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Investigating the role of ion channel activity of coronaviruses envelope protein in CD1d regulation

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Investigating the role of ion channel activity of coronaviruses envelope protein in CD1d regulation

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Content Investigating the role of ion channel activity of coronaviruses envelope protein in CD1d
regulation
by
Ramya Parandaman
A Thesis Presented to the
F ACUL TY OF THE USC KECK SCHOOL OF MEDICINE
In Partial Fulfillment of the
Requirements for the Degree in
MASTER OF SCIENCE
(MOLECULAR MICROBIOLOGY AND IMMUNOLOGY)
August 2023
Copyright 2023 Ramya Parandaman
Acknowledgements
I would like to take this opportunity to express my heartfelt appreciation to all
those who provided me with support and guidance during my two-year graduate study
at the University of Southern California.
First and foremost, I am immensely grateful to Dr . W eiming Y uan for his
mentorship and unwavering support throughout my Master's program and research. His
kindness, patience, and enthusiasm not only helped me grow as a scientist but also as
an individual.
I would also like to extend my gratitude to my master's thesis committee
members, Dr . Hyungjin Eoh and Dr . Zhaoxia Qu, for their valuable insights, constructive
comments, encouragement, and guidance during the course of my research.
My heartfelt thanks go to my lab colleagues, namely Xiangxue Deng, Xiaoting
Ren, Y i W ang, Lingxi Qui, Ruichen W ang, and Josue Hernandez, for their assistance,
collaborative ideas, and contributions. I would also like to acknowledge the previous lab
members, Hongjia Lu, Rongqi Zhao, and Zhewei Liu, for laying the foundation with their
fundamental work. Additionally , I am grateful for the support provided by our master's
graduate program and the staf f at the Department of Molecular Microbiology and
Immunology , particularly Dr . Axel Schonthal and Karina Recinos.
Last but certainly not least, I would like to express my deep appreciation to my
parents, brother and friends for their unwavering support and encouragement
throughout my academic journey . Their belief in me and the sacrifices they have made
have been crucial to my accomplishments. I am truly grateful for their constant love and
guidance.
ii
T able of Contents
Acknowledgements…………..………………………………………………………...……….ii
List of T ables………………………………………………………………………………….....v
List of Figures…………………………………………………………………………………...vi
Abbreviations………………………………………..………………………………………….vii
Abstract…………………………………………………………………………………..………ix
Chapter One: Introduction……………………………………………………………….……01
1.1 Coronavirus Genome Structure………………………….………………………01
1.2 iNKT/CD1d Antigen Presentation System………………………….…………..02
1.3 Envelope Protein……………………………………………..……….…………..03
1.4 V iroporin……………………………………………………….……….…………..04
Chapter T wo: Materials and Methods………………………………...……………………..06
2.1 Cell lines and Plasmids………………………………….…………………....….06
2.2 Reagents and Antibodies…………………………….…………………….…….06
2.3 T ransient T ransfection………………………………..…………………………..07
2.4 Cell L ysis and W estern Blot………………………….…………………………..07
2.5 Immunoprecipitation Assay…………………………..…………………………..08
2.6 W estern Blot…………………………………………...…………………………..08
2.7 Flow Cytometry and FlowJo………………………..…………………………….09
2.8 Immunofluorescence………………………………..…………………………….09
2.9 Cloning……………………………………………………………………………..10
2.10 Primer T able……………………………………………………………………....1 1
2.1 1 T emplate Sequence……………………………..……………………………….12
Chapter Three: Results………………….……………………..……………………………..13
3.1 Impact of dif ferent Coronavirus Envelope Proteins on CD1d Gene
Expression Levels……………………...…………..………………………………....13
3.2 Generation of SARS-CoV -2, OC43, 229E Envelope Protein
Constructs: Wildtype, T ransmembrane, and Plasma Membrane V ariants……...13
3.3 SARS-CoV -2 envelope protein ion channel function plays an important
role in downregulating APC surface CD1d expression…………………….....…...14
i i i
3.4 Ion channel function of SARS-CoV -2 Envelope Protein through
V oltage Patch Clamp……………………………………………………………..…...15
3.5 SARS-CoV -2, OC43, and 229E envelope protein cellular localization……...15
Chapter Four: Discussion……………….……………………………………………..……..16
References……………………………………………………………………………………..19
Figures…………………………………………………………………...………….………….24
i v
List of T ables
T able 1. List of Primers………………..………………………………………………………1 1
T able 2. T emplate Sequences………………………………………………………………..12
v
List of Figures
Figure 1. Selective Downregulation of CD1d Expression by E proteins from Highly
Pathogenic Coronaviruses……………………………………………………………………24
Figure 2. Generation of SARS-CoV -2, OC43, and 229E Envelope Protein Constructs:
Wild T ype, T ransmembrane, and Plasma Membrane V ariants…………………………...26
Figure 3. Comparison of Wild T ype, T ransmembrane and Plasma Membrane
constructs from SARS-CoV -2, OC43 and 229E plasmids…………………………….…..28
Figure 4. Ion channel function of SARS-CoV -2 Envelope (E) protein…………………...31
Figure 5. Localization of SARS-CoV -2, OC43, and 229E Envelope proteins in
Cells and Downregulation of Surface Expression………………………………………….32
v i
Abbreviations
APC Antigen-Presenting Cells
BSA Bovine Serum Albumin
CD1 Cluster of Dif ferentiation
CD1d Cluster of Dif ferentiation 1 (Class D)
CoVs Coronaviruses
Cy5 Cyanine %
DAMPS Damage associated molecular patterns
DC Dendritic Cell
DMEM Dulbecco’ s Modified Eagle Medium
E protein Envelope protein
ER Endoplasmic reticulum
ERGIC Endoplasmic reticulum Golgi intermediate compartment
Fc Fragment crystallizable
F ACS Fluorescence-activated cell sorting
FBS Fetal Bovine Serum
FITC Fluorescein Isothiocyanate
GFP Green fluorescent protein
Grp94 Glucose-regulated protein 94
HEPES 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
HRP Horseradish Peroxidase
IAA Iodoacetamide
MHC Major histocompatibility complex
NKT Natural Killer T cells
ORF Open reading frame
P AMPS Pathogen-associated molecular patterns
PBS Phosphate Buf fered Saline
PDZ PSD95(postsynaptic density protein 95)/Dlg1(Drosophila disc large tumor
suppressor)/zo-1(zonula occludens-1 protein
PBM PDZ-binding motif
PE Phycoerythrin
PEI Polyethyleneimine
PM Plasma Membrane
PMSF Phenylmethanesulfonyl fluoride
PS Ab Penicillin-Streptomycin antibody
PS buf fer Permeabilization solution
PVDF Polyvinylidene difluoride
RCF Relative centrifugal force
v i i
SDS Sodium Dodecyl Sulfate
SDS-P AGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
TBST T ris-Buf fered Saline with T ween 20
TBS T ris-Buf fered Saline
TCR T -cell receptor
TM T ransmembrane
TMD T ransmembrane Domain
TRITC T etramethylrhodamine Isothiocyanate
WT Wild T ype
β2 m β2- microglobulin
v i i i
Abstract
The emergence of severe acute respiratory syndrome (SARS) in 2003 and the
recent COVID-19 pandemic have underscored the devastating impact of coronaviruses
(CoVs) when they cross the species barrier and infect humans. Consequently , there has
been a renewed focus on studying coronaviruses, leading to the discovery of several
new human CoVs. Understanding the pathogenesis of CoVs is crucial for developing
ef fective antiviral treatments. One notable mechanism employed by these viruses
involves evading and suppressing the innate immune system through the
downregulation of host immune cell surface CD1d expression.
Our research has revealed that among the studied human coronaviruses, only
the E proteins of highly pathogenic viruses such as SARS-CoV -2, SARS-CoV , and
MERS are capable of suppressing CD1d expression. In contrast, E proteins of common
cold coronaviruses like HCoV -OC43, HCoV -229E, HCoV -NL63, and HCoV -HKU1 do not
possess this ability . This suggests that the evasion of NKT cell function through E
protein-mediated suppression of CD1d expression plays a crucial role in the
pathogenesis of highly pathogenic coronaviruses, contributing to their increased
virulence. W e identified the transmembrane (TM) domain of the E protein as a mediator
responsible for CD1d downregulation. Specifically , the TM domain reduces the levels of
mature CD1d proteins after their departure from the endoplasmic reticulum (ER),
indicating its involvement in suppressing CD1d traf ficking and promoting their
degradation. Through point mutations, we confirmed that the putative ion channel
function of the E protein is necessary for CD1d suppression, and we successfully
restored CD1d expression by inhibiting the ion channel function using small chemicals.
T o investigate the ion channel activity of the TM domain of the E protein, we
developed a strategy to target the protein to the plasma membrane. This involved
introducing mutations in the C-terminal domain of the E protein and incorporating a
Golgi-export signal. By employing this approach, we successfully directed both the
SARS-CoV -2 E protein and OC43 E protein to the plasma membrane. Our experiments
demonstrated the formation of a viroporin, a cation channel, when the E protein is
localized to the plasma membrane. Notably , we observed the passage of Na+ current in
cells transfected with the E protein compared to untransfected cells. This advancement
in studying viroporin activity provides a valuable tool for screening potential antiviral
drugs, enabling the identification of novel COVID-19 treatments by modulating the
function of viroporins. These discoveries hold promise for the development of ef fective
therapies against the virus.
i x
Chapter 1 : Introduction
1.1 Coronavirus Genome Structure
Coronaviruses (CoVs) are enveloped viruses with a positive single-stranded RNA
genome. Coronaviruses have the largest viral RNA genome known, ranging from
approximately 27 to 32 kilobases in length (Smith et al., 2013, Liu et al., 2021). They
belong to the Coronaviridae family and are classified into four genera: α-, β-, γ-, and δ-
