Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive-sense RNA genome that also serves as the mRNA for four nonstructural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed -replication systems， where production of replication-competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce noncapped RNA templates， which are poorly translated relative to CHIKV replicase-generated capped RNAs. It was found that， in human cells， constructs driven by RNA polymerase I promoters of human and Chinese hamster origin ... More
Chikungunya virus (CHIKV) is a mosquito-borne alphavirus. It has a positive-sense RNA genome that also serves as the mRNA for four nonstructural proteins (nsPs) representing subunits of the viral replicase. Coupling of nsP and RNA synthesis complicates analysis of viral RNA replication. We developed -replication systems， where production of replication-competent RNA and expression of viral replicase are uncoupled. Mammalian and mosquito RNA polymerase I promoters were used to produce noncapped RNA templates， which are poorly translated relative to CHIKV replicase-generated capped RNAs. It was found that， in human cells， constructs driven by RNA polymerase I promoters of human and Chinese hamster origin performed equally well. In contrast， RNA polymerase I promoters from mosquitoes exhibited strong species specificity. In both mammalian and mosquito cells， novel -replicase assays had exceptional sensitivity， with up to 10-fold higher reporter expression in the presence of replicase relative to background. Using this highly sensitive assay to analyze CHIKV nsP1 functionality， several mutations that severely reduced， but did not completely block， CHIKV replicase activity were identified: (i) nsP1 tagged at its N terminus with enhanced green fluorescent protein; (ii) mutations D63A and Y248A， blocking the RNA capping; and (iii) mutation R252E， affecting nsP1 membrane anchoring. In contrast， a mutation in the nsP1 palmitoylation site completely inactivated CHIKV replicase in both human and mosquito cells and was lethal for the virus. Our data confirm that this novel system provides a valuable tool to study CHIKV replicase， RNA replication， and virus-host interactions. Chikungunya virus (CHIKV) is a medically important pathogen responsible for recent large-scale epidemics. The development of efficient therapies against CHIKV has been hampered by gaps in our understanding of how nonstructural proteins (nsPs) function to form the viral replicase and replicate virus RNA. Here we describe an extremely sensitive assay to analyze the effects of mutations on the virus RNA synthesis machinery in cells of both mammalian (host) and mosquito (vector) origin. Using this system， several lethal mutations in CHIKV nsP1 were shown to reduce but not completely block the ability of its replicase to synthesize viral RNAs. However， in contrast to related alphaviruses， CHIKV replicase was completely inactivated by mutations preventing palmitoylation of nsP1. These data can be used to develop novel， virus-specific antiviral treatments.
