Comparison of intracellular and encapsidated viral RNA reveals selective constraints on reovirus recombination

Recombination can generate genetic diversity among RNA viruses; yet, mechanisms governing recombination for segmented, double-stranded RNA viruses remain poorly understood. Reovirus genomes contain ten segments of double-stranded RNA. Previous analyses suggested low rates of reovirus RNA recombination and led to a proposed mechanism of recombination during minus-strand synthesis. These analyses focused on recombination events detected in genomic double-stranded RNA extracted from purified viral particles. In the current study, we looked deeper into the origins of the observed recombination events by assaying total cellular material, which comprises viral genomic material, as well as replication intermediates, mRNAs, and products of recombination that may not be eligible for packaging into virus particles. Intracellular viral RNA, which is almost exclusively plus-strand RNA, exhibited substantially higher recombination frequency than particle-associated double-stranded RNA. The diversity and frequency of identified junctions were more similar among samples of a given type (intracellular or virion-encapsidated) from distinct infections than between distinct sample types from the same infection. Although potential recombination sites are widely distributed across the genome, a more limited set was used in junctions in packaged viral RNA than in intracellular viral RNA. Together, these observations suggest recombination may occur more frequently than previously recognized during reovirus infection, and many such events likely occur during plus-strand synthesis. However, recombination products generated during viral transcription may be selectively incorporated into viral particles.