coronaviruses. Β- coronaviruses, including SARS-CoV , MERS-CoV , and SARS-CoV -2
(which causes COVID-19), are highly pathogenic and infects mammals. These viruses
are transmitted through zoonotic transmission and can spread among humans through
close contact. There are also common human coronaviruses, such as HCoV -229E,
HCov-OC43, HCoV -NL63, and HCoV -HKU1, which generally cause mild respiratory
illnesses (Naqvi et al., 2020). The genome contains several genes that encode various
proteins essential for the virus’ s replication, assembly , and infection process.
The major structural proteins of coronaviruses include the spike (S) protein, membrane
(M) protein, envelope (E) protein, and nucleocapsid (N) protein. Each of these proteins
plays a crucial role in the structure, function, and life cycle of the virus.
1. Spike (S) Protein: The spike protein is responsible for facilitating the entry of the
coronavirus into host cells. It forms prominent protrusions on the viral surface,
giving the virus its characteristic crown-like appearance, hence the name
“corona” (Jackson et al., 2022). The S protein binds to specific receptors on the
host cell surface, allowing the virus to fuse with the cell membrane and enter the
host cell (W ang et al., 2020). This protein is a key target for vaccine development
and therapeutics, as it is essential for viral attachment and entry . (W alls et al.,
2020)
2. Membrane (M) Protein: M proteins are structural proteins that are 222 amino
acids long and work in conjunction with E, N, and S proteins (T ang et al., 2020).
They play a major role in RNA packaging, and their conserved amino acid
sequence suggests a common architecture among these proteins. M proteins are
the most abundant viral proteins in coronaviruses and contribute to the distinctive
shape of the virus. Notably , M proteins possess three transmembrane domains,
which is a distinct characteristic. (Naqvi et al., 2020)
3. Envelope (E) Protein: Envelope membrane (E) proteins are a group of small viral
proteins that contribute to the assembly and release of virions. In addition to its
role in assembly and release, the envelope protein of coronaviruses is involved in
viral pathogenesis and can impact the host’ s immune response (Schoeman et
al., 2019) . In the case of SARS-CoV -2, the E protein is considered a potential
1
target for antiviral drugs. This relatively small protein (75 amino acids) plays a
significant role in viral morphogenesis and assembly (Kuo et al., 2007). The E
protein acts as viroporins, forming protein-lipid pores in host membranes that are
involved in ion transport (Parthasarathy et al., 2008)
4. Nucleocapsid (N) Protein: N protein plays a critical role in packaging viral RNA
into ribonucleocapsid. It interacts with the viral genome and M protein, aiding in
viral assembly , RNA transcription, and replication (Neuman et al., 2016). As a
result, N proteins are considered potential targets for antiviral drugs (Bai et al.,
2021). In addition to its role in genome organization, packaging and replication,
the N protein is involved in immune evasion and can elicit a strong immune
response from its host (Fehr et al., 2015).
The structure proteins of coronaviruses are of significant interest in vaccine
development and therapeutic research. Understanding the structure and function of
these proteins aids in the development of strategies to target and neutralize the virus,
prevent viral entry , and stimulate immune responses to combat infection.
1.2 iNKT/CD1d Antigen Presentation System
The immune system is a powerful defense mechanism that protects the body by
identifying and eliminating foreign molecules. It consists of organs, tissues, cells, and
molecules working together . The immune system can be divided into innate immunity
and adaptive immunity (Lanier et al., 2013). Innate immunity provides a rapid response
and recognizes a range of pathogens without specificity . Dendritic cells, macrophages,
and natural killer cells detect pathogen-associated molecular patterns (P AMPs) and
damage-associated molecular patterns (DAMPs), triggering the innate immune
response. On the other hand, adaptive immunity is antigen-driven and involves
antigen-specific recognition by B cells and T cells (Akira et al., 2006). The adaptive
response has long-term memory , leading to a quicker and stronger response upon
re-exposure to the same pathogen (Rossjohn et al., 2012).
Natural killer T (NKT) cells, a subset of T cells, express markers of both T cells and
natural killer cells (Matsuda et al., 2008). Upon antigen stimulation, NKT cells can elicit
either activation or suppression of other immune cells through their rapid and robust
production of cytokines (T erabe et al., 2008). They recognize lipid-based antigens
presented by the CD1 family of major histocompatibility complex (MHC)-like molecules,
which are typically expressed by Antigen-presenting cells (APCs) (Girardi et al., 2012).
Due to their ability to modulate immune responses, NKT cells are considered
immunological modulators.
2
CD1-restricted T cells have important roles in infection, tumor immunity , and
autoimmunity . They can be categorized based on their recognition of antigens
presented by group 1 CD1 molecules (CD1a, CD1b and CD1c) or CD1d (Catia et al.,
2016). The extensively studied subset is CD1d-restricted natural killer T (NKT) cells,
which can be further divided into two subsets. T ype I NKT or invariant NKT (iNKT) cells
express a semi-invariant TCR (V α24 Jα18 V β1 1 in humans and V α14 Jα18 in mice.) that
recognizes the prototypic antigen α- galactosylceramide (α- GalCer). In contrast, type II
NKT cells have variable TCRs (Liao et al., 2013). iNKT cells rapidly respond to innate
signals and TCR engagement, producing large amounts of cytokines. Some type II NKT
cells exhibit adaptive-like immune functions (Pereira et al., 2019).
CD1d molecules are predominantly found on antigen-presenting cells (APCs) such as
dendritic cells (DCs), macrophages, and B cells. They have similar structure to that of
MHC-class-I molecules, and acquire self-antigens in the endoplasmic reticulum (ER)
(Chaudhry et al., 2014). Unlike MHC-class-I molecules, which require association with
β2- microglobulin (β2 m) to exit the ER, CD1d molecules can be transported to the cell
surface as free heavy chains alone in certain situations. CD1d protein is produced in the
endoplasmic reticulum (ER) and exists in two distinct forms within cells (Exley et al.,
2000, Kang et al., 2002). Approximately half of the newly synthesized CD1d molecules
remain in the ER in an immature state, while the other half undergo complete folding,
binding with β2- microglobulin, exit the ER, undergo complex glycosylation, and are
expressed on the cell surface as the mature form (Kang et al., 2002). V arious viruses,
such as HIV , HSV -1, KSHV , and LCMV , employ dif ferent strategies to inhibit the function
of CD1d and NKT cells (T essmer et al., 2009). One common approach used by viruses
to evade T cell activity is to reduce the expression of antigen-presenting molecules. In
the case of nearly all viruses investigated thus far , they accomplish the suppression of
NKT cell function by down-regulating the expression of CD1d (Hansen et al., 2009).
Given the aggressive nature of pathogens like the SARS-CoV -2 virus, it is conceivable
that the virus has developed mechanisms to inhibit the activity of innate lymphocytes,
including NKT cells.
1.3 CoV Envelope Protein
The CoV E protein, ranging from 76 to 109 amino acids and 8.4 to 12 kDa in size, is a
short integral membrane protein (Schoeman et al., 2019, Kuo et al., 2007). Its structure
consists of a hydrophilic amino terminus of 7-12 amino acids, followed by a hydrophobic
transmembrane domain (TMD) of 25 amino acids, and a hydrophilic carboxyl terminus
(Corse et al., 2000, Li et al., 2014). Within the hydrophobic region of the TMD, there is
at least one predicted amphipathic α- helix that forms an ion-conductive pore in
membranes (V erdia et al., 2012, Nieto et al., 2014). Although the E protein is the
3
smallest among the major structural proteins, it remains enigmatic. While it is
abundantly expressed inside infected cells during the replication cycle, only a small
fraction becomes part of the virion envelope. The majority of the E protein is localized in
intracellular traf ficking sites such as the endoplasmic reticulum (ER), Golgi, and ERGIC,
where it plays a role in various cellular processes including CoV assembly , budding and
intracellular traf ficking of the virus (Nieto et al., 201 1). Studies with recombinant CoVs
lacking E have demonstrated that its absence leads to significantly reduced viral titers,
impaired viral maturation, or the production of non-infectious progeny . This underscores
the importance of the E protein in virus production and maturation (DeDiego et al.,
2007, Ortego et al., 2007).
The transmembrane domain (TMD) of the E protein was found to be involved in
suppressing CD1d expression. It specifically targeted mature CD1d proteins that had
undergone post-ER processing, leading to their reduced levels and potential
degradation (Lu et al., 2023). Point mutations demonstrated that the ion channel
function of the E protein was essential for this suppression. Interestingly , inhibiting the
ion channel function using small chemicals rescued CD1d expression ((Lu et al., 2023).
Furthermore, similar to the full-length E protein, the transmembrane (TM) domain
decreased the levels of mature CD1d, while not af fecting the immature, ER-form of
CD1d. These findings highlight the critical role of the TM domain in the degradation and
downregulation of CD1d mediated by the E protein (Cabrera et al., 2021).
Computational predictions indicate that the C-terminus of β- and γ- CoVs' E protein
