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Mechanism of ethanol-mediated increase of SARS-COV-2 entry through GRP78 induction

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Mechanism of ethanol-mediated increase of SARS-COV-2 entry through GRP78 induction

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MECHANISM OF ETHANOL-MEDIATED INCREASE OF SARS-COV-2 ENTRY THROUGH
GRP78 INDUCTION
by
Nurzhan Mukashev

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 2022

Copyright 2022 Nurzhan Mukashev
ii

Acknowledgments
I want to sincerely thank the University of Southern California for letting me realize my desire to
attend this school. I also want to express my gratitude to the Molecular Microbiology and
Immunology Department for its support and advice throughout my time at USC. Without the
assistance of excellent mentors who pushed, encouraged, and advised me every step of the way, I
would not have been able to finish this thesis.
I express my sincere gratitude to Dr. Keigo Machida, the committee chair and principal
investigator (PI), for always encouraging me to reach my best potential. Being a member of Dr.
Machida's lab was a great honor, and I appreciate all the time he has invested in our one-on-one
meetings. His support and direction made finishing long, laborious days of work incredibly
rewarding. I would like to thank Dr. Hye Choi and Carlos Hernandez for instructing me in various
experiments and showing me how to use the lab's facilities. They were a huge help to me during
the assay procedure when it came to gathering data and ordering materials. I also want to express
my gratitude to the members of my committee, Dr. Jing-Hsiung James Ou, Dr. Weiming Yuan,
and Dr. Pinghui Feng, who deserve special mention.

iii

Table of Contents

Acknowledgments………………………………………………………………….…....…….…. ii
List of Tables……………………………….…………………………….……...……...…...……iv
List of Figures……………………………………………………….……………...…..…...….…v
Abstract……………………………………………………………………………...……....……vi
Chapter 1: Introduction ……………………………………………...……………..…………..….1
Chapter 2: Material and Methods…………………………………………..………...……………5
Chapter 3: Results and Discussion…….…………………...…..……………..…………………..11
Chapter 4: Summary…………….…………………...……………………………………..…….21
Chapter 5: Future Directions……………………………….……………..…………………...…22
References………………………………………………………………………………………..23

iv

List of Tables
Table 1. The components volume in restriction enzyme digestion…………......…….……………6
Table 2. The components volume in ligation reaction…………......………………………………7
Table 3: Primers used for amplification of hGRP78 promoter regions …………......…….…….15
Table 4: Plasmids used in the present study.……………………….………....……….…..…….16
Table 5: Primers for site-directed mutagenesis of GRP78 promoter cis-acting elements…….....20

v

List of Figures
Figure 1: Hypothetical model ……………………………………………...……………………. 4
Figure 2: Schematic diagram of SARS-CoV-2 pseudotyped virus production ………………....10
Figure 3. High-throughput screening of SARS-CoV-2 inhibitors identified nine potential
cyclic peptides for VSVpp-SARS-CoV-2 Spike envelope proteins ………………………….…13
Figure 4: Amplified regions of hGRP78 promoter ..……………………………………...…..…15
Figure 5: Example of colonies after ligation …………………………..………………………...16
Figure 6: Diagnostic restriction digestion of PCR products ………………………….…………17
Figure 7: Effect of ethanol on GRP78 promoter constructs …………………..……………….. 18
Figure 8. IPA based prediction of ethanol induced pathway and TF of HSPA5 gene …..…….. 19
Figure 9. TFBS of -189 bp … +33 bp region of GRP78 promoter sequence ………………..… 20

vi

Abstract
The continuing pandemic of coronavirus disease 2019 (COVID-19) poses an unmet medical need.
To address current global health crises, it is an urgent priority to find effective prevention and
treatment against COVID-19 caused by the severe acute respiratory syndrome coronavirus 2
(SARS-CoV-2). The virus utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for
entry and interacts through viral spike protein. As the initial stage of infection, the entrance of
SARS-CoV-2 into host cells represents a therapeutic intervention point. We identified and
optimized candidate compounds from the FDA-approved library that inhibit SARS-CoV-2 entry.
One of the co-factors of ACE2 is GRP78, which has been recently shown to facilitate the
infectivity of SARS-CoV-2.
Along with other factors causing ER stress inducers, ethanol is one of the most potent inducers.
We hypothesized that ethanol treatment could increase viral entry through ethanol mediated
GRP78 overexpression. Using promoter-reporter assay, we identified the minimum required
region for alcohol induction of the HSPA5 gene. Further bioinformatic analysis revealed potential
transcription factor binding sites. This study aims to demonstrate the role of alcohol in developing
SARS-CoV-2 infection.
1

