Mammalian orthoreovirus can exit cells in extracellular vesicles.

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.

Deciphering molecular mechanisms stabilizing the reovirus-binding complex.

Mammalian orthoreoviruses (reoviruses) serve as potential triggers of celiac disease and have oncolytic properties, making these viruses potential cancer therapeutics. Primary attachment of reovirus to host cells is mainly mediated by the trimeric viral protein, σ1, which engages cell-surface glycans, followed by high-affinity binding to junctional adhesion molecule-A (JAM-A). This multistep process is thought to be accompanied by major conformational changes in σ1, but direct evidence is lacking. By combining biophysical, molecular, and simulation approaches, we define how viral capsid protein mechanics influence virus-binding capacity and infectivity. Single-virus force spectroscopy experiments corroborated by in silico simulations show that GM2 increases the affinity of σ1 for JAM-A by providing a more stable contact interface. We demonstrate that conformational changes in σ1 that lead to an extended rigid conformation also significantly increase avidity for JAM-A. Although its associated lower flexibility impairs multivalent cell attachment, our findings suggest that diminished σ1 flexibility enhances infectivity, indicating that fine-tuning of σ1 conformational changes is required to successfully initiate infection. Understanding properties underlying the nanomechanics of viral attachment proteins offers perspectives in the development of antiviral drugs and improved oncolytic vectors.

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.

Intravesical oncolytic virotherapy and immunotherapy for non-muscle-invasive bladder cancer mouse model.

OBJECTIVES: To test if intravesical instillation of both an anti-programmed cell death protein 1 (PD-1) inhibitor and an oncolytic reovirus would demonstrate a greater effect than either treatment alone, as non-muscle-invasive bladder cancer that is refractory to intravesical bacillus Calmette-Guérin can be treated by systemic anti-PD-1 immunotherapy and we previously demonstrated improved overall survival (OS) with six once-weekly instillations of intravesical anti-PD-1 in a murine model. MATERIALS AND METHODS: Using an orthotopic syngeneic C3H murine model of MBT2 urothelial bladder cancer, groups of 10 mice were compared between no treatment, intravesical anti-PD-1, intravesical oncolytic reovirus, or intravesical reovirus + anti-PD-1. A single intravesical treatment session was given. The primary outcome was OS, and the secondary outcomes included long-term immunity and tumour-immune profile. RESULTS: With a median follow-up of 9 months, all mice that received no treatment died with a median survival of 41 days, while the comparison median OS was not reached for reovirus (hazard ratio [HR] 14.4, 95% confidence interval [CI] 3.9-32.6; P < 0.001), anti-PD-1 (HR 28.4, 95% CI 7.0-115.9; P < 0.001), and reovirus + anti-PD-1 (HR 28.4, 95% CI 7.0-115.9; P < 0.001). Monotherapy with anti-PD-1 or reovirus demonstrated no significant differences in survival (P = 0.067). Mass cytometry showed that reovirus + anti-PD-1 treatment enriched monocytes and decreased myeloid-derived suppressor cells, generating an immuno-responsive tumour microenvironment. Depletion of CD8+ T cells eliminated the survival advantage provided by the intravesical treatment. CONCLUSIONS: Treatment of murine orthotopic bladder tumours with a single instillation of intravesical reovirus, anti-PD-1 antibody, or the combination confers superior survival compared to controls. Tumour-immune microenvironment differences indicated myeloid-derived suppressor cells and CD8+ T cells mediate the treatment response.

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.

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 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.

Multiple Introductions and Antigenic Mismatch with Vaccines May Contribute to Increased Predominance of G12P[8] Rotaviruses in the United States.

