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.

Keywords: evolutionary conservation; genetic selection; ion channel; vulnerability mapping.

FIGURE 1

Mutation libraries analyzed in the study representing amino acid substitution occurrences for each of the protein's 275 residues. Panels (b) and (c) depict the results without and with protein induction, respectively. The color coding for each amino reflects their physicochemical properties according to the “shapely models” as implemented in Rasmol (Sayle & Milner‐White, 1995). The sequence of the wild‐type protein is given in panel a

FIGURE 2

Mutation over‐representation due to protein induction, presented as a function of amino acid mutation position, from 1 to 275. Values are calculated by dividing occurrences of specific mutations in the induced library (Figure 1c) by the occurrences of the same mutation in the uninduced library (Figure 1b). Amino acid color coding reflects their physicochemical properties according to the “shapely models” as implemented in Rasmol (Sayle & Milner‐White, 1995)

FIGURE 3

ORF3a monomeric (a) and dimeric (b) structures (Kern et al., 2021) colored according to the deleterious mutation hotspots identified in the study. The presumed locations of the lipid bilayer and cytoplasm are indicated. Each mutation in the induced library was multiplied by its PAM10 matrix score according to the corresponding position in the protein, and the sum of all the mutations in that position was calculated. The figure was generated by visual molecular dynamics (Humphrey et al., 1996)

FIGURE 4

Multiple sequence alignment of the ORF3a family. The sequences from top to bottom are: P0DTC3, A0A0K1Z045, A0A0U1WHG9, D3KDM6, E0XIZ4, P59632, Q3I5J4, Q3LZX0, A0A8F1CXK9, Q3ZTF2, A0A6G6A1N1, and A0A023PTR5. The yellow diagram represents the calculated alignment score, and at the bottom the logo at that position. Amino acids are color‐coded according to their physicochemical properties given by Jalview (Waterhouse et al., 2009), which was used to conduct the multiple sequence alignment using the default parameters

FIGURE 5

Mutations (relative to the P0DTC3 variant) found in 807,520 circulating variants of severe acute respiratory syndrome coronavirus 2 ORF3a. Particular abundant substitutions are indicated. The 20 most common mutations found in this study are presented as green dots with the mutation name. The database was downloaded from the NCBI virus database and analyzed by R studio (R Core Team, 2020)

FIGURE 6

Comparison between 3a conservation within the coronavirinae and the results of this study. Each point in the graph represents a single residue in the protein, whose distance from the abscissa and ordinate corresponds to its prevalence in our induced library and conservation, respectively. Individual data are depicted in Figures 2 and 4.