Chapter 1. Introduction
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible and
pathogenic representative of the Coronaviridae family that emerged in late 2019. SARS-Cov-2
has caused a worldwide pandemic of acute respiratory disease COVID-19), which threatens human
health and public safety. Many therapeutic targets, including the human ACE2 cell surface receptor,
play a crucial function in virus entry. SARS-Cov-2 is an enveloped virus with a large diameter
(60-140 nm) and a non-segmented, positive-sense, single-stranded (ss-(+))-RNA genome of 30 kb,
the most extended viral RNA genome observed to date. Cryo-electron microscopy (cryo-EM)
demonstrates that, like other coronaviruses, SARS-CoV-2 has a spherical shape, with Spike (S)
glycoproteins extending from the virion surface and providing it a crown-like look (1). Together
with envelope (E) and membrane (M) proteins, the S protein forms the viral envelope, whereas the
nucleocapsid (N) peptide, located within the envelope, connects, and preserves the RNA genome.
The connection between the receptor-binding domain (RBD) inside the S1 domain and the human
angiotensin-converting enzyme 2 (ACE2) receptor is essential for tissue tropism and attachment
of the SARS-CoV-2 S glycoprotein to the host cell. The S protein is a 1200 aa long homotrimeric
class I fusion protein, produced in the secretory pathway of the host cells, with three segments: a
large ectodomain with receptor-binding subunits S1 and S2, a single-pass transmembrane anchor,
and a short intracellular end [2]. The N-linked glycans projecting from the trimer side are
responsible for S folding and neutralizing antibody recognition (Abs) (3). This essential interaction
can be inhibited to prevent viral entry. Hsiang et al., using a biotinylated enzyme-linked
immunosorbent assay (ELISA), found that small peptides disrupt the interaction between the
SARS-CoV S protein and ACE2 (4).
2

Even though SARS-Cov-2 Spike and hACE2 interaction play a crucial role in viral entry, other
factors can serve as critical entry cofactors for viral infection (5, 6). Recent analyzes of molecular
docking have identified a putative interaction site between the 78 kDa glucose-regulated protein
(GRP78) and the receptor-binding domain (RBD) of SARS-2-Spike protein, increasing the
likelihood that GRP78 may serve as an additional receptor for SARS-CoV-2 entry (7).
Computer modeling shows that host-cell interaction through GRP78 enhanced in the mutated UK
variant of SARS-CoV-2 associated with higher transmissibility and the new 501.V2 South African
variant (8, 9). GRP78, also called BiP and encoded by the HSPA5 gene, is the primary HSP70
family member in the endoplasmic reticulum (ER), supporting essential protein folding tasks (10,
11). In addition, GRP78 is a master regulator of the unfolded protein response, allowing cells to
adjust to adverse stress circumstances targeting the ER (12, 13). GRP78 is extensively expressed
in various organs, particularly bronchial epithelial cells, and the respiratory mucosa. In relevant
case studies, serum GRP78 levels were higher in SARS-CoV-2 infections (14). Under pathological
situations such as cancer and viral infection, GRP78 can translocate from the ER to the cell surface,
acting as a coreceptor for different signaling molecules and viral entry (13, 15, 16). GRP78 is
reported to interact with bat coronavirus HKU9 and MERS CoV Spike proteins, facilitating cell-
surface attachment and viral entry (17, 18).
Furthermore, virus infection leads to ER stress and increased total and cell surface GRP78
(csGRP78) expression, further increasing viral infection in a positive feedback cycle. GRP78 can
form a complex with SARS-2-S and ACE2 on the surface, and at the perinuclear area typical of
the endoplasmic reticulum in VeroE6-ACE2 cells substrate-binding domain of GRP78 is necessary
for this interaction. Knockdown of csGRP78 dramatically decreased SARS-Cov-2 infection,
suggesting GRP78 as a possible therapeutic target (19).
3