Rotavirus is the leading global cause of diarrheal mortality for unvaccinated children under 5 years of age. The outer capsid of rotavirus virions consists of VP7 and VP4 proteins, which determine viral G and P types, respectively, and are primary targets of neutralizing antibodies. Successful vaccination depends upon generating broadly protective immune responses following exposure to rotaviruses presenting a limited number of G- and P-type antigens. Vaccine introduction resulted in decreased rotavirus disease burden but also coincided with the emergence of uncommon G and P genotypes, including G12. To gain insight into the recent predominance of G12P[8] rotaviruses in the United States, we evaluated 142 complete rotavirus genome sequences and metadata from 151 clinical specimens collected in Nashville, TN, from 2011 to 2013 through the New Vaccine Surveillance Network. Circulating G12P[8] strains were found to share many segments with other locally circulating strains but to have distinct constellations. Phylogenetic analyses of G12 sequences and their geographic sources provided evidence for multiple separate introductions of G12 segments into Nashville, TN. Antigenic epitopes of VP7 proteins of G12P[8] strains circulating in Nashville, TN, differ markedly from those of vaccine strains. Fully vaccinated children were found to be infected with G12P[8] strains more frequently than with other rotavirus genotypes. Multiple introductions and significant antigenic mismatch may in part explain the recent predominance of G12P[8] strains in the United States and emphasize the need for continued monitoring of rotavirus vaccine efficacy against emerging rotavirus genotypes.IMPORTANCE Rotavirus is an important cause of childhood diarrheal disease worldwide. Two immunodominant proteins of rotavirus, VP7 and VP4, determine G and P genotypes, respectively. Recently, G12P[8] rotaviruses have become increasingly predominant. By analyzing rotavirus genome sequences from stool specimens obtained in Nashville, TN, from 2011 to 2013 and globally circulating rotaviruses, we found evidence of multiple introductions of G12 genes into the area. Based on sequence polymorphisms, VP7 proteins of these viruses are predicted to present themselves to the immune system very differently than those of vaccine strains. Many of the sick children with G12P[8] rotavirus in their diarrheal stools also were fully vaccinated. Our findings emphasize the need for continued monitoring of circulating rotaviruses and the effectiveness of the vaccines against strains with emerging G and P genotypes.

Structural and Functional Features of the Reovirus σ1 Tail.

Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from ß-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of σ1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus σ1 and provide insight into the stabilization of a multidomain virus attachment protein.IMPORTANCE While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein σ1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large σ1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the σ1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus σ1 attachment protein.

Endogenous double-stranded Alu RNA elements stimulate IFN-responses in relapsing remitting multiple sclerosis.

Various sensors that detect double-stranded RNA, presumably of viral origin, exist in eukaryotic cells and induce IFN-responses. Ongoing IFN-responses have also been documented in a variety of human autoimmune diseases including relapsing-remitting multiple sclerosis (RRMS) but their origins remain obscure. We find increased IFN-responses in leukocytes in relapsing-remitting multiple sclerosis at distinct stages of disease. Moreover, endogenous RNAs isolated from blood cells of these same patients recapitulate this IFN-response if transfected into naïve cells. These endogenous RNAs are double-stranded RNAs, contain Alu and Line elements and are transcribed from leukocyte transcriptional enhancers. Thus, transcribed endogenous retrotransposon elements can co-opt pattern recognition sensors to induce IFN-responses in RRMS.

Structural Insights into Reovirus σ1 Interactions with Two Neutralizing Antibodies.

Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus. IMPORTANCE: Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses, such as HIV and influenza virus, have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein σ1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for σ1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the σ1 protein during viral cell entry.

Structural basis for 2'-5'-oligoadenylate binding and enzyme activity of a viral RNase L antagonist.

