The NSP6-L260F substitution in SARS-CoV-2 BQ.1.1 and XBB.1.16 lineages compensates for the reduced viral polymerase activity caused by mutations in NSP13 and NSP14.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants emerged at the end of 2021, and their subvariants are still circulating worldwide. While changes in the S protein of these variants have been extensively studied, the roles of amino acid substitutions in non-structural proteins have not been fully revealed. In this study, we found that SARS-CoV-2 bearing the NSP6-L260F substitution emerged repeatedly when we generated several SARS-CoV-2 variants by reverse genetics or when we passaged SARS-CoV-2 isolated from clinical samples and that it was selected under cell culture conditions. Although this substitution has been detected in BQ.1.1 and XBB.1.16 that circulated in nature, its effect on viral properties is unclear. Here, we generated SARS-CoV-2 with or without the NSP6-L260F by reverse genetics and found that NSP6-L260F promotes virus replication and by increasing viral polymerase activity and enhancing virus pathogenicity in hamsters. We also identified disadvantageous substitutions, NSP13-M233I and NSP14-D222Y, that reduced BQ.1.1 and XBB.1.16 replication, respectively. These adverse effects were compensated for by NSP6-L260F. Our findings suggest the importance of NSP6-L260F for virus replication and pathogenicity and reveal part of the evolutionary process of Omicron variants.IMPORTANCEAlthough the properties of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variants continue to change through the acquisition of various amino acid substitutions, the roles of the amino acid substitutions in the non-structural proteins have not been fully explored. In this study, we found that the NSP6-L260F substitution enhances viral polymerase activity and is important for viral replication and pathogenicity. In addition, we found that the NSP13-M233I substitution in the BQ.1.1 lineage and the NSP14-D222Y substitution in the XBB.1.16 lineage reduce viral polymerase activity, and this adverse effect is compensated for by the NSP6-L260F substitution. Our results provide insight into the evolutionary process of SARS-CoV-2.