Kristen M. Ogden

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. Summary: Recombination is an important means for RNA viruses to adapt and evolve, but recombination mechanisms for segmented, double-stranded RNA viruses are poorly understood. In previous work, we investigated recombination events in packaged viral RNA for the double-stranded RNA virus reovirus. We detected recombination relatively infrequently, and we proposed a mechanism of recombination during genomic RNA synthesis. Here, we report that recombination events are more frequent and potential recombination sites are used less selectively in viral plus-strand RNA transcripts isolated from infected cells than in viral double-stranded RNA packaged in matched purified virus particles. These findings suggest recombination is more promiscuous and widespread for double-stranded RNA viruses than previously recognized. However, the tightly coordinated assortment and packaging mechanisms of these viruses may selectively filter recombination products in the viral population.

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. IMPORTANCE: Viral 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.

Fusion-associated small transmembrane (FAST) proteins are viral nonstructural proteins that mediate cell-cell fusion to form multinucleated syncytia. We previously reported that human species B rotavirus NSP1-1 is a FAST protein that induces syncytia in primate epithelial cells but not rodent fibroblasts. We hypothesized that NSP1-1 proteins of other rotavirus species could mediate cell-cell fusion but might be limited to cell types derived from homologous hosts. To test this hypothesis, we predicted the structure and domain organization of NSP1-1 of species B rotavirus from a human, goat, and pig, species G rotavirus from a pigeon and turkey, and species I rotavirus from a dog and cat. We transiently expressed the NSP1-1 proteins in avian, canine, hamster, human, porcine, and simian cells. Regardless of the host origin of the virus, each NSP1-1 protein induced syncytia in primate cells, while few induced syncytia in other cell types. In some cell types, a lack of protein expression or RNA stability failed to explain the inability of NSP1-1 to mediate cell-cell fusion. To identify the domains that determine cell-specific fusion activity for human species B rotavirus NSP1-1, we engineered chimeric proteins containing domain exchanges with the p10 FAST protein from Nelson Bay orthoreovirus and found that the N-terminal and transmembrane domains were determinants. Together, these findings suggest that rotavirus species B, G, and I NSP1-1 are functional FAST proteins whose N termini contribute to specifying the cells in which they mediate syncytium formation. IMPORTANCE: Mechanisms of membrane fusion and determinants of host range for pathogens remain poorly understood. Improved understanding of these concepts could open new areas for therapeutic development and shed light on virus epidemiology. Our analyses of NSP1-1 proteins from species B, G, and I rotaviruses provide insights into the variability tolerated by functional FAST proteins. Further, the observation that all putative FAST proteins tested can induce syncytium formation in at least some cell types provides evidence that rotaviruses that encode NSP1-1 proteins are fusogenic viruses. Finally, although the criteria for their specificity remain unclear, our observations regarding fusion capacities of different NSP1-1 proteins and of chimeric FAST proteins suggest a potential role for rotavirus FAST proteins in determining the efficiency of viral replication within a given host or cell type.

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 genomes. Two strains of the double-stranded RNA 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 double-stranded RNA virus recombination in which the polymerase pauses RNA synthesis and re-initiates further along the same template at a specific junction stop site that has sequence homology to the junction start site. IMPORTANCE: Viral infection gives rise to defective viral genomes, which cannot complete a full replication cycle. Recombination facilitates defective viral genome generation but is understudied for RNA viruses with double-stranded genomes. We found that for a double-stranded RNA 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 double-stranded RNA viruses, likely through reinitiation after re-hybridization of the newly formed RNA product with the same RNA template molecule at a different location.

Rotavirus infections remain a significant cause of morbidity and mortality in infants. The viral surface proteins VP4 and VP7 are each classified into multiple genotypes (P[1]–P[58] for VP4 and G1–G42 for VP7), which differ in their susceptibility to neutralizing antibodies; however, detailed analyses of these differences remain limited. This study investigates the susceptibility of diverse VP4 and VP7 genotypes to neutralizing antibodies induced by vaccination or natural infection. A reverse genetics system based on the human rotavirus Odelia strain (G4P[8]) was used to create monoreassortant viruses by replacing the VP4 or VP7 genes with those from clinical isolates (obtained in Thailand) or prototype strains. We generated 11 VP4 and 19 VP7 reassortants, covering 7 and 17 genotypes, respectively. Neutralization tests using the monoreassortant viruses, immune sera, and MA104 cells indicate that the VP7 genotype, rather than the VP4 genotype, primarily influences susceptibility to neutralization. Furthermore, genotypes G2, bat-like G3, G11, G12, G25, and G33 were significantly less susceptible to sera from naturally infected Thai individuals, highlighting the importance of monitoring these genotypes. To identify the protein region responsible for susceptibility to neutralization, we generated chimeric viruses by exchanging the domains of high- and low-susceptibility VP7 genotypes and found that Domain I appears to play a substantial role in determining susceptibility. Thus, we identified multiple rotavirus genotypes with reduced susceptibility to neutralization, as well as the region primarily responsible for this reduction. These findings may help to predict possible future rotavirus outbreaks in humans and facilitate the development of effective rotavirus vaccines. IMPORTANCE: Rotavirus, the leading cause of severe acute gastroenteritis in infants, is responsible for approximately 128,500 infant deaths globally each year. The virus’s surface proteins are highly diverse, comprising approximately 100 genotypes. This diversity affects susceptibility to neutralizing antibodies and the efficacy of vaccines. Here, we found that certain genotypes are highly resistant to serum neutralizing antibodies induced by vaccination and natural infections. Furthermore, we identified a specific region in the viral outer capsid protein that plays a significant role in determining susceptibility to neutralization. These findings may be important for predicting outbreak-causing strains and for developing more effective vaccines, ultimately contributing to the prevention of future outbreaks and improving global infant health.