UNLABELLED: Synthesis of 2'-5'-oligoadenylates (2-5A) by oligoadenylate synthetase (OAS) is an important innate cellular response that limits viral replication by activating the latent cellular RNase, RNase L, to degrade single-stranded RNA. Some rotaviruses and coronaviruses antagonize the OAS/RNase L pathway through the activity of an encoded 2H phosphoesterase domain that cleaves 2-5A. These viral 2H phosphoesterases are phylogenetically related to the cellular A kinase anchoring protein 7 (AKAP7) and share a core structure and an active site that contains two well-defined HΦ(S/T)Φ (where Φ is a hydrophobic residue) motifs, but their mechanism of substrate binding is unknown. Here, we report the structures of a viral 2H phosphoesterase, the C-terminal domain (CTD) of the group A rotavirus (RVA) VP3 protein, both alone and in complex with 2-5A. The domain forms a compact fold, with a concave ß-sheet that contains the catalytic cleft, but it lacks two α-helical regions and two ß-strands observed in AKAP7 and other 2H phosphoesterases. The cocrystal structure shows significant conformational changes in the R loop upon ligand binding. Bioinformatics and biochemical analyses reveal that conserved residues and residues required for catalytic activity and substrate binding comprise the catalytic motifs and a region on one side of the binding cleft. We demonstrate that the VP3 CTD of group B rotavirus, but not that of group G, cleaves 2-5A. These findings suggest that the VP3 CTD is a streamlined version of a 2H phosphoesterase with a ligand-binding mechanism that is shared among 2H phosphodiesterases that cleave 2-5A. IMPORTANCE: The C-terminal domain (CTD) of rotavirus VP3 is a 2H phosphoesterase that cleaves 2'-5'-oligoadenylates (2-5A), potent activators of an important innate cellular antiviral pathway. 2H phosphoesterase superfamily proteins contain two conserved catalytic motifs and a proposed core structure. Here, we present structures of a viral 2H phosphoesterase, the rotavirus VP3 CTD, alone and in complex with its substrate, 2-5A. The domain lacks two α-helical regions and ß-strands present in other 2H phosphoesterases. A loop of the protein undergoes significant structural changes upon substrate binding. Together with our bioinformatics and biochemical findings, the crystal structures suggest that the RVA VP3 CTD domain is a streamlined version of a cellular enzyme that shares a ligand-binding mechanism with other 2H phosphodiesterases that cleave 2-5A but differs from those of 2H phosphodiesterases that cleave other substrates. These findings may aid in the future design of antivirals targeting viral phosphodiesterases with cleavage specificity for 2-5A.

Homologous 2',5'-phosphodiesterases from disparate RNA viruses antagonize antiviral innate immunity.

Efficient and productive virus infection often requires viral countermeasures that block innate immunity. The IFN-inducible 2',5'-oligoadenylate (2-5A) synthetases (OASs) and ribonuclease (RNase) L are components of a potent host antiviral pathway. We previously showed that murine coronavirus (MHV) accessory protein ns2, a 2H phosphoesterase superfamily member, is a phosphodiesterase (PDE) that cleaves 2-5A, thereby preventing activation of RNase L. The PDE activity of ns2 is required for MHV replication in macrophages and for hepatitis. Here, we show that group A rotavirus (RVA), an important cause of acute gastroenteritis in children worldwide, encodes a similar PDE. The RVA PDE forms the carboxy-terminal domain of the minor core protein VP3 (VP3-CTD) and shares sequence and predicted structural homology with ns2, including two catalytic HxT/S motifs. Bacterially expressed VP3-CTD exhibited 2',5'-PDE activity, which cleaved 2-5A in vitro. In addition, VP3-CTD expressed transiently in mammalian cells depleted 2-5A levels induced by OAS activation with poly(rI):poly(rC), preventing RNase L activation. In the context of recombinant chimeric MHV expressing inactive ns2, VP3-CTD restored the ability of the virus to replicate efficiently in macrophages or in the livers of infected mice, whereas mutant viruses expressing inactive VP3-CTD (H718A or H798R) were attenuated. In addition, chimeric viruses expressing either active ns2 or VP3-CTD, but not nonfunctional equivalents, were able to protect ribosomal RNA from RNase L-mediated degradation. Thus, VP3-CTD is a 2',5'-PDE able to functionally substitute for ns2 in MHV infection. Remarkably, therefore, two disparate RNA viruses encode proteins with homologous 2',5'-PDEs that antagonize activation of innate immunity.

Predicted structure and domain organization of rotavirus capping enzyme and innate immune antagonist VP3.