contains a conserved proline residue within a β- coil- β motif, which acts as a targeting
signal for the Golgi complex (Li et al., 2014). Experimental evidence demonstrates that
mutation of this proline disrupts the protein's localization to the Golgi complex,
redirecting it to the plasma membrane (Cohen et al., 201 1). Additionally , the C-terminus
of the SARS-CoV E protein contains a PDZ-binding motif (PBM) known as the
postsynaptic density protein 95 (PSD95)/Drosophila disc large tumor suppressor
(Dlg1)/zonula occludens-1 protein (zo-1) binding motif. This motif enables interactions
with cellular adapter proteins involved in host-cell processes relevant to viral infection
(T eoh et al., 2010).
1.4 V iroporin
V iroporins are viral proteins that can modify the host membrane by forming ion channels
or pores, aiding in the release of viral particles. Although the exact function of viroporins
is not fully understood and some aspects remain controversial, there is evidence
suggesting their involvement in viral propagation and pathogenesis. Coronavirus (CoV)
envelope (E) protein ion channel activity was determined in channels formed in planar
4
lipid bilayers by peptides representing either the transmembrane domain (TMD) of
SARS-CoV E protein or full length E protein. In coronaviruses, viroporins play a crucial
role in regulating the pH within intracellular organelles like the Golgi apparatus. By
altering ionic gradients across organelle membranes, viroporins can impact viral
assembly , propagation, and disrupt cellular homeostasis. Mutations B15A and V25F
disrupt the ion conductive property of the E protein transmembrane peptide. Studies
have demonstrated the permeability of the SARS-CoV -2 E protein to Na+ and K+ ions
and its sensitivity to changes in pH. Studies show that ion channel activity is inhibited by
the drug hexamethylene amiloride (HMA) in murine hepatitis virus and in human
coronavirus 229E and this inhibition was correlated with decreased viral replication in
these viruses. The absence of viroporin activity can significantly reduce viral replication
rate, although viroporins are not essential for viral replication.
5
Chapter 2 : Material and Methods
2.1 Cell lines and plasmids
T wo cell lines, 293T .CD1d and HeLa.CD1d, were utilized in this study to investigate
envelope protein mediated downregulation of CD1d molecules. The cell lines were
generated through lentiviral transduction for stable expression of CD1d molecules. The
293T .CD1d cell line, provided by Dr . W eiming Y uan, was cultured in Dulbecco’ s
Modified Eagle Medium (DMEM) supplemented with 5% Fetal Bovine Serum (FBS) and
0.05% puromycin. The inclusion of puromycin facilitated the selection and maintenance
of CD1d-positive cells, allowing for stable expression of high levels of human CD1d
molecules. Furthermore, HeLa.CD1d cell lines were grown in DMEM supplemented with
5% FBS and 100 μ g/ml of Penicillin-Streptomycin (PS) antibiotics.
Dr . W eiming Y uan provided the pT racer plasmid, which expresses GFP and serves as a
transfection indicator in the cell. The Wildtype SARS-CoV -2 gene expression library
which includes SARS2, OC43, 229E variants, was obtained from Dr . Krogan’ s lab at
UCSF . These genes were subsequently cloned into the pL VX vector incorporating a
2xStrep tag. (Gordon et al., 2020). T o investigate the impact of the T ransmembrane
domain (TMD) on viral pathogenicity and its role in CD1d gene downregulation, the
TMD was generated using the wildtype plasmids. Additionally , mutations were
introduced to the envelope proteins of the wildtype SARS2 and OC43 to redirect their
localization to the plasma membrane.
2.2 Reagents and Antibodies
The following primary antibodies were employed in this study: Monoclonal 51.1.3
antibody , provided by Dr . Steven Porcelli at Albert Einstein College of Medicine, Bronx,
NY , was used to detect the expression of mature human CD1d and β 2m complexes.
The D5 antibody was utilized to detect the CD1d heavy chain in its immature form. T o
serve as a loading control, the Grp94 antibody (rat monoclonal, Enzo) specifically binds
to the GRP94 protein. The anti-Strep tag mouse monoclonal antibody from Biolegend
was used for the detection of SARS-CoV -2 viral proteins carrying a 2XStrep tag. T o
detect the SARS-CoV -2 protein localized to the plasma membrane, the anti-Rabbit
polyclonal antibody specific to the SARS Envelope protein was employed.
For western blot analysis, secondary antibodies were prepared in TBST at a 1:5000
dilution. Goat anti-mouse-HRP (H+L) (Jackson) was utilized to detect the strep antibody
and D5 antibody . T o detect the Grp94 antibody , Donkey anti-rat-HRP (Jackson)
6
antibodies were employed. Additionally , Donkey anti-rabbit antibody was utilized to
detect the SARS envelope protein targeted to the plasma membrane.
In F ACS analysis, PE-conjugated goat anti-mouse polyclonal antibody (Biolegend) was
employed to stain cell surface CD1d. The antibody was used at a concentration of
1ug/ml in the PBS buf fer .
In immunofluorescence assays, the following secondary antibodies were utilized: Alexa
Fluor 488 goat anti-rabbit, Alexa Fluor 568 goat anti-mouse IgG1, and Alexa Fluor 647
goat anti-mouse IgG2b.
2.3 T ransient T ransfection
293t-CD1d cells were seeded onto a 10 cm or 6-well tissue culture plate and allowed to
reach 80-85% confluency . Subsequently , the cells were transiently transfected with
pT racer and other SARS-CoV -2 genes using the polyethyleneimine (PEI) method
(Kichler et al., 2001). After 48 hours of incubation at 37 °C, the cells were harvested and
washed with the PBS buf fer . For flow cytometry analysis, immediate staining and
analysis were performed, or the cells were stored at -20°C for subsequent W estern Blot
experiments.
HeLa-CD1d cells were seeded onto 24-well tissue culture plates at a density of 30,000
cells per well on coverslips. The following day , the cells were transfected with
SARS-CoV -2 genes using the PEI method. After a 48-hour incubation period, the cells
were fixed using a fixative solution and preserved for later staining and imaging
procedures.
2.4 Cell lysis and W estern Blot
Freshly prepared cell lysis buf fer was used in this study , comprising 1% T riton-X-100 in
T ris-buf fered saline (TBS) containing various components: 5 mM IAA, 0.1 mM PMSF , 1
mM NaVO
3
, 10 μ M Leupeptin, 1 mM NaF , 10 μ g/ml Pepstatin A, 1 mM
beta-glycerophosphate, 0.5 μ M Okadaic acid, and 1 mM NaPP . T o each 10 cm plate cell
sample, 1 ml of cell lysis buf fer was added and the cells were gently mixed using the
pipette. The mixture was placed in ice for 30 minutes, with the cells being mixed every
15 minutes during the incubation period. Subsequently , the cells were centrifuged at
1000 RCF at 4°C for 10 minutes to pellet the lysed cells. After centrifugation, 200 μ L of
supernatant was retained for the whole cell lysate, while 800 μ L was used for the
immunoprecipitation assay .
For the whole cell lysate sample, 40 μ L of 6X SDS loading buf fer was added to the 200
μ L supernatant, followed by heating at 95°C for 2 minutes before storing at 4°C. The
7
addition of SDS loading buf fer and boiling ensures denaturation of protein, disruption of
their interactions, and uniform coating of SDS, enabling accurate separation and
analysis of proteins based on size during SDS-P AGE.
2.5 Immunoprecipitation Assay
The immunoprecipitation assay consisted of two steps: pre-clearing and
immunoprecipitation. During the pre-clearing step, 1 μ L of normal rabbit serum was
combined with 40 μ L of Sepharose 4B and 40 μ L of Protein G beads, and added to the
remaining 800 μ L sample. The mixture was rotated at 4°C for 2 hours to reduce
non-specific binding and block Fc receptors, serving as a negative control for specificity .
The supernatant was collected by centrifugation, and 2 μ L of CD1d 51.1.3 antibody was
added along with 40 μ L of Sepharose 4B and 40 μ L of Protein G beads, The sample
was then rotated overnight at 4°C. The following day , the supernatant was collected and
saved for further use, while the beads were collected and subjected to three washings
with 0.1% T riton/TBS buf fer . Finally , 80 μ L of 2X SDS loading buf fer was added to each
eluted immunoprecipitated sample, which was then boiled at 95°C for 2 minutes before
storing at 4°C.
2.6 W estern Blot
T o ensure accurate estimation of molecular weight of the sample during SDS-P AGE, a
protein marker (Bio-Rad) was used as a reference. Dif ferent concentrations of SDS
polyacrylamide gels were employed based on the size of the proteins being analyzed.
For the SARS-CoV -2 E protein, a 15% SDS gel was utilized, while a 10% SDS gel was
used for CD1d protein and B-T ubulin protein. Each gel well was loaded with 20 μ L of
the respective samples, and electrophoresis was conducted at 80-120V for
approximately one hour .
Polyvinylidene fluoride (PVDF) membranes were activated using methanol prior to gel
transfer . The proteins were transferred onto the PVDF membranes using a constant
current of 0.12A for 90 minutes. Subsequently , the membranes were blocked with 5%
non-fat milk in TBST for 2 hours. After removing the excess blocking solution, the
membranes were incubated overnight at 4°C with the primary antibodies (Strep,
SARS2-Envelope, B-T ubulin and D5 antibodies) diluted at a ratio of 1:1000 in TBST .
Following incubation, the membranes were washed three times with TBST to remove
any unbound antibodies. Next, the membranes were incubated with secondary
antibodies conjugated with horseradish peroxidase (HRP) for one hour at room
temperature. Detection of chemiluminescence was achieved by using an HRP-based
detection fluid, and the membranes were images using the Chemi-Doc Imaging System
(Bio-Rad).
8
2.7 Flow cytometry and Flow-Jo
