Structural and Computational Insights into the Attenuated Innate Immune Recognition of the SARS-CoV-2 N15 Lineage, an Early-Pandemic Variant.

Functional diversification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages influences fitness and evasion, yet early-pandemic determinants remain incompletely characterized. In this study, we investigated the molecular basis of a weakened immune phenotype of a SARS-CoV-2 isolate, N15, which shares genetic backbone with the ancestral Wuhan-Hu-1 strain, using integrated experimental observations and comprehensive computational modeling. While N15 showed replication kinetics comparable to those of MA10, Beta, and Omicron in Calu-3 cells, it induced significantly lower cytokine and interferon responses, demonstrating that efficient replication can be maintained despite attenuated innate immune activation. To identify the viral determinants driving this phenotype, we systematically evaluated the thermodynamic and structural consequences of N15-specific mutations. Structural bioinformatics analysis revealed that mutations in nonstructural protein 13 (nsp13, H290Y) and the envelope protein are (E protein, T11M) expected to have notable effects on the attenuated phenotype of the N15 strain. Specifically, the H290Y substitution in nsp13 is predicted to enhance protein stability by physically shielding a key ubiquitination site, thereby potentially promoting intracellular viral persistence and delaying host immune sensing. Furthermore, the E protein T11M substitution is predicted to reduce its channel activity via altered monomer and pentamer stability. Together, these findings suggest a mechanistic model in which the degradation-resistant nsp13 and the dysfunctional E protein ion channel serve as putative contributors to the virus's ability to preserve replication while decreasing host innate immune responses. By generating plausible hypotheses, this work provides a structural framework and identifies specific candidate mechanisms that warrant future experimental validation to elucidate the molecular basis of attenuated viral pathogenesis.