Targeting conserved interaction surfaces of alphavirus nsP4 supports the rational design of live-attenuated vaccine candidates

ABSTRACT Alphaviruses are emerging arboviruses that pose a growing threat to global public health; however, effective interventions remain limited. Recent structural studies of the chikungunya virus (CHIKV) replicase core revealed that the viral RNA-dependent RNA polymerase nsP4 interacts with both nsP1 and nsP2 and is predicted to engage viral RNA. The conservation and multifunctionality of nsP4 make it a challenging but promising target for rational attenuation. Here, we evaluated 58 structure-guided mutations in nsP4 predicted to disrupt interactions with nsP1, nsP2, or viral RNA using a trans -replicase system. Sixteen selected mutations were further analyzed in a CHIKV-o’nyong-nyong virus chimera and Semliki Forest virus (SFV). Most mutants were viable but showed varying degrees of attenuation in mammalian and mosquito cells. Viruses carrying mutations in predicted RNA-interaction sites frequently acquired pseudoreversions and/or second-site compensatory changes, whereas those harboring mutations affecting nsP1 or nsP2 interfaces were more genetically stable. Three substitutions representing defects in nsP1 interaction (F164A), predicted RNA interaction (K174S), and nsP2 interaction (D393A) were further characterized in authentic CHIKV and displayed attenuation consistent with that observed in SFV. In vivo , all three variants showed reduced pathogenicity in the SFV mouse model, with F164A being avirulent, immunogenic, and conferring complete protection against lethal challenge. In a CHIKV mouse model, these mutations attenuated disease and reduced serum virus titers and inflammatory cell infiltration. Together, these findings provide a molecular framework for the rational design of live-attenuated alphavirus vaccine candidates and support broader evaluation of nsP4-based attenuation. IMPORTANCE Alphaviruses are an increasing global health concern, with hundreds of thousands of chikungunya cases reported worldwide in 2025; however, vaccine options remain limited, and no specific antiviral treatment is available. Recent structural studies have revealed conserved functional interfaces in the viral RNA polymerase nsP4, but their value for attenuation had not been systematically explored. Here, using structure-guided mutagenesis, validation in multiple viral systems, and in vivo analysis, we show that selected nsP4 mutations can strongly attenuate replication and pathogenicity while preserving immunogenicity. These findings identify conserved nsP4 interfaces as a shared vulnerability of alphaviruses and provide a molecular framework for the rational design of broadly applicable live-attenuated vaccines while also highlighting nsP4 as a functionally constrained target relevant to antiviral development.