The cells were transfected with plasmids as previously described. T o enable the
identification of transfected cells based on their green fluorescence, pT racer was
co-transfected along with the plasmids. In flow cytometry analysis, 200 μ L of cells were
taken from each 10cm plate, while the remaining cells were saved for western blot
analysis. The cells were washed with F ACS buf fer (1xdPBS with 0.5%BSA) and then
centrifuged at 1200 rpm for 3 minutes to remove the buf fer . T o the cells, 150 μ L of
diluted primary antibody 51.1.3 (5 μ g/ml) with F ACS buf fer was added and incubated on
ice for 30 minutes. The cells were then washed three times with a F ACS buf fer to
remove any excess unbound antibodies. Next, 150 μ L of secondary antibody PE goat
anti-mouse (1 μ g/ml) was added and incubated in the dark for 30 minutes, with the
samples covered by aluminum foil. The cells were washed three times with a F ACS
buf fer to remove any excess secondary antibodies. T o fix the stained cells, a 3.7%
formaldehyde solution was added for 10 minutes. The BD F ACSCanto II Flow
Cytometer was used to analyze the cell samples, and the F ACS data were processed
and analyzed using the FlowJo software.
2.8 Immunofluorescence
The assay utilized HeLa-CD1d cell lines, which were seeded at 30000 cells per well in a
24-well plate with microscope coverslips. The cells were transfected with SARS-CoV -2
viral proteins. After 48 hours, the cells were fixed using a 4% formaldehyde fixative
solution in DMEM with 10mM HEPES for 15 minutes at room temperature. The fixed
cells were washed three times with a wash buf fer of DMEM with 10 mM HEPES. At this
point, the cells could be stored overnight.
For permeabilization and staining, a permeabilization solution (PS) was added to each
well, and the cells were incubated at room temperature for 20 minutes. Primary
antibodies were diluted in PS at a concentration of 10 μ g/mL (1:2000). The diluted
antibodies were placed on a piece of parafilm with moist towels to create a moist
chamber and prevent the antibody from drying. The coverslips were placed cell side
down onto the antibody drops and incubated at room temperature for 30 minutes. The
coverslips were then washed three times with PS. The staining process was repeated
with secondary antibodies. Before mounting the coverslips, a quick water wash was
performed by dipping the coverslips in water and removing the residual water using
vacuum aspiration. The coverslips were mounted on glass microscope slides using
preheated 20% Mowiol. The samples were dried overnight at 4°C protected from light,
before viewing with a confocal microscope (Nikon) equipped with four laser channels.
The appropriate filter combinations were used to excite the stained dyes.
9
Primary antibodies used: anti-CD1d 51.1.3 mouse IgG2b, anti-Strep tag mouse IgG1,
anti-ERGIC rabbit.
Secondary antibodies used: Alexa Fluor 647 goat anti-mouse IgG2b, Alexa Fluor 568
goat anti-mouse mouse IgG1, Alexa Fluor 488 goat anti-rabbit.
(company ab)
For SARS-CoV -2-PM plasmid,
Primary antibodies used: anti-CD1d 51.1.3 mouse IgG2b, anti-SARS2-Envelope Rabbit,
Secondary antibodies used: Alexa Fluor 647 goat anti-mouse IgG2b, Alexa Fluor 568
goat anti-mouse mouse IgG1
2.9 Cloning
In this study , eight dif ferent constructs were used. The gene expression libraries for
SARS-CoV -2 WT , OC43 WT , and 229E WT were provided by Dr . Krogan's Lab at UCSF .
These libraries were cloned into a pL VX vector with a strep tag. The SARS-CoV -2 TM
construct, which contains only the TM domain of the envelope protein, was previously
generated in the lab to investigate its ef fect on CD1d cell surface expression. The
SARS-CoV -2 PM and OC43 TM constructs were chemically synthesized and cloned
into the pL VX vector . The OC43 PM and 229E TM constructs were generated using
custom-designed primers and PCR amplification with wild type E protein sequences as
templates. The amplified sequences were digested with EcoRI and BamHI restriction
enzymes. An empty pL VX vector was also created through restriction enzyme digestion.
The desired PCR product was isolated by purifying the resulting bands from the gel, and
the DNA was extracted using the QIAquick Gel Extraction Kit. The target DNA segment
was then joined with the empty vector using T4 ligase to produce the desired envelope
protein. Finally , one-shot T op-ten competent cells were used to create a stable bacterial
stock for the newly generated plasmids.
1 0
2.10 Primer T able.
Primer
Name
Sequence Sourc
e
Purpose
PM-OC43-
E-F
CTGGTGCTGAGCCCT AGCGCCGCCGCCGCC
AACAGAGGCAGACAGTTCT ACGAGTTCT A T AA
CGACGTGAAGCCCCCCGTGTT AGACCTGGAA
GGTGGCG
IDT T o generate
OC43 E
protein
PM-targetin
g
PM-OC43-
E-R
CGCCACCTTCCAGGTCT AACACGGGGGGCTT
CACGTCGTT A T AGAACTCGT AGAACTGTCTGC
CTCTGTTGGCGGCGGCGGCGCT AGGGCTCA
GCACCAG
IDT T o generate
OC43 E
protein
PM-targetin
g
OC43+Gol
gi-ES-Low
er
GT A TTCACCCTCACTCGTGA TTCTGCTCTTGCT
GCCGCTGCCGCTGCCGTCCAGCACGGGGGG
CTTCA
IDT T o add
Golgi ES to
PM-OC43-
E construct
OC43+Gol
gi-ES-Upp
er
AGAA TCACGAGTGAGGGTGAA T ACA T ACCCCT
GGA TCAGA TCGACA TCAACGTGCTCGAAGGC
GGCGGGGGA TG
IDT T o add
Golgi ES to
PM-OC43-
E construct
229E-TM-
N-Upper
GTGAA TTCGCCGCCACCA TGGACCACGCCCT
GGTGGTG
IDT T o generate
229E TM
construct
229E-TM-
C-Upper
CCA TCA TCAAGCTGA TCAAGCTCGAAGGCGG
CGGG
IDT T o generate
229E TM
construct
229E-TM-
C-
Lower
CCCGCCGCCTTCGAGCTTGA TCAGCTTGA TGA
TGG
IDT T o generate
229E TM
construct
1 1
2.1 1 T emplate Sequences.
Construct name Sequence
SARS-CoV -2-E-P
M-mKate2
TGTCGTGAGGA TCT A TTTCCGGTGAA TTCGCCGCCACCA TGT A
CAGCTTCGTGAGCGAGGAGACCGGCACCCTGA TCGTGAACA
GCGTGCTGCTGTTCCTGGCCTTCGTGGTGTTCCTGCTGGTG
ACCCTGGCCA TCCTGACCGCCCTGAGACTGTGCGCCT ACTG
CTGCAACGCCGCCAACGTGAGCCTGGTGAAGCCT AGCGCCG
CCGCCGCT AGCAGAGTGAAGAACCTGAACAGCAGCAGAGTG
CCCGGCAGCGGCAGCGGCAGCAAGAGCAGAA TCACGAGTG
AGGGTGAA T ACA T ACCCCTGGA TCAGA TCGACA TCAACGTGG
TGAGCGAGCTGA TCAAGGAGAACA TGCACA TGAAGCTGT ACA
TGGAGGGCACCGTGAACAACCACCACTTCAAGTGCACAAGC
GAGGGCGAGGGCAAGCCCT ACGAGGGCACACAGACCA TGA
GAA TCAAGGCCGTGGAGGGCGGCCCCCTGCCCTTCGCCTTC
GACA TCCTGGCCACAAGCTTCA TGT ACGGCAGCAAGACCTTC
A TCAACCACACCCAAGGCA TCCCCGACTTCTTCAAGCAGAGC
TTCCCCGAGGGCTTCACCTGGGAGAGAGTGACCACCT ACGA
GGACGGCGGCGTGCTGACCGCCACCCAAGACACAAGCCTG
CAAGACGGCTGCCTGA TCT ACAACGTGAAGA TCAGAGGCGT
GAACTTCCCT AGCAACGGCCCCGTGA TGCAGAAGAAGACCC
TGGGCTGGGAAGCT AGCACCGAGACGCTGT ACCCCGCCGAC
GGTGGTCTCGAGGGGCGCGCTGA T A TGGCCCTGAAGCTGGT
GGCGGCGGCCACCTGA TCTGCAACCTGANACCACCT ACAGA
OC43-E-TM+stre
p
GGTGAA TTCGCCGCCACCA TGGACACCGTGTGGT ACGTGGG
GCAGA TCA TCTTCA TCGTGGCCA TCTGCCTGCTGGTGACCA T
CGTGGTTGTGGCCTTCCTGGCCACCTTCAAACTGGAGGGTG
GTGGCGGCTGGTCGCA TCCCCAA TTTGAAAAAGGGGGGGGC
AGCGGTGGCGGCAGCGGTGGCGGCAGTTGGTCGCACCCTC
AGTTCGAGAAGT AAGGA TCCCG
1 2
Chapter 3 : Results
1. Impact of different Coronavirus E Proteins on CD1d Gene Expression
Levels
T o assess the impact of dif ferent coronavirus E proteins on CD1d gene expression,
293T -CD1d cells were transfected with various coronavirus E proteins alongside
pT racer , which served as a GFP positive control to measure transfection ef ficiency . After
48 hours, the transfected cells were collected and stained using the 51.1.3 primary
antibody to detect CD1d molecules on their cell surface. Flow cytometry was then
conducted to compare CD1d expression levels between transfected and untransfected
cells. The transfected and non-transfected cell populations were distinguished based on
GFP fluorescence intensity . Analysis of the F ACS results from previous studies revealed
that CD1d expression was reduced in cells transfected with SARS-CoV and
SARS-CoV -2 E proteins. However , MERS E protein caused a slight decrease in CD1d
expression, while 229E, OC43, NL63, and HKU1 coronaviruses did not exhibit any
downregulation of CD1d expression.
The protein expression was assessed using W estern blot analysis on the same set of
samples (Fig. 1C). Grp94 was used as a loading control. and similar band intensity of
Grp94 in each column indicated comparable total protein levels. The anti-Strep antibody
was employed to measure the expression levels of E proteins of dif ferent viral origins by
binding to the strep-tagged E protein. All samples showed bands with dif ferent
coronavirus E proteins, while the pT racer control did not show any band. The α- CD1d
51.1.3 antibody (mouse IgG2b) was utilized to specifically recognize the CD1d and β2 m
molecule complex, which represents the mature form of CD1d which is present on the
cell surface of antigen-presenting cells (APC) (Kang et al., 2002). On the other hand,
the α- CD1d D5 antibody (mouse IgG2b) can directly bind to the free heavy chain of
CD1d. This property enables the D5 antibody to attach to the precursor form of CD1d
within the cell. In Figure 1C, The immunoprecipitation blot which used α- CD1d 51.1.3
antibody revealed a consistent reduction in mature CD1d protein levels in cells
transfected with SARS-CoV -2 E protein. Conversely , there was no reduction in the
amount of immature or ER-form CD1d protein band observed in whole cell lysates using
the Cd1d:D5 antibody .
2. Generation of SARS-CoV -2, OC43, and 229E Envelope Protein Constructs:
W ildtype, T ransmembrane, and Plasma Membrane V ariants.
SARS-CoV -2 E protein is a viroporin, which is a small, hydrophobic protein that forms
hydrophilic pores in host cell membranes (Nieva et al., 2012). These pores allow the
passage of ions and small molecules. The E protein of SARS-CoV -2 can self-assemble
into oligomers and has ion channel activity . The transmembrane (TM) domains of
1 3
coronavirus E proteins belong to the Class I subclass A viroporins. A study
demonstrated that two crucial residues, N15 and V25, in the TM domain of SARS-CoV
E protein are necessary for its viroporin function ( Nieto-T orres et al., 2014 ). This
suggests that the ion channel function is essential for CD1d degradation and reduced
