as a potential drug candidate against HSV-1. Acyclovir is a purine analog
that selectively inhibits HSV-1 replication. However, it might have no effect on viruses
that are not replicating. Case studies have shown that patients who have had herpes
simplex encephalitis in the past have neurological deterioration and multiple relapses of
infection due to viral replication, in spite of antiviral treatment. These studies concluded
that the antiviral treatment available is not adequate and even after the treatment; the
possibility of relapse can not be excluded (Yamada et al., 2003). An anti-viral drug that
targets an inhibitor of antigen presentation like U
S
3 protein kinase could enhance
activation of T cell response to the latently infected neurons and prevent re-activation of
viruses, its spread to the central nervous system and recurrence of disease.

33
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Abstract (if available)

Abstract Herpes simplex virus 1 is a contagious human pathogen, which affects about 70% of the world population. After a productive infection phase on the skin/mucous membrane, a latency phase follows in the sensory ganglia, which is life-long. CD8 T cells and NKT cells play vital roles in both acute and latent herpes simplex virus 1 infections. Both these components of cell-mediated immunity are activated by antigen presentation through molecules like MHC-I and CD1d. HSV-1 has evolved to evade immune surveillance through mechanisms like latency and establishment of infection in the neurons. By transient and stable expression in mammalian cells, we found that US3 protein kinase and US3.5, its shorter transcript, significantly down-regulate MHC-I from the cell-surface and cause its degradation in the proteasome. Also, US3K220M is a loss of function mutant of this kinase and is not able to down-regulate MHC-I. These results reveal a new role for viral protein kinases in modulation of host antigen presentation. Further, through co-IP and Western blot analysis, we mapped the interaction of CD1d and HSV-1 glycoprotein B to their extracellular domains.

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Asset Metadata

Creator Kulkarni, Arpita (author)

Core Title Modulation of host antigen presentation by herpes simplex virus 1

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology

Publication Date 09/21/2010

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

Tag biochemistry,herpes simplex virus,immunology,microbiology,Molecular Biology,OAI-PMH Harvest,virology

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Yuan, Weiming (committee chair), Machida, Keigo (committee member), Schönthal, Axel (committee member)

Creator Email arpitak@gmail.com,arpitaku@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3462

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Legacy Identifier etd-Kulkarni-4083.pdf

Dmrecord 384126

Document Type Thesis

Rights Kulkarni, Arpita

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu