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The analysis and modeling of signaling pathways induced by the interactions of the SARS-CoV-2 spike protein with cellular receptors

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The analysis and modeling of signaling pathways induced by the interactions of the SARS-CoV-2 spike protein with cellular receptors

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THE ANALYSIS AND MODELING OF SIGNALING PATHWAYS INDUCED BY THE
INTERACTIONS OF THE SARS-CoV-2 SPIKE PROTEIN WITH CELLULAR RECEPTORS

By
Pingping Hou

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

August 2022

Copyright 2022 Pingping Hou
ii
ACKNOWLEDGEMENT
I would like to thank all people who provided generous support for my research and study
during my master program. Without their help, this dissertation would not be possible.
I would like to give my first thanks to my supervisor Dr. Jing-Hsiung James Ou. During
my master program, he always gives me the courage and confidence to try various ways to discover
unknown areas that I am interested in this topic without objection. In addition, I am very grateful
for all discussions with him about my results, all his suggestions regarding the detailed steps of
my experiments, and all his patient explanations of my questions, which gave me lots of insights
and helped me find a better strategy.
I also would like to thank my other thesis committee members: Dr. Keigo Machida and Dr.
Weiming Yuan, for their professional advice and suggestions. During the second year of my master
program, Dr. Keigo Machida provided helpful discussions and many valuable suggestions on my
experiments.
I also would like to thank all lab members in Dr. Ou’s lab. I am very thankful to Dr. Jiyoung
Lee, Dr. Jane (Yu Chen) Chuang and Dr. Ja Yeon Kim, who provide lots of guidance and
suggestions on my experiments and gave me lots of encouragements. Moreover, I also would like
to thank all other lab mates.
I also would like to thank my family and friends. They always support and encourage me
during these years, which is so important for an international student, especially during the
COVID-19 pandemic.

TABLE OF CONTENT

ACKNOWLEDGEMENT…...………………………………………………………………… ii
LIST OF FIGURES……………………………………………………………….…………….iii
LIST OF ABBREVIATIONS……………………..……………………………………………..v
ABSTRACT……………………………………………………………..…..………………….vii
CHAPTER 1: INTRODUCTION ................................................................................................ 1
1.1. COVID-19 pandemic ............................................................................................................... 1
1.2. SARS-CoV-2 spike protein...................................................................................................... 1
1.3. Recombinant Vesicular Stomatitis virus (VSV) with the SARS-CoV-2 spike protein ........... 2
1.4. Integrins: viral transportation and viral entry receptor ............................................................ 2
1.5. Focal Adhesion Kinase (FAK)................................................................................................. 3
1.5.1. FAK structure.................................................................................................................... 3
1.5.2. FAK/Src complex formation............................................................................................. 4
1.5.3. FAK/Src complex activity: cytoskeleton rearrangement .................................................. 4
1.5.4. FAK/Src complex activity: MAPK signaling pathway .................................................... 5
1.5.5. FAK/Src complex activity: innate immune responses ...................................................... 5
1.6. SARS-CoV-2 ........................................................................................................................... 6
1.6.1. SARS-CoV-2 alternative entry receptors.......................................................................... 6
1.6.2. SARS-CoV-2 spike protein interacts with integrins via the GRD motif .......................... 6
1.6.3. Clinical cases about the interactions between the spike protein and integrins ................. 7
1.6.4. Signaling pathways mediated by the interactions of SARS-CoV-2 spike protein with
integrins....................................................................................................................................... 8
CHAPTER 2: MATERIALS AND METHODS ........................................................................ 9
2.1. Cell culture ............................................................................................................................... 9
2.1.1. BHK21-T7 cells ................................................................................................................ 9
2.1.2. Huh-7 cells ........................................................................................................................ 9
2.1.3. Calu-3 cells ....................................................................................................................... 9
2.1.4. H1299 cells ....................................................................................................................... 9
2.2. Production of pseudoviral particles: VSV- G, VSV-G, VSV-SARS-CoV-2-S ..................... 9
2.3. Viral gene expression analysis ............................................................................................... 11
2.3.1. Luciferase expression in the viral particle production cell line (BHK-21-T7) ............... 11

2.3.2. Quantitative Real-Time PCR Analysis ........................................................................... 11
2.3.3. Immunofluorescence ....................................................................................................... 12
2.4. Infection assay using VSV- G, VSV-G, or VSV-S pseudoviral particles ............................ 13
2.5. Drug treatment assay.............................................................................................................. 13
CHAPTER 3: RESULTS ........................................................................................................... 14
3.1. Construction of the first generation of VSV-G particles ....................................................... 14
3.2. Construction of the next generation of pseudoviral particles ................................................ 15
3.3. Pseudoviral particles infection assay ..................................................................................... 16
3.3.1. Infection of Huh-7 cells .................................................................................................. 16
3.3.2. Infection of Calu-3 cells.................................................................................................. 17
3.3.3. Infection of H1299 cells.................................................................................................. 18
3.4. Immunofluorescence assay .................................................................................................... 19
3.5. Drug treatment assay.............................................................................................................. 21
3.5.1. U0126 treatment assay in the Huh-7 cell line ................................................................. 21
3.5.2. U0126 treatment assay in the Calu-3 cell line ................................................................ 22
3.5.3. U0126 treatment assay in the H1299 cell line ................................................................ 23
3.5.4. FAK inhibitor 14 treatment assay in the H1299 cell line ............................................... 24
3.5.5. Miltefosine treatment assay in the BSL-3 lab with the real SARS-CoV-2 infection ..... 26
3.6. The analysis and modeling of possible signaling pathways induced by the interactions of
SARS-CoV-2 spike protein with different human cellular receptors ........................................... 27
CHAPTER 4: DISCUSSION ..................................................................................................... 28
REFERENCES ............................................................................................................................ 32

iii

LIST OF FIGURES

Figure 1 The Construction of the First Generation of VSV-G Particles Inside the BHK21-T7
Cells
Figure 2 The Production of the next generation of Pseudoviral Particles, Including VSV-ΔG,
VSV-G, VSV-S
Figure 3 The Expression of Phosphorylation of ERK by Western Blot in Huh-7 Cells
Figure 4 The Expression of Phosphorylation of ERK by Western Blot in Calu-3 Cells
Figure 5 The Expression of Phosphorylation of ERK by Western Blot in H1299 Cells
Figure 6 Immunofluorescence Staining of Infected Huh-7, Calu-3, and H1299 Cells
Figure 7 Western Blot and Luciferase Expression Analysis for U0126 Drug Assay
Experiments in Huh-7 Cells
Figure 8 Fold Change of Luciferase Expression of Different Doses of U0126 Inhibitor
Treatment in Huh-7 Cells.
Figure 9 Western Blot and Luciferase Expression Analysis for U0126 Drug Assay
Experiments in Calu-3 Cells
Figure 10 Western Blot and Luciferase Expression Analysis for U0126 Drug Assay
Experiments in H1299 Cells
Figure 11 Fold Change Graph of Luciferase Expression of Different Doses of U0126 Inhibitor
Treatment in Huh-7 Cells.
iv
Figure 12 Western Blot and Luciferase Expression Analysis for FAK Inhibitor 14 Treatment
Assay Experiments in H1299 Cells
Figure 13 Fold Change Graph of Luciferase Expression of Different Doses of FAK Inhibitor
Treatment in H1299 Cell
Figure 14 Western Blot Analysis for the Miltefosine Treatment Assay Experiments in the
BSL-3 Lab with the Real SARS-CoV-2 Infection
Figure 15 The Analysis and Modeling of the Possible Signaling Pathways Induced by the
Interaction between SARS-CoV-2 Spike Protein and Different Host Cellular
Receptors.

