ORF3c is expressed in SARS-CoV-2-infected cells and inhibits innate sensing by targeting MAVS

Abstract

The respiratory virus SARS-CoV-2 is the causative agent for the COVID-19 pandemic. Transmitted by airborne droplets, once the virus reaches and infects its target cells in the respiratory tract, a battle for the cells’ resources ensues. Failure to prevent or contain viral replication allows the virus to mutate and spread further. Coronaviruses use an intricate way to encode multiple proteins within a single RNA molecule. During translation initiation, ribosomes may not recognize the first start codon they encounter and do so at an additional start further downstream (leaky scanning). In the viral ORF3a gene, this mechanism can lead to the expression of ORF3c from an alternative reading frame. Both, ORF3a and ORF3c, are accessory viral proteins, which often function as antagonists of the viral defense mechanisms of the host. Many proteins ins SARS-CoV-2 have been identified to interfere with the cellular immune response, oftentimes helping to avoid recognition of viral components or by directly targeting and disrupting antiviral signaling. Although ORF3c had previously been described in SARS-CoV, its expression and function have remained unknown. To characterize ORF3c, a SARS-CoV-2 variant expressing a tagged ORF3c was generated and subsequent infection experiments confirmed for the first time its expression in vitro. Furthermore, ORF3c was the only accessory protein encoded in ORF3a capable of efficiently suppressing activity of the IFN-β promoter, hinting at its possible function. Further analysis showed that ORF3c directly interacts with the mitochondrial antiviral signaling protein (MAVS) and leads to its degradation. In silico analysis showed, that the sequence of ORF3c is highly conserved across sarbecoviruses, which was reflected in the ability of different orthologs to also efficiently suppress IFN-β induction. Detailed investigation of naturally occurring mutations in ORF3c revealed that efficient viral replication is maintained in the absence of ORF3c. These observations could be confirmed in two human and a

bat-derived cell line. Taken together, ORF3c has been identified as an IFN antagonist of SARS-CoV- 2 and other sarbecoviruses. While ORF3c appears to be dispensable for efficient replication in vitro

and in vivo, its high degree of conservation might be indicative of an important role in SARS-CoV-2 natural host as well as its during co-evolution with other viral genes.