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Comparative studies of coronavirus interaction with CD1d-restricted iNKT cells
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Comparative studies of coronavirus interaction with CD1d-restricted iNKT cells
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Content
Comparative studies of coronavirus interaction with CD1d-restricted iNKT cells
by
Ruiting Zhou
A Thesis Presented to the
FACULTY OF THE USC KECK SCHOOL OF MEDICINE
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
Molecular Microbiology and Immunology
August 2021
Copyright 2021 Ruiting Zhou
ii
ACKNOWLEDGEMENTS
First, I would like to express the deepest appreciation to my supervisor, Dr. Weiming
Yuan. He has trained me to be a qualified master’s student and gave me encouragements
throughout the selection of project orientation, conducting a series of experiments,
sorting and analyzing data, and improving the organization and argumentation of my
thesis. Without his tremendous understanding and encouragement in the past two years, it
would be impossible for me to complete my study.
And I would also like to express my special thanks to my committee members, Dr.
Pinghui Feng and Dr. Stanley Tahara for their assistance at every stage of the research
project.
Lastly, I would like to thank my colleagues, Yingting Zhang, Siyang Chen, Zhewei Liu,
Rongqi Zhao and Hongjia Lu for their assistance and help, and also our master graduate
program and the Department of Molecular Microbiology and Immunology staff,
especially Dr. Axel Schönthal for their support.
iii
TABLE OF CONTENTS
ACKNOWLEDGEMENTS ............................................................................................................................. ii
LIST OF TABLES AND FIGURES .............................................................................................................. iv
ABBREVIATIONS ......................................................................................................................................... v
ABSTRACT ................................................................................................................................................... vi
CHAPTER 1: INTRODUCTION .................................................................................................................... 1
1.1 Current situation of COVID-19 .................................................................................................... 1
1.2 Classifications and symptoms of COVID-19 ............................................................................... 1
1.3 Structure of SARS-CoV-2 (coronaviruses) .................................................................................. 2
1.4 Life cycle of coronaviruses ........................................................................................................... 6
1.5 Human immune systems and components .................................................................................... 7
1.6 Natural Killer T cells and CD1d molecules .................................................................................. 8
CHAPTER 2: MATERIALS AND METHODS ............................................................................................. 9
2.1 Cell lines ....................................................................................................................................... 9
2.2 Plasmids and primer sequences .................................................................................................. 10
2.3 Antibodies ................................................................................................................................... 11
2.4 Transient transfection .................................................................................................................. 11
2.5 Western Blot ............................................................................................................................... 12
2.6 Flow cytometry ........................................................................................................................... 12
2.7 T cell stimulation Assay and ELISA Assay ................................................................................ 13
2.8 Immunofluorescence ................................................................................................................... 15
2.9 Plasmid construction ................................................................................................................... 16
CHAPTER 3 RESULTS ................................................................................................................................ 17
3.1 Potential modulation of CD1d expression level by envelope proteins of SARS-CoV and SARS-
CoV-2 ........................................................................................................................................................ 17
3.2 Strep-tagged SARS-CoV and SARS-CoV-2 E proteins downregulated CD1d-restricted iNKT
functions .................................................................................................................................................... 19
3.3 HCoV nucleocapsid proteins did not affect mature CD1d expression level, and Strep-tagged
SARS-CoV and SARS-CoV-2 N proteins activated CD1d-restricted iNKT functions ........................... 21
3.4 Mechanisms of SARS-CoV-2 N protein in activating CD1d-restricted iNKT cells functions .. 23
CHAPTER 4: DISCUSSION ........................................................................................................................ 25
REFERENCES: ............................................................................................................................................. 28
FIGURES ....................................................................................................................................................... 36
iv
LIST OF TABLES AND FIGURES
Table 1. The Plasmids and Primers used in the project. 10
Table 2. All the first and second antibodies used in the project. 11
Figure 1. Potential CD1d expression level modulation by E proteins of SARS-CoV and
SARS-CoV-2. 36
Figure 2. Strep-tagged SARS-CoV E and SARS-CoV-2 E protein downregulated CD1d-
restricted iNKT functions. 37
Figure 3. HCoV nucleocapsid proteins do not affect mature CD1d expression level, and
Strep-tagged SARS-CoV N and SARS-CoV-2 N can activate the CD1d-restricted iNKT
cells. 38
Figure 4. Mechanisms of SARS-CoV-2 N protein in activating CD1d-restricted iNKT
cells functions. 40
v
ABBREVIATIONS
AA Amino Acid
ACE2 angiotensin converting enzyme 2
APC Antigen-Presenting Cell
BSA Bovine Serum Albumin
CD Cluster of Differentiation
CD1d Cluster of Differentiation 1 (Class D)
CoV coronavirus
COVID-19 Coronavirus disease 2019
DMEM Dulbecco's Modified Eagle's Medium
DPBS Dulbecco′s Phosphate Buffered Saline
ER endoplasmic reticulum
ERGIC ER-Golgi intermediate compartment
FACS Fluorescence-Activated Cell Sorting
FBS Fetal Bovine Serum
HCoV Human coronavirus
hnRNP1
Heterogeneous nuclear ribonucleoprotein
A1
IL-2 Interleukin-2
IMEM Improved Minimum Essential Medium
MERS Middle East Respiratory Syndrome
NKT Natural Killer T cells
NSP Nonstructural Protein
iNKT Invariant Natural Killer T
ORF Open Reading Frame
PEI Polyethyleneimine
PVDF Polyvinylidene Fluoride
RBD Receptor Binding Domain
RNA ribonucleic acid
RTC Replicase-Transcriptase Complex
SARS Severe Acute Respiratory Syndrome
T cell Thymus Cell
TCR T-Cell Receptor
TMPRSS2
transmembrane protease/serine subfamily
member 2
α-GalCer Alpha-Galactosylceramide
β2m Β 2-microglobulin
vi
ABSTRACT
The outbreak of COVID-19 has become a global pandemic. Despite enormous
efforts being made, the pathogenetic mechanism of SARS-CoV-2 remains largely
unknown, including its interaction with the innate immunity system. Envelope (E) protein
and nucleocapsid (N) protein are the main structural proteins of coronaviruses that play
indispensable roles in the pathogenesis and life cycle of coronaviruses. The CD1d
molecule is expressed on the surface of many human antigen-presenting cells (APCs).