v
LIST OF ABBREVIATIONS
SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
COVID-19 Coronavirus Disease 2019
S protein Spike Protein
ACE2 Angiotensin-Converting Enzyme 2
RBD Receptor Binding Domain
VSV Vesicular Stomatitis Viruses
VSVG Vesicular Stomatitis Viruses with the Incorporation of Glycoprotein
VSVS Vesicular Stomatitis Viruses with the Incorporation of SARS-CoV-2 S protein
VSV- G Vesicular Stomatitis Viruses without Surface Protein Incorporation
AP-1 Activator Protein 1
MAPK Mitogen-Activated Protein Kinase
FAK Focal Adhesion Kinase
HIV Human Immunodeficiency Virus
HSV-1 Herpes Simplex Virus type 1
CXCR5 C-X-C Motif Chemokine Receptor 5
RGD Receptor Binding Domain
FERM Ezrin, Radixin, Moesin domain
FAT Focal Adhesion Targeting Domain
Y397 Tyrosine 397 site
SH2 Src Homology 2
Cdc42 Cell Division Control Protein 42 Homolog
mSOS Multiple Guanine Nucleotide Exchange Factors
vi
ERK Extracellular-signal-Regulated Kinase
IRF3 Interferon Regulatory Factor 3
IFN Interferons
MYD88 Myeloid Differentiation primary response 88
CPE Cytopathogenic Effect
RT-PCR Reverse Transcriptase Polymerase Chain Reaction
PFA Paraformaldehyde
SDS-PAGE Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

vii
ABSTRACT
SARS-CoV-2, as the causative pathogen of COVID-19, has caused one of the most severe
pandemics in human history. To date, the world has seen over 6 million deaths, and almost 60%
of Americans have been infected at least once. Although around 70% of the U.S. population has
been fully vaccinated, constantly emerging new variants pose new challenges and reduce current
vaccine effectiveness. These variants cause a surge of cases that overwhelm our healthcare system.
Due to the large number of people who have been infected, better antiviral compounds and more
convenient home-based therapeutic options are urgently needed for the treatment of post-COVID-
19 symptoms. So, it is crucial to investigate the host-virus interactions to deeply understand the
mechanisms of viral adaptability and infectivity. In this report, we mainly focus on studying the
virus-host signaling pathways mediated by the spike protein during the viral entry to discover small
intracellular molecules affected by the viral infection, which could be the novel potential antiviral
therapeutic targets. We used the recombinant vesicular stomatitis virus with the expression of
SARS-CoV-2 spike protein to infect different cell lines and compared them with the negative
control (VSV- G) and the positive control (VSV-G). We found the induction of phosphorylation
of FAK at tyrosine-397 (Y-397) and ERK 1/2 by the SARS-CoV-2 spike protein. To understand
these interactions between the spike protein and FAK or ERK, we performed the drug assay using
corresponding inhibitors to test their effects on viral entry. The results show that MAPK inhibitors
could enhance viral entry, and FAK inhibitor 14 could inhibit viral entry. So, the results
demonstrate that these two kinases can affect viral entry initiated by the spike protein. Additionally,
both p-FAK(Y-397) and p-ERK are critical components of the integrin-mediated signaling
pathway. Therefore, identifying this kinase upregulation will allow us to further investigate the
viii
entry mechanism of SARS-CoV-2, understand virus-host interactions, and discover novel potential
therapeutic targets against the COVID-19 infection.

