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Herpes Simplex virus-1 UL56 collaborates with Nedd4 E3 ubiquitin ligase to downregulate surface CD1d and facilitate immune evasion of NKT cell function
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Herpes Simplex virus-1 UL56 collaborates with Nedd4 E3 ubiquitin ligase to downregulate surface CD1d and facilitate immune evasion of NKT cell function
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Content
Herpes Simplex Virus-1 UL56 Collaborates with Nedd4 E3 Ubiquitin Ligase to Downregulate
Surface CD1d and Facilitate Immune Evasion of NKT Cell Function
by
Xinyue Shao
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 2022
ii
Acknowledgements
First, I would like to express my respect and thanks to my principal investigator Dr. Weiming
Yuan. The thesis would not have been completed without the valuable guidance and kind support
he provided throughout all stages of this project. Dr. Yuan cares so much about his lab members
that he never hesitates to provide encouragement, support and instructions when we need it the
most. Dr. Yuan has made this project a valuable learning process for me, not only in terms of this
specific research topic, but also in terms of scientific mindset, academic integrity and attitude
towards science.
My thanks and appreciation also goes to Xuedi Gao who worked alongside me in this project.
Xuedi contributed extensively to this project and provided many valuable ideas and constructive
suggestions.
I would also like to take this opportunity to thank my colleagues, Hongjia Lu, Xiangxue Deng,
Yi Wang and Xiaoting Ren, for their assistance and contributions, and previous lab members
Siyang Chen, Xiangshu Wen, Seil Kim, Ran Xiong, Ping Rao and Jae-Ho Lo for their
fundamental work in this area.
Finally, I would like to thank our master graduate program and the Department of Molecular
Microbiology and Immunology staff, especially Dr. Axel Schonthal, Dr. Hyungjin Eoh and Dr.
Pinghui Feng for their valuable support.
iii
Copyright 2022 Xinyue Shao
Table of Contents
I. Acknowledgements……………………………………………………..…………………ii
II. List of figures……………………………………………………….…………………….iv
III. Abstract…………………………………...……………….………………………………v
IV. Chapter One: Introduction……….……………………….……………………….….……1
1. iNKT cell and CD1d-restricted antigen presenting system……….………..…...….1
2. CD1d molecules…………………………………………………………………...2
3. Trafficking and recycling of CD1d….……………………………...……………..3
4. Herpes Simplex Virus…………………..…………………………………………4
5. The immune evasion of HSV……………………………………………….……..5
6. Nedd4 family E3 ubiquitin ligase…………………….……………………...…….7
V. Chapter Two: Materials and Methods…………….……...……………………………….10
1. Cell line……………………………………….………………………..…..…….10
2. Antibodies, reagents and inhibitors…….………………………………….……..10
3. Plasmids and Transient Transfection………….………………………………….11
4. Flow cytometry ……………………………………………………..……………12
5. Extraction of cell lysates and Western blotting……….…………………….…….12
6. Immunoprecipitation………………………….………………………………….13
7. Ubiquitination assay and inhibitor treatment………..………………..…………..13
8. Immunofluorescence……………………..……………………………..………..14
VI. Chapter Three: Results……………...………………………………………………...….15
1. Nedd4 E3 ligase downregulates mature form of CD1d………………………..15
2. Nedd4 ligase catalytic activity is required for CD1d downregulation…………...17
3. Nedd4 cooperates with UL56 to downregulate surface CD1d………………….18
4. Nedd4 does not downregulate CD1d through direct ubiquitination…………....19
VII. Chapter Four: Discussion…….………………………………………………………....22
VIII. References……………………………….………………………………………….…..25
IX. Figures………………………….…………………………………………………...…..31
iv
List of Figures
Figure 1. Nedd4 and Nedd4L E3 ligase downregulate mature CD1d expression…….…..……..31
Figure 2. Catalytic activity of Nedd4 is essential for downregulation of CD1d…………………34
Figure 3. Nedd4 cooperates with UL56 to downregulate CD1d expression in 293T.CD1d
cells………………………………………………………………………………...…...………..35
Figure 4. Nedd4 does not downregulate surface CD1d through direct ubiquitination……..……37
v
Abstract
Invariant Natural Killer T (iNKT) cells are a subset of Natural Killer T (NKT) cells that are
restricted by CD1d molecules. The CD1d-mediated-iNKT cell antigen presenting system plays
an important role in the immune defense against various virus infections. Herpes Simplex Virus
(HSV) has been shown to evade immune recognition by downregulating surface CD1d
expression in antigen-presenting cells, with the underlying mechanism under-illustrated. Here we
show that Nedd4, the prototype member of the Nedd4 family E3 ubiquitin ligase, contributes to
the downregulation of surface CD1d in 293T cells. With its catalytic activity essential in this
process, Nedd4 cooperates with HSV tegument protein UL56 to downregulate surface CD1d
molecules, which assists in the immune evasion strategy of HSV. However, such inhibition of
CD1d surface expression is not achieved by the direct ubiquitination of CD1d because the
decrease of CD1d persists in cell lines with mutant CD1d that cannot be ubiquitinated,
suggesting an indirect mechanism of CD1d downregulation.
1
Chapter One: Introduction
1. iNKT cell and CD1d-restricted antigen presenting system
Natural killer T cells (NKT cells) are a unique sublineage of T cells that express natural killer
lineage receptors (Bendelac, Savage, & Teyton, 2007). Functioning as a bridge between the
innate and adaptive immune systems, NKT cells are important mediators of immune response
against various pathogens (McEwen-Smith, Salio, & Cerundolo, 2015). There are two subsets
within NKT cells - type I NKT cells and type II NKT cells. The most studied subset of NKT
cells are type I NKT cells , also named invariant NKT cells (iNKT cells). Developed in thymus,
iNKT cells are restricted by CD1d which is a MHC class I like molecule (Gapin, 2018). As
implied in its name, iNKT cells express an invariant T cell antigen receptor (TCR) - Va14Ja18
chains in mice and Va24Ja18 chains in human, combined with a restricted but not invariant set of
β chains (Vb8.2, Vb7, Vb2 in mouse and Vb11 in human) (Kaer, 2004).