Human rotaviruses exhibit limited tropism and replicate poorly in most cell lines. Attachment protein VP4 is a key rotavirus tropism determinant. Previous studies in which human rotaviruses were adapted to cultured cells identified mutations in VP4. However, most such studies were conducted using only a single human rotavirus genotype. In the current study, we serially passaged 50 human rotavirus clinical specimens representing five of the genotypes most frequently associated with severe human disease, each in triplicate, three to five times in primary monkey kidney cells then ten times in the MA104 monkey kidney cell line. From 13 of the 50 specimens, we obtained 25 rotavirus antigen-positive lineages representing all five genotypes, which tended to replicate more efficiently in MA104 cells at late versus early passage. We used Illumina next-generation sequencing and analysis to identify variants that arose during passage. In VP4, variants encoded 28 mutations that were conserved for all P[8] rotaviruses and 12 mutations that were conserved for all five genotypes. These findings suggest there may be a conserved mechanism of human rotavirus adaptation to MA104 cells. In the future, such a conserved adaptation mechanism could be exploited to study human rotavirus biology or efficiently manufacture vaccines.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization.

Several egress pathways have been defined for many viruses. Among these pathways, extracellular vesicles (EVs) have been shown to function as vehicles of non-lytic viral egress. EVs are heterogenous populations of membrane-bound structures released from cells as a form of intercellular communication. EV-mediated viral egress may enable immune evasion and collective viral transport. Strains of nonenveloped mammalian orthoreovirus (reovirus) differ in cell lysis phenotypes, with T3D disrupting cell membranes more efficiently than T1L. However, mechanisms of reovirus egress and the influence of transport strategy on infection are only partially understood. To elucidate reovirus egress mechanisms, we infected murine fibroblasts (L cells) and non-polarized human colon epithelial (Caco-2) cells with T1L or T3D reovirus and enriched cell culture supernatants for large EVs, medium EVs, small EVs, and free reovirus. We found that both reovirus strains exit cells in association with large and medium EVs and as free virus particles, and that EV-enriched fractions are infectious. While reovirus visually associates with large and medium EVs, only medium EVs offer protection from antibody-mediated neutralization. EV-mediated protection from neutralization is virus strain- and cell type-specific, as medium EVs enriched from L cell supernatants protect T1L and T3D, while medium EVs enriched from Caco-2 cell supernatants largely fail to protect T3D and only protect T1L efficiently. Using genetically barcoded reovirus, we provide evidence that large and medium EVs can convey multiple particles to recipient cells. Finally, T1L or T3D infection increases the release of all EV sizes from L cells. Together, these findings suggest that in addition to exiting cells as free particles, reovirus promotes egress from distinct cell types in association with large and medium EVs during lytic or non-lytic infection, a mode of exit that can mediate multiparticle infection and, in some cases, protection from antibody neutralization. AUTHOR SUMMARY: The exit strategy that many viruses use to escape cells is unknown. Reovirus is a nonenveloped human virus and an ideal model system to understand virus exit strategies and their inluence on infection. We found that two different reovirus strains, one that disrupts cell membranes and one that leaves cells largely intact, increase the release of extracellular vesicles (EVs) from cells. Both reovirus strains are released from cells as free particles and in association with EVs, which are membrane-bound structures that function in cell-to-cell communication. Depending on cell type and virus type, EVs can act like an ‘invisibility cloak’ that shields reovirus from antibodies. EVs can also bundle and ferry reovirus particles between cells. Although we used cells to examine the effects of reovirus association with EVs, it is possible that in mammalian hosts, EVs may shield reovirus from immune defenses and promote more efficient transmission and infection through a ‘strength-in-numbers’ strategy. Future work building on these findings will test the biological significance of EV-enclosed reovirus and may inform delivery strategies for oncolytic reoviruses to tumor sites. Broadly, these findings enhance our understanding of virus egress strategies and infection principles that may apply to other viruses that travel in EVs.