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SARS-CoV-2 suppression of CD1d expression and NKT cell function

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SARS-CoV-2 suppression of CD1d expression and NKT cell function

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Content 1

SARS-CoV-2 Suppression of CD1d expression and NKT cell function

By
Rongqi Zhao

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 Rongqi Zhao
ii

Acknowledgments

First of all, I would like to thank my mentor and my chair of committee Dr. Yuan for his careful
and patient training and teaching over the past two years, which gave me the opportunity to study
and explore immunology. My progress and growth are inseparable from the guidance and
education of Dr. Yuan. I would like to express my sincere thanks to Dr. Yuan for helping me
with my master’s graduate degree and my future career.
Next, I would like to thank my committee members, Dr.Feng Pinghui and Dr. Keigo Machida.
Thanks to their professional and meticulous thinking and questioning, I have a better
understanding of future research.
Furthermore, I would like to thank my lab co-worker and members Zhewei Liu, Hongjia Lu,
Ruiting Zhou, Siyang Chen, Yingting Zhang, Amarjot Thind. Thank you for your help and
support.
In the end, I would like to thank my parents and friends for their love and support.

iii

TABLE OF CONTENTS

Acknowledgments ....................................................................................................................... ii
List of Figures ............................................................................................................................ iv
Abstract....................................................................................................................................... v
I. Introduction ............................................................................................................................. 1
1.1 COVID-19 and SARS-CoV-2 virus .................................................................................... 1
1.2 SARS-CoV-2 Virus structure............................................................................................. 1
1.3 SARS-CoV-2 Spike protein (S protein) ............................................................................. 2
1.4 SARS-CoV-2 Envelope protein (E protein) ....................................................................... 2
1.5 SARS-CoV-2 Membrane protein (M protein) ..................................................................... 3
1.6 SARS-CoV-2 Nucleocapsid protein (N protein) ................................................................. 3
1.7 Different coronaviruses ..................................................................................................... 4
1.8 NKT cell and CD1d molecule ............................................................................................ 4
II. Materials and methods ........................................................................................................... 6
2.1 Cell Lines .......................................................................................................................... 6
2.2 Construct plasmid for different coronavirus ....................................................................... 6
2.3 Antibodies ......................................................................................................................... 7
2.4 Transient transfection ....................................................................................................... 8
2.5 Cell lysis and Western blot ............................................................................................... 8
2.6 Immunoprecipitation assay ............................................................................................... 9
2.7 FACS and cell sorting ....................................................................................................... 9
2.8 Immunofluorescence ........................................................................................................ 9
III. Result ...................................................................................................................................11
3.1 Different coronavirus E proteins influence CD1d expression level differently ...................11
3.2 SARS E protein downregulation of CD1d expression level ..............................................12
3.3 Immunofluorescence result ..............................................................................................13
IV. Discussion ...........................................................................................................................15
References ...............................................................................................................................16
Figures ......................................................................................................................................21

iv

List of Figures

Figure 1. Different coronavirus E proteins transfected influence CD1d expression level ……...21
Figure 2. SARS E protein gradient test ………………………………………………………….23
Figure 3. Immunoprecipitation assay results for SARS E and SARS2 E protein transfection ….25
Figure 4. Immunofluorescence results for different coronavirus E proteins transfection ……....26

v

Abstract

The SARS-CoV-2 virus caused the COVID-19 pandemic explored all over the world. This
thesis focused on the influence of the SARS-CoV-2 virus on NKT cell function. The SARS-
CoV-2 virus has four main structural proteins, which are spike (S), envelope (E), membrane
(M), and nucleocapsid (N). Each of those structure proteins has its function and can lead to
different immune-modulating activities. CD1d molecular expression level is an essential clue
to direct NKT cell function. The primary purpose of this thesis is to investigate how different
coronavirus' proteins regulate NKT cell function. Our lab recently discovered that the E
protein of SARS-CoV-2 downregulates CD1d expression and suppresses iNKT cell function.
In this thesis, we discovered that SARS-CoV-2 and SARS virus E proteins could
downregulate CD1d molecular expression level but not the other types of common cold
coronaviruses. Our results suggested that the SARS coronaviruses have evolved to
specifically downregulation the function of CD1d and NKT cells.
1