cell surface expression. T reatment of E protein-expressing cells with viroporin inhibitors,
such as amantadine (known to inhibit influenza M2 viroporin and SARS-CoV -2 E
protein) or hexamethylene amiloride (HMA, a specific inhibitor of another viroporin,
HIV -1 vpu protein), confirmed the requirement of ion channel function for E
protein-mediated CD1d downregulation (Lu H et al., 2023).
T o investigate viroporin activity on cd1d ligand expression at the NKT cells, we
generated three variants of SARS-CoV -2 and OC43, two variants of 229E which is
illustrated in Figure 2. By targeting the E protein to the plasma membrane, we created
SARS-CoV -2 PM and OC43 PM constructs, which formed cation channels known as
viroporins. These viroporins exhibited pH-dependent modulation, allowing for
biophysical analysis using whole-cell patch clamp recording. Additionally , we generated
SARS-CoV -2 TM, OC43 TM, and 229E TM variants containing only the transmembrane
domain.
3. SARS-CoV -2 E protein ion-channel function plays an
important role in downregulating APC surface CD1d expression.
T o investigate the role of E protein’ s ion channel function in downregulating surface
CD1d expression on antigen-presenting cells (APCs), we generated eight dif ferent
constructs of SARS-CoV -2 WT , SARS-CoV -2 TM, SARS-CoV -2 PM, OC43 WT , OC43
TM, OC43 PM, 229E WT and 229E TM (Figure 2). T ransfection of 293T .CD1d cells with
these constructs was performed. After 48 hours, flow cytometry and W estern blot
analysis were conducted to assess changes in CD1d expression levels in the
transfected cells. From Figure 3A, Flow cytometry data revealed significant
downregulation of CD1d surface expression in cells transfected with SARS-CoV -2 and
SARS-CoV -2 TM constructs compared to control groups. Statistical analysis of F ACS
data using bar graphs shown in Figure 3B confirmed these findings, showing around
two-fold decrease in CD1d surface expression for SARS-CoV -2 and a greater two-fold
decrease for SARS-CoV -2 TM compared to control group (pT racer). W estern blot
results demonstrated protein expression of all the generated constructs (Fig. 3C).
Anti-Strep antibody was utilized to detect dif ferent E protein levels, with no band
observed for pT racer and SARS-CoV -2 PM since it lacks a Strep tag. In the case of
SARS-CoV -2 PM, an anti-Envelope protein Antibody was employed for protein
expression detection in the W estern blot analysis. β- T ubulin was used as a loading
control since it is ubiquitously expressed in 293T cells, Furthermore, CD1d:D5 antibody
was utilized to visualize the presence of immature or ER-form CD1d molecules in all the
samples.
1 4
4. Ion channel function of SARS-CoV -2 Envelope Protein through V oltage
Patch Clamp Experiments.
Our research aimed to investigate the involvement of ion channel function in the
molecular mechanism of E protein-mediated CD1d downregulation (Cabrera-Garcia et
al., 2021). In this regard, we conducted V oltage Patch clamp experiments (Cang et al.,
2014, Cang et al., 2015) on SARS-CoV -2 PM E protein. The results revealed the
presence of a Na+ current in cells transfected with SARS-CoV -2 PM E protein (Fig. 4),
indicating its potential ion channel activity . However , we did not observe a proton
current, and further characterization, such as inhibition studies, was not performed due
to challenges with low pH and the presence of endogenous proton-gated Na+ channels
in HEK cells. Additionally , it remains uncertain whether the observed current is specific
or a result of non-specific leakage, possibly influenced by the health status of the cells.
5. SARS-CoV -2, OC43 and 229E E protein cellular localization
Immunofluorescence assay was employed to determine the localization of E proteins
and their mutants from SARS-CoV -2, OC43, and 229E plasmids (Fig. 5). The E protein
plays a crucial role in virus assembly , budding, envelope formation, and pathogenesis,
and it is primarily expressed in the endoplasmic reticulum (ER) and ER-Golgi
intermediate compartment (ERGIC) region (Schoeman et al., 2019). HeLa.CD1d cells
were used in this immunological assay as previously mentioned under cell lines in
material and methods. Three proteins were immunostained: CD1d 51.1.3 labeled with
Alexa647 Cy5, strep-tagged SARS-CoV -2 E protein and its mutants labeled with
Alexa568 TRITC, and ERGIC complex labeled with Alexa488 FITC. The CD1d staining
revealed the presence of CD1d on the cell surface, and due to cell extrusion, the 51.1.3
CD1d was distributed throughout the entire cell. T ransfection with dif ferent coronavirus
E proteins resulted in varying degrees of CD1d downregulation. The ERGIC location
was revealed by 488-FITC, and colocalization with ERGIC was observed for
strep-tagged E protein using 568-TRITC in the immunofluorescence analysis. In the
case of Wild T ype variants of SARS-CoV -2, OC43, and 229E envelope proteins,
immunofluorescence images demonstrated co-localization with ERGIC staining (fig.
5A). However , the TM variants exhibited CD1d protein localization in intracellular
organelles, mainly in the ERGIC region and other cellular areas (fig. 5B). The PM
variants displayed E protein localization in intracellular organelles as well as on the
plasma membrane of the cells (Fig. 5C).
1 5
Chapter 4 : Discussion
The suppression of NKT cells through the reduction of CD1d antigen presentation has
been extensively documented in various DNA and RNA viruses, including HIV , HSV -1,
KHSV and other viruses (Lu H et al., 2023). Previous studies aimed to investigate how
dif ferent forms of the E protein from coronaviruses af fect the expression levels of the
CD1d gene. However , this study aimed to understand the role of E protein in decreasing
the expression of CD1d on antigen presenting cells (APCs) through ion channel
formation. Additionally , the study explores the ion channel functionality of the
SARS-CoV -2 E protein by specifically targeting the protein to the plasma membrane. T o
further analyze the ion channel activity of the envelope proteins, voltage patch clamp
experiments were conducted as shown in Figure 4. The patch clamp technique of fers
several advantages, allowing us to modify voltage or current changes within cell
membranes by applying compounds that either block or open channels. Through these
techniques, we can gain a comprehensive understanding of how ion channels behave in
both normal and disease states, and how various drugs, ions, or other analytes can
influence these conditions.However , it's essential to consider a limitation of the patch
clamp experiment. In natural conditions, the E proteins are localized in the ERGIC
region, not the PM. Consequently , their electrophysiological properties may dif fer to
some extent from those observed in the PM. Nonetheless, conducting patch clamp
studies at the PM serves as a valuable starting point to compare the
electrophysiological properties of dif ferent E proteins.
The study revealed that the E protein of SARS-CoV -2 has a significant impact on the
downregulation of CD1d expression on the cell surface. This downregulation
mechanism may contribute to the suppression of the innate immune system and the
functionality of iNKT cells, which are responsible for the production of type I interferons.
Clinical reports have shown that severe COVID-19 patients exhibit reduced levels of
innate lymphocytes in their blood compared to healthy or mild patients. This overall
decrease in NKT cells and other innate-like T cells supports the notion that T cells play
a crucial role in combating the disease during the early stages.
The CD1d protein is synthesized in the endoplasmic reticulum (ER) and exists in two
distinct forms. Roughly half of the newly synthesized CD1d molecules remain in the ER
as an immature form, while the other half undergoes complete folding, associates with
β2- microglobulin, exits the ER, and undergoes complex glycosylation to express itself
on the cell surface as the mature form. Interestingly , the expression of the envelope
protein led to a significant reduction in the mature form of CD1d, but not in the immature
form present in the ER.
1 6
T o gain a deeper understanding of how the E protein induces CD1d degradation and
downregulates its expression on the cell surface, we examined the dif ferent domains of
the E protein. The E protein is composed of three domains: the N-terminal domain
(N-TM), the transmembrane domain (TMD), and the C-terminal domain (C-TM). It was
found that both the N-TM and TMD domains were equally ef fective in downregulating
CD1d, suggesting that the N-terminal short peptide is not crucial for this function.
Therefore, similar to the full-length E protein, it was determined that the transmembrane
domain (TMD) is responsible for mediating CD1d degradation and downregulation.
Through immunofluorescence staining of CD1d in cells expressing the E protein, it was
clearly observed that both the full-length E protein and the TMD domain of the E protein
caused a noticeable reduction in CD1d staining. This further supports the role of the E
protein, specifically its transmembrane domain, in the degradation and downregulation
of CD1d expression.
Previous computational models and biochemical analyses of the SARS ‐CoV ‐1 E
protein have proposed that it forms a pentamer of 9 kDa subunits, resulting in a 45 kDa
protein complex (Pervushin et al., 2009). Electrophysiological recordings conducted in
artificial planar bilayer membranes have also been employed to investigate the
functional properties of the E protein. As a viroporin, the E protein plays a crucial role in
the viral morphogenesis of coronaviruses by facilitating virion packaging and envelope
formation. However , the exact functions of viroporins are not fully understood.