CD1d molecule presents lipid antigen to iNKT cells. This study mainly focused on the
interaction between E protein and N protein of HCoVs and the CD1d-restricted iNKT
cells. The first part was to investigate whether both SARS-CoV and SARS-CoV-2 E
proteins can downregulate the mature CD1d expression level and affect the function of
CD1d-restricted iNKT cells. The second was to examine whether N proteins of different
HCoVs can stimulate the functions of CD1d-restricted iNKT cells and explore the
mechanisms therein. We generated the plasmid constructs expressing E and N proteins
from different coronaviruses and expressed the proteins in 293T.CD1d cells then
analyzed the CD1d expression and iNKT cell stimulation by flow cytometry and ELISA,
respectively. Our results showed Strep-tagged SARS-CoV and SARS-CoV-2 E proteins
similarly downregulated the mature CD1d expression level and blocked functions of
iNKT cells. In addition, Strep-tagged SARS-CoV N and SARS-CoV-2 N proteins can
stimulate the functions of iNKT cells and the C-terminal domain of SARS-CoV-2 N was
required for this stimulation. Our results suggested that SARS coronaviruses have
specifically evolved to precisely modulate the function of CD1d-restricted iNKT cells.
1
CHAPTER 1: INTRODUCTION
1.1 Current situation of COVID-19
The outbreak of the coronaviruses disease 2019 (COVID-19) began in December
2019 and was caused by the respiratory viral pathogen severe acute respiratory syndrome
coronavirus 2 (SARS-CoV-2). The first known case was identified in Wuhan, China in
late December 2019 (Hu B et al., 2020). Later, the World Health Organization (WHO)
announced the standard definition of the disease syndrome of COVID-19 and its
nomenclature on February 11, 2020. By May 2021, the virus has caused over 150 million
cases and more than 3 million people died globally (Jee Y et al., 2020). Although the
symptoms of COVID-19 vary by individual, it is common that many cases include cough,
fever, fatigue, breathing difficulties, and loss of smell and taste (Saniasiaya J, Islam MA
et al., 2021; Saniasiaya J, Islam MA et al., 2020). Sepsis is the most frequently observed
complication, followed by respiratory failure, acute respiratory distress syndrome
(ARDS), heart failure, and septic shock (Fei Zhou et al., 2020).
1.2 Classifications and symptoms of COVID-19
Coronaviruses are a group of enveloped viruses with non-segmented, single-
stranded, and positive-sense RNA genomes (V'kovski P, Kratzel A et al., 2020). The
coronaviruses can be classified into four subfamilies, α, β, γ, and δ-CoVs. The α-
2
coronaviruses and β-coronaviruses infect only mammals. The γ-coronaviruses and δ-
coronaviruses infect birds, but some of them can also infect mammals (Woo, P. C. et al.,
2012). According to the initial sequencing results, SARS-CoV-2 belongs to the family of
β-coronaviruses (Zhou, P et al., 2020). Human coronaviruses (HCoV) infection causes
respiratory diseases with mild to severe outcomes (Fung TS et al., 2019). Scientists have
identified two strains of high-pathogenic human coronaviruses, the severe acute
respiratory syndrome coronaviruses (SARS-CoV and SARS-CoV-2) and Middle East
respiratory syndrome coronavirus (MERS-CoV) (Fung TS et al., 2019). These HCoVs
usually are responsible for severe and fatal respiratory syndrome diseases, whereas the
other four human coronaviruses, including HCoV-OC43, HCoV-NL63, HCoV-229E and
HCoV-HKU1 more often induce mild upper respiratory diseases, such as the common
cold. According to the database, all human coronaviruses can be traced to their natural
hosts. SARS-CoV, SARS-CoV-2, MERS-CoV, HCoV-NL63 and HCoV-229E originated
from bats; HCoV-OC43 and HKU1 may have come from rodents (Corman VM et al.,
2018). Furthermore, the coronaviruses can also be carried by intermediate hosts such that
they may be transmitted to humans. For example, the intermediate host of SARS-CoV is
Masked palm civet (Paguma larvata) and camels are the intermediate host of MERS
(Nadeem MS et al., 2020).
1.3 Structure of SARS-CoV-2 (coronaviruses)
Based on previous studies, the virion of CoVs consists of genome RNA, four
structural proteins and sixteen nonstructural proteins (nsp1-16) and other accessory
3
proteins, such as HE protein (Chen Y et al., 2020). The genome RNA is associated with
phosphorylated nucleocapsid (N) proteins and is inside of the virion envelope. The other
three structural proteins, envelope (E) protein, membrane (M) protein and spike (S)
glycoprotein, are anchored at the surface of viral envelope.
Spike glycoprotein
The spike glycoproteins play an indispensable role in entry of the coronaviruses
into host cells by recognition of host cell receptor called angiotensin-converting enzyme
2 (ACE2) (Li, W., Moore et al., 2003). The spike glycoproteins are comprised of two
subunits, the S1 subunits that function to associate with ACE2 and S2 subunits that are
responsible for fusion of the viral and host cell membranes (Kirchdoerfer, R., Cottrell, C.,
Wang, N. et al., 2016; Glowacka I et al., 2011). Before entering the host cell membrane,
the S proteins are cleaved by a protease from host cells called transmembrane
protease/serine subfamily member 2 (TMPRSS2) that is primarily expressed in the
respiratory tract. This process called TMPRSS2-mediated cleavage, enables S proteins to
bind with ACE2 and begin the process of vial entry into the host cells (Sternberg A et al.,
2020). Interestingly, the S proteins of SARS-CoV-2 possess a distinctive structure named
S1/S2 furin cleavage site, but its function remains unclear (Xia, S. et al., 2020).
4
Membrane protein
Membrane (M) protein (25-30 kDa) is the most abundant structural protein in
coronaviruses (Alharbi SN et al., 2020). More importantly, it has been considered that M
protein plays a central role in interaction with other structural proteins.