1
CHAPTER 1: INTRODUCTION
1.1. COVID-19 pandemic
Since late 2019, the COVID-19 pandemic has caused an unprecedented crisis worldwide at all
levels. Lacking effective treatments allows the pandemic to evolve continuously, so understanding
its pathogenesis and exploring the therapeutics are becoming increasingly important. Although
most people have already been vaccinated, antiviral compounds that can effectively treat COVID-
19 infection and reduce clinical symptoms are urgently needed to protect immunocompromised
patients or patients with severe allergies, and to control emerging variants, and treat post-infection
symptoms or complications for recovered COVID-19 patients.
Therefore, it is crucial to investigate the mechanism of viral infection and the molecular pathway
of virus-induced pathologies in patients.
1.2. SARS-CoV-2 spike protein
SARS-CoV-2 is the cause of COVID-19 pandemic. It is a single-stranded and positive-sense RNA
virus that belongs to the genus beta-coronavirus genus of the Coronaviridae family (Hasan et al.,
2021). The SARS-CoV-2 spike protein (S), the main envelop protein present on the surface of the
virion, is a multifunctional protein that plays a vital role in host receptor binding, cell tropism, and
pathogenesis, and as a result, is an important target of the host immune response (Sigrist et al.,
2020). Without the presence of the spike protein, the whole viral infection process could not be
initiated, so it is crucial to study the signaling pathway during viral infection to define the
interaction between the spike protein and host cells and thus reveal cellular molecules used by
SARS-CoV-2 entry to discover novel targets for the development of antiviral therapeutics.
2
1.3. Recombinant Vesicular Stomatitis virus (VSV) with the SARS-CoV-2 spike protein
In the BSL-3 lab, Dr. James Ou’s lab found that two hours of authentic SARS-CoV-2 infection
could cause the activation of cellular kinases, including ERK and FAK, in Calu-3 cells, a lung
epithelial carcinoma cell line. To simplify the studies, I used vesicular stomatitis virus (VSV) to
develop pseudotyped SARS-CoV-2 particles with the expression of the SARS-CoV-2 spike
protein on the surface. Specifically, in this study, lentiviruses with vesicular stomatitis virus -spike
protein (VSV-S) was used to investigate the signaling pathway at a cellular level in different cells,
including Huh-7 hepatoma cells, H1299 non-small cell lung carcinoma cells, and Calu-3 cells. In
addition, vesicular stomatitis viruses with glycoprotein (VSV-G) and without glycoprotein (VSV-
G) were used to serve as the positive control and the negative control, respectively.
1.4. Integrins: viral transportation and viral entry receptor
Integrins are composed of heterodimeric glycoprotein α and β subunits and could serve as
transmembrane linkers and cellular receptors to mediate cell movement, adhesion, and migration
(Teoh et al., 2015). Integrins could tightly connect the extracellular matrix and intracellular actin
cytoskeleton and interact with various ligases to mediate the essential cellular signaling
transduction, thereby regulating the rearrangement of extracellular actin-based structures and
dynamic cytoskeleton (Spear & Wu, 2014). This reorganization has a crucial role in viral migration
towards the entry site on the cell surface, viral entry, viral intracellular migration, viral nucleus
translocation, and release. For example, extracellular human immunodeficiency virus (HIV) could
surf along filopodia in an actin-dependent mechanism to get close to host cells for the entry
initiation and interact with integrin-mediated and Rac1-dependent signaling pathway to induce
actin dynamics to further proceed with its entry and nuclear migration (Makowski et al., 2021).
Also, herpes simplex virus type 1 (HSV-1) activates the integrin-mediated and Rho-PISK
3
dependent pathway to mediate the filopodia formation and cellular cytoskeleton rearrangement to
facilitate viral infection (Hussein et al., 2015).
Furthermore, integrins could function as entry receptors for many different viruses with integrin-
recognition motifs to promote viral pathogen internalization, including human herpesvirus, Ross
River virus (RRV), and human immunodeficiency virus (HIV) (Spear & Wu, 2014). A tripeptides
sequence motif of Arg-Gly-Asp (RGD) is the most common motif presented on the viral surface
protein, which enables interaction with more than half of known integrins, including α5β1, αVβ5,
α2β1, αVβ6, α6β1, etc. (Lv et al., 2019).
1.5. Focal Adhesion Kinase (FAK)
After integrin ligation, focal adhesion kinase (FAK) might get autophosphorylated, followed by
the cascade of downstream signaling molecules to regulate a series of cellular functions, like
cytoskeleton reorganization, cell proliferation, migration, and immune responses (Jakhmola et al.,
2021).
1.5.1. FAK structure
Focal adhesion kinase (FAK) functions as a critical regulator of cellular communication, especially
for the integrin-mediated signaling pathway. The structure of FAK is composed of the FERM
domain, FAT domain, and the central catalytic domain (Parsons et al., 2008). First, the FERM
domain located in the NH2 terminus of FAK is composed of around 300 amino acids residues and
can directly bind to the integrin cytoplasmic portion(Zhou et al., 2019). Second, the FAT domain
in the COOH-terminus serves as the scaffold of FAK and contains several proline-rich regions that
could bind to the SH3 domain, like p130Cas, and mainly interact with focal adhesion proteins, like
paxillin, talin, and Grab2(Parsons et al., 2008). Third, as the key to FAK signaling, the central
4
kinase domain comprises around 600 conserved amino acids and six tyrosine phosphorylation
sites(Parsons et al., 2008).
1.5.2. FAK/Src complex formation
For the subsequent transphosphorylation of FAK, the autophosphorylation of tyrosine 397 (Y397)
provide a high-affinity binding site for the SH2 domain to form the activated FAK/Src complex
(Guan, 1997). The FAK/Src association could phosphorylate other tyrosine residues on FAK to
mediate FAK activity and phosphorylate paxillin and p130cas to regulate the cytoskeleton
arrangement (Cary, 1999). Different cytoskeleton proteins start to colocalize and aggregate in focal
adhesions upon FAK activation. For example, talin can directly bind to the cytoplasmic domain of
integrin β and then interact with FAK, and paxillin or tensin with talin can stimulate and sustain
FAK activation by directly interacting with FAK in the colocalization manner.
1.5.3. FAK/Src complex activity: cytoskeleton rearrangement
FAK/Src complex activates downstream Rac through the p130Cas pathway and then modulates
the activities of Rho GTPases, then results in the rearrangement of the cytoskeleton. Rho family
proteins are divided into different groups based on their homology sequence, including Rho (RhoA,
RhoB), Rac (Rac1, Rac2, Rac3), Cdc42. Since harboring the G domain that is a conserved GDP-
/GTP-binding domain, Rho family proteins could switch signals between “OFF” ( the inactivated
state, Rho GTPases) and “ON” ( the activated state, Rho GTPases) (Tomar & Schlaepfer, 2009).
As the cellular molecule switch, Rho GTPases can regulate directional cell movement, lead edge
organization, and rearrange the actin cytoskeleton (Mosaddeghzadeh & Ahmadian, 2021). For
example, Rac induces the formation of actin filaments and promotes lamellipodia protrusion to
reduce contractility at the leading edge; Cdc42 could trigger cell polarity and actin polymerization;
Rho could sustain lamellipodia growth and decrease actin polymerization.
5
1.5.4. FAK/Src complex activity: MAPK signaling pathway
Activated FAK-Src complexes could phosphorylate p130Cas and thus form FAK-Src-p130Cas
complexes that provide a binding site for the adaptor protein, Crb2, that can further bind to
exchange factors, multiple guanine nucleotide exchange factors (mSOS) (Cary, 1999). This is
followed by activating RAS, GTP binding proteins, at the plasma membranes to initiate subsequent
MAPK signaling cascades. Specifically, RAS will go through conformational change and switch
to GTP bound RAS (active), which causes the recruitment and activation of RAF, which actives
MEK, leading to the phosphorylation of ERK (Whitney et al., 2012). Eventually, Activated ERK
phosphorylates multiple substrates ranging from kinases to transcription factors because it can
translocate to the nucleus and finally transmit the extracellular signals to gene expression
mechanisms by phosphorylation of transcription factors and thus regulate critical cellular functions,
such as cell proliferation, cell cycle arrest, differentiation, and apoptosis. For example, one of
ERK’s targets is fos, an AP-1 transcription factor heterodimer member. AP-1 family transcription
factors induce the transcription of cyclin D1, which promotes cell-cycle entry (Peyssonnaux &
Eychène, 2001).
It has been reported that the MAPK cascades can be hijacked by many DNA and RNA viruses for
the sake of their infection. For example, the herpes virus activates the MAPK pathway to rearrange
the cytoskeleton for its entry and Ebola virus inherits Raf/MEK/ERK pathway to trigger
glycoprotein-mediated cytotoxicity, and influenza A virus and hepatitis C virus hijacks
Raf/MEK/ERK activity for efficient viral replication (Ghasemnejad-Berenji & Pashapour, 2021).
1.5.5. FAK/Src complex activity: innate immune responses
For innate immune responses, Integrin-activated FAK/Src complex could stimulate downstream
targets, Syk, Card9, and thus IRF3, leading to translocation of phosphorylated IRF3 into the
6
nucleus along with AP-1 and NF-kB to induce the transcription of cytokines genes, including IFN-
α, IFN-β, IL-2, and IL-10 (Gianni & Campadelli-Fiume, 2014). As to the Interferon regulatory
transcription factor (IRF3), it is the critical regulator of the innate immune system to produce the
type I IFN that plays a crucial role in protecting the host from a viral infection, such as herpes
simplex virus type 1 (HSV-1) infection and Encephalomyocarditis viruses (EMCV) infection
(Menachery et al., 2010).
1.6. SARS-CoV-2
1.6.1. SARS-CoV-2 alternative entry receptors
According to clinical results of onset of diseases among COVID-19 patients, SARS-CoV-2
infection can cause not only lung damage but also multiple organ injuries, including heart, kidney,
liver, brain, intestines, and central nervous system (Dakal, 2021). Moreover, scientists found that
in the normal respiratory system, the initial expression of ACE-2 receptor is relatively low, but
later viral infection starts to boost the ACE2 receptor expression in the airway and lung and induce
interferons production (Hikmet et al., 2020). Therefore, we hypothesize that there is an alternative
entry route for SARS-CoV-2 to facilitate its entry.
Many viruses utilize their RGD motif, a known integrin-binding tripeptides sequence, presented
on viral surface proteins that could interact with the extracellular domain of integrins to enter host
cells and then hijack cellular signaling pathways for their sustenance, such as adenoviruses and
herpesviruses (Dakal, 2021).
1.6.2. SARS-CoV-2 spike protein interacts with integrins via the GRD motif
The NCBI virus database analysis reveals a conserved RGD (Arg-Gly-Asp tripeptide sequence)
motif in the receptor-binding domain of the spike proteins (Jakhmola et al., 2021). In addition, the
7
functional domain analysis by ScanPROSITE demonstrates that spike protein could interact with
various members of integrin family receptors, including α5β1, α8β1, αvβ1, αvβ3, αvβ8, αvβ5, and
αvβ6 (Jakhmola et al., 2021). This interaction may aid in initial binding, thereby realizing viral
internalization and enhancing the S protein's binding potential to ACE-2. So, we hypothesize that
SARS-CoV-2 may also use integrins as cell co-receptors to broaden cell tropism and potentially
affect viral pathogenicity and transmission.
1.6.3. Clinical cases about the interactions between the spike protein and integrins
The analysis of pathological characteristics from severe COVID-19 patients through biopsy
demonstrates that the most devastating complication is the SARS-CoV-2-associated coagulation
disorders that appear in most organs, including the heart, liver, lungs, kidneys, etc. (Makowski et
al., 2021). It is known that activated platelet integrins, including α IIbβ3, α5β1, αvβ3, and α6β1, are
responsible for the clot-forming process, which converts fibrinogen to fibrin fibers around the
platelet surface, thereby causing platelet aggregation and adhesion. A real example of the severe
coagulopathy upon virions spreading is Hantaviruses, which can bind to integrin αvβ3 on
endothelial cells and integrin α IIbβ 3 on platelets, working as the connector between endothelial
cells and intensive platelets, and thus triggering the severe hemostasis (Koskela et al., 2021). It has
been reported that SARS-CoV-2 can interact with endothelial cells along the blood vessels during
its spreading inside the human body (Koskela et al., 2021). Therefore, the coagulation pathogenesis
among COVID-19 patients could be caused by the interaction between the RGD motif of spike
protein and integrins on endothelial cells and platelets after entering the blood vessel.
An autopsy study on the lung tissue shows that excessive angiogenesis is the most common
complication among patients who died from COVID-19 (Makowski et al., 2021). It is known that
activated integrins, such as α5β3 and αvβ5, play an essential role in promoting devastating
8
angiogenesis in pulmonary tissue (Weis & Cheresh, 2011). It is possible that the RGD motif of
SARS-CoV-2 spike protein could activate integrins and enhance the growth of new blood vessels
for the sake of their replication and infection. Since integrins are the key regulator for these two
cellular processes and the SARS-CoV-2 spike protein harbors the integrin-binding RGD motif, we
hypothesize that during the early viral infection, integrins might be the effective co-receptor or the
alternative gate for enhancing SARS-CoV-2 entry and infection.
1.6.4. Signaling pathways mediated by the interactions of SARS-CoV-2 spike protein with
integrins
The continuous COVID-19 pandemic may prove that SARS-CoV-2 has evolved a robust entry
mechanism to initiate host cell colonization and then impair the homeostasis for their replication
and sustenance. This whole process starts with the binding of viruses to cells, which binding
capability with different cellular receptors, like integrins, could ensure viral proximity to cells and
thus realize the most crucial step, the eventual internalization and infection. In our study, we found
that spike protein can activate p-ERK and p-FAK at Y397 in both Huh7 and H1299 cells, which
are the main downstream molecules in the integrin-mediated cellular signaling pathway. Moreover,
MEK inhibitor (U0126) administration enhances the SARS-CoV-2 spike protein mediated cellular
entry, and FAK inhibitor 14 could inhibit viral entry. Therefore, our findings support the idea that
the small molecular inhibitors, such as integrins blockers or FAK autophosphorylation inhibitors,
maybe a potential therapeutic target for anti-SARS-CoV-2 treatment.

