Chikungunya virus live-cell reporter systems reveal cell-type-dependent antiviral responses to ribavirin
Chikungunya virus (CHIKV) remains a significant global public health threat, yet antiviral discovery is limited by experimental systems that provide little mechanistic resolution and rely heavily on transformed cell lines of uncertain physiological relevance. Here, we developed complementary live-cell CHIKV reporter systems comprising a dual-fluorescent subgenomic replicon and fluorescent infectious viruses that enable real-time monitoring of distinct stages of the viral replication cycle. By coupling an nsP3-tagged reporter to genomic RNA translation and replication with a subgenomic reporter of structural gene expression, we achieved temporal resolution of early and late replication events in living cells. Functional validation using replication-defective and subgenomic RNA-deficient mutants confirmed that the system distinguishes between viral RNA replication and downstream structural gene expression. Application of these reporters revealed distinct antiviral responses across transformed and physiologically relevant human cell models. Ribavirin and the nsP2 inhibitor RA-0002034 potently inhibited CHIKV replication in transformed cell lines. Unexpectedly, ribavirin activity was markedly diminished in primary human skeletal muscle myoblasts and dermal fibroblasts, despite robust antiviral effects in commonly used screening cell lines. Mechanistic studies showed that ribavirin-mediated inhibition was rescued by guanosine but not inosine supplementation and was recapitulated by mycophenolic acid, consistent with guanine nucleotide depletion as the predominant antiviral mechanism under the conditions tested. Notably, inhibition through this pathway was substantially less effective in primary human cells. Together, these findings establish versatile live-cell CHIKV reporter systems for mechanistic studies of viral replication and reveal striking cell-type-dependent differences in response to guanine nucleotide depletion. Our results demonstrate that host-cell context can profoundly influence both CHIKV replication and antiviral susceptibility, highlighting the importance of physiologically relevant models for understanding virus-host interactions and antiviral mechanism.