Alcohol considerably enhances GRP78 expression (20). Reports also indicate that people with
alcohol use disorder (AUD) are susceptible to contracting this virus and developing severe COVI-
19 illness consequences (21, 22). Current investigations reveal that many people with mild and
severe COVID-19 disease consume alcohol. Alcohol abuse is estimated to be responsible for 3,3
million fatalities annually, or around 6 percent of all deaths worldwide. Alcohol intake has been
connected to more than 200 life-threatening diseases. Significantly increasing the chance of death
by 1.8-fold, alcoholic liver disease is an important predictor of COVID-19 mortality. Both Covid-
19 infection and AUD are relatively prevalent conditions. There are 2.4 billion alcoholics globally,
with 1.5 billion (1.4–1.6 billion) male drinkers and 0.9 billion (0.8–1.0 billion) female drinkers. In
the western world, per capita consumption ranges from 8 to 12 liters, although in Asia, there is a
gradual growth (such as in China, 6–8 liters) (23). The prevalence of AUD in the adult population
ranges from 8.8 percent (Europe) to 0.8 percent (Eastern Mediterranean Region) throughout the
previous several years (24). In addition, the media claim an increase in alcohol intake during this
pandemic due to social alienation and self-isolation, providing a sense of isolation and possible
depression.
We hypothesized that a conserved aromatic cluster of SARS-CoV (RBD ridge patch) binds its
cellular receptor hACE2 to develop SARS-CoV-2 entry inhibitors. We determined the molecular
mechanism of entry inhibition. Additionally, we suggest that ethanol treatment can increase the
susceptibility of a cell to SARS-Cov-2 infection through ethanol-mediated GRP78 overexpression
(Figure 1).
We focused on the unique structural features of the receptor-binding domain (RBD) of SARS-
CoV-2 to develop small molecule inhibitors that can block the entry and replication of SARS-
CoV-2. These studies complement the ongoing efforts of developing therapeutics and vaccines
4

against COVID-19 and generated a unique set of small molecule inhibitors that can be further
developed into COVID-19 therapeutics. These studies also can open new avenues for antiviral
drug design. The next aim is to reveal the mechanism of alcohol induction of GRP78 expression
and its effect on SARS-CoV-2 entry. For this purpose, we used GRP78 promoter deletion analysis
to determine a minimum required sequence within a GRP78 promoter responsible for
transcriptional regulation by ethanol.

Figure 1. Hypothetical model. Entry inhibitors can block the viral Spike and ACE2 interaction. Alcohol treatment can
increase the susceptibility of a cell to SARS-CoV-2 entry through GRP78 overexpression.
5

Chapter 2. Material and Methods
2.1 Primer design.
There were nine primer pairs designed to truncate 9 evenly dispersed regions of GRP78 promoter.
Primers were designed manually, using the following principles: length of 18-24 bases, G/C
content - 40-60%, without exact nucleotide repetition, 3’ end nucleotide should be G or C. Leader
Sequence (extra base pairs on the 5' end of the primer assist with restriction enzyme digestion (6bp)
and Restriction Site: restriction site for cloning (6-8 bp) were added to Hybridization Sequence.
The primers were analyzed for self-complementarity and dimer formation on https://primer3.ut.ee/.
The oligonucleotides are shown in Table 1. Primers were ordered from IDT. Diluted for 100
pmol/μL of stock solution and then diluted to 20 pmol/μL as a work concentration.
2.2 PCR.
hGRP78 promoter fragments were amplified on a C100 Thermal Cycler (Bio-Rad). The
amplification program consisted of the following steps: initial denaturation at 95°C for 1 min; 35
cycles followed, consisting of the stage of denaturation (95°C, 30 sec.), primer annealing (from
54°C to 59°C depending on the primer set, 45 sec.), and elongation (68°C, from 1 min. to 2 min
30 sec, depending on amplicon size); then the final elongation was carried out at 68°C for 5 min.
The results of PCR were separated by electrophoresis in 1% agarose gel, while ethidium bromide
was used as an intercalating agent for analytical samples. The size of the DNA amplification
products was determined based on the size of the fragments of the GeneRuler molecular weight
marker (Thermo Fisher Scientific, USA). The electrophoresis results were visualized using a
transilluminator or Gel Doc™ EZ Gel Documentation System (BioRad, USA).

6

2.3 Plasmid construction.
The DNA fragments generated by restriction digestion were then put into an expression vector.
Gene overexpression was carried out using the pGL3 Basic vector as a backbone. The pGL3 Basic
vector (produced by Promega company) and GRP78 promoter inserts are cut at particular locations
using the necessary volume of the restriction enzymes KpnI and XhoI, both made by the BioLabs.
The restriction cut enzyme is inactivated by heating tubes in 65°C for 20 minutes after 2 hours of
incubation in 37°C.
Deionized Water 33.8 μL
10x Cut smart buffer 5 μL
KpnI (20U/μL) 0.5 μL
XhoI (20U//μL) 0.5 μL
pGL3 Basic vector 10 μL
Total 50 μL
Table 1. The components volume in restriction enzyme digestion
After 20 minutes of running on a 1% agarose gel, the sample is analyzed by electrophoresis. UV
light can be used to locate the band (GRP78 promoter fragment sizes in bp are listed in Table 2,
and the pGL3 Basic vector size is 4818 bp). A 1.5 ml Eppendorf tube is weighed, and the results
are recorded. The band is then removed using a razor, placed in an Eppendorf tube, and weighed
once more. The weight of the gel makes up the difference between the two weights.
The target fragments were extracted using the QIAquick Gel Extraction Kit (QIAGEN). By
analyzing the band intensity on a 1% agarose gel and comparing it to the ladder (Thermofisher),
one may determine the amount of DNA present. In a 3:1 molar ratio, the vector fragment and
GRP78 promoter sequence are combined. A cut pGL3 Basic vector without any inserts is utilized
for negative control. The reaction mix was incubated for 16°C overnight and transformed into
competent cells (BioLab). The cells are incubated for 10 minutes on ice, followed by 30 seconds
in 42°C and 2 minutes on ice. The tube is filled with 200 μL of SOC media and then incubated for
7