9
CHAPTER 2: MATERIALS AND METHODS
2.1. Cell culture
2.1.1. BHK21-T7 cells
BHK21-T7 cells were maintained in the DMEM media supplemented with 10% fetal bovine serum
(FBS), 10mM HEPES, and 1% penicillin/streptomycin and placed in the 5% CO2 incubator at
37°C.
2.1.2. Huh-7 cells
Huh-7 cells were maintained in the DMEM media supplemented with 10% fetal bovine serum
(FBS), 10mM HEPES, and 1% penicillin/streptomycin and placed in the 5% CO2 incubator at
37°C.
2.1.3. Calu-3 cells
Calu-3 cells were cultured in EMEM media supplemented with 20% fetal bovine serum (FBS) and
1% penicillin/streptomycin and placed in the 5% CO2 incubator at 37°C.
2.1.4. H1299 cells
H1299 cells were purchased from ATCC (company name), cultured in the RPMI 1640 media
supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, and placed in
the 5% CO2 incubator at 37°C.
2.2. Production of pseudoviral particles: VSV- G, VSV-G, VSV-SARS-CoV-2-S
Seed BHK21-T7 cells to be 90% confluent before transfection. Wash 10cm plate with 5ml of pre-
warmed serum-free-DMEM and add 9ml of serum-free-DMEM. Dilute lipofectamine 3000
reagents in Opti-MEM and mix well. Prepare plasmid master mix. Dilute packaging mix and pBS
10
expression vector in Opti-MEM, add P300 Enhancer Reagent, and mix well. Specifically, add 6,
10, 16, and 2ug of pBS-N, P, G, and L, respectively, to Opti-MEM. Briefly vortex, add 15ug of
ΔG-Luc (genome) and 35ul P3000 reagent. Gently vortex. Combine plasmid master mix and
transfection reagent solution. Incubate for 15minutes at room temperature. Add the transfection
mixture to a 10cm plate containing BHK21-T7 cells at 90% confluency. Rock the plate to the side
and back and forth to mix evenly. Plate the plate back to 37 ˚C, 5% CO2 incubator. 6 hours later,
remove the combined mixture and add 10ml of DMEM containing 5% FBS. 24hours later, check
the cells for CPE and harvest the supernatant containing rVSV-ΔG-Luc from the 10cm plate and
transfer it to a centrifuge tube. Centrifuge the pseudoparticle solution (1000RPM for 4 minutes) to
pellet any cells and cell debris. Transfer the supernatant to 1ml cryovials and can be stored at -
80 ℃.
Instead of performing plasmid master mix transfection every time, rVSV-ΔG-Luc from this
process can be amplified. It can be done by completing the detailed protocol from step2(Fig.1).
Transfection: To transfect BHK21-T7 cells in a 10cm plate, add dilute lipofectamine 3000
reagents in 1.5ml Opti-MEM media to 8ug pBS-G vector and P3000 enhancer reagent in 1.5ml
Opti-MEM media or 24ug p-CAGGS-SARS-Cov-2-S vector and P3000 enhancer reagent in 1.5ml
Opti-MEM media and mix well. Incubate for 15minutes at room temperature. Then add 3ml
transfection mixture to the BHK21-T7 VSVG and VSVS production cell line. Six hours later,
change media to 10ml warm complete DMEM. VSVG plate should show strong CPE. Infection:
24hours later, discard 5ml from 10cm plate and add 100ul rVSVG to cell media. One 1hour later,
gently wash the cells 3times with warm DPBS and add 10ml of 10% FBS DMEM to the cells.
Harvest: Collect the cell media containing VSV- G, VSV-G, or VSV-S and filter it through a
11
45um pore size filter. Then transfer the supernatant to 2ml cryovials, and either uses it immediately
to perform a titration or store it at -80 ˚C.
2.3. Viral gene expression analysis
2.3.1. Luciferase expression in the viral particle production cell line (BHK-21-T7)
After harvesting viral particles, add 2ml 1x Passive lysis buffer. Put the plate in a shaker for 5
minutes at room temperature. Add 100ul luciferase substrate and 20ul lysis buffer to each well of
the 96-well plate and mix well. Measure bioluminescence using the Promega machine.
2.3.2. Quantitative Real-Time PCR Analysis
2.3.2.1. RNA extraction
Add 0.75ml Trizol LS Reagent to 0.5ml pseudoviral particle media and mix well by pipetting and
then leave it at room temperature for 5min for complete dissociation of nucleoproteins complex.
Add 200ul chloroform, then incubate for 2 minutes at room temperature. After centrifugation at
12k rpm for 15 min at 4 °C, transfer the aqueous phase containing RNA to a new tube and add
0.5ml of isopropanol. After incubating at room temperature for 10min, total RNA was pelleted by
centrifugation for 10 min at 12k rpm at 4°C. Then the RNA pellet was resuspended with 1ml of
75% ethanol and washed by centrifugation at 73k rpm for 5min at 4°. Lastly, the pellet was dried
in the air, and the RNA was eluted with 20 ul RNase-free water.
2.3.2.2. DNA free
Add 0.1 volume 10x TURBO DNase Buffer and 1ul of TURBO DNase Enzyme to the RNA and
then mix gently. Incubate the sample in the 37°C incubator for 30min. Add 0.1 total volume of
resuspended DNase Inactivation reagent, then mix well by vertexing. Then transferring the
supernatant containing RNA to a fresh tube after centrifugation at 10k RCF for 90min.
12
2.3.2.3. Reverse transcription PCR
Anneal primer to template RNA: prepare RNA-primer mix: 1ul of 50uM random hexamers, 1ul
of 10mM dNTP mix, 11ul of template RNA (contain Nulease-free H2O), then mix well and briefly
centrifuge it. The RNA-primer mixture was heated at 65°C for 5minutes and then incubated on ice
for 2 minutes. Prepare RT reaction mix: the following components were combined: 4ul of 5x
SSIV buffer, 1ul of 100mM DTT, 1ul of RNsse OUT Recombinant RNase inhibitor, and 1ul of
superscript IV Reverse transcriptase, then mixed well and briefly centrifuged. Combine annealed
RNA and RT reaction mix: add a 7ul RT reaction mix to each well-containing RNA sample.
Incubate the reaction: incubate the combined mixture at 23°C for 10min, then at 50-55°C for
10min, followed by 80°C for 10min.
2.3.2.4. Quantitative Real-Time PCR
Total RNA was reversely transcribed using SuperScript™ III Reverse Transcriptase
(Invitrogen) with three ug of input RNA and 1 ul Oligo(dT)12-18 primers (Invitrogen). Quantitative
real-time PCR reactions were performed in 96-well plates with Power SYBR Green PCR Master
Mix using 4 ul of the 1:20 cDNA dilutions (in DEPC water) in a volume of 20 ul with the specific
primers. The qPCR was run with the following parameters: denaturation at 95°C for 10 min,
followed by 40 cycles of denaturation at 95°C for 1 min, and annealing at 60°C for 1 min. Each
reaction was performed in duplicate. All results were normalized to GAPDH mRNA level and
calculated using the ddCt method.
2.3.3. Immunofluorescence
Huh-7, H1299, or Calu-3 were seeded to 8well chamber slides overnight. Cells were infected with
different amounts of VSV- G, VSV-G, or VSV-S particles for one hour. Cells were washed with
13
DPBS three times, and then fresh culture media was added to each well. 24hours later, the cells
were washed with 1x PBS and fixed using 4% paraformaldehyde, followed by the permeabilization
with 0.3% Triton X-100 detergent in 1x PBS. The fixed cells were blocked using the blocking
buffer (PBS with 1% BSA, 0.02% saponin, 0.05% sodium azide). The cells were then incubated
with mouse anti-VSVN antibody (1:200) at 37°C. After 1h, discard the primary antibody solution
and wash the cells with wash buffer three times. Incubate the cells with 1:100 dilution of secondary
antibody in the dark. Then the cells were washed three times with the wash buffer. Remove the
media chamber carefully, scrape the glue off and then mount the cells with DAPI Fluoromount-G
(SouthernBiotech; 0100-20). Image the slide the following day using the fluorescence microscope.

