A novel interaction between the 5′ untranslated region of the virus genome and Musashi homolog 2 is essential for Chikungunya virus genome replication

Chikungunya virus (CHIKV) is a single-stranded, positive-sense alphavirus of the Togaviridae family and is transmitted among humans via Aedes spp. mosquitos. Typical symptoms of CHIKV infection include debilitating arthralgia which can persist for months or years. The recent re-emergence of CHIKV raises serious global health concerns due to high rates of morbidity and the lack of licensed antiviral drugs or clinically approved vaccines. Current knowledge about the molecular mechanisms controlling CHIKV replication and virus-host interactions is limited. Previous studies from our group have mapped six stem-loops within the 5′ untranslated region (5′ UTR) and the first ~200nt of ORF-1. Phenotypic analysis demonstrated that they are RNA replication elements (RREs) required for virus genome replication through structuredependent mechanisms, which involve vertebrate and invertebrate-specific factors. However, the aspect of molecular virology of how the RREs function or through what interactions are yet to be investigated. In this study, reverse genetics and biochemical approaches were used to identify and confirm a specific interaction between cellular RNA binding protein Musashi homolog 2 and this structured region of the CHIKV genome. Using electromobility shift assay, I confirmed the direct interaction between MSI2 and the 5′ UTR of the CHIKV genome, with the binding site being the single-stranded region upstream of the AUG start codon. Using infectious virus and sub-genomic replicon systems, combined with RNA silencing and drug inhibition assays, it was demonstrated for the first time that MSI2 is required for CHIKV genome replication. A CHIKV trans-complementation system and strand-specific qRT-PCR were used to show that MSI2 is required for the initiation of negative-strand synthesis, possibly by functioning as a molecular switch for translation and replication as MSI2 also interacts directly or indirectly with viral non-structural proteins nsP1 and nsP3 – both are essential components of the viral replication complex. These findings provide novel insights into how CHIKV exploits cellular components for its replication and identify potential targets for antiviral therapy.