an hour on the thermoshaker. The mixtures are then equally spread out on the plates with LB agar
containing ampicillin and incubated at 37°C overnight.
Linearized pGL Basic vector (50ng//μL) 1.0 μL
GRP78 promoter sequence 0.5 μL
10x T4 DNA ligase buffer 1.0 μL
Deionized water 6.5 μL
T4 DNA ligase (40U//μL) 1.0 μL
Total 10.0 μL
Table 2. The components volume in ligation reaction
The colony numbers on the plates were counted, compared to the negative control, and used to
determine whether the insertion and ligation were successful. Once the insertion and ligation were
confirmed, single colonies were picked up and incubated in 5 ml of LB broth containing ampicillin
for overnight incubation at 37°C. On the second day, the plasmid was extracted using a QIAprep
Spin Miniprep Kit (QIAGEN), and the concentrations were then determined using a Nanodrop
spectrophotometer.
2.4 Transfection of GRP78 promoter deletion constructs
The cells are placed on a 24-well plate after reaching 70–80 percent confluency on the T75 flask.
Per well, 40,000 cells are seeded (the surface area of each well is 1.9 sq. cm). Cell Counter
(Thermofisher) counts the number of cells. The promoter deletion constructs were transfected into
the mammalian cells using the calcium-phosphate method. There are three repeats of each variant.
Solution A, including 1.0 g plasmid DNA and 1.5 μL of 2.5 M CaCl 2, and Solution B (HEPES-
buffered saline containing sodium phosphate), were the mixture's components for the 24-well plate.
The process is based on gradually mixing solutions A and B. After being equally distributed over
the culture plate, the mixture was overnight incubated at 37°C.
The media are changed with new media containing 40 mM ethanol on the first post-transfection
day. Alcohol is not added to the media for the control group. Since the ethanol on the plates quickly
8

evaporated, the media in two groups were replaced daily. The cells are collected after 48 hours
have passed since transfection.
2.5 Cell culture.
Dulbecco's Modified Eagle's Medium was used to culture and maintain the Huh7 and HEK29T
cell lines (Genclone). NCI-H1299 cells were cultured in RPMI 1640 medium. To make complete
media, 10% heat-inactivated fetal bovine serum (Gemini), 1% non-essential amino acids, 1%
L-glutamine, and 1% antibiotic-antimycotic (Gibco) were added. Cells were cultivated and kept
in a humidified incubator at 37°C and 5% CO 2. In T25 flasks, cell stock from the -80°C freezer is
seeded and cultivated in an incubator. 1 ml of trypsin is added to the cells and incubated for 3
minutes, to detach the cells from the flask. The collected cell culture is spun at 1300 rpm for 5
minutes, after which the supernatant is discarded. Cell pellets are resuspended in 1 ml of pre-
warmed media, and each new T25 flask is seeded with 0.5 ml of the resuspended cells to complete
the passage.
2.6 Luciferase assay.
The media is removed, and cells are washed by PBS. Cells are lysed and collected by addition of
100 μL of 1X PLB buffer to each well. The measurement of firefly luciferase activity is performed
on the Omega Fluostar Plate reader on a 96-well plate with white bottom and 10 μL of cell lysate.
50 μL of Luciferase Assay Reagent (#E151, Promega) is added to 10 μL of cell lysate to measure
the activity of Firefly luciferase. The following measurement parameters are used: filter 670 +/-
10 nm, long pass; gain - 2000. The results are normalized by measurement of total protein
concentration (DC Protein Assay Kit, Bio-Rad). The samples are treated by Solution A, containing
Reagent A and Reagent S in ratio 1:50. 25 μL of solution A is added to each well, then mixed with
9

