Diverse role of ubiquitylation in SARS-CoV-2 infected mammalian cells

Background:

The pandemic of coronavirus disease 2019 (COVID-19) is a global health emergency concern due to severe
acute respiratory syndrome (SARS) caused by the novel coronavirus. Coronaviruses are positive-sense,
single-stranded RNA viruses that can infect a wide range of avian and mammalian hosts [1]. The proteins
encoded by coronaviruses include 4 main structural proteins, spike (S), envelope (E), membrane (M), and
nucleoprotein (N), 16 major proteins[2]. The spread of the COVID-19 infection initially originated from
animal to human transmission, which was followed by sustained human to human spread.

Post-translational modifications (PTMs) are chemical modifications that are essential for precise regulation
of the properties and functions of proteins and are key mechanisms that increase proteomic diversity in
cells. Lysine ubiquitination is a PTM in eukaryotic cells that is involved in diverse biological activities.
Research on virus infection showed that ubiquitination modification plays a key role in the innate immune
response to viral infections by inducing antiviral defenses and facilitating virus replication [3].

The following research will perform for understanding of ubiquitylation pattern mapping in SARS-CoV-2,
investigating how viruses escape immunity through ubiquitylation and to explore the chance of viral
proteins to be degraded by ubiquitin proteasome system.

Aims of Study

The study aims for following things:

• To map for ubiquitylation pattern in Covid19 infected mammalian cells.

• To investigate the ubiquitination changes that occur in cells during Covid19 infection.
• To investigate the inhibition of Covid19 replication through ubiquitination and promoting its
degradation.
• To identify potential therapeutic targets considering ubiquitylation as basis.
Experimental Design

• Replication of SARS-CoV-2 in cell lines:

I propose SARS-CoV-2 cell-culture model in Caco-2 cells. Inoculation of virus isolated from Covid-
19 patient will be performed. The monolayers of cell will be inoculated with SARS-CoV-2 at a
multiplicity of infection (MOI) of 5.0. This will lead to fast progression of virus and cytopathogenic
effects will be visualized after around 24 hours using microscopy. Viral infection will further be
assessed by staining of SARS-CoV-2 nucleoprotein.

• Ubiquitome analysis in host cell infection:

Following the strategy which includes determination of ubiquitylation of protein by liquid

chromatography (LC) with Mass Spectrometry (MS), ubiquitome analysis will be performed [4]. After
infection with SARS-CoV-2, total cellular proteins will be extracted and will treated with ubiquitin
protein enrichment. Mock infected cells will be used as control. The protein-protein interaction will be
analyzed using STRING database which is very popular protein-protein interaction prediction database.
Based on the interaction generated, I will identify proteins which shows positive sign on binding
ubiquitin molecule and will quantify. From this interaction, various ubiquitination sites will be mapped
to different proteins.

• Determination of subcellular localization of different ubiquitylated proteins:

As subcellular localization of proteins is important parameter in identifying drug target, I will locate
the subcellular location for ubiquitinylated protein determined from Experiment 2. As there are various
tools to determine subcellular localization, my research will be based on BaCelLo bioinformatics tool
which is widely used for eukaryotic cellular localization [5]. From the information generated from this
technique, I can even predict the activity of these ubiquitylated proteins in cell.

• Determining whether SARS-CoV-2 infection changes ubiquitination pattern in host cell:

Literatures have showed changes in ubiquitylation pattern in host proteins after SARS-CoV-2 infection
[6]. Major changes can be determined through this experiment. In the Caco-2 cells, I will inoculate
actual virus and the mock protein to investigate how SARS-CoV-2 hijacks host immune system and
what changes does it bring in ubiquitylation of host proteins to evade innate immune response from
host. Depth understanding of this parameter can bring new therapeutics.

• Protein-Protein interaction changes in ubiquitylation after SARS-CoV-2:

Protein interaction study is important to understand functional links between proteins. I will use gene
ontology enrichment analysis on interacting cell line proteins and viral proteins and identify the major
change in cell processes of interacting protein [7]. I will screen topmost interacting proteins using
bioinformatics and will compare the changes in viral infected cell lines and in control mock protein
incorporated cell lines. I will also try to determine underlying molecular mechanism on the changes in
protein-protein network after SARS-CoV-2 infection in cell. For example, SARS-CoV-2 takeover host
Cul5-RING E3 ubiquitin ligase and protect itself from degradation and other such takeover will be
exploited in this research to know more about protein-protein interaction.
• Ubiquitylation of functional domain of SARS-CoV-2:

I will perform experiment to determine the presence of ubiquitylated residue on viral proteins. By
screening possible protein candidate containing lysine residues will be selected. Identification of
ubiquitin in protein will be readily accomplished using co-immunoprecipitation protocol. The degree
of ubiquitination of the protein of interest will be assessed by comparing the ratio of
ubiquitinated/unmodified target protein in several experiment conditions. I will specifically check for
ubiquitylation in Receptor Binding Domain (RBD) of SARS-CoV-2 virus which interacts with ACE2
in host cells upon entry[8]. The degree of ubiquitylation will also be assessed.

• Searching for the domains of SARS-CoV-2 susceptible to degradation by ubiquitin-proteasome

system:

Initially it has been showed that SARS-CoV-2 nsp16 serves as important gene involved in genome
replication [9]. I will search for the all possible Ubiquitin E3 ligase that act upon virus nssp16. I will
use yeast two hybrid system to screen a cDNA library of human interacting with nsp16. Positive clones
will be identified and will see for inhibition of SARS-CoV-2 replication. Based on the literature review,
E3 ligase which can degrade other viral proteins will be further studied. To confirm the ubiquitylation,
the target E3 Ligase will be knockdown or downregulated using appropriate techniques and the effect
will be visualized in cell culture.

Potential Outcomes

Initially after infecting cells with SARS-CoV-2, in ubiquotome analysis, I expect mapping of numerous
protein-protein interaction prediction sites and ubiquitylation hotspots. From the subcellular
localization experiment, I will know the ubiquitination location in cell which is important for therapy.
For generating new therapeutics, study of ubiquitylation pattern change in cells by SARS-CoV-2 is
important so the determination of protein-protein interaction changes will provide new insights. I
assume SARS-CoV-2 undergoes ubiquitylation in Receptor Binding Domain (RBD) to interact with
ACE2 receptor, so the experiment mentioned above regarding the check for ubiquitylation in RBD will
confirm the result. In the experiment named ‘Searching for the domains of SARS-CoV-2 susceptible
to degradation by ubiquitin-proteasome system’ I assume various host proteins will be found which
builds immunity towards SARS-CoV-2. This assumption is based on fact that every person does not
suffer equally with SARS-CoV-2, so there must be degradation of SARS-CoV-2 by innate immunity
system.

Clinical Significance

Ubiquitin plays an important role in regulating protein on the cellular level and it has promising
potential for a variety of targeted cellular medicine treatments. After successful mapping of
ubiquitylation pattern, numerous sites can be found. Some of the ubiquitylation are incorporated by
viruses to evade immune system so proteasome inhibitors could block viruses from ubiquitinating host
cells and could not evade immune system. It is known that upon SARS-CoV-2 infection, there is
generation of cytokine storm in the cell which has detrimental effects, successful ubiquitylation
inhibitors could alleviate this effect. Similarly, deubiquitylating enzymes could act on those substrates
and reverses the effects. On the other hand, if this research as proposed could find potential host E3
 ligase enzyme which can degrade the viral proteins during entry or during replication, this would be
beneficial to hit the viral target.

References

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