From Generation to Selection: Defining the Recombination Landscape of Reovirus

Abstract

Defective viral genomes (DVGs) are noncanonical RNA products frequently generated through recombination during RNA virus replication. The mechanisms governing recombination and the accumulation of DVGs remain poorly understood for segmented double-stranded RNA (dsRNA) viruses. In this dissertation, I investigated the factors that shape recombination and DVG diversity for reovirus, a model dsRNA virus with potential as an oncolytic therapeutic. I demonstrate that the viral polymerase is not the primary determinant of strain-specific DVG diversity or recombination specificity, as exchanging polymerase complexes between reovirus strains does not alter DVG populations or recombination profiles. Instead, recombination profiles segregate with the viral gene segment identity, indicating that intrinsic RNA features play a role in directing recombination. I further show that recombination junctions frequently occur at regions of microhomology and that, although microhomology sites are abundant throughout the viral genome, only a subset are utilized, demonstrating that recombination is selective. Finally, I show that the recombination landscape detected in virions is more limited than the population generated during infection, suggesting that recombinant RNAs undergo an additional layer of selection during downstream stages of the viral life cycle. Collectively, these findings support a model in which reovirus genetic diversity is shaped by selection at multiple stages, including both recombination and assembly.