Through decades, CD1d-mediated iNKT cells have been extensively studied for their ability to
respond to various viral infections, including but not limited to KSHV (Diana & Lehuen, 2009),
HSV (Grubor-Bauk et al., 2003), HIV (Juno et al., 2019), MCMV (Wesley et al., 2008), RSV
(Johnson et al., 2002) and influenza virus (De Santo et al., 2008). Once activated, iNKT cell can
rapidly produce large amount of cytokines, including interferon-γ (IFNγ), interleukin-4 (IL-4),
IL-10, IL-13, IL-17, IL-21, TNF) and GM–CS, through which it signals to downstream immune
cells such as NK cells, conventional T cells, Tregs, B cells and dendritic cells (Novak & Lehuen,
2011; Reilly, Wands, & Brossay, 2010). As a result, iNKT cells are able to stimulate and amplify
downstream immune systems in response to viral infections.
2
2. CD1d molecules
CD1d belongs to a family of transmembrane glycoproteins - CD1 family - that structurally and
functionally resemble major histocompatibility complex (MHC) class I molecules. Similar to
MHC class I molecules, CD1 proteins are expressed as heterodimers which consist of a
transmembrane heavy chain paired with beta-2-microglobulin (Brigl & Brenner, 2004). In
humans, CD1 family has 5 isoforms - CD1a, CD1b, CD1c, CD1d, and CD1e. The antigen-
binding groove of CD1 proteins shows a two-pocket structure, an A-pocket and a F-pocket,
which varies among each CD1 isoform to allow for distinct antigen binding (Zajonc & Wilson,
2007). Based on their structure and functionality, CD1 family members are classified into two
groups - group I CD1 (CD1a, CD1b, CD1c and CD1e) which interact with T cells of diverse
TCR repertoire, and group II CD1 (CD1d) which interact with iNKT cells.
After the report of the first crystal structure of the CD1d protein, CD1d was found to be highly
conserved in almost all mammals (Dutronc & Porcelli, 2002). In contrast of MHC molecules
which bind to peptide antigens, CD1d is characterized by a hydrophobic antigen-binding cleft
which are ideal for binding of lipid-based antigens, including lipids, glycolipids, and lipopeptides
(Brigl & Brenner, 2004; Wilson, 1997; Godfrey, 2012).
The formation of CD1d-lipid antigen complex allows for the direct recognition by TCR and
activates T cells (Moody & Porcelli, 2003). Despite the similarity of CD1d to MHC class I
molecules and the similarity of iNKT cell TCR to peptide TCR, the molecular interaction model
of CD1d to iNKT cell TCR differs significantly from typical peptide recognition. The binding
between CD1d and iNKT cell TCR is parallel to the binding groove, relying solely on CDR 1
3
and 3 of the TCRα chain (Rossjohn, 2007). This interaction model contributes to the cross
reactivity of mice and human CD1d and iNKT cell TCRs (Speak, Cerundolo & Platt, 2008).
3. Trafficking and recycling of CD1d
The intracellular trafficking of CD1d molecules starts in endoplasmic reticulum (ER) and Golgi
apparatus. The folding of CD1d heavy chains takes place in ER, followed by the assembly of
beta2-microglobulin (beta2m) (Sugita, Porcelli, & Brenner, 1997). Different from its MHC class
I counterparts, the assembly of CD1d is independent from the transporter-associated-with-
antigen-processing(TAP) mechanism(Sugita, Porcelli, & Brenner, 1997). CD1d is then
transported to Golgi and trafficked to the cell surface through secretory pathway (Salio et al.,
2013).
As a non-polymorphic antigen presenting molecule, CD1d has to avoid being overwhelmed by
the large amount of lipids in cellular membranes. In order to solve this problem, CD1d adopts an
endosomal trafficking method, which allows for its trafficking into discrete intracellular
compartments where lipid ligands are loaded (Moody & Porcelli, 2001). Multiple studies have
shown that CD1d cytoplasmic tail is the primary mediator of endosomal trafficking of the
molecule, with the YXXf motif (“Y” denotes tyrosine, “X” denotes any amino acid, and ‘f’
denotes hydrophobic amino acid) playing an important role in its endosomal sorting (Robinson
& Bonifacino, 2001; Jayawardena-Wolf et al., 2001), while the interaction with MHC class II
molecules is also proven to be a potentially significant factor (Kang & Cresswell, 2002).
4
The recycling of CD1 molecules goes through an endocytic pathway which is dependent on a
cytoplasmic tyrosine-based endocytic motif YXXf (Joyce, 2001). Compared with their MHC
class I counterparts, CD1d are relatively long lived molecules with a half life longer than 15
hours (Jayawardena-Wolf et al, 2001). Studies show that the molecule may undergo intensive
recycling between cellular membrane and intracellular compartment such as MHC class II
compartments (MIIC), which allows for the repeat loading and exchanging of glycolipid antigens
(Moody & Porcelli, 2001).
4. Herpes Simplex Virus
Herpes Simplex Virus (HSV) is one of the most prevalent herpesviruses in humans. Its infection
may lead to a wide range of pathological symptoms from mile mucocutaneous infection to life
threatening conditions. Among the various types of HSV, HSV-1 and HSV-2 are the most
serious human pathogens, with HSV-1 generally leading to orofacial infections and HSV-2
associated with genital infections (Whitley & Roizman, 2001). An important character of both
HSV-1 and HSV-2 is their ability to establish life-long latency within the host. During primary
infection, HSV-1 enters sensory neurons and is transported to neuronal cell bodies where latency
is established. Upon reactivation, the virus can efficiently generate new virus progeny which are
transported back to the periphery, resulting in recurrent lesions on the host near the point of entry
(Nicoll, Proença, & Efstathiou, 2012). Many researches have shown that this latency is crucial to
the survival and pathogenicity of the virus (Thompson & Sawtell, 2001; Efstathiou, & Preston,
2005).
5
The immune response against HSV involves both the innate and the adaptive immune system.
The innate immune response to HSV-1 involves natural killer (NK) cells which recognize and
kill virus infected cells and produce cytokines, and plasmacytoid dendritic cells (pDCs) which
produce type I interferon includes IFNα and IFNβ (Biron & Brossay, 2001; Lund et al., 2003).
On the other hand, the adaptive antiviral response is important in interfering with the latency,
reactivation rate, and disease severity of the virus (Chew, Taylor, & Mossman, 2009). Both
CD8+ T cells and CD4+ T cells have been shown to play important roles in the defense against
HSV infection, largely through the production of IFNγ (Dobbs et al., 2005; Johnson, Chu, &
Milligan, 2008). iNKT cells also play an important role in anti-HSV immune response, acting as
an early source of cytokines and a bridge between innate and adaptive immune systems.