V iroporins, such as the E protein, are small hydrophobic proteins capable of
oligomerization within the membranes of host cells. This oligomerization process leads
to the formation of hydrophilic pores that allow the passage of ions and small molecules.
The ion channel activity associated with the viroporin function of the E protein is
hypothesized to be linked to the downregulation of CD1d.T o investigate this hypothesis,
a study was conducted utilizing N15A and V25F mutants of the E protein. The results
obtained from these mutant proteins confirmed the existence of a relationship between
the ion channel activity of the SARS-CoV -2 E protein and the degradation of mature
CD1d. By studying the underlying mechanisms through which the envelope protein
downregulates CD1d, it may be possible to develop strategies that can reverse immune
evasion, leading to more ef fective therapeutic treatments for viral infections.
In addition to the previous findings, this study investigated the envelope proteins of two
coronaviruses: the severe SARS-CoV -2 and the mild OC43. The results showed that
the severe coronavirus (SARS-CoV -2) forms a viroporin that is modulated by current.
From Figure 4, Patch clamp experiments using the SARS-CoV -2 plasma membrane
construct demonstrated the presence of Na+ current in cells transfected with the
envelope protein. The change from NMDG to Na+ resulted in an inward current. Further
1 7
characterization is needed to determine if this current is af fected by low pH and to
explore the possibility of non-specific leakage in the cells.
F ACS results from Figure 4A revealed a significant decrease in CD1d expression in
SARS-CoV -2 wild-type (WT) and SARS-CoV -2 transmembrane (TM) plasmid
constructs, consistent with findings in severe coronaviruses. However , downregulation
in the SARS-CoV -2 plasma membrane (PM) construct was observed inconsistently ,
suggesting that the downregulation of CD1d in the PM construct might be weaker or
af fecting a smaller percentage of cells. In contrast, there was no downregulation of
CD1d observed in constructs of mild coronaviruses such as OC43 WT , OC43 TM, OC43
PM, 229E WT , or 229E TM, which aligns with previous studies.
Immunofluorescence results from Figure 5 indicated that the envelope proteins of both
severe (SARS-CoV -2) and mild coronaviruses (OC43 and 229E) localize in the
endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The transmembrane
domain of the envelope protein exhibited localization in the ERGIC region and other
cellular areas, suggesting similar or slightly stronger expression compared to the
wild-type construct. The SARS-CoV -2 and OC43 PM constructs successfully targeted
the envelope protein to the plasma membrane, in addition to the ERGIC localization,
allowing the study of the envelope protein's ion channel properties through voltage
patch clamp experiments. This approach of studying viroporin activity by directing
membrane localization and employing electrophysiological recordings holds promise for
identifying potential antiviral drugs. By screening for inhibitors of the E protein using this
targeted method, researchers can investigate their potential antiviral activity against
SARS-CoV -2 in vitro, with the ultimate goal of developing novel treatments for
COVID-19.
1 8
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2 3
Figures
Figure 1
A
B
Figure 1: Selective Downregulation of CD1d Expression by E proteins from Highly
Pathogenic Coronaviruses.
A. Amino acid sequence alignment of E proteins from seven dif ferent human
coronaviruses.
B. Whole cell lysates of the transfected cells were subjected to immunoprecipitation
using an anti-CD1d antibody (CD1d.51.1.3), and the levels of the mature CD1d
2 4
protein were compared through the western blotting. Additionally , whole cell
lysates were western blotted with anti-Strep antibodies to assess the expression
of individual E proteins from human coronaviruses.
2 5
Figure 2
A.
SARS-CoV -2 WT - RMYSFVS EET GTLIVNSVLL FLAFVVFLL V TLAIL T ALR
LCA YCCNIVNVSL VKPSFYVYSR VKNLNSSR VPDLL V
SARS-CoV -2 TM - EET GTLIVNSVLL FLAFVVFLL V TLAIL T ALR
SARS-CoV -2 PM - RMYSFVS EET GTLIVNSVLL FLAFVVFLL V TLAIL T ALR
LCA YCCNIVNVSL VKPSFYVYSR VKNLNSSR VP GSGSGS
KSRITSEGEYIPLDQIDINV
B.
OC43 WT - MADA YL ADT VWYVGQIIFI V AICLL VTIV VV AFLA TFK
LCIQLCGMCNTL VLSPSIYVFNRGRQFYEFYNDVKPPVLDVDDV
OC43 TM - ADT VWYVGQIIFI V AICLL VTIV VV AFLA TFK
OC43 PM - MADA YL ADT VWYVGQIIFI V AICLL VTIV VV AFLA TFK
LCIQLCGMCNTL VLSPSIYVFNRGRQFYEFYNDVKPPVLD GSGSGS
KSRITSEGEYIPLDQIDINV
2 6
C.
229E WT - MFLKL V DDH AL VVNVLL WC VVLIVILL VC ITIIKLIKLC
FTCHMFCNRTVYGPIKNVYHIYQSYMHIDPFPKR VIDF
229E TM - DDH AL VVNVLL WC VVLIVILL VC ITIIKLIKLC
Figure 2: Generation of SARS-CoV -2, OC43, and 229E Envelope Protein
Constructs: W ildtype, T ransmembrane, and Plasma Membrane V ariants.
A. Three dif ferent constructs of the SARS-CoV -2 envelope protein are depicted. The
first construct (SARS-CoV -2 WT) represents the wildtype version without any
mutations. It displays the amino acid sequences, including the C-terminal ER
retention signal (DLL V). The construct is tagged with a strep tag for protein
expression analysis via western blot. The second construct (SARS-CoV -2 TM)
consists only of the transmembrane domain and is tagged with the strep tag. The
third construct (SARS-CoV -2 PM) is mutated to direct the expression of the E
protein to both the plasma membrane and intracellular structures. This construct
incorporates the envelope protein from the SARS-CoV -2 WT construct fused with
a carboxyl(C)-terminal fluorescent tag mKate2. The fusion is separated by an
intermediate spacer (GS) composed of glycine-serine repeats (GSGSGS).
Additionally , a consensus Golgi export signal (ES) from Kir2.1
( (KSRITSEGEYIPLDQIDINV) is inserted. Mutations in the PDZ-binding motif
(PBM) in the C-terminal domain involve replacing six residues (46-47 and 56-59)
with alanine (Ala6), along with the deletion of the C-terminal ER retention signal
DLL V .
B. Three dif ferent constructs of the OC43 envelope protein are depicted. The first
construct (OC43 WT) represents the wildtype version without any mutations. It
displays the amino acid sequences, including the C-terminal ER retention signal
(VDDV). The construct is tagged with a strep tag for protein expression analysis
via western blot. The second construct (OC43 TM) consists only of the
transmembrane domain and is tagged with the strep tag. The third construct
(OC43 PM) is mutated to direct the expression of the E protein to both the
plasma membrane and intracellular structures. This construct incorporates the
2 7
envelope protein from the OC43 WT construct fused with the strep tag. The
fusion is separated by an intermediate spacer (GS) composed of glycine-serine
repeats (GSGSGS). Additionally , a consensus Golgi export signal (ES) from
Kir2.1 ( (KSRITSEGEYIPLDQIDINV) is inserted. Mutations in the PDZ-binding
motif (PBM) in the C-terminal domain involve replacing four residues (56-59) with
alanine, along with the deletion of the C-terminal ER retention signal VDDV .
C. T wo dif ferent constructs of the 229E envelope protein are depicted. The first
construct (229E WT) represents the wildtype version without any mutations. It
displays the amino acid sequences. The construct is tagged with a strep tag for
protein expression analysis via western blot. The second construct (229E TM)
consists only of the transmembrane domain and is tagged with the strep tag.
2 8
Figure 3
A.
2 9
B.
C.
3 0
Figure 3: Comparison of W ild T ype, T ransmembrane, and Plasma Membrane
constructs generated from SARS-CoV -2, OC43, and 229E plasmids.
A. 293T .CD1d cells were subjected to transfection with various envelope proteins
constructs including the pT racer employed as a positive control. After a 48-hour
incubation, flow cytometry was performed to assess the level of CD1d cell
surface expression.
A. The relative CD1d expression level was calculated, as depicted. Statistical
analysis was performed using unpaired Student’ s t-test and one-way ANOV A test
for F ACS results.
B. T o evaluate the expression of individual E proteins from various constructs
generated, whole cell lysates of the transfected cells were analyzed through
western blotting using anti-Strep antibodies. However , for the SARS-CoV -2 PM
construct that lacks the Strep tg, an anti-Envelope antibody specific to the
envelope protein was utilized. Anti-T ubulin antibody served as the cell loading
control, while the levels of both mature and immature CD1d protein were
assessed using the anti-CD1d:D5 antibody .
3 1
Figure 4
A.
Figure 4: Ion channel function of SARS-CoV -2 PM Envelope (E) protein.
C. T o investigate the molecular mechanism and involvement of ion channel function
in E protein-mediated CD1d downregulation, patch clamp experiments involving
SARS-CoV -2 PM constructs were performed.
3 2
Figure 5
A.
B.
3 3
C.
Figure 5: Localization of SARS-CoV -2, OC43, and 229E Envelope Protein in Cells
and Downregulation of Surface Expression
A. In the context of Wild T ype variants of SARS-CoV -2, OC43 and 229E Envelope
protein localization, mature CD1d staining is represented in green, while E
protein expression is shown in red. Additionally , ERGIC staining is displayed in
purple. The transfected cells exhibited a downregulation in surface expression
compared to the untransfected cells.
B. In the context of T ransmembrane variants of SARS-CoV -2, OC43 and 229E
Envelope protein localization, mature CD1d staining is represented in red, while
E protein expression is shown in orange. Additionally , ERGIC staining is
displayed in green.
C. In the context of Plasma Membrane variants of SARS-CoV -2, OC43 and 229E
Envelope protein localization, mature CD1d staining is represented in red, while
E protein expression is shown in green.
3 4