Envelope protein
Envelope (E) protein (8-12 kDa) is a small integral membrane protein, and it is
present in very small amounts in the virion. E protein greatly affects the major life cycle
steps of coronaviruses, including virion entry, virus assembly, budding, viral envelope
formation, virus release, and pathogenesis. (Schoeman D et al., 2019). E protein can be
divided into three main parts, the N-terminal domain, the transmembrane domain, and the
C-terminal domain (Malik Ya et al., 2020). Importantly, E proteins are vital to viral
pathogenesis. It has been recently found that SARS-CoV E protein contains a binding
motif called PDZ-binding motif (PBM) that is in the last four amino acids of the C
terminus (Lo Cascio E et al., 2021). The PDZ domain is commonly found in signaling
proteins, and it binds to a short region of the C-terminus of other specific proteins. It has
been suggested that E proteins of some coronaviruses, including SARS-CoV, SARS-
CoV-2, act as a cation selective channel that influences infectivity and virulence (K.
Pervushin et al., 2009). Studies have shown that when the ion channel function of SARS
E proteins are blocked, the viruses will largely lose the ability of infection (Singh Tomar
PP et al., 2020).
5
Nucleocapsid protein
The nucleocapsid (N) protein is encoded by the 9
th
ORF of SARS-CoV, and the
molecular weight is about 46 kDa (Surjit M et al., 2007). N protein consists of two
independent domains, the N-terminus and C-terminus, and a linker domain. The N-
terminal domain mainly binds to RNA because it contains most of the positively charged
amino acids (Surjit M et al., 2007). On the other hand, the C-terminus domain is
responsible for N protein oligomerization. Additionally the linker domain serves as a
connection for membrane (M) protein and human cellular hnRNPA1 protein (Fang, X. et
al., 2006); N also has a SR-rich motif present in the middle part of N protein. One
potential application related to N protein is that the N protein can be used to produce
vaccine against COVID-19 for two major reasons (Dutta NK et al., 2020). The first is that
N protein is one of the major antigens of SARS-CoV-2. The other reason is that the gene
variation among N proteins is low, which is an important prerequisite for a potential
vaccine. Therefore, after S protein that has been utilized for vaccine production, N
protein may be the next candidate for a new vaccine application.
6
1.4 Life cycle of coronaviruses
The life cycle of coronaviruses can be broken down into several steps: attachment
to the host cell, entering the host cell and uncoating, viral RNA genome replication and
viral gene expression, formation of virion particles and budding into the trans Golgi for
virion release (Trougakos IP et al., 2021).
First, coronaviruses infect host cells by binding to appropriate receptors on the
membrane of host cells. The S1 subunit binds to the receptors and the S2 subunit is
designed for fusion of viruses and cell membranes. There is a variable part of S1 called
the receptor binding domain (RBD), and it can bind to different cell surface receptors due
to its high flexibility (Li F et al., 2005). For example, the RBDs of SARS-CoV and
SARS-CoV-2 both can bind to ACE2 protein. After the attachment, the S protein will
undergo a conformational change that helps the viral genome enter the cytoplasm.
After entry the viral genome serves as a messenger RNA that undergoes
translation by host ribosomes, and it is translated into two large polyproteins, pp1a and
pp1ab. After that process, there are a series of proteases to cleave the polyproteins. The
pp1ab yields sixteen nonstructural proteins (nsp1-nsp16), and many NSPs will combine
to form a replicase-transcriptase complex (RTC) (Harrison AG et al., 2002). The
formation of RTC is considered as the starting point of viral RNA synthesis (Hartenian E
et al., 2020). Then the RTC can directly bind to the viral genome and initiate viral
genomic RNA replication, transcription and translation that will generate four structural
7
proteins and other accessory proteins. It should be emphasized that the structural proteins
(except N protein) and accessory proteins that interact with membrane are synthesized in
the endoplasmic reticulum (ER), whereas other proteins are translated by ribosomes that
are in the cytoplasm. Furthermore, most structural proteins will go through
posttranslational modification. Then, the structural proteins and accessory proteins are
inserted into the ER-Golgi intermediate compartment (ERGIC) (Venkatagopalan P et al.,
2015).
1.5 Human immune systems and components
The human immune system inside our body can be divided into two main
categories, innate immune system, and adaptive immune system. Firstly, the innate
immune system usually provides our body with instant protection with nonspecific
defenses. For instance, many pathogens can be detected by pattern recognition receptors
expressed by host cells. (Sonnenberg GF et al., 2019). Besides, there are a variety of
immune cells, including neutrophils, macrophages, and natural killer cells, belong to the
innate immune system. On the other hand, the adaptive immune system requires
recognition of non-self-antigens that needs an antigen presentation process by some
antigen presenting cells and is tailored to specific immune responses (Bonilla FA et al.,
2019). Usually, T cells and B cells take indispensable roles in the antigen-specific
immune responses. However, the two immune systems are not independent of each other.
Instead, the two systems complement each other. It is commonly believed that T cells
serves as a bridge between two systems (Rabb H et al., 2002).
8
1.6 Natural Killer T cells and CD1d molecules
Natural killer T (NKT) cells are a subpopulation of T cells that exist in both
humans and mice and link the innate immune system and adaptive immune system. NKT
cells share the properties of both NK cells and T cells because NKT cells have T cell
receptors (TCR) and NK cell receptors, and the cells are CD1d-restricted T cells. Among
all families of NKT cells, the best-known subset is the invariant NKT (iNKT) cells or
Type 1 NKT cells in that they express invariant T cell receptor α chain (Godfrey, DI et al.,
2004). CD1d is a non-polymorphic major histocompatibility complex class I-like antigen
presenting molecule (Brennan, Patrick J. et al., 2013). NKT cells utilize CD1d molecules
to recognize lipid antigens from variable pathogens (Kriegsmann M et al., 2018).
9
CHAPTER 2: MATERIALS AND METHODS
2.1 Cell lines
293T cell lines and HeLa.CD1dcell lines were provided by Professor Yuan. 293T
cell lines and HeLa.CD1d cell lines were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) (Corning Cellgro) with 4.5 g/L glucose, L-glutamine, sodium pyruvate, 5%
Fetal Bovine Serum (FBS) (HyClone) and 1% Penicillin/Streptomycin antibiotics (100X,
cell culture core, Pen 5000 u/ml and Strep 5000 μg/ml). Cells were incubated at 37°C
with 5% CO2.