2.4. Infection assay using VSV- G, VSV-G, or VSV-S pseudoviral particles
Huh-7, H-1299, or Calu-3 were seeded to the 12 well plate overnight. Cells were infected with the
same M.O.I. of VSV- G, VSV-G, or VSV-S particles for 30 minutes. Then wash each well three
times with 1X PBS, adding 100ul 1X passive lysis buffer for 5 minutes at room temperature. Then
perform the centrifugation at 14k RCF for 5 minutes at 4
o
C. Then the samples were denatured in
sodium dodecyl sulfate (SDS) buffer and boiled for 5 min. Samples were electrophoresed on 8%
or 10% polyacrylamide gels, followed by semi-dry transfer. The transfer membrane was incubated
in a cool room overnight with a 1 in 10000 ratios of primary antibody to blocking buffer.
2.5. Drug treatment assay
H7, H1299, or Calu-3 were seeded to the 12 well plate overnight. Cells were infected the same
M.O.I. of VSV- G, VSV-G, or VSV-S particles for 30 minutes. Then wash each well three times
with 1X PBS, adding 100ul 1X passive lysis buffer for 5 minutes at room temperature.
14
CHAPTER 3: RESULTS
3.1. Construction of the first generation of VSV-G particles
The pBS-ΦT vector was used to encode different vesicular stomatitis virus (VSV) proteins, including
Nucleocapsid(N), phosphoprotein(P), matrix(M), luciferase (Luc), polymerase (L). This vector
contains a bacteriophage T7 promoter that allows the expression of the recombinant protein in BHK21-
T7 cells. It also includes a T7 terminator sequence and an ampicillin resistance gene. The luciferase
expression gene, which served as a reported gene, provides the convenience of measuring viral
replication inside the cells. The generation of VSV-G particles requires the transfection of these five
different plasmids into the BHK-21-T7 cell line with the expression of the G receptor on the cell
surface, thereby leading to the transcriptionally active VSV template.