I. Introduction
1.1 COVID-19 and SARS-CoV-2 virus
The SARS-CoV-2 virus caused respiratory syndrome COVID-19 spread rapidly all around the
world since December 2019. COVID-19 has caused over 1.5 billion confirmed cases and over 3
million death cases. According to the WHO, SARS-CoV-2 Virus frequently spreads through
contact, droplet, airborne, fomite, fecal-oral, bloodborne, mother-to-child, and animal-to-human
will also lead to the virus transmission. Therefore, research about SARS-CoV-2 structure and
function makes essential sense for disease treatment and prevention. For now, there are more
than 38% of the population is fully vaccinated in California. However, there still be much
research that needs to be done about the pathogenesis of the SARS-CoV-2 virus.

1.2 SARS-CoV-2 Virus structure
The SARS-CoV-2 virus is an enveloped virus that belongs to the genus beta coronavirus (order
Nidovirales, family Coronaviridae, subfamily Coronaviridae), as same as SARS, OC43, HKU1,
and MERS (Middle East respiratory syndrome coronavirus). (Kim et al., 2020, Zhou et al., 2020)
NL63 and 229E coronaviruses belong to alpha coronavirus (order Nidovirales, family
coronavirus, subfamily Orthocoronavirinae)(Lau et al., 2012). Like other coronaviruses, the
2

SARS-CoV-2 virus has a 29.9 kb RNA genome which contains two large ORFs, which are
ORF1a and ORF1b. Structure proteins that are encoded in the genome are spike (S), envelope
(E), membrane (M), nucleocapsid (N), and several small accessory proteins. (Davidson, Andrew
D., et al.,2020)

1.3 SARS-CoV-2 Spike protein (S protein)
Compared with other structure proteins, the spike protein gene is longer, and the protein is1273
aa long and more complicated than other structural proteins. Plenty of spike proteins are located
on the SARS-CoV-2 virus surface to recognize and bind to the host cell receptor angiotensin-
converting enzyme 2 (ACE2) to help viruses get into the host cell. After the virus enters the host
cell, the viral genome will be released, translated, and replicated. That is why inhibiting the spike
protein binding with the host cell is one of the strategies for the COVID-19 vaccine (Yuan
Huang, et al.,2020).

1.4 SARS-CoV-2 Envelope protein (E protein)
Envelope protein is the smallest, minor structural protein, which has 76–109 amino acids. This
small protein plays an essential role in the viral life cycle for assembly, budding, envelope
formation, and pathogenesis (Schoeman, D., et al.,2019). The E protein is mainly expressed in
ER and Golgi-complex. Accordingly, delete E proteins will weaken the virus and even abolish
virulence (Cao, Y., et al.,2021). The mutation of E protein will lead to apoptosis (Parthasarathy
3

K, Ng L, Lin X, et al.2008). The offspring of mutant SARS-CoV-2 display a significant decrease
in viral titer and maturity (Curtis, K., 2002). In addition, E protein can inhibit the activity of
inflammasomes through blocking ion channel activity (Schoeman, D., et al.,2019). Protein
structure and the amino acid sequence of the SARS and SARS-CoV-2 E proteins are highly
homologous, and the multimer formation mechanisms of those two coronaviruses are likely
similar (Schoeman, D., et al.,2019).

1.5 SARS-CoV-2 Membrane protein (M protein)
The most abundant protein of the SARS-CoV-2 virus is M protein, which can adapt a region of
membrane for virus assembly and captures other structural proteins at the budding site (Neuman,
B., et al.,2011). Interestingly, M protein can bind and associate with different structural proteins.
For instance, M protein bind with N proteins can help the N protein-RNA complex stay stable
and promote viral assembly (Astuti, I., et al.,2020); M protein bind with S proteins can lead to
mutation, which will influence the virus binding and get into the host cell (Bianchi, M., et
al.,2020); M protein and E protein make up the viral envelope and their interaction can lead to
VLP formation (Corse, E., et al.,2003, Baudoux, P., et al.,1998)