200 μL of Reagent B. For standard curve Bovine Saline Albumin is diluted in the following
concentrations: 2.0, 1.0, 0.5, 0.25, and 0.125 mg/mL. Relative luciferase units are calculated to
each sample by the formula: Promoter activity (RLU) = Firefly luciferase luminescence / Protein
concentration.
2.7 Pseudoparticle virus production
Pseudotyped viruses are useful virological tools because of their safety and versatility. Based on a
vesicular stomatitis virus (VSV) pseudotyped virus production system, we can test assay against
SARS-CoV-2 in biosafety level 2 facilities. This assay uses VSV packaging vector system
pseudotyped with SARS-COV-2 spike proteins on the viral envelope (while retaining a VSV
replicon). The different strain S proteins have either an HA or not [SARS-S (derived from the
Frankfurt-1 isolate or Wuhan strain) with or without a C-terminal HA epitope tag. These are
individually co-transfected with the VSV replicon plasmid with a T7 promoter to make the VSV
genome in BHK21 cells (25). The latter cell line expresses phage T7 RNA polymerase and is
transfected with the VSV-SARS-CoV-2 spike. The produced virus was used for infection of lung
epithelial cells for SARS-CoV-2 entry assays. Cell viability assays in the absence of virus were
performed in parallel to exclude the artifactual reduction of virus entry.
10

Figure 2. Schematic diagram of SARS-CoV-2 pseudotyped virus production
The algorithm of pseudotyped virus production is described below.
Day 1. To transfect HEK 293T cells in a T175 flask, mix 2 mL serum-free DMEM with 60 μL
BioT transfection reagent and 40 μg plasmid DNA encoding SARS-CoV-2 spike protein; incubate
for 5 minutes at room temperature. Add the transfection mixture to a T175 flask containing HEK
293T cells at ~70% confluency; rock the flask side to side and back and forth to mix evenly.
Incubate the cells at 37°C, 5% CO2 for 24 hours.
Day 2. Remove the media and replace it with fresh, warm DMEM containing 100 μL VSV-ΔG
virus particles encoding luciferase reporter. Rock the flask to mix the solution. Incubate the cells
at 37°C, 5% CO2 for 1 hour. Remove the cell media, wash the cells twice with warm PBS, then
add fresh, warm DMEM (just enough to cover the surface, ~12 mL). Seed the cells in a 96-well
11

plate (white bottom) at 18,000 cells/well; rock plate back and forth and side to side to evenly
distribute cells. Incubate the plates at 37°C, 5% CO2 overnight.
Day 3. The day after adding the VSV-Δ-G virus particles, harvest the cell media containing the
pseudo particles from the T175 flask and transfer it to a centrifuge tube. Centrifuge the
pseudoparticle solution (1000 rpm for 4 minutes) to pellet any cells and cell debris. Transfer the
supernatant to 2 mL cryovials and either use immediately to perform a titration or store at -80°C.
In a 96-well round-bottom plate, add 100 μL cell media to each well and 100 μL pseudoparticle
solution. Rock the plate back and forth and side to side to evenly distribute the pseudoparticles.
Incubate at 37°C, 5% CO2 for 24 hours.
Day 4. Place luciferase substrate (One-Glo) and plates at room temperature for ~30 minutes to
equilibrate to room temperature. Remove cell media from each well of the 96-well plate containing
cells. Add 100 μL luciferase substrate to each well and mix. Measure bioluminescence using
Fluostar Omega plate reader. Increase gain or measurement interval time to increase signal if
necessary.
Chapter 4. Results and Discussion
4.1 Small cyclic peptides inhibited SARS-CoV-2 entry.
Through published structural data analyses (26-30), our research identified a unique structural
characteristic of the S protein of SARS-CoV-2 at its binding interface to the host receptor ACE2.
Based on the binding mechanisms of neuronal nicotinic acetylcholine receptors (nAChR) loop C
to various natural and synthetic small molecule ligands, we conducted a knowledge-based docking
by focusing on aromatic clusters and a cation-p interaction mode and subsequently identified
several small molecule compounds with promising activities in inhibiting viral entry. Systematic
12

structural and bioinformatic analyses identified potential drug target site between SARS-CoV-2
and hACE2 receptor interface. We used these structures to perform an initial in silico screen of
compound libraries and identified leading compounds that showed specific activities in SARS-
CoV-2 entry assays. Virtual screen identified top 16 SARS-CoV inhibitor candidates that bind the
SARS-CoV-2 aromatic cluster. We have selected 64 compounds for the viral entry assay. High-
throughput screening based on pseudotyped virus infection revealed three hit compounds
inhibiting SARS-CoV-2 viral entry. Conotoxin MVIIA and a-Conotoxin PIA blocked all SARS-
CoV-2, CoV-1 and MERS pseudoparticle entries. The results are shown on Figure 3.
Pseudoparticle luciferase assays confirmed the broad pan-Coronavirus inhibitory effects for
SARS-CoV-2, UK variant and MERS (Fig. 3B). The top cyclotide candidates (Fig. 3) have longer
stability/half-life of serum protein levels since cyclic peptides are not incorporated into cells and
circulate in blood stream like those of other small molecules.
13