Figure 1. construction of the first generation of VSV-G particles inside the BHK21-T7 cells. (A) the plasmid master
mix: (N): Nucleocapsid; (L): polymerase; (P): Phosphoprotein; (M); matrix; (Luc): luciferase. (B) p-CAGGS-VSV G
was put into cells to encode cell membrane with VSV-Glycoprotein. (C) all input plasmids were expressed and
assembled to produce a transcriptional template.
A
15

3.2. Construction of the next generation of pseudoviral particles
After the first step, VSV-ΔG-Luc can be amplified by performing the below-detailed steps.
BHK21-T7 cells were transfected with p-CAGGS-VSV G or p-CAGGS-SARS-CoV-2-S plasmid.
After 24 hours, VSV-G particles were added to acquire the next generation of VSV-pseudoviral
particles, VSV-G or VSV-S, respectively. After collecting the media, the cells were lysed with 1x
passive lysis buffer to measure luciferase protein expression inside the transformed cells.

Figure 2. The production of pseudoviral particles, including VSV- G, VSV-G, and VSV-S.

16
3.3. Pseudoviral particles infection assay
3.3.1. Infection of Huh-7 cells
Our study focused on the cellular signaling pathway caused by the SARS-CoV-2 entry into host
cells and how we can find a novel therapeutic target to block viral entry and further transmission
to neighboring cells. For this, we first investigate the MAPK signaling cascades. We observed the
bands of the expression of phosphorylation of ERK in vesicular stomatitis virus (VSV) particles
bearing SARS-2-S during both 30min and 1-hour infection, compared to negative group (VSV-
ΔG) and positive group (VSV-G). Moreover, we further confirmed this activation result by using
different particles. This consistent result indicated that p-ERK and p-FAK at Y397 is upregulated
by the spike protein of SARS-CoV-2.

A B
17

Figure 3. The expression of phosphorylation of ERK by Western Blot. (A) Huh-7 cells were infected by the negative
control pseudoviral particles (VSV-ΔG), the positive control pseudoviral particles (VSV-G), and the experiment group
(VSV-S) for different time points including 30min and 1hour. (B) The same experiment was conducted again to
confirm the left-figure result. (C) Huh-7 cells were infected by the negative control pseudoviral particles (VSV-ΔG),
the positive control pseudoviral particles (VSV-G), and the experiment group (VSV-S) for different time points
including 30min and 1hour.
3.3.2. Infection of Calu-3 cells
Since most COVID-19 patients get severe complications in the lung cells, known as the main host
cells for SARS-CoV-2 infection, we use Caul-3 cells, the lung epithelial carcinoma cell line, as
the viral entry study model. However, the western blot result shows that the infection of VSV-
SARS-CoV-2-S did not activate p-ERK during 30min, 1 hour, and 2 hours infection.
C
18

Figure 4. The expression and phosphorylation levels of target proteins were analyzed by western blotting. Calu-3 cells
were infected by three different pseudoviral particles, including the negative control pseudoviral particles (VSV-ΔG),
the positive control pseudoviral particles (VSV-G), and the experiment group (VSV-S), for different time points,
including 30min, 1hour, and 2 hours.

3.3.3. Infection of H1299 cells
In order to understand why VSV-SARS-CoV-2-S infection cannot elicit the expression of p-ERK
as it does in H7 cells, we next examined it in H1299, another lung epithelial carcinoma cell line.
The results show that VSV-SARS-CoV-2-S particles activate the p-FAK at Y397.
19

Figure 5. The expression and phosphorylation levels of target proteins were analyzed by western blotting. H1299 cells
were infected by three different pseudoviral particles, including the negative control pseudoviral particles (VSV-ΔG),
the positive control pseudoviral particles (VSV-G), and the experiment group (VSV-S), for different time points,
including 30min and 1hour.

3.4. Immunofluorescence assay
In order to keep different particles-driven infection at the same M.O.I., Huh-7, Calu-3, and H1299
cells were stained with anti-VSV-N mouse primary antibody and FITC anti-mouse secondary
antibody. We found that the amount of VSV-S particles must be 100-fold more than VSV-G to
achieve the same infection percentage. Intriguingly, even for VSV-G with high infection efficiency,
the percentage of infected Calu-3 cells is relatively low, which could answer why the expression
of p-ERK did not change in all different infection time points, including 30 minutes, 1 hour, and
2 hours, and also explain why there are no differences of luciferase expression in VSV-SARS-
CoV-2-S particles-driven entry group with the U0126 treatment. Moreover, immunofluorescent
staining in H1299 cells shows high infection efficiency for VSV-S-drive entry.
20

Calu-3
H1299
VSVG-H7 VSVS-H7
Huh-7
Blue: DAPI
Green: VSV-N (VSV-Nucleocapsid) Ab

21
Figure 6. Immunofluorescence staining of infected Huh-7, Calu-3, and H1299 cells. After Huh-7 cells were infected
by three different pseudoviral particles, including the negative control pseudoviral particles (VSV-ΔG), the positive
control pseudoviral particles (VSV-G), and the experiment group (VSV-S). The amount of VSV-S is 100-fold more
than VSV-G. VSV-N protein was stained with anti-VSV-N mouse antibody and FITC anti-mouse secondary antibody.

3.5. Drug treatment assay
3.5.1. U0126 treatment assay in the Huh-7 cell line
Our previous results show that the spike protein of SARS-CoV-2 could activate p-ERK in both
Huh-7 and H1299 cell lines. So, we want to further test whether the treatment of cells with ERK
inhibitor prior to infection would prevent the activation of p-ERK by the VSV-SARS-CoV-2-S
pseudoviral particles. U0126, a known MAPK pathway inhibitor, was used to treat cells first,
followed by the viral infection. Then luciferase activities were measured 24 hours later to evaluate
the effects on viral entry. However, opposite to our hypothesis, the luciferase results suggested
that MAPK inhibitor administration could enhance viral access. U0126 inhibitor treatment resulted
in a more than three-fold increase of VSV-SARS-CoV-2-S pseudoviral entry in Hu7 cells.

A B
22
Figure 7. The expression and phosphorylation levels of target proteins were analyzed by western blotting. Huh-7 cells
were treated with U0126 for 1-hour prior to infection and then were infected with different pseudoviral particles,
including the negative control pseudoviral particles (VSV-ΔG), the positive control pseudoviral particles (VSV-G),
and the experiment group (VSV-S). (B) After 24 hours, the luciferase assay was conducted to analyze the viral entry.

