PROJECT SUMMARY For viruses with multi-segmented double-stranded RNA (dsRNA) genomes, the correct assortment of segments must be selectively packaged into an assembling viral capsid to form an infectious particle, but packaging mechanisms are incompletely understood. This gap in knowledge limits the ability to genetically modify segmented dsRNA viruses for vaccines and therapies. The packaging mechanisms of oncolytic dsRNA virus mammalian orthoreovirus (reovirus) remain largely understudied. Thus, genetic modification to increase its oncolytic potential has been limited. Packaging of reovirus RNA is thought to be a highly ordered process. Signals required for packaging are thought to be located at the segment termini, which are predicted to interact and form RNA secondary structures. The observation that reovirus can package defective viral genomes (DVGs) presents new opportunities to identify minimal reovirus packaging determinants, since DVGs are nonfunctional forms of the viral genome that retain packaging and replication signals. The goal of my proposed research is to elucidate mechanisms of reovirus RNA packaging and identify elements within the viral genome that play a role in this process. Preliminary and published data indicate that reovirus can package deletion DVGs that lack most of the internal region of a segment but retain both termini. I will take advantage of reovirus DVG selection to elucidate minimal packaging requirements. I hypothesize that reovirus DVGs contain minimal RNA recognition elements and that structures at the viral RNA segment termini mediate packaging. To test this hypothesis, I propose two specific aims. In Specific Aim 1, I will use ClickSeq, Illumina next-generation short-read sequencing, Oxford nanopore third-generation long-read sequencing, and molecular virology approaches to define DVG characteristics that allow preferential RNA packaging. In Specific Aim 2, I will use 2’-hydroxyl acylation followed by primer extension (SHAPE) and reverse genetics to determine the structures of reovirus RNA segments and identify components within the viral RNA that are essential for selective reovirus segment packaging. The findings generated by the proposed aims will reveal selective reovirus RNA packaging mechanisms, which may apply to other segmented viruses. Continued studies building on these findings will facilitate reovirus genetic modification to improve its effectiveness as an oncolytic therapy.