Abstract (if available)

Abstract The emergence of severe acute respiratory syndrome (SARS) in 2003 and the recent COVID-19 pandemic have underscored the devastating impact of coronaviruses (CoVs) when they cross the species barrier and infect humans. Consequently, there has been a renewed focus on studying coronaviruses, leading to the discovery of several new human CoVs. Understanding the pathogenesis of CoVs is crucial for developing effective antiviral treatments. One notable mechanism employed by these viruses involves evading and suppressing the innate immune system through the downregulation of host immune cell surface CD1d expression.

Our research has revealed that among the studied human coronaviruses, only the E proteins of highly pathogenic viruses such as SARS-CoV-2, SARS-CoV, and MERS are capable of suppressing CD1d expression. In contrast, E proteins of common cold coronaviruses like HCoV-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1 do not possess this ability. This suggests that the evasion of NKT cell function through E protein-mediated suppression of CD1d expression plays a crucial role in the pathogenesis of highly pathogenic coronaviruses, contributing to their increased virulence. We identified the transmembrane (TM) domain of the E protein as a mediator responsible for CD1d downregulation. Specifically, the TM domain reduces the levels of mature CD1d proteins after their departure from the endoplasmic reticulum (ER), indicating its involvement in suppressing CD1d trafficking and promoting their degradation. Through point mutations, we confirmed that the putative ion channel function of the E protein is necessary for CD1d suppression, and we successfully restored CD1d expression by inhibiting the ion channel function using small chemicals.

