Furusawa Yuri, Iwatsuki-Horimoto Kiyoko, Yamayoshi Seiya, Kawaoka Yoshihiro
The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Shinjuku, Tokyo, Japan.
Division of Virology, Institute of Medical Science, University of Tokyo, Bunkyo, Tokyo, Japan.
J Virol. 2025 Jun 17;99(6):e0065625. doi: 10.1128/jvi.00656-25. Epub 2025 May 13.
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.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）奥密克戎变种于2021年底出现，其亚变种仍在全球传播。虽然这些变种的刺突（S）蛋白变化已得到广泛研究，但非结构蛋白中氨基酸替代的作用尚未完全揭示。在本研究中，我们发现，当我们通过反向遗传学方法构建多个SARS-CoV-2变种，或对从临床样本中分离的SARS-CoV-2进行传代时，携带NSP6-L260F替代的SARS-CoV-2会反复出现，且它是在细胞培养条件下被选择出来的。虽然在自然界传播的BQ.1.1和XBB.1.16中已检测到这种替代，但它对病毒特性的影响尚不清楚。在此，我们通过反向遗传学方法构建了携带或不携带NSP6-L260F替代的SARS-CoV-2，发现NSP6-L260F通过提高病毒聚合酶活性促进病毒复制，并增强病毒在仓鼠中的致病性。我们还鉴定出了不利替代，即NSP13-M233I和NSP14-D222Y，它们分别降低了BQ.1.1和XBB.1.16的复制能力。这些不利影响被NSP6-L260F抵消。我们的研究结果表明NSP6-L260F对病毒复制和致病性的重要性，并揭示了奥密克戎变种进化过程的一部分。
重要性
虽然严重急性呼吸综合征冠状病毒2（SARS-CoV-2）奥密克戎变种的特性通过获得各种氨基酸替代而不断变化，但非结构蛋白中氨基酸替代的作用尚未得到充分探索。在本研究中，我们发现NSP6-L260F替代增强了病毒聚合酶活性，对病毒复制和致病性很重要。此外，我们发现BQ.1.1谱系中的NSP13-M233I替代和XBB.1.16谱系中的NSP14-D222Y替代降低了病毒聚合酶活性，而这种不利影响被NSP6-L260F替代抵消。我们的结果为SARS-CoV-2的进化过程提供了见解。