Figure 8. Different doses of U0126 inhibitor treatment in Huh-7 cells. Graphs represent the fold change of viral entry
measured by luciferase protein expression. Statistical significance is determined with a P-value.

3.5.2. U0126 treatment assay in the Calu-3 cell line
We also evaluated the effects of M.E.K. inhibitor, U0126, on viral entry in Calu-3 cells. The no
differences in luciferase results revealed that all these three viral particles entries did not be
affected by M.E.K. inhibitor treatment. The reason could be the low infection efficiency of
pseudoviral particles in Calu-3 cell lines, which showed in Figure3.
23

Figure 9. The expression and phosphorylation levels of target proteins were analyzed by western blotting. Calu-3 cells
were treated with U0126 for 1-hour prior to infection and then were infected with different pseudoviral particles,
including the negative control pseudoviral particles (VSV-ΔG), the positive control pseudoviral particles (VSV-G),
and the experiment group (VSV-S). (B) After 24 hours, the luciferase assay was conducted to analyze the viral entry.

3.5.3. U0126 treatment assay in the H1299 cell line
In order to evaluate the transferability of the result of M.E.K. inhibitor treatment in Huh-7 cells in
the lung cell system, we also performed the drug treatment experiments in H1299 cells. We
observed the viral infection exacerbation after 10uM U0126 treatment, which is consistent with
results from the Huh-7 model.
A B
24

Figure 10. The expression and phosphorylation levels of target proteins were analyzed by western blotting. H1299
cells were treated with U0126 for 1-hour prior to infection and then were infected with different pseudoviral particles,
including the negative control pseudoviral particles (VSV-ΔG), the positive control pseudoviral particles (VSV-G),
and the experiment group (VSV-S). (B) After 24 hours, the luciferase assay was conducted to analyze the viral entry.

Figure 11. Different doses of U0126 inhibitor treatment in Huh-7 cells. Graphs represent the fold change of viral entry
measured by luciferase protein expression. Statistical significance is determined with a P-value.

3.5.4. FAK inhibitor 14 treatment assay in the H1299 cell line

A B
25
We further explore the viral entry mechanism with the FAK inhibitor treatment assay in H1299
cells using recombinant VSV- ΔG, VSV-G and VSV-S particles. The western blot results show
the specific inhibition of FAK inhibitor 14 at the tyrosine 397 of p-FAK, and the luciferase
expression analysis results show that FAK inhibitor 14 has the specific inhibition effect towards
viral entry mediated by the SARS-CoV-2 spike protein, compared to negative group (VSV-ΔG)
and positive group (VSV-G).

Figure 12. (A) The expression and phosphorylation levels of target proteins were analyzed by western blotting. H1299
cells were treated with FAK inhibitor 14 for 2-hour prior to infection and then were infected with different pseudoviral
particles, including the negative control pseudoviral particles (VSV-ΔG), the positive control pseudoviral particles
(VSV-G), and the experiment group (VSV-S). (B) After 24 hours, the luciferase assay was conducted to analyze the
viral entry.
A B
26

Figure 13. Different doses of FAK inhibitor treatment in H1299 cells. Graphs represent the fold change of viral entry
measured by luciferase protein expression. Statistical significance is determined with a P-value.

3.5.5. Miltefosine treatment assay in the BSL-3 lab with the real SARS-CoV-2 infection
Caul-3 cells were infected by the real SARS-CoV-2 in the BSL-3 lab with the different time
courses of Miltefosine treatments, including one-hour pretreatment (-1h), and no pretreatment (0h),
and one-hour post-treatment (+1h). Compared to the uninfected group, the expression of p-ERK1/2,
p-FAK Y397, and p-FAK Y925 get upregulated in the group infected by the real SARS-CoV-2.
Next, we would go to the BSL-3 lab to test viral entry activities affected by the FAK inhibitors.

27
Figure 14. The expression and phosphorylation levels of target proteins were analyzed by western blotting.

3.6. The analysis and modeling of possible signaling pathways induced by the interactions
of SARS-CoV-2 spike protein with different human cellular receptors
Based on our results from this project and our analysis and screening of the signaling pathways
affected by the viral entry driven by the SARS-CoV-2 Spike protein, we summarize and model the
possible host signaling pathways induced by the interactions of SARS-CoV-2 Spike protein with
different cellular receptors.

Figure 15. The summary and modeling of the possible signaling pathways induced by the interactions between SARS-
CoV-2 Spike protein and different cellular receptors.

