Amir Rooddehghan, University of Saskatchewan/VIDO

Amirhossein Rooddehghan1,2, Zeineb M'hamdi2, Angela L. Rasmussen1,2 1 University of Saskatchewan, Department of Biochemistry, Microbiology and Immunology, Saskatoon, Canada 2 Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan

Suppressing & Conquering

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Although much research on functional significance has focused on mutations in the spike protein, adaptation is also dependent upon changes in other proteins. Many SARS-CoV-2 accessory proteins are involved in antiviral response inhibition or invasion. Variant-specific differences in interferon (IFN) induction and signaling mediated by nonstructural accessory proteins may reveal insights into the mechanisms regulating host antiviral response evasion. First, we identified the distinct non-synonymous mutations among different variants of SARS-CoV-2 (alpha, beta, gamma, delta, and Omicron BA.1, BA.2, BA.4, BA.5). We generated plasmids expressing ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF9b from the ancestral B.1 virus or variant-specific mutations that have emerged since 2020. Protein expression was confirmed by immunoblotting. Luciferase Reporter assay data indicates that ORF6 WT inhibits ISG expressions less than wild type up to 70%. Mutations in ORF3a Q57H (Beta) and S253P(Gamma) suppress ISG expression more than wild type ORF3a. IFIT1 and ISG54 expression were quantified using qPCR to confirm ISG induction. Data showed these mutations do not impact IFIT1 and ISG54 expression inhibition compared to wild type proteins. Immunoblotting data confirmed consistent expression of ORF3a Q57H, and ORF3a S253P. It may be concluded that ORF3a Q57H, and ORF3a S253P are actual phenotypes. The results highlight the functional role of variant-specific mutations in non-structural accessory proteins play in suppressing the innate immune response. Further research and work are needed to specify and address these accessory proteins' role in pathogenesis and transmission.