Thoner, T. W. | bioRxiv Science

bioRxiv2022

Reovirus efficiently reassorts genome segments during coinfection and superinfection

Reassortment, or genome segment exchange, increases diversity among viruses with segmented genomes. Previous studies on the limitations of reassortment have largely focused on parental incompatibilities that restrict generation of viable progeny. However, less is known about whether factors intrinsic to virus replication influence reassortment. Mammalian orthoreovirus (reovirus) encapsidates a segmented, double- stranded RNA genome, replicates within cytoplasmic factories, and is susceptible to host antiviral responses. We sought to elucidate the influence of infection multiplicity, timing, and compartmentalized replication on reovirus reassortment in the absence of parental incompatibilities. We used an established post-PCR genotyping method to quantify reassortment frequency between wild-type and genetically-barcoded type 3 reoviruses. Consistent with published findings, we found that reassortment increased with infection multiplicity until reaching a peak of efficient genome segment exchange during simultaneous coinfection. However, reassortment frequency exhibited a substantial decease with increasing time to superinfection, which strongly correlated with viral transcript abundance. We hypothesized that physical sequestration of viral transcripts within distinct virus factories or superinfection exclusion also could influence reassortment frequency during superinfection. Imaging revealed that transcripts from both wild-type and barcoded viruses frequently co-occupied factories with superinfection time delays up to 16 hours. Additionally, primary infection dampened superinfecting virus transcription with a 24 hour, but not shorter, time delay to superinfection. Thus, in the absence of parental incompatibilities and with short times to superinfection, reovirus reassortment proceeds efficiently and is largely unaffected by compartmentalization of replication and superinfection exclusion. However, reassortment may be limited by superinfection exclusion with greater time delays to superinfection. IMPORTANCEReassortment, or genome segment exchange between viruses, can generate novel virus genotypes and pandemic virus strains. For viruses to reassort their genome segments, they must replicate within the same physical space by coinfecting the same host cell. Even after entry into the host cell, many viruses with segmented genomes synthesize new virus transcripts and assemble and package their genomes within cytoplasmic replication compartments. Additionally, some viruses can interfere with subsequent infection of the same host or cell. However, spatial and temporal influences on reassortment are only beginning to be explored. We found that infection multiplicity and transcript abundance are important drivers of reassortment during coinfection and superinfection, respectively, for reovirus, which has a segmented, double-stranded RNA genome. We also provide evidence that compartmentalization of transcription and packaging is unlikely to influence reassortment, but the length of time between primary and subsequent reovirus infection can alter reassortment frequency.

microbiology↗

bioRxiv2020

Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage

For viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5' and 3' terminal sequences and featured large internal deletions, while similar segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral gene segments. Recombination junction sites were frequently associated with short regions of identical sequence. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites. IMPORTANCEViruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

microbiology↗