Trans-complementation of chikungunya virus replicase mutants reveals alphavirus replication complexity and supports antiviral tool development.

Chikungunya virus (CHIKV) is a reemerging alphavirus responsible for large-scale outbreaks in tropical regions. Its RNA replication depends on the assembly of functional replication complexes using the P123 and P1234 polyprotein precursors and their cleavage products, the nonstructural proteins (nsP1-nsP4). To dissect this process, we developed a trans-complementation assay using either plasmid-based expression or tetracycline-inducible stable cell lines expressing individual nsPs to rescue the activities of defective replicases. CHIKV nsP1, as well as nsP1 from closely related alphaviruses such as Ross River virus, successfully complemented CHIKV replicases carrying RNA capping-deficient mutations in nsP1. However, no complementation was observed for a replicase with an nsP1 mutation that completely disrupted membrane association. CHIKV and Eastern equine encephalitis virus (EEEV) nsP4 formed functional replication complexes with matching P123, as well as with P123 from most of alphaviruses. Genomes of CHIKV and EEEV lacking the nsP4 region remained infectious in cells expressing the corresponding nsP4 and could be propagated under these conditions. CHIKV replicase containing a mutation in the protease active site of nsP2 was also rescued by transient expression of wild-type nsP2. In contrast, replicases with mutations in the active site of the NTPase/RTPase/helicase domain of nsP2, or in nsP3 affecting phosphorylation or ADP-ribose binding/hydrolysis, could not be complemented. These results reveal key functional interdependencies among CHIKV nonstructural proteins. The inducible cell lines and trans-complementation platform for CHIKV and EEEV lacking nsP4 represent valuable tools for generating conditionally infectious virus systems and for facilitating high-throughput antiviral and neutralizing antibody screening under lower biosafety conditions.