Human Rotaviruses of Multiple Genotypes Acquire Conserved VP4 Mutations during Serial Passage.

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.

Multiple rotavirus species encode fusion-associated small transmembrane (FAST) proteins with cell type-specific activity.

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 the NSP1-1 proteins of other rotavirus species could also mediate cell-cell fusion and that fusion activity might be limited to cell types derived from homologous hosts. To test this hypothesis, we predicted the structure and domain organization of NSP1-1 proteins 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 cloned these sequences into plasmids and transiently expressed the NSP1-1 proteins in avian, canine, hamster, human, porcine, and simian cells. Regardless of host origin of the virus, each NSP1-1 protein induced syncytia in primate cells, while few induced syncytia in other cell types. To identify the domains that determined 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. Using the chimeric proteins, we found that the N-terminal and transmembrane domains determined the cell type specificity of fusion activity. Although the species and cell type criteria for fusion activity remain unclear, these findings suggest that rotavirus species B, G, and I NSP1-1 are functional FAST proteins whose N termini play a role in specifying the cells in which they mediate syncytia formation.

Mammalian orthoreoviruses exhibit rare genotype variability in genome constellations.

Mammalian orthoreoviruses (reoviruses) are currently classified based on properties of the attachment protein, σ1. Four reovirus serotypes have been identified, three of which are represented by well-studied prototype human reovirus strains. Reoviruses contain ten segments of double-stranded RNA that encode 12 proteins and can reassort during coinfection. To understand the breadth of reovirus genetic diversity and its potential influence on reassortment, the sequence of the entire genome should be considered. While much is known about the prototype strains, a thorough analysis of all ten reovirus genome segment sequences has not previously been conducted. We analyzed phylogenetic relationships and nucleotide sequence conservation for each of the ten segments of more than 60 complete or nearly complete reovirus genome sequences, including those of the prototype strains. Using these relationships, we defined genotypes for each segment, with minimum nucleotide identities of 77-88% for most genotypes that contain several representative sequences. We applied segment genotypes to determine reovirus genome constellations, and we propose implementation of an updated reovirus genome classification system that incorporates genotype information for each segment. For most sequenced reoviruses, segments other than S1, which encodes σ1, cluster into a small number of genotypes and a limited array of genome constellations that do not differ greatly over time or based on animal host. However, a small number of reoviruses, including prototype strain Jones, have constellations in which segment genotypes differ from those of most other sequenced reoviruses. For these reoviruses, there is little evidence of reassortment with the major genotype. Future basic research studies that focus on the most genetically divergent reoviruses may provide new insights into reovirus biology. Analysis of available partial sequences and additional complete reovirus genome sequencing may also reveal reassortment biases, host preferences, or infection outcomes that are based on reovirus genotype.

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 (dsRNA) 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 h. Additionally, primary infection progressively dampened superinfecting virus transcript levels with greater 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. IMPORTANCE Reassortment, 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.

Monoreassortant Rotaviruses of Multiple G Types Are Differentially Neutralized by Sera From Infants Vaccinated With ROTARIX and RotaTeq.

BACKGROUND: Rotavirus is a leading cause of pediatric diarrheal mortality. The rotavirus outer capsid consists of VP7 and VP4 proteins, which, respectively, determine viral G and P type and are primary targets of neutralizing antibodies. METHODS: To elucidate VP7-specific neutralizing antibody responses, we engineered monoreassortant rotaviruses each containing a human VP7 segment from a sequenced clinical specimen or a vaccine strain in an identical genetic background. We quantified replication and neutralization of engineered viruses using sera from infants vaccinated with monovalent ROTARIX or multivalent RotaTeq vaccines. RESULTS: Immunization with RotaTeq induced broader neutralizing antibody responses than ROTARIX. Inclusion of a single dose of RotaTeq in the schedule enhanced G-type neutralization breadth of vaccinated infant sera. Cell type-specific differences in infectivity, replication, and neutralization were detected for some monoreassortant viruses. CONCLUSIONS: These findings suggest that rotavirus VP7, independent of VP4, can contribute to cell tropism and the breadth of vaccine-elicited neutralizing antibody responses.

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 relatively 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 similarly fragmented 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 genome segments. Recombination junctions were frequently but not always characterized by short direct sequence repeats upstream and downstream that spanned junction sites. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites.IMPORTANCE Viruses 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.

Reovirus σ1 Conformational Flexibility Modulates the Efficiency of Host Cell Attachment.

Reovirus attachment protein σ1 is a trimeric molecule containing tail, body, and head domains. During infection, σ1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows σ1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in σ1 from a compact form on virions to an extended form on ISVPs. To determine the importance of σ1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can cross-link σ1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that cross-link the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride, which blocks virion-to-ISVP conversion. Together, these data suggest that σ1 conformational flexibility modulates the efficiency of reovirus host cell attachment.IMPORTANCE Nonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, σ1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that cross-link σ1 monomers in nonreducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of σ1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that cross-linking the σ1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that σ1 conformational rearrangements mediate the efficiency of reovirus host cell binding.

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein.

Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.