Research has shown that mice lacking CD1d or iNKT cells displayed more severe outcomes for
infection of both HSV-1 and HSV-2 (Grubor-Bauk et al., 2003; Grubor-Bauk, Arthur, &
Mayrhofer, 2008).
5. The immune evasion of HSV
All viruses that establish life-long infections have to solve the problem of achieving persistence
under the attack of host immune response. Through hundreds and thousands of years in
evolution, HSV-1 co-evolved with humans and developed strategies to evade both innate and
adaptive immune responses in order to establish effective latency and lifelong infections.
Researches have shown that HSV-1 can evade early antiviral responses through various
mechanisms, such as interfering with Toll-like receptors (TLRs) recognition of viral
compartment, interrupting host interferon response, and suppressing the activity of innate
immune cells (Tognarelli et al., 2019). At the same time, the evasion of adaptive immunity by
6
HSV has also become an increasingly popular researched topic in recent years, mostly through
the downregulation of cell surface CD1d (Yuan et al., 2006; Rao et al., 2011).
In general, it is a common strategy for viruses to evade immune recognition by downregulating
surface expression of antigen-presenting molecules. Many viruses have been shown to target the
maturation, assembly and export of MHC class I molecules (Alcami & Koszinowski, 2000).
Researches found that Kaposi’s sarcoma-associated herpesvirus(KSHV) K3 and K5 zinc finger
membrane proteins are able to downregulate cell surface MHC class I molecules (Ishido, 2000;
Coscoy & Ganem, 2000). The downregulation of MHC class I molecules reduces the recognition
and activity of CD8+ T cells. Some other viruses, including adenovirus, MCMV and HCMV,
were proven to downregulate MHC class II molecules, largely through transcription and post-
translational regulation (Alcami & Koszinowski, 2000; Früh, 1999). Moreover, HSV ICP-47 and
HCMV US6 have been shown to turn off transporter associated with antigen processing (TAP)
by inhibiting peptide binding to TAP and transportation through TAP pore (Hill et al., 1995;
Hengel, Brune, & Koszinowski, 1998).
As an antigen-presenting molecule, CD1d is also a popular target for virus molecules
(Opasawatchai & Matangkasombut, 2015). Research has shown that KSHV proteins K3 and K5
(MIR-1, MIR-2) molecules can ubiquitinated CD1d on the lysine residue in its cytoplasmic tail
(Sanchez, Gumperz, & Ganem, 2005; Coscoy, Sanchez, & Ganem, 2001). By downregulating
cell surface CD1d, the virus is able to evade immune recognition and attack by NKT cells.
Similarly, HIV protein Nef accelerates the internalization of CD1d (Chen et al., 2006), and HIV
VpU interferes with CD1d trafficking from endosome (Moll et al., 2010).
7
HSV-1 viral molecules are also extensively researched on their ability to downregulate CD1d.
An early research of our lab showed that HSV-1 infection led to downregulation of cell surface
CD1d in antigen presenting cells by interfering with its recycling (Yuan et al., 2006). The study
showed that HSV-1 infection causes endocytosed CD1d to be redistributed to the lysosome-
limiting membrane, reducing their chance of reappearing in cellular membrane (Yuan et al.,
2006). It was then proven that HSV-1 proteins gB and US3 downregulate surface CD1d through
lysosomal degradation (Rao et al., 2011). However, knock out of gB or US3 genes failed to
completely rescue surface CD1d expressing, indicating additional players remain to be
discovered in this pathway (Rao et al., 2011). A recent study by my lab coworker Siyang Chen
showed that HSV-1 tegument protein UL56 can contribute to the downregulation of CD1d
(Chen, 2019). As the viral tegument protein, UL56 is essential for the pathogenicity of HSV-1
(Berkowitz, 1994). In Chen’s study, 293T.CD1d cells transfected with UL56 showed
downregulation of mature forms of CD1d, which may contribute to the immune evasion of HSV-
1 (Chen, 2019). Chen also investigated the potential role of Nedd4 family E3 ligases in CD1d
downregulation, indicating that UL56 may interact with Nedd4 family ubiquitin ligase to achieve
degradation of surface CD1d (Chen, 2019).
6. Nedd4 family E3 ubiquitin ligase
As a form of post-translational modification, ubiquitination is a predominant method of
modulating protein expression, localization, activity and degradation. The covalent attachment of
ubiquitin to target proteins involves the regulation of a three level cascade. The initial step is
carried out by E1 activating enzymes in an ATP-dependent manner, resulting in the formation of
8
a thioester on ubiquitin C terminus(Pickart, 2001). The intermediate step is carried out by E2
conjugating enzymes, resulting in a E2-Ub thioester intermediate (Berndsen & Wolberger,
2014). The final step is carried out by E3 ligases who bind to both E2 and the substrate, where
ubiquitin is transferred from E2 cysteine to the substrate lysine or N terminus (Berndsen &
Wolberger, 2014). In contrast to the small number of E1 observed in eukaryotes, there is a large
set of E2 and a larger number of E3 enzymes. This provides multiple levels of regulation, and
allows for the large diversity and specificity of ubiquitination (Pickart & Eddins, 2004).
Neuronal precursor cell-expressed developmentally downregulated 4 (Nedd4) is a family of E3
ubiquitin ligase that shares some common characteristics. Nedd4 family members include
Nedd4, Nedd4L (Nedd4-2), NedL1, NedL2, WWP1, WWP2, Smurf1, Smurf2, and Itch. The
construct of Nedd4 family E3 ligases includes a C-terminal HECT domain, a N-terminal C2
domain, and WW domains that vary in number among family members (Ingham, Gish, &
Pawson, 2004). The HECT domain is responsible for catalytic activity; the N-terminal C2
domain binds to Ca2+, lipids, and proteins; and WW domains recognize PY motifs and provide
sites for substrate binding (Shearwin‐Whyatt, 2006).
Because of their role as ubiquitin ligase, Nedd4 family members are commonly involved in
targeted degradation of proteins that possess PPxY motifs for WW domain recognition (Ingham,
Gish, & Pawson, 2004). Some viruses develop strategies to downregulate host immune
molecules with the assistance of Nedd4 family E3 ligases. For example, Nedd4 was shown to
degrade antiviral protein IFITM3, which facilitates the infection of influenza virus (Chesarino,
McMichael, & Yount, 2015). On the other hand, some antiviral methods have been discovered
9
targeting Nedd4 family E3 ligases. Research has identified small-molecule probes that can block
egress of many RNA viruses by interfering with viral PPxY-host Nedd4 interaction (Han et al.,
2014). HSV tegument protein UL56 was also found to interact with Nedd4 family members.