Figure 3. High-throughput screening of SARS-CoV-2 inhibitors identified nine potential cyclic peptides for VSVpp-
SARS-CoV-2 Spike envelope proteins. (A) SARS-COV2 VSV pseudoparticle entry inhibition assay system. High-
throughput screening identified several hit compounds. Conotoxin inhibited SARS-CoV-2 pseudoparticle entry. (B,
C) H1299 and VERO-E6 ACE2 cells ectopic expressing SARS-CoV-2 spike were pre-treated with various
concentration of Conotoxin as indicated 2 hours before pseudoparticles infection. 16 hours post-infection, relative
luciferase activities were determined. Data represent mean± S.D. from three independent experiments. *P<0.05. The
concentration giving 50% of inhibition (IC50) in H1299 cells was calculated through nonlinear regression using Excel
software. (D) Several Conotoxins share similar chemical structures.

14

Hit compound FDA-approved w-conotoxin (sub-nano molar IC 50: 28 nM) belong to a class of
disulfide-crosslinked cyclic peptides from cone snail (conotoxins) or plant (cyclotides), which are
physiologically stable, orally bioavailable and able to cross cellular membranes to modulate
protein–protein interactions (PPIs) via structure-activity-relationship (SAR) (Fig. 3D) with a
unique topology containing a head-to-tail cyclized backbone stabilized by three disulfide bonds
that form a cystine knot (31,32). Natural and engineered cyclic peptides could be an attractive class
of molecules for developing inhibitors of the Cys loop aromatic cluster of the SARS-CoV-2 S1
protein.
The results of entry inhibitor assay complemented the many ongoing efforts of developing
therapeutics and vaccines against COVID-19 and will generate a unique set of cyclic peptide
inhibitors that can be further developed into COVID-19 therapeutics. These studies also opened
new avenues for antiviral drug design for all Coronavirus, mutant SARS-CoV-2 (D614G), mutant
SARS-CoV-1 (Frankfurt strain), mutant MERS and vaccine resistant strains

4.2 GRP78 promoter plasmid construction.
The human GRP78 promoter sequence was downloaded from The Eukaryotic Promoter Database
(https://epd.epfl.ch//index.php). A total of nine forward primers and one reverse primer were
designed (Table 1). The primers were checked for specificity on the NCBI BLAST database.

15

Table 3. Primers used for amplification of hGRP78 promoter regions
Primers were checked for self-complementarity (hairpin formation) and dimer formation. After a
series of optimization of PCR parameters (annealing temperature, elongation time, primer
concentration), distinctive and specific amplicons were obtained (Figure 4).

Figure 4: Amplified regions of hGRP78 promoter
hGRP78 primers Sequence
hGRP78-2438-KpnI-F 5'-CGGGGTACCCGTAAGTGACTGTGCTTTGG-3'
hGRP78-1951-KpnI-F 5'-CGGGGTACCGGTATCTGGACACAGTATACCC-3'
hGRP78-1644-KpnI-F 5'-CGGGGTACCGGATGGTCTTGATCTCCTGACC-3'
hGRP78-1304-KpnI-F 5'-CGGGGTACCGGTTGCAGTGAGCTGAGATTGC-3'
hGRP78-1031-KpnI-F 5'-CGGGGTACCGGTAGGCTTTCAGCAAATAGTGG-3'
hGRP78-946-KpnI-F 5'-CGGGGTACCCCTATCAAATGTGTTGTCTGCG-3'
hGRP78-620-KpnI-F 5'-CGGGGTACCGGGACATAACTGGCAGGAAGG-3'
hGRP78-396-KpnI-F 5'-CGGGGTACCGTGAGGGATGGAGGAAGGGAG-3'
hGRP78-189-KpnI-F 5'-CGGGGTACCGGCGGATGTTATCTACCATTGG-3'
hGRP78+33-XhoI-R 5'-CGGCTCGAGCGCCTACTCGGCTTATATACC-3'
16

The eight amplicons and pGL3 Basic vector were cut at the exact cutting locations by the
restriction enzymes KpnI and XhoI. The target fragments were extracted using the Gel Extraction
Kit from QIAGEN. Then, a volume-appropriate mixture of inserter and vector fragments was made
in a 3:1 ratio. These three mixes were put into competent cells, where they proceeded to transform
on the LB plate. Figure 4 shows clone growths of recombinant plasmids on plates.

