PROJECT SUMMARY Flaviviruses are single-stranded positive-sense RNA viruses that include dangerous human pathogens like dengue, West Nile, Yellow Fever, Zika, and many others. During infection, these viruses produce a set of non- coding RNAs called ‘subgenomic flavivirus RNAs’ (sfRNAs) that interact with cellular proteins to manipulate the cellular environment, to include inhibiting the antiviral response. sfRNAs have been directly linked to cytopathic and pathogenic outcomes, and viruses that cannot produce sfRNAs are attenuated, motivating efforts to understand the mechanism of xrRNA production. sfRNAs are made when cellular 5’à3’ exoribonucleases (in particular, Xrn1) processively degrade the viral genomic RNA but then halt at specifically structured RNA elements in the viral 3’ UTR called exoribonuclease resistant RNAs (xrRNAs). By solving the structures of multiple xrRNAs by x-ray crystallography and combining this with biochemistry, biophysics, and virology, we showed that xrRNAs fold into a unique ring-like topology that creates a mechanical block the exoribonuclease cannot pass through. Furthermore, we used our discoveries to classify xrRNAs and to find new examples associated with both non-coding and coding RNAs. These successes now define several new questions. First, xrRNAs are often found in multiple copies ‘in tandem’ where their function is coupled in some way, but the structural basis of this coupling, and the effects of breaking the coupling on both sfRNA formation and viral infection kinetics, are unknown. Second, although we have a good understanding of several classes of xrRNAs, we have yet to solve the structure of an xrRNA from a tick-borne flavivirus, which appear to have interesting and unique properties. Third, although we have found many new examples of xrRNAs, it appears there are many more that are undiscovered, and we also do not understand how the various classes of xrRNA relate evolutionarily. How do these structures diversify and evolve in 3-D given the tight constraints on their folding? Here, we propose to answer these questions in three aims, employing a strategy that combines biochemistry, x- ray crystallography, cryo-EM, virology, and in vitro selections coupled with computational tools. The research described here will contribute significant basic knowledge regarding an important molecular process of broad applicability to viral disease, a necessary step between the discovery of a mechanism and the targeting of it for therapeutic intervention.

PROJECT NARRATIVE Flaviviruses produce non-coding ‘subgenomic flavivirus RNAs’ (sfRNAs) during infection that interact with cellular proteins and manipulate the cellular environment. These sfRNAs are formed through partial degradation of the viral genomic RNA, when highly structured RNA elements block the progression of cellular 5’ to 3’ exoribonucleases in a defined location. We seek to understand the mechanism of these exoribonuclease- resistant RNA structures, how they regulate sfRNA production, and find more examples using a combination of structural biology, biochemistry, and virology.