1.6 SARS-CoV-2 Nucleocapsid protein (N protein)
The essential role of N protein is to pack the viral genome into nucleocapsids and play essential
roles to maintain correct replication and reliable transmission (McBride, R., et al.,2014). Interact
with RNAs and the resulting complex to oligomerize are two critical functions for the N protein
4

function. SARS vaccine research indicated that N protein representing antigen could induce
SARS-specific T-cell proliferation and cytotoxic activity (Dutta, N., et al.,2020). The SARS-
CoV-2 virus has a similar and substantial conserved sequence of N protein as SARS and other
coronaviruses (Cubuk, J., et al.,2021), suggesting that the N protein can be a target for COVID-
19 vaccine development.

1.7 Different coronaviruses
Infection by different coronavirus have varying symptoms. For instance, SARS and MERS cause
severe infections that lead to high mortality rates, coronavirus 229E, OC43, NL63, and HKU1
are milder and cause common colds, pneumonia, and bronchiolitis (Rucinski, S., et al.,2020,
Schoeman, D., et al.,2019, Gaunt, E., et al.,2010). 229E and OC43 were first discovered in 1960.
In 2003, SARS-associated coronavirus was first identified, then in 2004, NL63 and HKU1 were
found, and the outbreak disease MERS exploded in 2012 (Bruning, A., et al.,2018, Al-Sharif, E.,
et al.,2021).

1.8 NKT cell and CD1d molecule
iNKT cell is one of the subtypes of NKT cell, which has both T cell and NK cell characters.
iNKT cells have a different antigen-presenting mechanism than other subtypes of T cells, such as
CD4+ and CD8+. Conventional CD4+ can recognize peptide antigens presented by MHC II,
while CD8+ can recognize peptide antigens through recognizing MHC I bind with peptides.
5

NKT cell are stimulated by lipidic antigens presented by CD1d, a non-polymorphic CD1
antigen-presenting protein family (Juno, J., Keynan, et al.,2012). In general, the expression level
of CD1d can be a clue for iNKT cell activation, and the higher CD1d level, the higher NKT cell
activation. For the experiment level, iNKT cell function can be activated by the model ligand, -
GalCer isolated from the marine sponge, which has been used as a vaccine adjuvant to activate
iNKT cells (Kumar, A., et al.,2017).
iNKT cells recognize lipid agonists and are activated quickly, and then iNKT cells secret
different kinds of cytokines and chemokines in a short time. For cancer therapy, iNKT cells play
an essential role in controlling tumor immunity, and the activation of iNKT cells can trigger
innate immunity for different kinds of cancers. Based on those characteristics, iNKT is attractive
for tumor immunotherapy as a target (Bollino, D., et al.,2017, Waldowska, M., et al.,2017,
Kumar, A., et al.,2017).

6

II. Materials and methods
2.1 Cell Lines
293T.CD1d cell lines (provided by Dr.Yuan) were cultured in Dulbecco's Modified Eagle
Medium (DMEM), with 5% Fetal Bovine Serum (FBS) (HyClone) and 0.05% puromycin
antibiotics. Puromycin was used to select and maintain 293T cell express human CD1d
molecule. Hela.CD1d cell lines (provided by Dr.Yuan) were cultured in Dulbecco's Modified
Eagle Medium (Invitrogen) with 5% Fetal Bovine Serum (FBS) (Gibco) and 1% Penicillin
antibiotics. A549.hACE2 cell lines (provided by Dr. Makino) were cultured in Dulbecco's
Modified Eagle Medium (DMEM) with 5% Fetal Bovine Serum (FBS) (HyClone) 0.05%
Kanamycin and 0.02%Blasticidin-S HCl. Blasticidin-S HCl is used to select and maintain
A549.hACE2 cells that express human ACE2 molecules. Hela.CD1d.hACE2 cell lines were
cultured in Dulbecco's Modified Eagle Medium (Invitrogen) with 5% Fetal Bovine Serum (FBS)
(Gibco) and 0.02%Blasticidin-S HCl. Trypsin 0.25% EDTA was used to digest Hela.CD1d,
A549.hACE2 and Hela.CD1d.hACE2.