Figure 5: Example of colonies after ligation
No growth was noted on the plates with plasmids pGRP78-2438/+33 and pGRP78-1951/+33. The
ligation step was repeated with ratios 5:1 and 10:1 without any successful result. Further
experiments were performed using six GRP78 promoter deletion constructs, as shown in Table 2.
Table 4: Plasmids used in the present study.
Restriction enzymes BstEII and PflMI were used to digest the transformation product for 2 hours
and then loaded in 1% DNA agarose gel for 20 minutes of electrophoresis. The result (Figure 5)
Plasmid Name Backbone Insertion
pGRP78-1644/+33 pGL3 Basic Vector Plasmid (Promega) GRP78 promoter (-1644/+33)
pGRP78-1031/+33 pGL3 Basic Vector Plasmid (Promega) GRP78 promoter (-1031/+33)
pGRP78-946/+33 pGL3 Basic Vector Plasmid (Promega) GRP78 promoter (-946/+33)
pGRP78-620/+33 pGL3 Basic Vector Plasmid (Promega) GRP78 promoter (-620/+33)
pGRP78-396/+33 pGL3 Basic Vector Plasmid (Promega) GRP78 promoter (-396/+33)
hGRP78-189/+33 pGL3 Basic Vector Plasmid (Promega) GRP78 promoter (-189/+33)
17

indicated two bands consistent with expected lengths of GRP78 promoter sequence and linear
pGL3 Basic vector. The electrophoresis results further verified the digestion and ligation process.

Figure 6: Diagnostic restriction digestion of PCR products
4.3 Luciferase assay results.
The plasmids were co-transfected with the SV40 Renilla vector to Huh7 and NCI-H1299 cells.
The calcium-phosphate method was performed on 24-well plates in two variants: alcohol treatment
and DMSO treatment. The cell lysates were harvested after 36 hours of treatment to detect
luminescence by a luciferase assay. The results are shown in Figure 6.
18

Figure 7. Effect of ethanol on GRP78 promoter constructs
The luciferase assay results demonstrate that ethanol treatment induces the activity of GRP78
promoter constructs. Therefore, we suggest that at least the HSPA5 promoter region from -189 to
+33 bp contains transcription factor binding sites (TFBS) and activates overexpression under
alcohol treatment.
4.4 Bioinformatic analysis of transcription binding sites of GRP78 promoter.
As a next step, we performed bioinformatic analysis to find TFBS within region -189 …+33 bp of
the GRP78 promoter sequence. We used Ingenuity Pathway Analysis (IPA) online tool to analyze
published data for transcriptional regulation of the HSPA5 gene (Figure 7).
19

Figure 8. IPA based prediction of ethanol induced pathway and TF of HSPA5 gene
GRP78 is known to be upregulated during ER stress as a response to a high number of unfolded
proteins. UPR is caused by different factors, including ethanol and viral infection. Previously
reported that transcriptional induction of mammalian GRP78 is mediated by multiple redundant
cis-acting elements and that among these, the most critical regulatory regions are ERSE, so-called
ER stress response elements (33, 34). There are cis-binding elements (CCAAT(N9)CCACG) that
are triplicated and located near (-140…-40 bp) to the transcription start site (TSS) (35). The
HSPA5 promoter in plasmid construct hGRP78-189/+33, along with the other five constructs, is
induced by ethanol, and three ERSE are within the analyzed sequence. There are also other
conserved regions in the GRP78 promoter sequence, such as a CCAAT box and a cAMP-
responsive element CRE (36, 37). Transcription factors bind to these regulatory elements,
including CBF/NF-Y, CREB, activating transcription factor 2 (ATF-2), YY1, YB1, Sp1, ATF4,
TFII, ATF6, and XBP1, regulate the HSPA5 gene expression (38-44).
20

We have selected several transcription factors that induce the HSPA5 gene, then searched to
predict potential TFBS. For this purpose, we used TRANSFAC, a database of eukaryotic
transcription factors, their genomic binding sites, and DNA binding profiles. The results of the
search of TFBS of GRP78 promoter are shown in Figure 8.

Figure 9. TFBS of -189 bp … +33 bp region of GRP78 promoter sequence (TRANSFAC analysis)
After the cut-off of false-positive results, five potential TFBS for ATF2, ATF6, and XBP-1 are
identified. These TFBSs are located within the -167 and -143 bp regions of the GRP78 promoter.
To further the determination of cis-acting elements, we designed primers for site-directed
mutagenesis by using an online tool (https://www.agilent.com/store/primerDesignProgram.jsp).
The primers are listed in Table 3.
Transcription
factor
Binding site Primer Name Primer Sequence (5' to 3')
ATF6

aaaCGTCAc t1541c_t1542g_t1543c_ 5'-ggccccctccgcaatgcgcgtcactgctccgcc-3'
5'-ggcggagcagtgacgcgcattgcggagggggcc-3'
ATF4

aaACGTCa t1541c_t1542g_t1543c_ 5'-ggccccctccgcaatgcgcgtcactgctccgcc-3'
5'-ggcggagcagtgacgcgcattgcggagggggcc-3'
XBP-1 gcaataAACGTcactgc t1546a_g1547t_c1548c_ 5'-ggccccctccgatataaacgtcactgctccgc-3'
5'-gcggagcagtgacgtttatatcggagggggcc-3'
Table 5. Primers for site-directed mutagenesis of GRP78 promoter cis-acting elements
21

