Mutations in the nsP1 dodecamerization surface impair Semliki Forest virus RNA synthesis in mosquito cells

Positive-sense RNA (+ssRNA) viruses remodel host membranes to form replication organelles (ROs), a process largely driven by the oligomerization of virus-encoded membrane-binding proteins. In the case of Togaviruses, non-structural protein 1 (nsP1) assembles into dodecameric rings that serve as scaffolds bridging viral replication spherules with the host plasma membrane. However, the precise role and necessity of nsP1 oligomerization in viral replication remain incompletely understood. In this study, we employed structure-based computational modeling to design nsP1 mutants predicted to disrupt monomer-monomer interactions within the dodecamer. Molecular dynamics simulations further indicated that these mutations may impair the membrane-binding affinity of nsP1. Western blotting was performed under denatured reducing conditions, and confocal microscopy was used to assess the expression levels and subcellular localization of mutant nsP1, respectively. Mutant proteins appear to not be stably retained at the plasma membrane, instead being mislocalized to endosomal compartments. Three-component trans-replicase assays and viral challenge experiments revealed that nsP1 dodecamerization-surface mutants are deficient in their ability to support viral replication. This study highlights the critical role of nsP1 dodecamerization in Togavirus replication and proposes a potential antiviral strategy targeting oligomeric protein structures in +RNA viruses. IMPORTANCE: Togaviruses are emerging arboviruses capable of causing severe disease and sporadic outbreaks. During viral replication, viral nsP1 assembles into dodecameric rings that anchor replication organelles called spherules to host membranes; however, the functional necessity of the oligomeric organization remains poorly defined. In this study, we combined structure-based modeling, molecular dynamics simulations, and cell-based assays to investigate how mutations that are predicted to destabilize nsP1 oligomers affect viral replication. We found that these mutations reduced membrane-binding affinity, altered subcellular localization, and nsP1 harboring these mutations failed to support viral RNA replication. These findings indicate that nsP1 dodecamerization is required for its stabilization on plasma membranes. More broadly, our findings suggest that targeting the oligomerization of membrane-associated replication proteins represents a viable antiviral strategy for Togaviruses and potentially other positive-sense RNA viruses.