2.2 Construct plasmid for different coronavirus
p-Tracer as a GFP expressed plasmid was used for transient transfection, which can identify the
transfection efficiency. SARS, SARS2, MERS, OC43, NL63, HKU-1, and 229E plasmid for
different coronaviruses E protein. The protein sequence of envelope protein from severe acute
respiratory syndrome coronavirus two was retrieved from the NCBI database. The NCBI
7

Reference Sequence is YP_009724392.1. The protein sequence of envelope protein from OC43
coronavirus was retrieved from the NCBI database, and the GenBank number is LC315648.1.
The protein sequence of envelope protein from HKU1 coronavirus was retrieved from the NCBI
database, and the NCBI Reference Sequence is NC_006577.2. The protein sequence of envelope
protein from NL63 coronavirus was retrieved from the NCBI database, and the GenBank number
is JX504050.1. The protein sequence of envelope protein from 229E coronavirus was retrieved
from the NCBI database, and the NCBI Reference Sequence is NC_002645.1. The protein
sequence of envelope protein from MERS and SARS coronavirus was retrieved reference gene
from the NCBI database. The NCBI Reference Sequence for SARS is NC_004718.3. The NCBI
Reference Sequence for MERS is YP_009047209.1. The NCBI Reference Sequence for SARS-
CoV-2 is YP_009724392.1. Construction plasmid with different coronavirus E proteins, the
plasmid was retrieved and add strep tag to a vector. All protein sequences were recoded for
optimal expression in human cells (Genewiz Inc.). Sequencing and alignment were performed to
verify the amino acid sequences.

2.3 Antibodies
For the first antibody, the human CD1d expression level was detected by Monoclonal 51.1.3
(provided by Dr. Steven Porcelli at Albert Einstein College of Medicine, Bronx, NY). The linear
form of human CD1d was detected by D5, Grp94 as a loading control. For the secondary
antibody, Goat-anti-Mouse detects HRP, Goat-anti-Rat HRP, and Goat-anti Rabbit HRP
antibodies were diluted in TBST with 1:5000 dilution.
8

2.4 Transient transfection
Seed 293T.CD1d/Hela.CD1d in six well-plates or 10cm dish. Polyethyleneimine (PEI) was used
for plasmid transient transfection when cell density was between 70%-90%. Maximum culture
for 48 hours in a 37℃ incubator. Harvest cell sample and wash with DPBS three times. For
FACS, staining cells immediately. For Western blot, samples can be stored in a -20℃ fridge.

2.5 Cell lysis and Western blot
Cell lysis buffer which needs to prepare freshly contains 1% Triton-X-100 in Tris-buffered
Saline buffer (TBS) with IAA(5mM), PMSF(0.1mM), NaVO3(1mM), NaF(1mM),
Leupeptin(10uM), Pepstatin A(1 μg/ml), β-glycerophosphate(1mM), Okadaic acid(0.5uM) and
NaPP(1mM). 10 cm dish cells need 1ml cell lysis buffer, lysis for 30 mins on ice. Centrifuge cell
sample for 10 mins with 1000 rcf. Add 40ul 6X SDS page to 200ul lysis sample and boiling
sample for two mins. The remaining 800ul lysis sample can be used for immunoprecipitation
assay. For Western blot, boiling samples before loading samples. Protein marker (Bio-Rad) as a
template for accurate molecular weight estimation of SDS-PAGE. Run Gels at 80-120 Volts for
about 2 hours. Methanol was used to activate Polyvinylidene Fluoride (PVDF) membranes for
gel transfer. Gel transfer with 12 Volts for 90 minutes. 5 % milk with TBST (Tris-Buffered
Saline, 0.1% Tween) block transformed gel minimum for 30 mins. Wash membranes and
incubate primary antibodies overnight under 4℃. Wash membranes four times total for 20 mins.
Incubate second antibodies for 1 hour under room temperature. Wash membranes three times
total for 30 mins. Develop chemiluminescence using Bio-Rad developer.
9

