Probing the role of mechanical rigidity in viral xrRNAs as the key to preventing their degradation by host RNases

RNase enzymes are part of the cellular defenses against viral infection, digesting RNA from invading viruses. Some viruses evade RNases by partially blocking digestion so that the RNases produce viral RNA fragments, called xrRNAs, that interfere with cellular processes to enhance infectivity. We recently showed that a Zika virus xrRNA acts as a mechanical roadblock preventing RNases from digesting the viral RNA because of its rigidity. It is unclear if the same is true for other xrRNAs, and it remains to be determined what mechanical and/or structural features of xrRNAs are required in general to produce RNase resistance. We will answer these questions by surveying xrRNAs from viruses like dengue, Zika, West Nile, and yellow fever. We will use laser tweezers to measure the mechanical rigidity and probe how it relates to RNase resistance. We will test if rigidity is sufficient to explain RNase resistance in each case and determine the features required for this resistance, by knocking out specific interactions in the xrRNA with mutations or binding short oligomers to specific parts of the xrRNA to prevent them from folding. Next, we will study the interactions between xrRNAs and RNases by using lasers tweezers to watch single enzyme molecules as they digest RNA messages containing xrRNAs, verifying directly that xrRNAs block RNase motion and observing how RNases respond when they encounter xrRNAs. Finally, by mapping xrRNA folding pathways using these measurements, we will identify key intermediates in which xrRNAs can be 'trapped' to prevent resistance and test if using oligomers to do so is effective at reducing virus pathogenicity in cultured cells. This work will clarify how resistant structures form in xrRNAs, how they interact with RNases, and what are the critical components that could be targeted as a potential therapeutic strategy, and it will provide proof-of-principle that targeting xrRNA mechanical resistance may be effective as an antiviral treatment.