Structural determinants of viral RNAs resistant to exoribonucleases in the alphavirus supergroup

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Summary Structured elements within genomes of RNA viruses have critical roles during infection. Examples are exonuclease-resistant RNAs (xrRNAs) that resist degradation by host cellular exoribonucleases, leading to the production of viral subgenomic RNAs. Detailed three-dimensional structural studies of xrRNAs from flaviviruses and a subset of plant viruses led to a mechanistic model in which xrRNAs block enzymatic digestion using a ring-like structure that braces against the protein surface. Very recently, a new xrRNA class was found in the more distantly related and diverse alphavirus supergroup, which includes several important human pathogens. Initially found in the Benyviridae and several other virus families, this xrRNA has no obvious similarity to those previously studied. Thus, we do not know how it folds, how it blocks the host exonuclease, and how widespread it is distributed. These fundamental gaps in knowledge block progress in understanding what could be a pervasive and important RNA element in the alphavirus supergroup and beyond. In addition, because xrRNAs have been found in several major superfamilies of viruses, they may represent a `molecular clock' that could help us understand virus evolution and pathogenicity. To that end, understanding the fold of the xrRNA from Benyviridae is critical. Here, we propose to address these key unknowns and enable future studies by pursuing two aims. First, we will determine the structural basis of exonuclease resistance by the new class of xrRNA from Benyviridae. This aim will use advanced structural biology methods to inform not only on the mechanism for resistance to degradation, but also enable accurate searches for similar motifs throughout the alphavirus supergroup. Second, we will determine the distribution of the xrRNA fold from Benyviridae across the viral world. This aim combines computational and biochemical approaches, which has previously led to successfully characterizing xrRNAs that were also considered `exotic' at the time. Exploring how widespread this new class of xrRNAs is has the potential to illuminate the biology of viruses of diverse but related alphavirus supergroup members, expand our knowledge of RNA structure in the viral world, and motivate futures studies into the roles of xrRNAs in virus-induced disease.