HSV-2 UL56 was found to interact with Nedd4 and regulate its localization in order to facilitate
transport and release of virions (Ushijima et al., 2008; Ushijima et al., 2009). Dr. Ushijima also
found UL56 to interact with Nedd family member Itch and regulate its ubiquitination (Ushijima
et al., 2010).
Putting the pieces together, we summarize our present knowledge of this topic into three points:
1) HSV achieve immune evasion of CD1d-iNKT antigen presenting system by interfering with
the recycling of surface CD1d. 2)HSV tegument protein UL56 can downregulate cell surface
CD1d, possibly through ubiquitination. 3) UL56 is able to interact with and regulate various
Nedd4 family members. In this study, we sought to determine the role of Nedd4 E3 ubiquitin
ligase in the regulation of cell surface CD1d, and investigate possible underlying mechanisms of
how Nedd4 participates in the immune evasion of HSV.
10
Chapter Two: Materials and Methods
1. Cell line
293T CD1d cell line and HeLa CD1d cell line (provided by Dr. Yuan), both overexpressing
CD1d molecules, were used in this project. 293T CD1d cells were cultured in Dulbecco’s
Modified Eagle Medium (DMEM) with 5% Fetal Bovine Serum (FBS) and 0.05% puromycin
antibodies. HeLa CD1d cells were cultured in DMEM with 5% FBS and 1% penicillin
antibodies.
In order to meet the needs of the mutation experiment, two stable cell lines were constructed
with 293T cells by retrovirus and cloning. 293T-KR-mutant cell line was constructed to
overexpress CD1d with single point mutation from Lysine to Arginine of the only Lysine on the
cytoplasmic tail of CD1d. 293T-TD10 cell line was constructed by deleting 10 amino acids on
the cytoplasmic tail of CD1d, preventing its ubiquitination. The two stable cell lines were
developed by transducing host 293T cells with retroviruses encoding mutant CD1d proteins.
Transducted cells were selected in DMEM with 10% FBS and 1% puromycin antibodies in order
to generate stable cell lines.
2. Antibodies, reagents and inhibitors
Primary antibodies: Monoclonal mouse anti-51.1.3 (from Dr. Steven Porcelli at Albert Einstein
College of Medicine, Bronx, NY) was used to detect mature CD1d expression. Anti-D5 antibody
was used to detect linear forms of CD1d. Anti-Grp94 antibody was used as loading controls.
Anti-UL56 Ab was used to detect un-tagged UL56. Anti-flag mouse Ab was used to detect flag-
Nedd4, flag-Nedd4L and other Nedd4 family members. Anti-HA-HRP Ab was used to detect
HA-Nedd4 DD and HA-Ub to show ubiquitination status.
11
Secondary antibodies: Goat-anti-Mouse IgG(H+L) HRP, Goat-anti-Rabbit IgG HRP, Goat-anti-
Rat HRP was used as secondary antibodies in Western blotting. PE Goat-anti-mouse IgG was
used in flow cytometry.
Inhibitors: MG132 was used as a proteasome inhibitor at a concentration of 50uM; Chloroquine
(CQ) was used as a lysosome inhibitor at a concentration of 30uM.
3. Plasmids and Transient Transfection
Various plasmids were used for transient transfection. pTracer (provided by Dr. Yuan) is a
plasmid with GFP expression. pTracer-UL56 is an untagged plasmid with UL56 and GFP
coexpression. Flag-Nedd4 and Flag-Nedd4LpCI are plasmids expressing flag-tagged Nedd4 and
Nedd4L E3 ligase, respectively. HA-Nedd4-DD is a HA-tagged plasmid with a mutated form of
Nedd4 on which the catalytic activity is eliminated. V5-his-K3 and V5-his-K5 are plasmids with
both V5 and 6*his at the C-terminal of K3 and K5 proteins of KSHV. Plasmids were
transformed into bacteria and prepared using miniprep or maxiprep techniques.
293T CD1d cells were cultured in 10 cm plates, 15 cm plates, or 6 well plates, and were
transfected at 60%-70% confluency using transient transfection technique. The ratio between
DNA (1λ) and polyethylenimine (PEI) was kept as 1:3. In 10 cm plates, 10ug plasmid were
transfected; in 20 cm plates, 30uL plasmid were transfected; and in 6 well plates, 2ug plasmid
were transfected. In co-transfection experiments, the amount of each plasmid was kept
equivalent, and the total amount of plasmid was kept equivalent across samples, supplemented
with pcDNA. Cells were harvested 48 hours after transfection.
12
4. Flow cytometry
After being harvested, 293T cells were centrifuged at 1200 rpm for 3 minutes, and washed 3
times with PBS. When fixed with 3.7% paraformaldehyde/PBS, cells were washed with PBS
with 0.4M Glycine. Cells were transferred into 96 well plates and were stained with first
antibodies at a concentration of 10 ug/mL. Primary antibodies used for FACS staining include
MHC class I W6/32 mouse antibody, LAMP-1 H5G11 rabbit antibody, and anti-CD1d 51.1.3
mouse antibody. After 30 minutes on ice, cells were washed twice in PBS/0.05%BSA. Cells
were then incubated on ice for 15 minutes with secondary antibodies at 0.4ug/mL. Secondary
antibodies include PE goat-anti-mouse IgG (from BD), and PE goat-anti-rabbit (from Thermo
Fisher). Stained cells were suspended in PBS and loaded to BD FACSCanto II for analysis.