2.6 Immunoprecipitation assay
Preclear each lysis sample with 40:40:1 protein A, protein G, and rabbit serum. Rotate samples 1
hour under 4℃ (up to overnight). Pellet beads with 5000 rpm for 1 min. Recover supernatant and
add 40:40:1 protein A, protein G, and 51.1.3 or D5. Rotate samples for 2 hours under 4℃ (up to
overnight). Wash with 1 ml of 0.1% Triton/TBS three times. Add 80ul 4X SDS loading buffer to
elute immunoprecipitated proteins.

2.7 FACS and cell sorting
For FACS, wash and resuspend cells in 1% BSA in DPBS, add 50 ul of primary antibody dilute
1:2000 with DPBS/1% BSA. Incubate on ice for 30 mins. Wash cells with DPBS/1% BSA three
times. Add 100 ul of secondary antibody dilute 1:5000 with DPBS/1% BSA. Wash three times
and last wash with just DPBS. Suspend cells in 100ul 3.7% formaldehyde in DPBS, transfer the
cell suspensions to FACS tubes, and store them at 4°C in the dark. For cell sorting, staining
conditions are the same as FACS. Culture sorted cell in 96-well plate and amplify cell line.

2.8 Immunofluorescence
Place coverslips in a 24-well plate and seed Hela CD1d cell with 40k/ml. Transfect cell and
culture for 48 hours. Aspirate media and quickly add 400 ul fixative solution for 20 min at room
temperature. Wash coverslips twice with about 500 ul sacrum-free media with 10 mM Hepes.
10

Samples can be stored overnight at this point. For permeabilization and staining, aspirate and add
300-500 ul permeabilization solution (PS) per well and incubate for 15 min at room temperature.
Dilute primary antibody 1:2000 in PS and vortex to mix well. Place 20 ul drops of dilute
antibody on a flat piece of parafilm stuck to the benchtop with a film of water. Place coverslips
with the cell side onto the antibody drop. Put moist towels around the parafilm and cover
everything with aluminum foil to create a moist chamber and prevent antibodies from drying.
Incubate at room temperature for 30 min and wash 3 times, each 5 min with 400ul PS. Repeat
staining step with secondary antibody. Before mounting coverslips, wash them quickly with
water by passing through a break filled with water to rinse. Aspirate the residual water from the
coverslips by vacuum aspiration as much as possible. Turn coverslips onto 5 ul drops of Mowiol
on a glass microscope slide. Before viewing, dry samples 30 min at room temperature or
overnight at 4℃. Keep slides protected from light.

11

III. Result
3.1 Different coronavirus E proteins influence CD1d expression
level differently

Based on our lab's previous study and screening, transfection of SARS-Cov-2 E protein can
downregulate CD1d expression in 293T CD1d cells. E Protein as a structural protein has high
homology within different types of coronavirus, which gives us an idea to find out if different
coronavirus E proteins will influence CD1d expression level similarly. This study can help us
understand the relationship between different coronavirus E protein structures and functions. We
use 293T CD1d cell line and transfected different coronavirus for 48 hours to test the influence
of different coronavirus E protein downregulation levels. p-Tracer was used as a GFP positive
control and as a clue to test plasmid transfection efficiency. FACS and Western Blot were used
to check CD1d expression levels. From the FACS result, there was a downregulation of CD1d
expression after transfection with SARS E protein and SARS-Cov-2 E protein. 229E, OC43,
NL63, and HKU1 coronavirus did not show downregulation of CD1d expression level. MERS E
protein transfection shows a minor downregulation of CD1d expression. Western blots were used
with the same group of samples to detect protein expression. For the results of the Western Blots,
GRP94 was used to compare the total protein levels. Among different coronavirus transfection
samples, there was a similar amount of GRP94, which indicates the total protein level for
different dishes of cells is similar and comparable. The anti-strep antibody can bind with strep-
tagged E protein to compare the expression level of different E proteins. All samples showed
similar band intensities after transfection with different E protein, and the sample transfection
12

with only p-Tracer did not show band. The Western Blots result proved that cell sample
transfection with different E proteins does produce E proteins comparably, so the FACS can be
used to detect CD1d surface expression level.