28
CHAPTER 4: DISCUSSION
In this study, we aimed to study the host signaling pathway initiated by the SARS-CoV-2 spike
protein by utilizing recombinant pseudoviral particles with VSV-SARS-CoV-2-spike protein
expression. After the infection of VSV-SARS-CoV-2-S pseudoviral particles, we found that p-
ERK and p-FAK (Y397) get activated in both Huh-7 and H1299 cell lines compared to the negative
control (VSV- G) and positive control (VSV-G). This activation could reveal the entry
mechanism of action of spike proteins through the integrin-mediated FAK signaling pathway and
could be the potential therapeutic target against SARS-CoV-2 infection. We further explore the
viral entry mechanism with a drug treatment assay by measuring luciferase protein expression. It
demonstrated that U0126 (MAPK inhibitor) could enhance viral entry, and FAK inhibitor 14 could
inhibit viral entry into H1299 and Huh7 cell lines. Currently, we are attempting to figure out the
antiviral mechanism of FAK inhibitor 14 and to study if integrin blockers could decrease viral
entry to discover if integrins could act as the alternative entry receptor for SARS-CoV-2.
Although multiple COVID-19 vaccines have been available to effectively protect the public from
getting severe symptoms, hospitalization, and dying from SARS-CoV-2 infection, frequently
emerging SARS-CoV-2 variants still challenge our vaccine effectiveness. It has been reported that
the latest variant, Omicron, harbors over 30 mutations in the spike protein alone and over half of
these mutations appear in the receptor-binding domain of the spike protein (Kim et al., 2021).
Since the Omicron variant causes suddenly arouse surges of cases around the world even in the
third year of the pandemic, it is crucial to know whether this highest transmission rate is associated
with these mutations sites in spike protein and to further investigate how mutant spike protein
facilitate viral infection of host cells, like through broadening its entry receptors choices or through
manipulating cellular signaling cascades.
29
In this study, we found that the expression of p-ERK gets upregulated after the infection of VSV-
SARS-CoV-2-S pseudoviral particles. Based on the infection mode of SARS-CoV-1, which is that
the treatment of MAPK inhibitors can restrain viral infection, we hypothesized that the spike
protein could manipulate this kinase activation, so if we treat infected cells with the MAPK
inhibitor (U0126), it will block viral entry. But our drug treatment results show that the MAPK
inhibitor administration can enhance viral entry, in contrast to our hypothesis. However, this result
is consistent with the authentic SARS-CoV-2 study (Mirabelli et al., 2021). It has been reported
that administration of all MAPK inhibitors, including cobimetinib, trametinib, binimetinib, and
U0126, can exacerbate the viral infection (Mirabelli et al., 2021). They concluded that the
mechanism could be MAPK inhibition could accelerate SARS-CoV-2 spread to neighboring cells
by developing more multinucleated cells and promoting the intracellular diffusion of viral RNA.
and S protein(Mirabelli et al., 2021). Thus, it is still important to further investigate this cellular
pathway caused by viral infection because it could discover more infection modes of SARS-CoV-
2.
It has been shown that SARS-CoV-2 possesses a very high transmission ability and can cross a
variety of animal species(Rodrigues et al., 2020). This robust adaptability is initiated from the
receptor-mediated entry process. So, studying the interaction of spike protein and cellular receptors
is crucial for inhibiting viral infection and preventing further transmission. It has been reported
that, unlike SARS-CoV-1, SARS-CoV-2 spike protein contains R.G.D. tripeptide sequence near
the ACE-2 interaction site and could bind to integrin molecules by the computational analysis of
the structural databases.(Jakhmola et al., 2021). Moreover, our results show that the SARS-CoV-
2 spike protein could activate the FAK-Src complex (Y397) and its downstream MAPK pathway,
both of which are involved in the integrin-mediated cell signaling pathway.
30
Thus, it is plausible that integrins could be the entry receptors for SARS-CoV-2 and could enhance
the interaction between spike protein and other known cell receptors, like ACE-2, thereby
accelerating viral internalization. Therefore, it is necessary to figure out if the integrin-binding
blockers, such as the antibody natalizumab against α4β1 and tirofiban against αIIbβ3, could inhibit
SARS-CoV-2 spike protein-mediated cell entry. Since there are currently very limited anti-SARS-
CoV-2 small-molecule blockers, these specific integrin-binding inhibitors could provide a novel
therapeutic target for COVID-19 treatment and reveal a new biological mode of SARS-CoV-2
infection. Besides this, to synthesize the R.G.D. derivatives to block integrins could also be the
alternative way to inhibit viral attachment. However, since integrins are composed of
transmembrane αβ heterodimers through the dynamic association of at least eighteen α and eight
β subunits, certain integrin blockers could have difficulties to specifically inhibit the interaction
between integrins and spike protein(Campbell & Humphries, 2011).
The ACE2 receptor is a known cell entry receptor for SARS-CoV-2, but the data showed that it
only exists in limited types of lung cells with low expression(Aboudounya & Heads, 2021). In
addition, over 60% of COVID-19 death cases suffer from devastating liver damage where was
tested high SARS-CoV-2 infection (Makowski et al., 2021). So, it is very curious how SARS-
CoV-2 broaden their cellular attachment options and launches the intensive infection rapidly in
cells with limited presence of ACE-2 receptors. Recently, studies have found that SARS-CoV-2
spike protein binds and activates the TLR4, which could be a reason for the acute hyper
inflammation complication (Gadanec et al., 2021). Moreover, the interaction between spike protein
and TLR4 enables to boost of the cellular ACE2 expression to rapidly establish more colonies for
SARS-CoV-2 and sustain their survival. Furthermore, integrins could cooperate with toll-like
receptors to initiate proinflammation rather than antiviral responses(Aboudounya & Heads, 2021).
31
For example, the attachment of HSV envelop proteins (gH/gL) with αvβ3 and TLR2 enables the
broadening of its cellular spectrum. So, it is suggested that further study of other cellular receptors
except ACE-2 used by SARS-CoV-2 spike protein is necessary to discover more viral entry
mechanisms.
Our results demonstrate that the FAK signaling pathway mediated by integrins is essential for viral
entry. It will thus be interesting to determine if integrins blockers or Src inhibitors could be
potential therapeutic options for SARS-CoV-2 treatment. And it will be interesting to extend this
study to primary human lung epithelial cells or immune cells, like macrophages, to explore more
host-virus interactions. Also, it would help us understand more the transmission mechanism of
SARS-CoV-2 by mutating the RGD motif of SARS-CoV-2 to the KGD motif of SARS-CoV-1.
Additionally, it is still necessary to compare the surface receptor expression levels of ACE2 and
other receptors in Huh-7, H1299 and Calu-3 cell lines after SARS-CoV-2 infection. Moreover, it
is also essential to use real SARS-CoV-2 to test our findings in the BSL-3 lab.

32
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Abstract (if available)

Abstract SARS-CoV-2, as the causative pathogen of COVID-19, has caused one of the most severe pandemics in human history. To date, the world has seen over 6 million deaths, and almost 60% of Americans have been infected at least once. Although around 70% of the U.S. population has been fully vaccinated, emerging new variants pose new challenges and reduce current vaccine effectiveness. These variants cause a surge of cases that overwhelm our healthcare system. Due to the large number of people who have been infected, better antiviral compounds and more convenient home-based therapeutic options are urgently needed for the treatment of post-COVID-19 symptoms. So, it is crucial to investigate the host-virus interactions to deeply understand the mechanisms of viral adaptability and infectivity. In this report, we mainly focus on studying the virus-host signaling pathways mediated by the spike protein during the viral entry to discover small intracellular molecules affected by the viral infection, which could be novel potential antiviral therapeutic targets. We used the recombinant vesicular stomatitis virus with the expression of SARS-CoV-2 spike protein to infect different cell lines and compared them with the negative control (VSV-G) and the positive control (VSV-G). We found the induction of phosphorylation of FAK at tyrosine-397 (Y-397) and ERK 1/2 by the SARS-CoV-2 spike protein. To understand these interactions between the spike protein and FAK or ERK, we performed the drug assay using corresponding inhibitors to test their effects on viral entry. The results show that MAPK inhibitors could enhance viral entry, and FAK inhibitor 14 could inhibit viral entry. So, the results demonstrate that these two kinases can affect viral entry initiated by the spike protein. Additionally, both p-FAK(Y-397) and p-ERK are critical components of the integrin-mediated signaling pathway. Therefore, identifying this kinase upregulation will allow us to further investigate the entry mechanism of SARS-CoV-2, understand virus-host interactions, and discover novel potential therapeutic targets against COVID-19 infection.

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Asset Metadata

Creator Hou, Pingping (author)

Core Title The analysis and modeling of signaling pathways induced by the interactions of the SARS-CoV-2 spike protein with cellular receptors

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2022-08

Publication Date 07/29/2022

Defense Date 05/05/2022

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

Tag ACE-2,cellular receptors,ERK,FAK,integrin,molecular interactions,OAI-PMH Harvest,RGD motif,SARS-CoV-2,signaling pathways,spike protein

Format application/pdf (imt)

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Ou, Jing-Hsiung James (committee chair), Machida, Keigo (committee member), Yuan, Weiming (committee member)

Creator Email houp@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC111375554

Unique identifier UC111375554

Legacy Identifier etd-HouPingpin-11039

Document Type Thesis

Format application/pdf (imt)

Rights Hou, Pingping

Type texts

Source 20220729-usctheses-batch-963 (batch), University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

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