Evolution of BA.2.86 to JN.1 reveals that functional changes in non-structural viral proteins are required for fitness of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is still circulating among humans, leading to the continuous evolution. SARS-CoV-2 Omicron JN.1 evolved from a distinct SARS-CoV-2 lineage, BA.2.86, and spread rapidly worldwide. It is unclear why BA.2.86 did not become dominant and was quickly replaced by JN.1, which possesses one amino acid substitution in the spike protein (S:L455S) and two in the non-spike proteins NSP6 and ORF7b (NSP6:R252K and ORF7b:F19L) compared to BA.2.86. Here, we utilized recombinant viruses to elucidate the impact of these mutations on the virological characteristics of JN.1. We found that the mutation in the spike attenuated viral replication, while the non-spike mutations acted synergistically to enhance replication. This suggests that the mutations in the non-spike proteins compensate for the one in the spike, improving viral fitness, as the mutations in the spike contribute to further immune evasion. Our findings suggest that functional changes in both the spike and non-spike proteins are necessary for the evolution of SARS-CoV-2, enabling evasion of adaptive immunity within the human population while sustaining replication. IMPORTANCE: Because the spike protein is strongly associated with certain virological properties of SARS-CoV-2, such as immune evasion and infectivity, most previous studies on SARS-CoV-2 variants have focused on spike protein mutations. However, the non-spike proteins also contribute to infectivity, as observed throughout the evolution of Omicron subvariants. In this study, we demonstrate a "trade-off" strategy in SARS-CoV-2 Omicron JN.1 in which the reduced infectivity caused by spike mutation is compensated by non-spike mutations. Our results provide insight into the evolutionary scenario of the emerging virus in the human population.