3.2 SARS E protein downregulation of CD1d expression level

Since the SARS E protein transfection sample showed downregulation as SARS-Cov-2 E protein
transfection sample, the SARS E protein transfection gradient test was for further verification.
For the SARS E protein gradient test, transfected different amounts of SARS E protein to 293T
CD1d cells for 48 hours. Same culture, staining, and cell lysis condition as transfection with
different E protein. The FACS result showed that different SARS E protein transfection amounts
influenced the downregulation of CD1d molecular expression levels. The higher amount
transfected, the more downregulation of the CD1d molecular expression level. Compared with p-
Tracer transfected control, transfected with 12.5% 2.5ul plasmid per dish, the total
downregulation level of CD1d molecular expression was about15%. Transfected with a general
amount of 10ul plasmid per dish, the total downregulation level of CD1d molecular expression
was about 33%. For Western Blot treated with strep antibody, E protein expression level was
increased when transfection increased the SARS E protein plasmid amount. The higher amount
transfected, the darker band showed. For Western Blot treated with GRP94 antibody, there
showed a similar protein expression level.

13

In order to study the mechanism of SARS E protein-mediated CD1d downregulation,
immunoprecipitation was used to assay the total amount of 51.1.3-reactive CD1d. Preclear cell
lysis samples with rabbit serum and then IP with 51.1.3 anti-CD1d Ab. 51.1.3 Antibody reacts
with CD1d molecule in a mature form. On the other hand, the D5 antibody recognizes an
immature form of the CD1d molecule (Kang, S., et al.,2002). After boiling the sample, both
51.1.3- and D5-reactive forms of CD1d are denatured into the SDS-PAGE buffer. Blotting with a
D5 antibody can detect all kinds of CD1d molecules. After transfection with SARS E protein and
SARS-Cov-2 E protein and IP with 51.1.3, the western blot results showed the amount of 51.1.3-
reactive CD1d was downregulated. There were lighter bands with SARS E protein and SARS-
Cov-2 E protein transfected samples than p-Tracer only samples. D5 IP did not show obvious
downregulation. Anti-strep and anti-GRP94 antibodies were treated to the whole-cell lysis
samples, and the results showed consistent comparison with former results about transfection
with different coronavirus. FACS also showed the same result.

3.3 Immunofluorescence result
E protein was highly involved with virus assembly, budding, envelope formation, and
pathogenesis (Schoeman, D., et al.,2019) and was mainly expressed in ER and ERGIC region.
Transfected different coronavirus E proteins to HeLa CD1d cells and stained with Alexa488 for
51.1.3-reactive CD1d, Alexa568 for strep Alexa647 for ERGIC. 647-cyer.5 showed the location
of ERGIC, and 568-TRIC for strep showed colocation with ERGIC in the immunofluorescence.
Strep protein accumulated at a point and around ERGIC. 488-FICT for 51.1.3 CD1d showed the
CD1d located on the cell surface, and since the cell sample was extruded the location of 51.1.3
14

CD1d was all over the cell. Transfection with different coronavirus E proteins showed different
levels of CD1d downregulation. The brighter of Alexa-568 for strep (and E protein), the darker
of Alex 488 for 51.1.3-reactive CD1d.

15

IV. Discussion

Different coronavirus E proteins influence CD1d molecular expression at different levels. SARS
and SARS-CoV-2 E protein showed a clear downregulation of CD1d, and other coronavirus E
proteins downregulate CD1d molecular expression level slightly. The interesting result was that
MERS E protein transfected samples have unstable downregulation of CD1d molecular
expression level within several tries. More research should be done in future studies to figure out
the reason, and one hypothesis was about the different forms of ion channel in the E proteins
may influence the CD1d-downregulation function (Verdiá-Báguena, C., et al.,2012).

