PROJECT SUMMARY/ ABSTRACT RNA viruses encode the information required to usurp cellular metabolism and gene regulation and to enable their own replication in two ways: in the linear sequence of their RNA genomes and in complex higher order structures. Although structured RNA elements are pervasive throughout viral genomes and have complex regulatory effects on all stages of the virus life cycle, little is known about the extent to which RNA structures occur across viral genomes or how critical structures function mechanistically. Post-transcriptional RNA genome chemical modifications such as N6-methyladenosine (m6A) are regulators of infection in diverse viruses and can have profound impacts on, or be impacted by, RNA structure. However, the link between RNA structure- and m6A-mediated regulation of viral infectivity remains uncharacterized because we lack a comprehensive structural understanding of RNA genomes and studies mapping m6A modifications have been imprecise. During the mentored phase of this proposal, I will gain new training in virology, RNA epigenetics, and RNA chemical biology to define how RNA elements and m6A modifications regulate viral replication and infection. In Aim 1, I will characterize RNA structure interrelationships with the m6A post-transcriptional RNA chemical modification by creating high-resolution RNA structure models and m6A modification site maps for the dengue virus (DENV) and the respiratory enterovirus EV-D68 RNA genomes. No broadly effective vaccines or therapeutics are available to prevent or treat the serious infections caused by these two single-stranded, positive-sense RNA viruses from distinct virus families. Through Aim 2, I will establish the functional importance of m6A-related RNA genome structures in DENV and EV-D68 viral life cycle stages using cell culture-based virus functional assays. Finally, in Aim 3, I will define structure-function relationships in m6A-related RNA structures in DENV and EV-D68 genomes by evaluating dynamic changes in RNA structure, m6A modification, and protein binding. Together, these studies will reveal the complex roles that interrelationships between RNA structure and m6A modification play in regulating the life cycles of diverse RNA viruses and will identify novel RNA regulatory motifs that might be exploited in the design of anti-DENV and anti-EV-D68 therapeutics and vaccination strategies. I have assembled an expert team of mentors and collaborators and plan to attend workshops, seminars, and conferences that will result in the training necessary to achieve the research goals proposed here and to transition into a successful career as an independent research scientist. The excellent training environments in the Weeks and Horner laboratories, along with hands-on training from my collaborator Dr. Cameron, will provide me with a solid foundation on which to build a successful independent research program.