5. Extraction of cell lysates and Western blotting
After harvesting, cells were lysed on ice in a freshly made lysis buffer for 30 minutes. The lysis
buffer was made with 1% Triton-X-100 in Tris-buffered Saline buffer (TBS) with inhibitors
100x IAA, 10 mM PMSF, 200 mM NaVO3, 200 mM Leupeptin, 200 mM Pepstatin A, 1 mM
NaF, 100 uM Okadaic Acid, 1 mM β-glycerophosphate and 0.5 mM NaPP. The mixture was
centrifuged at 1000 rcf for 10 min and the supernatant was collected. 200 uL out of 1mL
supernatant were used directly as whole cell lysate samples, and the remaining were proceeded
with immunoprecipitation. The whole cell lysates were boiled in 1x Sodium Lauryl Sulfate
(SDS) buffer, and loaded on 10% SDS polyacrylamide gels at 15-20 uL per well. Bio-Rad
Precision Plus Protein All Blue Standards was used as the protein marker. Gels were run under
80 Volts for approximately 2 hours. Semi-wet transfer technique was used to transfer gels onto
Polyvinylidene Fluoride (PVDF) membranes, running under 12 Volts for 90 minutes. After
13
transferring, PVDF membranes were blocked in 5% non-fat milk in TBST for 30 minutes. The
membranes were incubated at 4C overnight in 1ug/mL primary antibodies in 1% BSA/TBS-T.
Secondary incubation took 1 hour at room temperature in TBS-T. Membranes were washed for 3
times in TBS-T in between each incubation and at the end of secondary antibody incubation.
Chemiluminescence images were obtained using the Bio Rad ChemiDoc Imaging System. Image
band signal intensity was quantified using Image Lab software, and bar graphs were constructed
based on quantification data.
6. Immunoprecipitation
Before performing immunoprecipitation (IP), a preclearance step was adopted in order to remove
nonspecific bindings. Whole cell lysate samples were precleared with 1uL normal rabbit (or
mouse) serum mixed with 40uL protein G and Sepharose 4B beads at a ratio of 1:1. After rotated
in 4C for 2 hours, the mixture was centrifuged at 5000 rpm for 1 minute. Supernatant was
recovered for immunoprecipitation with 2uL 51.1.3 antibodies mixed with 40uL protein G and
Sepharose 4B beads. The mixture was then rotated overnight in 4C. After overnight incubation,
beads were washed three times with 0.1% Triton/TBS, and boiled in a 2xSDS buffer to prepare
for gel electrophoresis.
7. Ubiquitination assay and inhibitor treatment
In the ubiquitination assay, HA-Ub was used as an extra source of Ubiquitin, and was co-
transfected into 293T CD1d cells together with flag-Nedd4, UL56, HA-Nedd4-DD or V5-his-
K3/K5. Cells were cultured in 10cm plates and transfected at 70-80% confluency. After 36
hours, cells were treated with MG132 at the concentration of 50uM and Chloroquine (CQ) at the
14
concentration of 30uM. Cells were harvested 12 hours after the inhibitor treatment. During cell
lysis, N-Ethylmaleimide (NEM) was added to the lysis buffer at a concentration of 0.024mg/mL
in order to protect ubiquitinated proteins. During Western blotting, HA-HRP antibody (from Dr.
Yuan) was used to detect protein ubiquitination.
8. Immunofluorescence
HeLa cell line overexpressing CD1d is used for immunofluorescence imaging. Cultured on
coverslips in 24 well plates, HeLa.CD1d cells were transfected with flag-Nedd4 at an initial
confluency of 30000 cells/L, and harvested after 48 hours. At room temperature, cells were fixed
with 4% paraformaldehyde/PBS for 15 minutes and permeabilized with permeabilization
solution (PS) for 15 minutes. Permeabilized cells were stained with primary antibodies diluted in
PS at a concentration of 10 ug/uL - 20uL diluted antibodies were used for the staging of one
coverslip. After 30 minutes of incubation at room temperature, coverslips were washed in PS for
three times. The incubation of secondary antibodies followed similar protocols as that of primary
antibodies, except that it must take place in a dark environment to protect fluorescent antibodies
from light. After incubation for 30 minutes, coverslips were rinsed in water and placed face
down on a glass microscope slide with 5uL drop of Mowiol. Samples were dried at 4C overnight
and protected from light before imaging. Primary antibodies used for immunofluorescence
staining include anti-flag M2 IgG antibody, anti-CD1d 51.1.3 antibody, and anti-Lamp1 rabbit
Antibody. Secondary antibodies include Alex fluor 488 Goat-anti-Mouse IgG antibody, Alex
fluor 568 Goat-anti-Mouse lgG antibody, and Alex fluor 647 Goat-anti-Rabbit antibody.
15
Chapter Three: Results
1. Nedd4 E3 ligase downregulates mature form of CD1d
Our lab has a history of researching on the iNKT cell immune evasion mechanism of HSV-1.
Previous works have shown that HSV-1 evades iNKT cell recognition by suppressing the
recycling of CD1d, since the virus reduces APC cell surface CD1d expression without interfering
with CD1d protein synthesis or enhancing CD1d endocytosis (Yuan et al., 2006). It is also
proven that HSV-1 viral protein UL56 can downregulate cell surface CD1d through degradation,
and that Nedd4 E3 ubiquitin ligases may have a potential role in this downregulation (Chen,
2019). In the light of these previous works, here we sought to determine whether Nedd4 family
E3 ligase contribute to the downregulation of CD1d.
In order to screen out the strongest candidate, all Nedd4 E3 ligase family members were co-
transfected into 293T cells overexpressing CD1d (293T.CD1d cells), together with pTracer
plasmids expressing GFP fluorescence signal. pTracer was used as the negative control and
UL56 as the positive control. The candidates included hITCH, NedL1, NedL2, Nedd4, Nedd4L,
Smurf1, Smurf2, WWP1, and WWP2 (Fig. 1C). After 48 hours, cells were harvested and stained
for surface CD1d with PE fluorescence signal. Flow cytometry was then performed on the
stained samples, separating transfected and untransfected cells based on fluorescence intensity of
GFP, and measuring cell surface CD1d expression based on PE level. In this screening
experiment, Nedd4 and Nedd4L were reported to cause the most significant surface CD1d
downregulation in GFP-positive populations compared to GFP-negative populations (Fig. 1C
1D), which is consistent with Chen’s research (Chen, 2019). Therefore, Nedd4 and Nedd4L were
chosen to be the primary targets for further research.
16
The cellular distribution of Nedd4 was also verified through immunofluorescence imaging. HeLa
cells overexpressing CD1d (HeLa.CD1d cells) were cultured in 24 well plates and transfected
with Nedd4. After 48 hours, cells were harvested and stained for Nedd4, Lamp1, and CD1d, with
the fluorescent signal Alexa488/FITC, Alexa568/TRITC, and Alexa647/Cy-5 respectively. As
shown in Figure 1A, Nedd4 and CD1d molecules partially collocalize at ER and cellular
membrane, indicating the two proteins have the potential to interact with each other at these
locations.