To investigate the ion channel activity of the TM domain of the E protein, we developed a strategy to target the protein to the plasma membrane. This involved introducing mutations in the C-terminal domain of the E protein and incorporating a Golgi-export signal. By employing this approach, we successfully directed both the SARS-CoV-2 E protein and OC43 E protein to the plasma membrane. Our experiments demonstrated the formation of a viroporin, a cation channel, when the E protein is localized to the plasma membrane. Notably, we observed the passage of Na+ current in cells transfected with the E protein compared to untransfected cells. This advancement in studying viroporin activity provides a valuable tool for screening potential antiviral drugs, enabling the identification of novel COVID-19 treatments by modulating the function of viroporins. These discoveries hold promise for the development of effective therapies against the virus.

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Creator Parandaman, Ramya (author)

Core Title Investigating the role of ion channel activity of coronaviruses envelope protein in CD1d regulation

School Keck School of Medicine

Degree Master of Science

Degree Program Molecular Microbiology and Immunology

Degree Conferral Date 2023-08

Publication Date 08/09/2023

Defense Date 06/07/2023

Publisher University of Southern California. Libraries (digital)

Tag coronavirus,envelope protein,iNKT-CD1d,ion-channel,OAI-PMH Harvest,plasma-membrane,viroporin

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Yuan, Weiming (committee chair), Eoh, Hyungjin (committee member), Qu, Julia (committee member)

Creator Email parandam@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-oUC113297699

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