Seven different T cell hybridomas (CD1d-restricted iNKT cells) were provided by
Professor Yuan, and they were cultured in 1X RPMI-1640 Medium with L-glutamine
(Cellgrow #10-040-cv), 10% Fetal Bovine Serum (FBS) (HyClone), 5% essential amino
acids (GIBCO#11130-051, 50X MEM amino acids solution), 5% nonessential amino
acids (GIBCO#11140-050, 100X, 10 mM), 55 μM β-mercaptoethanol (Sigma#M3148,
14.3M) and 1% Penicillin/Streptomycin antibiotics (100X, cell culture core, Pen 5000
u/ml and Strep 5000 μg/ml). T cell Hybridomas were incubated at 37°C with 5% CO2.
10
2.2 Plasmids and primer sequences
Plasmid Protein Tag Comments
pTracer N/A GFP protein
HCoV-OC43 N Myc tag Addgene#151960
HCoV-NL63 N Myc tag Addgene#151939
SARS-CoV N Flag tag From Howard University
SARS-CoV N Strep tag Sequence from Genewiz
®
SARS-CoV-2 N Strep tag
From University of California-
San Francisco
SARS-CoV-2 N NTD-
deletion
Strep tag Synthesized from Dr. Yuan
SARS-CoV-2 N CTD-deletion Strep tag Synthesized from Dr. Yuan
SARS-CoV E Flag tag From Howard University
SARS-CoV E Strep tag Sequence from Genewiz
®
SARS-CoV-2 E Strep tag
From University of California-
San Francisco
Primer Name Primer Sequence
HCoV-OC43-N-NotI-N CCAGCGGCCGCCACCATGTCCTTCACC
HCoV-OC43-N-KpnI-C GCTGGTACCGAGATCTCCGAAGTGTCCTCGG
HCoV-NL63-N-NotI-N CCAGCGGCCGCCACCATGGCATCAGTG
HCoV-NL63-N-KpnI-C GCTGGTACCGAGTGAAGGACCTCATTCACAA
N-SARS-CoV-2 N-
NTD-Del-N-up
CTCTAAACAGAGGCGACCACAGGGATCACGCGGCG
GCAGCCAAG
N-SARS-CoV-2 N-
NTD-Del-C-down
CTTGGCTGCCGCCGCGTGATCCCTGTGGTCGCCTCT
GTTTAGAG
N-SARS-CoV-2 N-
CTD-Del-N-up
CAAGAAAAGCGCTGCAGAAGCTCCCACCGAACCCA
AGAAGGACA
N-SARS-CoV-2 N-
CTD-Del-C-low
TGTCCTTCTTGGGTTCGGTGGGAGCTTCTGCAGCGC
TTTTCTTG
C-AfeI-for SARS-CoV-
2 N-NTD-D
CGTTTTTGTCGAGGTTTTTTAGAAGC
C-BamHI-SARS-CoV-2
N
AGGGGCGGGATCCTTACTTTTCAAACTGC
N-EcoRI-SARS-CoV-2
N
GAGGATCTATTTCCGGTGAATTCG
11
Table 1. The Plasmids and Primers used in the project. All the protein tags were located
at the C-terminus of the target proteins.
2.3 Antibodies
Antibody Name species
1
st
antibody ⍺-Strep mouse
1
st
antibody ⍺-Myc mouse
1
st
antibody ⍺-SARS-CoV-2 N rabbit
1
st
antibody ⍺-CD1d D5 mouse
1
st
antibody ⍺-CD1d 51.1.3 mouse
1
st
antibody ⍺-GRP94 Goat
1
st
antibody ⍺-Albumin Goat
2
nd
antibody ⍺-mouse-HRP Goat
2
nd
antibody ⍺-rabbit-HRP Goat
2
nd
antibody ⍺-rat-HRP Goat
Table 2. All the first and second antibodies used in the project.
2.4 Transient transfection
293T.CD1d cells were cultured in 10 cm culture dishes until the confluency was
between 80% to 90%. For Western Blot and FACS assay, pTracer with HCoV-OC43-N,
HCoV-NL63-N, SARS-CoV-N, SARS-CoV-2-N, MERS-N, SARS-CoV-2-N-NTD-
deletion, SARS-CoV-2-N-CTD-deltion, SARS-CoV-E, SARS-CoV-2-E plasmids with 1
mg/ml Polyethyleneimine (PEI) were added to DMEM Medium and the solutions then
were transferred to each cell dish. Transfected cells were collected after 48 h transfection.
12
2.5 Western Blot
Twenty microliters of samples were loaded on the 10%/15% SDS-PAGE gels.
Protein molecular weight marker (Bio-Rad) was loaded for accurate molecular weight
estimation of SDS-PAGE. Gels were run at 80 V through the stacking gel and switched to
120 V through the separating gels for 2 hours. Proteins were transferred from gels to
polyvinylidene fluoride (PVDF) membranes pre-activated in absolute methanol before.
Electrophoretic transfer was performed at constant current at 0.12 A for 1.5 hours. The
membranes were washed in 1X TBST solution for 10 min. Next, the membrane was
incubated with primary antibody overnight at 4°C. The membrane was washed 3 times
in 1X TBST wash butter and blotted with secondary antibody for at least 1 hour at room
temperature. The membrane was washed 3 times. Antigen bands were visualized with
Bio-rad Imaging developer to develop chemi-illuminescence.
2.6 Flow cytometry
Transfected cells were collected into Eppendorf tubes and spun down at 5000 rpm
for 1 minute. The medium was discarded and replaced with 1X DPBS to wash cells.
Cells were collected at 2400 rpm for 2 min. The supernatant was discarded, and cells
13
were resuspended in 1ml 1X DPBS/0.05% BSA, added to each tube Primary antibody
was diluted into 1X DPBS/0.05% BSA solution and 100 μl primary antibody solution
was added to each tube and incubated on ice for 40 min. Cells were washed three times
with 1X DPBS/0.05% BSA solution. Secondary FACS antibody was diluted 1:5000 into
1X DPBS/0.05% BSA solution and 100 μl was added to each tube, incubated on ice
covered by aluminum foil to avoid light for 30 min. Cells were washed 3 times, and then
100 μl or 3.7% formaldehyde/DPBS solution was added to fix cells for 10 min. Then, 300
μl 1X DPBS was also added to each tube. Stained cells were transferred to FACS sample
tube and stored at 4°C in the dark.