As shown by the FACS assay, both Nedd4 and Nedd4L can significantly downregulate cell
surface CD1d in 293T.CD1d cells (Fig. 1D; Fig. 1E). We followed up with immunoprecipitation
and Western blot analysis in order to study the effect of the two E3 ligases on total cellular
CD1d. 48 hours after transfection, 293T.CD1d cells were harvested and lysed. A small portion of
whole cell lysate samples were saved for Western blot, while the others were followed up with
immunoprecipitation. After preclearance, WCL were immunoprecipitated with anti-CD1d (mAb
51.1.3). Notably, 51.1.3 antibodies were adopted here in order to precipitate CD1d in its mature
form, since 51.1.3 can only bind to folded form of CD1d due to its conformation-dependent
nature. On the other hand, D5 antibodies were used during Western blot in order to detect CD1d
after the samples were boiled in SDS. As shown in the result, both Nedd4 and Nedd4L
transfected cells showed decrease in 51.1.3-reactive form, indicating that Nedd4 and Nedd4L
downregulate not only cell surface CD1d, but also total amount of mature cellular CD1d in 293T
cells (Fig. 1F; Fig. 1G). Since the Western blot of WCL samples didn’t show significant decrease
when blotted with anti-D5, it is possible that the down-regulating effect of Nedd4 is limited to
17
mature forms of CD1d (Fig. 1G). This is consistent with our previous findings that HSV-1
interferes with the recycling of CD1d without disrupting its synthesis (Yuan et al, 2006). This
finding directed our research focus to the trafficking and recycling of post-ER form of CD1d.
2. Nedd4 ligase catalytic activity is required for CD1d downregulation
After confirming the effect of Nedd4 on downregulation of cellular CD1d, we sought to
determine whether Nedd4 ligase activity is required for CD1d downregulation. A mutation
experiment was performed by constructing a Nedd4 DD plasmid with a mutated catalytic
domain, depleting catalytic activities of the original Nedd4 molecule. In the same batch, Nedd4
and Nedd4 DD plasmids were transfected into different 293T.CD1d samples. pTracer-transfected
sample was used as negative control whose innate fluorescence signal was observed under
microscope to estimate transfection efficiency. KSHV K3 and K5 (also named MIR-1 and MIR-
2) were used as positive control in this experiment because of their ability to downregulate
CD1d (Sanchez, Gumperz, & Ganem, 2005). Cells were harvested 48 hours after transfection
and a small portion stained for FACS assay. Lysis and immunoprecipitation was performed on
the rest of samples, and the results analyzed by Western blotting.
As shown in Figure 2A, the results of FACS in Nedd4 transfected cells were able to replicate
previous results, with the transfected population expressing less CD1d molecules on cell surface.
However, in the Nedd4 DD transfected sample, FACS failed to detect any difference in surface
CD1d expression in transfected and untransfected populations (Fig. 2A; Fig. 2B). As we would
assume, both K3 and K5 transfected cells showed significant CD1d downregulation in
18
transfected populations, which is consistent with previous research (Sanchez, Gumperz, &
Ganem, 2005).
Similarly, Western blot analysis showed no significant downregulation of CD1d in IP samples of
Nedd4 DD compared with that of Nedd4 or K3 K5 positive controls (Fig. 2C). Notably, whole
cell lysate blot didn’t show significant difference among different samples since neither Nedd4
nor K3 K5 downregulate unfolded forms of CD1d molecules which makes up the majority of
intracellular CD1d. This adds to the reasons that we chose to perform immunoprecipitation with
anti-51.1.3 instead of direct blotting with anti-D5. To put together, since Nedd4 DD failed to
downregulate CD1d in both FACS and Western blot assays, we conclude that the Nedd4 ligase
catalytic activity is essential for CD1d downregulation in 293T cells.
3. Nedd4 cooperates with UL56 to downregulate surface CD1d.
Since UL56 and Nedd4 family E3 ligases were shown to interact with each other (Ushijima et
al., 2008; Ushijima et al., 2009), we hypothesized that UL56 and Nedd4 may cooperate in some
way to downregulate surface CD1d and therefore assist in the immune evasion of HSV. In order
to investigate the combined effect of UL56 and Nedd4, we conducted a co-expression
experiment and utilized Flow cytometry and Western blotting to analyze the result. In
293T.CD1d cells, flag-Nedd4/4L were co-transfected with UL56 with transient transfection.
Single expression groups of Nedd4, Nedd4L, and UL56 were also included, with the total DNA
amount balanced with empty plasmid DNA. pTracer was co-transfected in each group to provide
fluorescent signal, and pTracer single expression group was used as negative control. After 48
19
hours, a small portion of cells were used for FACS assay, and the rest of cells were lysed and
analyzed with Western blotting.
As shown in Figure 3A, flow cytometry results showed more significant CD1d downregulation
in Nedd4/UL56 co-transfected 293T.CD1d cells, in comparison with Nedd4 or UL56 transfected
alone. This result was verified by quantification of FACS results based on repeated
measurements of independent trials (Fig. 3B). This was also observed in Western blot results,
where total expression of mature CD1d in co-expression samples was decreased to a larger
extent compared with single expression counterparts (Fig. 3C) The explanation to this
observation may vary since no evidence has shown whether this effect of co-expression is
additive or synergic. However, taken the fact the both Nedd4 and UL56 exist in relatively small
amounts in human system, and that the transfection provides way more Nedd4 and UL56
plasmids than necessary to achieve CD1d downregulation, we tend to believe that Nedd4 and
UL56 interact and cooperate in the pathway to downregulate cell surface CD1d in order to
facilitate immune evasion of HSV.
4. Nedd4 does not downregulate CD1d through direct ubiquitination
After confirming the downregulation of CD1d by Nedd4, we took one step further to investigate
the mechanism of CD1d downregulation. Early works of our lab have shown that HSV infection
was able to decrease surface CD1d expression by interfering with the recycling of CD1d and
trapping CD1d in lysosome-like structures(Yuan et al., 2006). Subsequently, it was found that
HSV-1 proteins gB and US3 downregulate surface CD1d through lysosomal degradation (Rao et
al., 2011). Taking the fact that Nedd4 participates in the ubiquitin cascade as a E3 ubiquitin
20
ligase, we hypothesized that Nedd4 may cause the ubiquitination of CD1d which lead to the
degradation of the molecule.
