Betacoronavirus Internal protein as a determinant of pathogenicity

Human betacoronaviruses encode an Internal (I) protein via an alternative reading frame within the nucleocapsid (N) gene, namely ORF8b for Middle-East Respiratory Syndrome Coronavirus (MERS-CoV) and ORF9b for Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and SARS-CoV-2. Previous reports suggested that ORF8b and ORF9b are involved in evading innate immune responses, but their roles in mediating pathogenesis in infected animals has not been determined. In this study, we abrogated the expression of ORF8b in MERS-CoV (rMERS-Δ8b) and ORF9b in SARS-CoV-2 (rSARS2-Δ9b) and found that rMERS-Δ8b was more virulent while rSARS2-Δ9b was attenuated compared to their respective wild-type (WT) viruses in mice. Upon further analysis, we detected high levels of IFN-I expression and enhanced infiltration of immune cells to the lungs of rMERS-Δ8b-infected mice. In addition, mutations identified in previously circulating SARS-CoV-2 variant of concern (VOC) that were proposed to enhance ORF9b expression did not lead to significant changes in virulence. These data suggest the disparate and virus-specific functions of ORF8b and ORF9b which potentially explain the different changes in virulence.