2.7 T cell stimulation Assay and ELISA Assay
Cultured 293T.CD1d cells were transfected with desired plasmids for 48 hours. In
the meantime, T cell hybridomas were cultivated in 6-well plates. After 48 hours, diluted
α-GalCer (220 μg/ml in saline Tween, from P. Askanese) was added to DMEM Medium
with 5% FBS. The final concentration of α-GalCer was 100 pg/ml. The medium was
sonicated for 10 min at room temperature using a bench-top waterbath sonicator. The
medium in 293T.CD1d dishes was replaced with α-GalCer medium and incubated at
37°C, in 5% CO2 for 2 hours. The 293T.CD1d cells were washed three times (by
centrifugation) with T cell hybridoma medium. Cells were carefully counted using a
hemocytometer and diluted to 2 X 10
5
cells/ml (2 X 10
4
/100 μl) in T cell hybridoma
medium. T cells were counted and plated at 100 μl T cells/well (10
6
/ml). 100μl of
14
293T.CD1d cells were added to the T cell containing wells and incubated at 37°C, 5%
CO2 for 48 hours.
After 48 hours of incubation of T cell assay, plates were sealed and stored at -20°C.
Corning Costar 9018 ELISA plate was coated with 100 μl/well of capture antibody in
coating buffer. The plates were sealed and incubated overnight at 4°C. Supernatants were
aspirated and washed 5 times with wash buffer (1X PBS with 0.05% Tween-20). Time
was allowed for soaking during each wash step to increase the effectiveness of the
washes. Assay diluent was diluted from 5X stock (1 part) with deionized water (4 parts).
Wells were blocked with 200 μl/well of 1X Assay Diluent and incubated at room
temperature for 1 hour. Afterwards wells were washed 5 times. Standards were diluted in
1 X Assay Diluent and 100 μl/well were added to the appropriate wells. Two-fold serial
dilutions of the top standards were prepared to make the standard curve for quantitation.
Cell sample supernatants of 100 μl were added to the appropriate wells. Plates were
sealed and incubated at room temperature overnight at 4°C. Supernatants were removed
and wells were washed 5 times in 1X assay diluent followed by addition of 100 μl/well of
detection antibody diluted in 1 X Assay Diluent. Plates were incubated at room
temperature for 1 hour. The supernatant was aspirated, and wells were washed 5 times in
1X assay diluent followed by addition of 100 μl/well of Avidin-HRP (Catalog# N200)
diluted in 1X Assay Diluent. Plates were incubated at room temperature for 30 min.
Unreacted reagent was removed by aspiration and wells were washed 7 times with wash
buffer. Antigen was visualized by addition of 100 μl/well of substrate solution and
15
incubated at room temperature for 5 minutes. Reactions were stopped by adding 50 μl of
stop solution to each well. Plates were read by absorbance at 450 nm.
2.8 Immunofluorescence
Place microscope coverslips to each well of 24-well plates. HeLa.CD1d cells
(20000/well) were added in in DMEM Medium with 5% FBS, 1%
Penicillin/Streptomycin antibiotics. Cells were incubated for 24 hours to allow cells to
attach and spread. Cells were transfected as described earlier and incubated at 37°C, 5%
CO2 for 48 hours. Afterwards the media was aspirated and quickly 400 μl of fixative
solution (90% IMEM Medium+10% 37% formaldehyde) was added and incubated at
room temperature for 20 min. Coverslips were washed twice with 400 μl DMEM media.
Cells were permeabilized by addition of 400 μl permeabilization solution (PS) per well
and incubated further for 15 min at room temperature. Diluted primary antibody in PS
was placed on parafilm (20 μl drop), which is attached to the bottom of a plastic square
box above a film of water. Coverslips (cell side down) were placed onto the antibody
solution. Moist towels were placed around the parafilm in the container to create a moist
chamber and prevent the antibody from drying. Coverslips were incubated at room
temperature for 30 min followed by washing 3 times, 5 min each with 400 μl PS.
Secondary antibody was added similarly and incubated with coverslips. Care was taken
to protect coverslips from light during incubation by covering the plastic container with
aluminum foil. Finally, coverslips were wash 3 times as before and one time with pure
16
water. The coverslips were mounted on microscope slides. Coverslips were placed cell
side down onto 5 μl Mowiol on each glass microscope slide. The glass microscope slides
were stored in the dark at room temperature for 20 min and then kept at 4°C before
microscopy.
2.9 Plasmid construction
The target gene sequence was synthesized from gene synthesis company (Genewiz,
Inc.). Then, the specific DNA sequence was digested with restrictive enzymes and the
digested DNA sequence (insert DNA fragment) ligated by T4 DNA ligase to the vector
plasmid that was also digested with the same restriction enzymes.
17
CHAPTER 3 RESULTS
3.1 Potential modulation of CD1d expression level by envelope proteins of SARS-
CoV and SARS-CoV-2
The envelope (E) protein is a small intact membrane protein that is involved in many
biological activities, including virion assembly, budding, and pathogenesis (Schoeman D
et al., 2019). According to the phylogenetic analysis, SARS-CoV and SARS-CoV-2 both
belong to β-coronaviridae. After comparing the sequences of E proteins of SARS-CoV
and SARS-CoV-2, it is known to us that the two E proteins share very high similarities
with three amino acid differences in the C-terminal domain. Interestingly, E proteins can
oligomerize and become an ion-channel protein called a viroporin (Singh Tomar PP et al.,
2020). The ion channel of E protein is regarded as the main contributor affecting the host
immune response, such as a cytokine storm (Wong NA et al., 2021). More importantly,
many studies found that deleting the E protein was sufficient to attenuate the virulence of
HCoVs (Cao Y et al., 2021).
