Reovirus recombination is highly selective, and its profiles are primarily dictated by viral gene segment identity

Recombination facilitates the generation of defective viral genomes (DVGs), truncated derivatives of the parental genome that require a helper virus to replicate. Recombination mechanisms are poorly understood for viruses with double-stranded RNA (dsRNA) genomes. Two strains of the dsRNA virus reovirus differ in the pattern of packaged DVGs during serial passage. To determine whether the polymerase complex or gene segment sequence contributes to these differences, we exchanged polymerase complexes between the two reovirus strains. We identified DVG patterns using RT-PCR and recombination junction profiles using ClickSeq. Reoviruses synthesized DVGs that maintained the 5' and 3' termini and contained large central deletions. The polymerase complex did not detectably affect DVG pattern following reovirus serial passage or viral recombination junction profiles. Instead, recombination junction profiles correlated with the identity of viral RNA gene segments, even in the presence of a non-native polymerase complex or virus background. Reovirus recombination junction start and stop sites often occur in regions of sequence microhomology. While we observed many instances of short stretches of identical nucleotides within a viral gene segment, only a select few positions were incorporated into recombination junctions. Overall, these data suggest that recombination events that can mediate reovirus DVG formation are highly selective, and properties of viral gene segments primarily dictate where recombination occurs. These observations suggest a model for dsRNA virus recombination in which the polymerase pauses RNA synthesis and reinitiates further along the same template at a specific junction stop site that has sequence homology to the junction start site.IMPORTANCEViral infection gives rise to defective viral genomes (DVGs), which cannot complete a full replication cycle. Recombination facilitates DVG generation but is understudied for RNA viruses with double-stranded genomes. We found that for a double-stranded RNA (dsRNA) virus, reovirus, recombination occurs preferentially at specific sites in the genome that correspond with the identity of the gene segment. Recombination tends to initiate and terminate at sites sharing identical sequences. However, even if these short nucleotide sequences appear multiple times in a gene segment, only specific sites are used for recombination. Our results indicate that reovirus recombination is a highly orchestrated event in which individual gene segments contain the characteristics that drive recombination. These findings suggest that RNA properties, such as sequence and structure, drive recombination for dsRNA viruses, likely through reinitiation after re-hybridization of the newly formed RNA product with the same RNA template molecule at a different location.