DECIPHERING CHIKUNGUNYA VIRUS REPLICATION: THE ROLE OF RNA STRUCTURAL AND SEQUENCE ELEMENTS IN GENE EXPRESSION AND REPLICATION

Chikungunya virus (CHIKV) is a significant global pathogen, causing illness and disabilitythroughout tropical regions. It is therefore important to understand how chikungunya virus replicates sothat we can identify targets for antiviral drugs. Here, we studied how chikungunya virus uses features ofits subgenomic promoter and subgenomic RNA to promote the production of its structural proteins. Wehypothesized that CHIKV may use RNA structure in the sgRNA 5’ untranslated region (UTR) to promotesgRNA translation. Indeed, we used SHAPE-MaP to identify a highly stable hairpin, which we call sgSL1,in the sgRNA 5’ UTR. Mutagenesis studies identified sequences within sgSL1 that are important forstructural protein production; however, we did not find evidence that RNA structure is important forsupporting sgRNA translation. Instead, we found that the sequence of nucleotides in the loop of sgSL1has a particularly strong role in promoting sgRNA transcription; its role in translation remains unclear.Therefore, these results suggest that the primary sequence of paired nucleotides (6-10 and 17-21) in thesgRNA 5’ UTR promotes viral structural protein expression and the primary sequence of unpairednucleotides in the intergenic region (11-15) promotes transcription. We also describe a protocol for usingreverse transcription-PCR in combination with commercial nanopore sequencing services to performcompetition assays on untagged RNA viruses. Our assay, called the Universal Competition Assay byNanopore Sequencing (U-CAN-seq), is relatively cheap and highly sensitive. We used this approach toshow that mutations to the CHIKV 5’ conserved sequence element that disrupt sequence but not structuredid not affect the fitness of CHIKV. However, similar mutations to an adjacent CHIKV stem loop (SL3) didcause a fitness disadvantage compared to wild-type CHIKV, suggesting that structure-independent,primary sequence determinants in this loop play an important role in CHIKV biology. Together, ourresults illuminate the subtle ways in which CHIKV uses noncoding features of its RNA to promote viralreplication.