Exhaustive mutational analysis of severe acute respiratory syndrome coronavirus 2 ORF3a: An essential component in the pathogen's infectivity cycle

Abstract

Detailed knowledge of a protein's key residues may assist in understanding its function and designing inhibitors against it. Consequently, such knowledge of one of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)'s proteins is advantageous since the virus is the etiological agent behind one of the biggest health crises of recent times. To that end, we constructed an exhaustive library of bacteria differing from each other by the mutated version of the virus's ORF3a viroporin they harbor. Since the protein is harmful to bacterial growth due to its channel activity, genetic selection followed by deep sequencing could readily identify mutations that abolish the protein's function. Our results have yielded numerous mutations dispersed throughout the sequence that counteract ORF3a's ability to slow bacterial growth. Comparing these data with the conservation pattern of ORF3a within the coronavirinae provided interesting insights: Deleterious mutations obtained in our study corresponded to conserved residues in the protein. However, despite the comprehensive nature of our mutagenesis coverage (108 average mutations per site), we could not reveal all of the protein's conserved residues. Therefore, it is tempting to speculate that our study unearthed positions in the protein pertinent to channel activity, while other conserved residues may correspond to different functionalities of ORF3a. In conclusion, our study provides important information on a key component of SARS-CoV-2 and establishes a procedure to analyze other viroporins comprehensively.

A Novel Polymer Nanoparticle Polydimethyl Diallyl Ammonium Chloride as An Adjuvant Enhances the Immune Response of SARS‐CoV‐2 Subunit Vaccine

“…The S protein is expressed as a trimer on the viral envelope. [ 4,5 ] It consists of two subunits, namely S1,S2, with the receptor‐binding domain (RBD) located in the S1 subunit. [ 6–9 ] Due to the ongoing spread of SARS‐CoV‐2, multiple variants of the virus emerged, such as Alpha, Beta, Gamma, Delta, and Omicron.…”