An ubiquitination experiment was performed to investigate the effect of Nedd4 on the
ubiquitination status of CD1d. In 293T.CD1d cells, HA-Ub was co-transfected with flag-Nedd4,
V5-K5, or HA-Nedd4 DD. The K5 co-expressed group was used as a positive control, since K5
was proven to directly ubiquitinate CD1d (Sanchez, Gumperz, & Ganem, 2005; Coscoy,
Sanchez, & Ganem, 2001). Nedd4 DD with defective catalytic activity was used as negative
controls. In order to research the pathway of ubiquitination, proteasome inhibitor MG132 and
lysosomal inhibitor Chloroquine were added to Nedd4 and Nedd4DD transfected groups 36
hours after transfection. Cells were harvested 12 hours after inhibitor treatment, and the
ubiquitination status of CD1d was analyzed by immunoprecipitation and Western blotting.
Consistent with our previous findings, CD1d D5 expression in 51.1.3 immunoprecipitated
samples was downregulated in Nedd4 transfected groups compared to pTracer or Nedd4DD
transfected groups (Fig. 4A). However, the treatment with MG132 and CQ both failed to rescue
the expression of mature CD1d. In addition, although the total ubiquitination level in whole cell
lysates increased with Nedd4 transfection, the 51.1.3 immunoprecipitated samples failed to show
similar trends. In 51.1.3 IP blotted with HA-HRP, the three Nedd4 transfected groups showed
about the same level of ubiquitination as Nedd4 DD groups, which is in contrast with K5
positive control who displayed significant ubiquitination on CD1d (Fig. 4A). This result
indicated that Nedd4 catalytic activity does not directly contribute to the ubiquitination of CD1d.
21
In order to confirm our result, we generated two 293T cell lines stably expressing mutated forms
of CD1d. 293T.CD1d.KR was constructed to overexpress CD1d with single point mutation from
Lysine to Arginine of the only Lysine residue on the cytoplasmic tail of CD1d;
293T.CD1d.TD10 was constructed by deleting 10 amino acids on the cytoplasmic tail of CD1d.
In both cell lines, CD1d was mutated to prevent it from being ubiquitinated. Together with
293T.CD1d.WT, three cell lines were transfected in parallel with Nedd4/4L and UL56 co-
expressed (Fig. 4B). UL56 single expression groups were utilized as a quantitative reference. K5
and pTracer were used as positive and negative controls respectively. Cells were harvested 48
hours after transfection and analyzed by flow cytometry. As shown in Figure 4C, surface CD1d
expression in both KR and TD10 cell lines were rescued in K5 transfected samples, because the
ubiquitination of CD1d by K5 is inhibited in these cell lines. However, persistent CD1d
downregulation was observed in Nedd4/4L and UL56 co-transfected samples in KR mutant cell
line and TD10 cell lines, which was about the same level of decrease as in the WT cells (Fig. 4C;
Fig. 4D). This indicates that the downregulation of CD1d by Nedd4 and UL56 is not achieved by
ubiquitination of the molecule itself. Although surface CD1d can be ubiquitinated and degraded
by K5 in 293T cells, its regulation by Nedd4 and UL56 seems to follow a different path which
remains to be discovered.
22
Chapter Four: Discussion
The CD1d-mediated-iNKT cell antigen presenting system plays essential roles in various
antiviral immune responses. Herpes Simplex Virus (HSV) has been shown to evade immune
recognition by downregulating surface CD1d expression in antigen presenting cells by
interfering with CD1d recycling (Yuan, Dasgupta & Cresswell, 2006). This study identified
Nedd4 as a contributor to the inhibition of surface CD1d expression, and took a step further to
show that a Nedd4-UL56 cooperation system may be in place for HSV evasion of iNKT cell
recognition. However, a few points remain to be discussed: 1) What is the model of cooperation
between Nedd4 and UL56; 2)How surface CD1d is downregulated by Nedd4 and UL56 if not
through direct ubiquitination.
This study provided evidence that Nedd4 E3 ligase cooperates with HSV tegument protein UL56
to downregulate surface CD1d in 293 cells. However, the exact model of collaboration remains
to be discussed. Taken the fact that UL56 performs in small amount during viral infection, and
that both Nedd4 and UL56 are overexpressed in transient transfected cells, we tend to
hypothesize that the two molecules can achieve synergic effect on the downregulation of CD1d.
Further research can be done to investigate this issue. For example, a titration experiment could
be done with different concentrations of UL56 to determine the minimum amount of UL56
needed for CD1d downregulation. Knock out experiments could also be performed to study
whether Nedd4 is necessary for the inhibition of CD1d expression. These future experiments
may provide more insights into the mechanism underlying CD1d downregulation, and which role
Nedd4 and UL56 play respectively in this pathway.
23
In addition, this experiment was conducted predominantly with 293T cells overexpressing CD1d,
which was proven to be reliable and efficient in studies of iNKT cells. However, results from
mice in-vivo virus infection situations may exhibit higher levels of replicability in the human
system, as it mimics the iNKT antiviral immune response to a larger extent. A recent study from
our lab reported that higher resistance to HSV-1 ocular infection was observed in WT mice
compared with CD1d knock-out mice, indicating that CD1d is specifically targeted by HSV for
immune evasion purposes (Rao et al., 2018).
Previous work by my colleague showed that UL56 can cause surface CD1d downregulation, and
UL56 interacts with Nedd4 family E3 ligase (especially Nedd4 and Nedd4L) (Chen, 2019).
Taken the fact that CD1d can be ubiquitinated by HSV-1 proteins gB and US3 through
lysosomal degradation ((Rao et al., 2011), it was reasonable for us to hypothesize that Nedd4
ubiquitinates surface CD1d, tagging it for recognition of lysosomal degradation. However,
contradictory to our hypothesis, Western blot results from our ubiquitination experiment shows
no significant ubiquitination on CD1d molecule, though the ubiquitination level in whole cell
lysates increased (Fig. 4A). Considering the role of Nedd4 as a E3 ubiquitin ligase, the elevated
levels of ubiquitination in whole cell lysate samples are more likely b explained by the board
ubiquitination effect of Nedd4 on various cellular proteins. This finding was then confirmed by
mutation experiments, as both 293T.CD1d.KR and 293T.CD1d.TD10 cell lines exhibited
persistent CD1d downregulation in Nedd4/UL56 co-expressed samples. These findings suggest
that the CD1d downregulation in this case is not dependent on its ubiquitination status, with the
exact mechanism of its downregulation remains to be determined. It is possible that Nedd4 and
UL56 interrupts the normal reuptake of CD1d by trapping CD1d in intravesicular structures after
24
endocytosis. Another direction to look for would be ubiquitin-independent degradation systems
in which Nedd4 may direct the ubiquitination of some other proteins that interact with CD1d in
this pathway. Unraveling of this mystery will provide more insights into the immune evasion of
HSV, which is of great interest to the prevention and treatment of this prevalent virus.
