Distinct chikungunya virus polymerase palm subdomains contribute to virus replication and virion assembly

Alphaviruses encode an error-prone RNA-dependent RNA polymerase (RdRp), nsP4, required for genome synthesis, yet how the RdRp functions in the complete alphavirus life cycle is not well-defined. Previous work using chikungunya virus (CHIKV) has established the importance of the nsP4 residue cysteine 483 in maintaining viral genetic fidelity. Given the location of residue C483 in the nsP4 palm domain, we hypothesized that other residues within this domain and surrounding subdomains would also contribute to polymerase function. To test this hypothesis, we designed a panel of nsP4 variants via homology modeling based on the Coxsackievirus B3 3 polymerase. We rescued each variant in both mammalian and mosquito cells and discovered that the palm domain and ring finger subdomain contribute to polymerase host-specific replication and genetic stability. Surprisingly, in mosquito cells, these variants in the ring finger and palm domain were replication competent and produced viral structural proteins, but they were unable to produce infectious progeny, indicating a yet uncharacterized role for the polymerase in viral assembly. Finally, we have identified additional residues in the nsP4 palm domain that influence the genetic diversity of the viral progeny, potentially via an alteration in NTP binding and/or discrimination by the polymerase. Taken together, these studies highlight that distinct nsP4 subdomains regulate multiple processes of the alphavirus life cycle, placing nsP4 in a central role during the switch from RNA synthesis to packaging and assembly. Author SummaryChikungunya virus (CHIKV) is a re-emerging alphavirus transmitted to humans by mosquitoes and causing frequent explosive outbreaks. Its replication relies on a polymerase that incorporates a significant number of errors in the new genomes, making it a good candidate to develop vaccines or antiviral strategies. However, little is known on alphavirus polymerase function in alternate hosts. To begin to understand how the CHIKV polymerase nsP4 functions, we designed a panel of nsP4 variants taking advantage of the conservation of polymerase structure across positive strand RNA viruses. We discovered that the palm domain and ring finger of the polymerase were involved in host-specific RNA replication, genetic stability, and virus assembly. In addition, we demonstrated that the palm domain directly impacted the generation of viral genetic diversity. Taken together, these findings add further evidence to the crucial impact of the core palm domain of CHIKV polymerase not only on the replication of the RNA itself, but also on the genetic stability of the protein, as well as its involvement in viral assembly.