293T.CD1d cell line is a good tool to study the pathogenetic effects of envelope
protein because it overexpresses CD1d molecules that is a member of the glycoproteins
expressed on the surface of many human antigen-presenting cells and are related to the
presentation of lipid antigens of T cells. The CD1d molecule is produced in the
endoplasmic reticulum (ER) and forms a complex with β2m, loaded with endogenous
lipids then locate to the surface of the cell. From there the CD1d can re-enter the
18
cytoplasm and be loaded with other endogenous or exogenous antigens. Finally, the
CD1d molecule returned the cell surface and could be recognized by iNKT cells
(Brutkiewicz RR et al., 2018). The mature form of CD1d can be detected by an antibody
called 51.1.3, whereas immature CD1d and denatured mature CD1d can be detected by
an antibody called D5. Based on previous studies in the Yuan lab, it was found that
SARS-CoV-2 E protein can downregulate the mature CD1d molecule located on the
surface of 293T.CD1d cells as shown by flow cytometry. Thus, it’s necessary to evaluate
whether SARS-CoV E protein (Flag-tag) can also have a similar effect. Firstly, the
SARS-CoV E plasmid with Flag-tag was received from a lab at Howard University. The
nucleotide sequence of Flag-tagged E protein was originated from viral cDNA. The
293T.CD1d cells were transfected with SARS-E Flag-tag plasmid. pTracer plasmid was
co-transfected to track the transfection efficiency. After 48 hours, the harvested and lysed
cell samples were used to perform Western Blot for detecting protein expression. Besides,
the whole cell lysate was used to do IP in which the antibody 51.1.3 and D5 were used to
purify mature CD1d samples and immature CD1d samples, respectively. To confirm the
result of Western Blot, flow cytometry was performed to detect the mature CD1d
expression level.
The result showed that the 293T.CD1d cells transfected with SARS-CoV-2 E
expressed decreased amounts of mature CD1d, whereas the group of Flag-tagged SARS-
CoV E did not show a significant downward trend of mature CD1d (Figure 1A). Because
of this reason, the SARS-CoV E gene was re-coded and was expressed as a Strep-tagged
version. Interestingly both Strep-tagged SARS-CoV E and SARS-CoV-2 E can decrease
19
the mature CD1d expression of 293T.CD1d (Figure 1B). From the result of flow
cytometry, the conclusion was made that only the expression level of mature form CD1d
were decreased in SARS-CoV-2 E and Flag-tagged SARS-CoV E groups compared to
that of Control (pTracer Only) group (Figure 1C).
To figure out why the result of the two SARS-CoV E plasmids was not consistent, a
Western Blot assay was designed to detect the expression level of E protein. The primary
antibody obtained from Thermo Fisher (Catalog # PA1-41158) specifically bind to a
short amino acid region of SARS-CoV E protein. Specifically, the peptide recognized by
the antibody corresponded to the sequence Y 59 S R V K N L N S S E G 70 of the
putative SARS E protein. Without these amino acids, only a background protein band
could be seen. This antibody also binds to the E protein of SARS-CoV-2. As a result, the
expression level of Strep-tagged SARS-CoV E plasmid may be higher than Flag-tagged
SARS-CoV E plasmid (Figure 1D).
3.2 Strep-tagged SARS-CoV and SARS-CoV-2 E proteins downregulated CD1d-
restricted iNKT functions
Our lab mainly focused on CD1d-restricted iNKT cells that served as the link
between the innate immune system and the adaptive immune system. The glycolipid, α-
galactosylceramide (α-GalCer) is a prototype ligand that stimulates iNKT cells (Zhang Y
et al., 2019). Two ELISA assays were designed to test the hypothesis that the functions of
CD1d-restricted iNKT cells may be inhibited if the mature CD1d molecules were
20
downregulated by E proteins of SARS-CoV and SARS-CoV-2. One contained exogenous
lipid (⍺-Galcer) and the other was autoreactivity assay that examines the presentation of
autologous lipid antigens. The functions of CD1d-restricted iNKT cells were evaluated
by detecting the mIL-2 secretion level since it was the cytokine secreted by iNKT cell
hybridomas. 293T.CD1d cells were transfected with SARS-CoV E and SARS-CoV-2 E
plasmids respectively. Besides, an untransfected group was designed as a negative
control.
From the bar graphs shown in Figure 2A, compared to the control group, both E
proteins could downregulate the secretion level of mIL-2 significantly in a total of seven
different T cell hybridomas. However, the result of the autoreactivity assay was not
consistent with that of ⍺-GalCer assay (Figure 2B). Although the KI-2 and DN32.D3
hybridomas suggested that the mIL-2 secretion was decreased, the degree of
downregulation was too faint. This may be due to the fact that the autoreactivity is close
to the lower detection limit of ELISA assays we are using. In such low signals, the
decrease in mIL-2 due to the lowered CD1d expression cannot consistently detected by
ELISA. More sensitive methods to detect NKT cell activation, for example, the influx of
calcium or the upregulation of mIL-2 gene expression may need to be examined.
21
3.3 HCoV nucleocapsid proteins did not affect mature CD1d expression level, and
Strep-tagged SARS-CoV and SARS-CoV-2 N proteins activated CD1d-restricted
iNKT functions
Nucleocapsid (N) proteins of human coronaviruses are the most abundant viral
protein in HCoV-infected cells (Chang CK et al., 2013). The main function of N protein
is to bind the viral genome and form a ribonucleoprotein (RNP) complex. N proteins also
have been shown to deregulate the host cell cycle (Wurm, T et al., 2001). Unlike SARS-
CoV, SARS-CoV-2 and MERS that can induce severe symptoms, other HCoVs, such as
HCoV-OC43, usually generate mild symptoms like the common cold. Thus, finding out
whether the functions of nucleocapsid proteins between the two groups was quite
intriguing.
Previously, my lab colleagues, Siyang Chen and Hongjia Lu did an ELISA
autoreactivity assay and found that SARS-CoV-2 N protein could increase the mIL-2
secretion of CD1d-restricted iNKT cells compared to other proteins of SARS-CoV-2. In
other words, SARS-CoV-2 N protein could stimulate the CD1d-restricted iNKT functions
since one of the most efficient ways to evaluate the functions of T cell hybridomas
(CD1d-restricted iNKT cells) is to measure mIL-2 secretion. The possible reason is that
SARS-CoV-2 N protein can upregulate the expression of mature CD1d expression level
in 293T.CD1d cells, and N protein from different human coronaviruses can also
potentially affect the expression level of mature CD1d. The Western Blot result indicated
all N proteins cannot affect the mature CD1d level (Figure 3A). To replicate the result
22
from Siyang Chen and Hongjia Lu, an autoreactivity assay was conducted in which the
293T.CD1d cells were transfected with four N protein expression plasmids mentioned
before. According to the bar graphs, only five different hybridomas, Hyb1.2, Hyb1.4, KI-
2, KI-15, and KI-16, showed that SARS-CoV-2 N protein could increase the mIL-2
secretion level (Figure 3B). Since Flag-tagged SARS-CoV N protein cannot stimulate the
iNKT functions, the gene of SARS-CoV N was re-coded and was expressed as a Strep-
tag version. So, this time, the Strep-tagged SARS-CoV N was added to the previous test
group and the same western blot and autoreactivity assays were repeated. To eliminate
the possible influence of glycosyl-residues on the interaction of CD1d protein and
primary antibody, PNGase F (Catalog#P0704S) was utilized to deglycosylate expressed
N proteins. The western blot showed that all nucleocapsid proteins still cannot affect the
mature CD1d expression level (Figure 3C). Furthermore, the flow cytometry result
confirmed that none of the tested N proteins could increase or decrease the mature CD1d
level (Figure 3D).