25
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31
Figures
A.
B.
C.
32
D.
E.
F. G.
Figure 1. Nedd4 and Nedd4L E3 ligase downregulate mature CD1d expression. (A).
HeLA.CD1d cells transfected with Nedd4, stained for Nedd4, CD1d and Lamp1, and imaged
with immunofluorescence microscope. Green color denotes the localization of Nedd4, red color
denotes CD1d, and purple color denotes Lamp1. Yellow color represents areas where Nedd4 and
33
CD1d collocalize. (B). Fluorescent images show an estimate of transfection efficiency. (C)
FACS assay performed on 293T.CD1d cells transfected with Nedd4, Nedd4L, UL56, and
pTracer. Gated with GFP signal, the transfected population is represented in red, and the
untransfected population is represented in blue. X-axis is cellular surface expression of CD1d; Y-
axis is cell count normalized to mode. (D) Quantification of FACS data based on statistics from
4 independent trials. Y-axis represents the expression of surface CD1d of the transcripted
population divided by that of the untransfected population, with pTracer sample normalized to 1.
(E) Western blot result shows downregulation of mature CD1d in IP samples of both Nedd4 and
Nedd4L transfected 293T.CD1d cells. (F) Quantification of Western blot data of
immunoprecipitated samples by Image Lab software based on statistics from 5 independent
trials. Y-axis represents the relative quantity of CD1d expression with pTracer as reference.
34
A.
B.
C.
35
Figure 2. Catalytic activity of Nedd4 is essential for downregulation of CD1d. (A) Flow
cytometry shows no significant decrease in surface CD1d in Nedd4 DD transfected 293T.CD1d
cells compared with pTracer negative control. X-axis is cellular surface expression of CD1d; Y-
axis is cell count normalized to mode. (B) Quantification of FACS data based on 3 independent
trials. Y-axis represents the expression of surface CD1d of the transcripted population divided by
that of the untransfected population, with pTracer normalized to 1. (C) Western blot shows no
mature CD1d expression in Nedd4 DD transfected 293T.CD1d cells immunoprecipitated with
anti-51.1.3, compared with pTracer negative control.
A.
36
B.
C.
Figure 3. Nedd4 cooperates with UL56 to downregulate CD1d expression in 293T.CD1d cells.
(A) Flow cytometry results show more significant CD1d downregulation in Nedd4/UL56 co-
transfected 293T.CD1d cells, in comparison with Nedd4 or UL56 transfected alone. The
transfected population is represented in red, and the untransfected population is represented in
blue. X-axis is cellular surface expression of CD1d; Y-axis is cell count normalized to mode.
(B). Quantification of FACS data based on 3 independent trials. Y-axis represents the expression
of surface CD1d of the transcripted population divided by that of the untransfected population,
with pTracer normalized to 1. (C) Western blot result shows more significant decrease of mature
CD1d in Nedd4/UL56 co-transfected 293T.CD1d cells immunoprecipitated with anti-51.1.3, in
comparison with Nedd4 or UL56 transfected alone.
37
A.
B.
38
C.
D.
Figure 4. Nedd4 does not downregulate surface CD1d through direct ubiquitination. (A) Western
blot result of ubiquitination experiment shows no significant ubiquitination in 51.1.3
immuprecipted samples compared with K5 positive control. 293T.CD1d cells were treated with
50uM MG132 or 30uM CQ 36 hours after transfection and were harvested 12 hours after
inhibitor treatment. (B) Western blot results of mutant cell line experiment. Nedd4 and Nedd4L
were co-transfected into 293T.CD1d cells with UL56 plasmid. K5 and UL56 single expression
groups were used as reference, and pTracer group was used as negative control. (C) FACS
results of mutant cell line experiment shows persistent CD1d downregulation in Nedd4, Nedd4L
39
and UL56 transfected samples in KR mutant cell line and TD10 cell line.The transfected
population is represented in red, and the untransfected population is represented in blue. X-axis
is cellular surface expression of CD1d; Y-axis is cell count normalized to mode. (D).
Quantification of FACS results of mutant cell line experiment based on data from 4 independent
trials.
Abstract (if available)
Abstract
Invariant Natural Killer T (iNKT) cells are a subset of Natural Killer T (NKT) cells that are restricted by CD1d molecules. The CD1d-mediated-iNKT cell antigen presenting system plays an important role in the immune defense against various virus infections. Herpes Simplex Virus (HSV) has been shown to evade immune recognition by downregulating surface CD1d expression in antigen-presenting cells, with the underlying mechanism under-illustrated. Here we show that Nedd4, the prototype member of the Nedd4 family E3 ubiquitin ligase, contributes to the downregulation of surface CD1d in 293T cells. With its catalytic activity essential in this process, Nedd4 cooperates with HSV tegument protein UL56 to downregulate surface CD1d molecules, which assists in the immune evasion strategy of HSV. However, such inhibition of CD1d surface expression is not achieved by the direct ubiquitination of CD1d because the decrease of CD1d persists in cell lines with mutant CD1d that cannot be ubiquitinated, suggesting an indirect mechanism of CD1d downregulation.
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Asset Metadata
Creator
Shao, Xinyue
(author)
Core Title
Herpes Simplex virus-1 UL56 collaborates with Nedd4 E3 ubiquitin ligase to downregulate surface CD1d and facilitate immune evasion of NKT cell function
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Degree Conferral Date
2022-12
Publication Date
10/01/2022
Defense Date
07/22/2022
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E3 ubiquitin ligase,herpes simplex virus,natural killer T cells,OAI-PMH Harvest
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Yuan, Weiming (
committee chair
), Eoh, Hyungjin (
committee member
), Feng, Pinghui (
committee member
)
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3066982287@qq.com,xinyuesh@usc.edu
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Tags
E3 ubiquitin ligase
herpes simplex virus
natural killer T cells