Accordingly, the IF image for different coronavirus E proteins transfected samples showed
several phases of the cell. Matured form CD1d molecules expressed on the cell surface. The
images of CD1d (FITC channel) can indicate the shape of cells. The cell with low-level CD1d
expression showed several points of signal and cannot indicate the cell shape. Under this
situation, ERGIC and Dapi were used to check the cell. Based on the E protein function about
viral assembly, budding, and envelope formation, E protein should accumulate at the ERGIC of
the cell. The E protein (TRITC channel) and ERGIC (Alexa647 channel) image showed the
exact location, which proved the E protein localization was the same as ERGIC. Expected the
regular pattern, the higher expression level of E protein, the lower expression level of CD1d. All
different coronavirus E proteins transfected samples showed the inverse relationship between E
protein and CD1d expression level. However, only SARS and SARS 2 showed a high CD1d
downregulation ratio of about 10%-30% amount 100 cells. Other coronavirus E proteins have a
low CD1d downregulation ratio of about 2%-5% amount 100 cells.

16

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21

Figures
A1.

A2.

22

B.

C.

Figure 1. Different coronavirus E proteins transfected influence CD1d
expression level
SARS-CoV-2 and SARS E protein downregulate CD1d expression level. Other kinds of
coronavirus E proteins influence CD1d expression level lightly. Figure 1.C showed the
homology between different coronavirus E proteins.

23

A.

B

0.25µg/well
0.5µg/well
1µg/well
2µg/well
0
2000
4000
6000
2777.5 2445.0 2292.5 1177.5
Decresae of CD1d MFI
1µg/well
2µg/well
0.5µg/well
0.25µg/well
24

C

D
0.25µg/well
0.5µg/well
1µg/well
2µg/well
0.0
0.5
1.0
1.5
1.000 0.910 0.490 0.165
Relative Quantity of CD1d Moleculers
0.25µg/well
0.5µg/well
1µg/well
2µg/well

Figure 2. SARS E protein gradient test.
The gradient test for SARS E protein transfection showed the increased amount of plasmid
transfected, the CD1d downregulated level increased. The Western Blots result showed a
darker band with a higher amount of plasmid transfection, and the total protein expression was
at the same level.

25

A.

Figure 3. Immunoprecipitation assay results for SARS E and SARS2 E
protein transfection
After immunoprecipitation assay with 51.1.3 protein, SARS-Cov-2 and SARS E protein
transfected samples showed downregulation. The total protein expression level is the same,
and the strep protein expression of SARS-Cov-2 and SARS E protein transfected samples are
at the same level.

26

27

Figure 4. Immunofluorescence results for different coronavirus E proteins
transfection
CD1d molecule was expressed on the cell surface, and the E protein was accumulated around
ERGIC. The higher expression level of strep always showed with the lower expression level of
the CD1d molecule.

Abstract (if available)

Abstract The SARS-CoV-2 virus caused the COVID-19 pandemic explored all over the world. This thesis focused on the influence of the SARS-CoV-2 virus on NKT cell function. The SARS-CoV-2 virus has four main structural proteins, which are spike (S), envelope (E), membrane (M), and nucleocapsid (N). Each of those structure proteins has its function and can lead to different immune-modulating activities. CD1d molecular expression level is an essential clue to direct NKT cell function. The primary purpose of this thesis is to investigate how different coronavirus' proteins regulate NKT cell function. Our lab recently discovered that the E protein of SARS-CoV-2 downregulates CD1d expression and suppresses iNKT cell function. In this thesis, we discovered that SARS-CoV-2 and SARS virus E proteins could downregulate CD1d molecular expression level but not the other types of common cold coronaviruses. Our results suggested that the SARS coronaviruses have evolved to specifically downregulation the function of CD1d and NKT cells.

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Asset Metadata

Creator Zhao, Rongqi (author)

Core Title SARS-CoV-2 suppression of CD1d expression and NKT cell function

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2021-08

Publication Date 08/02/2021

Defense Date 06/03/2021

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

Tag CD1d,NKT cell,OAI-PMH Harvest,SARS-CoV-2

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

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

Creator Email rongqiizhao@hotmail.com,rongqizh@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC15673809

Unique identifier UC15673809

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