Next, an autoreactivity assay was used to test whether Strep-tagged SARS-CoV N
protein can increase mIL-2 secretion. The expression of each protein was confirmed
through western blot (Figure 3E). What was interesting was that in the autoreactivity
assay, Strep-tagged SARS-CoV N and SARS-CoV-2 N could increase the secretion level
of mIL-2 significantly (Figure 3F). Compared to the positive control of Strep-tagged
SARS-CoV N and SARS-CoV-2, the MERS-N protein could not significantly activate
the functions of T cell hybridomas (Hyb1.2 and DN32.D3). To figure out whether Flag-
tagged and Strep-tagged SARS-CoV N produced inconsistent results, the
23
immunofluorescence assay was conducted. However, the result was that the expression
level of Strep-tagged N protein was higher than Flag-tagged N protein (Figure 3G), and
further work should be done to solve this problem.
3.4 Mechanisms of SARS-CoV-2 N protein in activating CD1d-restricted iNKT cells
functions
To explain the mechanism, or in other words to identify which part of nucleocapsid
protein of SARS-CoV-2 elicited CD1d-restricted iNKT functions, it was necessary to
break down the whole protein into different parts and study each separately. The N-
terminal domain and C-terminal domain of SARS-CoV-2 N protein are responsible for
RNA genome binding. The NTD domain may undergo conformational adaptation upon
RNA binding, and the CTD domain exists in a dimeric form and plays an important role
in protein-protein interaction (Satarker S et al., 2020). The SARS-CoV-2 NTD-deletion
and SARS-CoV-2 CTD-deletion plasmids were designed by Dr. Yuan and amplified by
my colleague Hongjia Lu, and the diagram of two partially cleaved proteins is shown in
Figure 4A. Firstly, the Western Blot result confirmed the expression of each protein
(Figure 4B).
Next, the flow cytometry results also confirmed that SARS-CoV-2 NTD-deletion
and CTD-deletion proteins can not increase or decrease the mature CD1d expression
level (Figure 4C). Then, the ELISA autoreactivity assay illustrated that compared to
Strep-tagged SARS-CoV N and SARS-COV-2 N proteins, the SARS-CoV-2 NTD-
deletion protein can still induce CD1d-restricted iNKT functions, but the CTD-deletion
24
protein lost the ability to activate CD1d-restricted iNKT cells (Figure 4D). However, the
result was not consistent since the result of KI-15 indicated that both NTD-deletion and
CTD-deletion N mutants retain stimulation of the KI-15 iNKT clone, suggesting that the
NTD-deletion N protein may still have the function of stimulating production of some
specific endogenous lipids recognized by the TCR of KI-15 iNKT cell clone.
25
CHAPTER 4: DISCUSSION
Human coronaviruses are a group of coronaviruses, which mainly cause human
respiratory diseases varying from the common cold to severe pneumonia. From the
outbreak of SARS in 2003, MERS in 2012 to COVID-19, scientists have spared no effort
in pursuing effective treatments to combat coronaviruses, and many vaccines have been
developed recently, preventing millions of individuals from SARS-CoV-2 infection or
severe diseases. The mortality rate has been significantly reduced in infected persons.
However, the coronaviruses are more likely to mutate so that they may not be wiped out
by existing anti-viral methods in the future if we do not make any progress in coronavirus
research.
This project was aimed to find out the possible immune-modulating functions of
envelope and nucleocapsid proteins of different coronaviruses and exploit the association
between the functions of CD1d-restricted iNKT cells and these viral proteins. As for
envelope protein, I mainly focused my attention on the genetic and functional similarities
between SARS-CoV and SARS-CoV-2. From the results, I concluded that both viral E
proteins (Strep-tagged SARS-CoV E and SARS-CoV-2 E proteins) significantly
downregulated mature CD1d expression and harmed CD1d-restricted iNKT functions.
The possible explanation for this result was that they can inhibit the antigen presentation
process by downregulating mature CD1d expression level, such that iNKT invariant
ligand cannot interact efficiently with lipid ligands presented by mature CD1d molecules
on antigen-presenting cells.
26
As for nucleocapsid protein, it serves an important role in virion packaging and
binding to the viral genome. Studying the role of N protein can yield some insights into
COVID-19 treatment or vaccination development. My primary goal was to compare
different nucleocapsid proteins from human coronaviruses concerning their effects on
mature CD1d expression levels and compare their potential effects on CD1d-restricted
iNKT cells. As a result, I found that none of these nucleocapsid proteins affected the
mature form of CD1d expression. In fact, efficient expression of SARS-CoV N and
SARS-CoV-2 N proteins by Strep-tagging significantly stimulated CD1d-restricted iNKT
functions based on the increased secretion level of mIL-2. However, some ELISA results
from T cell hybridomas were not consistent with other hybridomas. One possible reason
is due to the diversity of these individual iNKT cell clones. N protein modulation of
iNKT cells may be specific for some iNKT cells but not others.
So, from my perspective, the reason why both N proteins stimulated CD1d-restricted
iNKT cells was that they had nothing to do with the expression level of mature CD1d but
could potentially affect the mature CD1d trafficking processes. Furthermore, I identified
that the CTD domain of SARS-CoV-2 N protein may be essential to the stimulation of at
least some CD1d-restricted iNKT functions. For further work, the first thing will be to
construct different mutations of the CTD domain and explore these sites within this
domain to determine their roles for inducing the immune response of CD1d-restricted
iNKT cells. It is very necessary to use primary cells for this analysis, which natively
express CD1d molecules to replace the 293T.CD1d cell lines used in this study, to better
mimic the natural situation. If possible, I would inject certain mice with SARS-CoV-2 N
27
protein to explore whether the N protein can also stimulate CD1d-restricted iNKT cells in
vivo.