Chapter 5. Summary
Systematic structural and bioinformatic analyses identified a potential drug target site between
SARS-CoV-2 and the hACE2 receptor interface. We used these structures to perform an initial in
silico screen of compound libraries and identified leading compounds that showed specific
activities in SARS-CoV-2 entry assays. We used a unique structure-based approach to search for
small molecule inhibitors that can bind SARS-CoV-2 S1 RBD and block its interaction with host
receptor ACE2. The SARS-CoV-2 Spike pseudoparticle virus production is performed. After
performing this study, GRP78 promoter deletion constructs are designed and transfected to
mammalian cells to identify the minimum required promoter region for ethanol-mediated
induction of the HSPA5 gene. The following bioinformatics analysis revealed potential
transcription binding sites on the promoter sequence. Further experiments are needed to specify
the transcription factor binding sites and verify the results.
22

Chapter 6. Future Directions
6.1 Promoter assay with site-directed mutagenesis: The above deletion construct analysis
suggested the regions responsible for ethanol-mediated GRP78 transcriptional induction. To test
the role of specific sequence elements within these regions, six mutant-luciferase plasmids will be
constructed by in vitro mutagenesis.
6.2 ChIP assay for protein-DNA interactions in the GRP78 enhancer/promoter: If certain cis-
regulatory elements are shown to be essential for ethanol-mediated GRP78 induction by the
reporter assay above, we will examine whether predicted trans-acting factors bind to the cis-
elements by ChIP analysis of cross-linked DNA from untreated or ethanol-stimulated Huh7 cells
transduced with ethanol.
6.3 Electromobility shift assay (EMSA): To validate protein-DNA interactions shown by ChIP
analysis, we will perform EMSA using a labeled putative cis-element from the GRP78 promoter.
Nuclear extracts from ethanol transduced Huh7 cells will be analyzed with or without ethanol
stimulation. We will also use probes with mutations in core sequences for cis-elements to support
the specific binding. Identification of proteins forming a shifted protein-DNA complex will be
made by super shift assay using specific antibodies against a trans-acting factor of interest.
6.4 Effects of lentiviral shRNA-knockdown on GRP78 promoter activity: To confirm the role of
a trans-acting factor identified to be critical for ethanol-mediated induction of GRP78, a lentiviral
expression vector for shRNA specific for this factor will be used to knock down its expression and
to determine the effect on the GRP78 promoter activity and protein-DNA binding in ethanol-
transduced Huh7 cells by the reporter and ChIP assays, respectively.
Real-time PCR or immunoblot analysis will be performed to select the best shRNA sequence and
confirm knockdown efficiency prior to the promoter-reporter or ChIP assay.
23

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Abstract (if available)

Abstract The continuing pandemic of coronavirus disease 2019 (COVID-19) poses an unmet medical need. To address current global health crises, it is an urgent priority to find effective prevention and treatment against COVID-19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus utilizes the host receptor angiotensin-converting enzyme 2 (ACE2) for entry and interacts through viral spike protein. As the initial stage of infection, the entrance of SARS-CoV-2 into host cells represents a therapeutic intervention point. We identified and optimized candidate compounds from the FDA-approved library that inhibit SARS-CoV-2 entry. One of the co-factors of ACE2 is GRP78, which has been recently shown to facilitate the infectivity of SARS-CoV-2.
Along with other factors causing ER stress inducers, ethanol is one of the most potent inducers. We hypothesized that ethanol treatment could increase viral entry through ethanol mediated GRP78 overexpression. Using promoter-reporter assay, we identified the minimum required region for alcohol induction of the HSPA5 gene. Further bioinformatic analysis revealed potential transcription factor binding sites. This study aims to demonstrate the role of alcohol in developing SARS-CoV-2 infection.

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Asset Metadata

Creator Mukashev, Nurzhan (author)

Core Title Mechanism of ethanol-mediated increase of SARS-COV-2 entry through GRP78 induction

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2022-08

Publication Date 08/08/2022

Defense Date 05/31/2022

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

Tag ethanol,GRP78,OAI-PMH Harvest,SARS-CoV-2

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

Creator Email nkmukashev@gmail.com,nmukashe@usc.edu

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