The ongoing coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This virus is a zoonotic pathogen belonging to the family Coronaviridae of the genus betacoronaviruses. It is around 30kb in size and is an extremely infectious single-stranded and positive-sense RNA virus.
The Functional Roles of SARS-CoV-2 Proteins
SARS-CoV-2 encodes structural (spike, envelope, membrane, and nucleocapsid) and non-structural (Nsp1-Nsp16) proteins. The non-structural proteins are transcribed from two major open reading frames (ORF1a and ORF1b), which produce Nsps. It also contains around six accessory proteins, i.e., ORF3a, ORF6, 60 ORF7a, ORF7b, ORF8, and ORF10.
The primary function of the spike (S) protein of SARS-CoV-2 is to establish the viral infection. The S protein consists of two functionally distinct subunits, S1 and S2. The receptor-binding domain (RBD), present in the S1 domain, interacts with the host cell’s angiotensin-converting enzyme 2 (ACE2) and the S2 domain fuses the cell membranes. In this way, the virus gains entry into the host cell. The primary function of the Nsps is initiating viral replication and, thus, promoting viral RNA synthesis by the RNA-dependent RNA polymerase Nsp12.
Both structural proteins, as well as Nsps, take part in immune evasion. In the case of the Nsps, it suppresses interferon response and, thereby, aids in the host’s innate immune system evasion. Accessory proteins are non-conserved and are highly variable among different coronavirus species. Although functional roles of these proteins are mainly unknown, they are known to take part in the evasion of immune responses and disease severity.
SARS-CoV-2 Proteins and Development of Therapeutics
To date, SARS-CoV-2 has claimed more than 4.85 million lives worldwide. Therefore, to protect individuals from this virus, there is a continuing need for more effective therapeutics. To develop SARS-CoV-2 therapeutics, understanding the intraviral and viral-host protein-protein interactions (PPIs) associated with COVID-19 infection is essential.
Previous studies have shown that Nsp10 and Nsp8 are potential inducers of the “cytokine storm“. Cytokine storm is a dysregulated and hyperactive immune response that occurs in severe COVID-19 disease and often causes death. These studies have indicated that Nsp8 and Nsp10 could be efficacious targets for drug development. In addition to its role in viral infection, RBD is also a critical determinant of viral tropism, which is a major target for SARS-CoV-2-neutralizing antibodies.
Mutations have led to the emergence of several SARS-CoV-2 variants which have been classified as variants of interest (VOI) and variants of concern (VOC). All the variants have shown mutations in the S protein, including in the RBD region. The mutation rate of SARS-CoV-2 is found to be relatively low when compared with other RNA viruses. In comparison to the original strain, SARS-CoV-2 VOCs have shown increased transmissibility, high virulence, and a decrease in the effectiveness of available vaccines and diagnostics.
A New Bacterial Two-hybrid (B2H) System
The development of novel methods for identifying and dissection of the interactions of virally encoded proteins is essential for understanding basic viral biology. Moreover, as stated above, these also provide a foundation for therapeutic advances. Recently, scientists have developed a bacterial two-hybrid (B2H) system to study the PPIs of SARS-CoV-2 in a heterologous non-eukaryotic system. This study is available on the bioRxiv* preprint server. Put simply, the B2H system could be used to analyze the SARS-CoV-2 proteome.
In this study, sixteen distinct intraviral protein-protein interactions (PPIs) were identified, involving sixteen proteins, and many of the recently identified proteins were found to interact with more than one partner. Researchers also carried out genetic dissection of these interactions via their model and were able to identify selectively disruptive mutations. Thus, this system aids the genetic dissection of protein interactions and discovers their functional roles.
Researchers demonstrated that a modified B2H system could detect disulfide bond-dependent PPIs in the normally reducing Escherichia coli cytoplasm. Additionally, the spike RBD-ACE2 interaction was studied, and the effect of mutations found in VOCs was investigated using this system. Researchers found that the RBD-ACE2 interaction got perturbed by several RBD amino acid substitutions in circulating VOCs. Thus, in principle, this system could also facilitate the identification of potential therapeutics targeting the interactions of virally encoded proteins.
Importance of the Study
The study highlights the use of the bacteria-based system for studying the interactions of the proteins encoded by SARS-CoV-2. For the study of viral PPIs, B2H is a convenient and economical genetic tool. According to the authors, this is the first bacteria-based viral interactome that describes sixteen different intraviral PPIs from SARS-CoV-2. In addition, one of the advantages of using a non-eukaryotic system (the B2H assay) is the lack of bridging factors that typically complicate the interpretation of positive results.
A limitation of this bacterial system is the lack of machinery for promoting potentially post-translational modifications, e.g., protein phosphorylation and protein glycosylation.
The current study revealed that the newly developed oxidizing B2H reporter strain was able to detect the SARS-CoV-2 spike RBD-ACE2 interaction. Furthermore, it could also characterize the effects of several RBD substitutions present in circulating variants.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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