In addition, from the results, there remained a question why Flag-tagged proteins
cannot perform the expected consequences. The first possible reason was that the
expression level of Flag-tagged proteins was lower than the Strep-tagged ones. The
second reason was that maybe the Strep-tag can help the N protein interaction with
293T.CD1d cells and can influence the antigen-presenting process that affects the
functions of CD1d-restricted iNKT cells. In the future, it was necessary to generate no-
tag protein expression constructions to eliminate the possible effects of tags. Instead of
using tags to detect the proteins, it was easy to use antibodies specific to the proteins
themselves.
28
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of probable bat origin. Nature 2020, 1–4. [CrossRef]
36
FIGURES
A. B.
C.
D.
Figure 1. Potential modulation of CD1d expression level by envelope proteins of SARS-CoV and SARS-CoV-2.
A. The Western Blot result showed that only SARS-CoV-2 E protein downregulated the mature CD1d expression level.
B. The Western Blot assay suggested that Strep-tagged SARS-CoV and SARS-CoV-2 E protein can decrease the
mature CD1d expression level. C. The flow cytometry indicated that Strep-tagged SARS-CoV and SARS-CoV E
protein downregulated the expression level of mature form of CD1d. D. The Western Blot assay indicated that the
expression level of the Strep-tagged SARS-CoV E plasmid may higher than that of Flag-tagged SARS-CoV E plasmid.
37
A.
B.
Figure 2. Strep-tagged SARS-CoV E and SARS-CoV-2 E protein downregulated CD1d-restricted iNKT
functions. A. The result of the α-GalCer assay showed the significant downregulation of mIL-2 secretion level of
CD1d-restricted iNKT on Strep-tagged SARS-CoV E and SARS-CoV-2 E samples compared to the control sample. B.
The ELISA result of the autoreactivity assay showed that there was no significant difference of mIL-2 secretion level
between SARS-CoV E, SARS-CoV-2 E, and control group in different CD1d-restricted iNKT cells.
38
A. C.
B.
D.
E.
39
F.
G:
Figure 3. Human Coronaviruses nucleocapsid proteins does not affect mature CD1d expression level of
293T.CD1d cells, and Strep-tagged SARS-CoV N and SARS-CoV-2 N protein can stimulate the CD1d-restricted
iNKT cells. A. Western Blot result indicated that the mature form of CD1d expression didn’t change after 293T.CD1d
was transfected with different N proteins. B. ELISA result showed that SARS-CoV-2 N protein increased the mIL-2
secretion level significantly. C. Western Blot assay indicated that all tested N proteins cannot influence on mature
CD1d expression level. D. Flow cytometry result showed that the mature CD1d expression levels were not affected in
all different N proteins-transfected 293T.CD1d cells. E. The existence of tested N protein expression by Western Blot.
F. The ELISA assay indicated Strep-tagged SARS-CoV N and SARS-CoV-2 N increased the mIL-2 secretion level
significantly. G. The Immunofluorescence image showed the expression of mature CD1d level in four groups and the
existence of N proteins in three experimental groups.
40
A. B.
C.
D.
Figure 4. C-terminal domain of SARS-CoV-2 N protein may play an indispensable role in activating CD1d-
restricted iNKT cells. A. the diagram of N-terminal domain and C-terminal domain deletion of SARS-CoV-2 N
protein. B. Western Blot showed the expression of N proteins, including NTD deletion and CTD deletion of SARS-
CoV-2 N protein. C. Flow cytometry results of all nucleocapsid proteins reassured that nucleocapsid protein cannot
affect the CD1d level expressed in 293T.CD1d cells. D. ELISA result showed that in Hyb1.2, Hyb1.4 and KI-2 groups,
CTD-deletion group cannot increase the mIL-2 secretion level, but NTD-deletion group can still increase the mIL-2
secretion level.
Abstract (if available)
Abstract
The outbreak of COVID-19 has become a global pandemic. Despite enormous efforts being made, the pathogenetic mechanism of SARS-CoV-2 remains largely unknown, including its interaction with the innate immunity system. Envelope (E) protein and nucleocapsid (N) protein are the main structural proteins of coronaviruses that play indispensable roles in the pathogenesis and life cycle of coronaviruses. The CD1d molecule is expressed on the surface of many human antigen-presenting cells (APCs). CD1d molecule presents lipid antigen to iNKT cells. This study mainly focused on the interaction between E protein and N protein of HCoVs and the CD1d-restricted iNKT cells. The first part was to investigate whether both SARS-CoV and SARS-CoV-2 E proteins can downregulate the mature CD1d expression level and affect the function of CD1d-restricted iNKT cells. The second was to examine whether N proteins of different HCoVs can stimulate the functions of CD1d-restricted iNKT cells and explore the mechanisms therein. We generated the plasmid constructs expressing E and N proteins from different coronaviruses and expressed the proteins in 293T.CD1d cells then analyzed the CD1d expression and iNKT cell stimulation by flow cytometry and ELISA, respectively. Our results showed Strep-tagged SARS-CoV and SARS-CoV-2 E proteins similarly downregulated the mature CD1d expression level and blocked functions of iNKT cells. In addition, Strep-tagged SARS-CoV N and SARS-CoV-2 N proteins can stimulate the functions of iNKT cells and the C-terminal domain of SARS-CoV-2 N was required for this stimulation. Our results suggested that SARS coronaviruses have specifically evolved to precisely modulate the function of CD1d-restricted iNKT cells.
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Asset Metadata
Creator
Zhou, Ruiting
(author)
Core Title
Comparative studies of coronavirus interaction with CD1d-restricted iNKT cells
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Molecular Microbiology and Immunology
Degree Conferral Date
2021-08
Publication Date
07/23/2021
Defense Date
06/03/2021
Publisher
University of Southern California
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Tag
?-GalCer,CD1d molecule,envelope protein,human coronavirus,invariant NKT cell,nucleocapsid protein,OAI-PMH Harvest,SARS-CoV-2
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English
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Yuan, Weiming (
committee chair
), Feng, Pinghui (
committee member
), Tahara, Stanley (
committee member
)
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ruitzhou@gmail.com,zrt19960921@163.com
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Tags
?-GalCer
CD1d molecule
envelope protein
human coronavirus
invariant NKT cell
nucleocapsid protein
SARS-CoV-2