Early Events in RNA Virus Infection: Profiling the Pre-Replicated Interactome of Chikungunya Virus

Abstract

The race between RNA virus replication and the host innate immune response begins immediately after viral infection. While awaiting transcriptional upregulation of interferons and antiviral genes, cells must rely on mRNAs and proteins that already exist in the cytoplasm. RNA viruses also must co-opt host machinery for replication before the innate immune response primes cellular defenses. However, the molecular interactions that underly this early communication between host protein and viral RNA are not well understood. We developed a method to identify proteins that directly bind to viral RNA genomes pre-replication that we term VIR-CLASP (Viral Cross-Linking And Solid-phase Purification). Our approach can be used with essentially any RNA virus and can capture protein-RNA interactions within minutes of viral infection. We used VIR-CLASP to identify hundreds of host RNA-binding proteinss that interact with the pre-replicated Chikungunya virus (CHIKV) genome, including the YTHDF family of N6-methyladenosine (m6A) reader proteins. We established that m6A RNA modifications are abundant on the 5’ end of CHIKV genomic RNA. We discovered that the effect of m6A on CHIKV viral replication is subject to combinatorial regulation by YTHDFs: knockdown and over-expression studies revealed that YTHDF1 strongly restricts viral replication, while YTHDF2 and YTHDF3 have the opposite effect. Upon mapping of YTHDF1 sites, we found that YTHDF1 binds to non-m6A-modified RNA sites on the CHIKV genome. Analysis of published datasets revealed that YTHDF2 also binds non-m6A-modified sites on mRNA to regulate mRNA translation. There are currently no direct therapeutic options available to those at risk for CHIKV infection and the debilitating, long-lasting joint pain that follow. VIR-CLASP aims to increase the known repertoire of drug targets for CHIKV, and other RNA viruses, in order to advance current efforts to design vaccines and anti-viral compounds.