UNLABELLED: Rotaviruses and orbiviruses are nonturreted Reoviridae members. The rotavirus VP3 protein is a multifunctional capping enzyme and antagonist of the interferon-induced cellular oligoadenylate synthetase-RNase L pathway. Despite mediating important processes, VP3 is the sole protein component of the rotavirus virion whose structure remains unknown. In the current study, we used sequence alignment and homology modeling to identify features common to nonturreted Reoviridae capping enzymes and to predict the domain organization, structure, and active sites of rotavirus VP3. Our results suggest that orbivirus and rotavirus capping enzymes share a domain arrangement similar to that of the bluetongue virus capping enzyme. Sequence alignments revealed conserved motifs and suggested that rotavirus and orbivirus capping enzymes contain a variable N-terminal domain, a central guanine-N7-methyltransferase domain that contains an additional inserted domain, and a C-terminal guanylyltransferase and RNA 5'-triphosphatase domain. Sequence conservation and homology modeling suggested that the insertion in the guanine-N7-methyltransferase domain is a ribose-2'-O-methyltransferase domain for most rotavirus species. Our analyses permitted putative identification of rotavirus VP3 active-site residues, including those that form the ribose-2'-O-methyltransferase catalytic tetrad, interact with S-adenosyl-l-methionine, and contribute to autoguanylation. Previous reports have indicated that group A rotavirus VP3 contains a C-terminal 2H-phosphodiesterase domain that can cleave 2'-5' oligoadenylates, thereby preventing RNase L activation. Our results suggest that a C-terminal phosphodiesterase domain is present in the capping enzymes from two additional rotavirus species. Together, these findings provide insight into a poorly understood area of rotavirus biology and are a springboard for future biochemical and structural studies of VP3. IMPORTANCE: Rotaviruses are an important cause of severe diarrheal disease. The rotavirus VP3 protein caps viral mRNAs and helps combat cellular innate antiviral defenses, but little is known about its structure or enzymatic mechanisms. In this study, we used sequence- and structure-based alignments with related proteins to predict the structure of VP3 and identify enzymatic domains and active sites therein. This work provides insight into the mechanisms of rotavirus transcription and evasion of host innate immune defenses. An improved understanding of these processes may aid our ability to develop rotavirus vaccines and therapeutics.

Both genome segments contribute to the pathogenicity of very virulent infectious bursal disease virus.

Infectious bursal disease virus (IBDV) causes an economically significant disease of chickens worldwide. Very virulent IBDV (vvIBDV) strains have emerged and induce as much as 60% mortality. The molecular basis for vvIBDV pathogenicity is not understood, and the relative contributions of the two genome segments, A and B, to this phenomenon are not known. Isolate 94432 has been shown previously to be genetically related to vvIBDVs but exhibits atypical antigenicity and does not cause mortality. Here the full-length genome of 94432 was determined, and a reverse genetics system was established. The molecular clone was rescued and exhibited the same antigenicity and reduced pathogenicity as isolate 94432. Genetically modified viruses derived from 94432, whose vvIBDV consensus nucleotide sequence was restored in segment A and/or B, were produced, and their pathogenicity was assessed in specific-pathogen-free chickens. We found that a valine (position 321) that modifies the most exposed part of the capsid protein VP2 critically modified the antigenicity and partially reduced the pathogenicity of 94432. However, a threonine (position 276) located in the finger domain of the virus polymerase (VP1) contributed even more significantly to attenuation. This threonine is partially exposed in a hydrophobic groove on the VP1 surface, suggesting possible interactions between VP1 and another, as yet unidentified molecule at this amino acid position. The restored vvIBDV-like pathogenicity was associated with increased replication and lesions in the thymus and spleen. These results demonstrate that both genome segments influence vvIBDV pathogenicity and may provide new targets for the attenuation of vvIBDVs.

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.

Optimum length and flexibility of reovirus attachment protein σ1 are required for efficient viral infection.

Reovirus attachment protein σ1 is an elongated trimer with head-and-tail morphology that engages cell-surface carbohydrate and junctional adhesion molecule A (JAM-A). The σ1 protein is comprised of three domains partitioned by two flexible linkers termed interdomain regions (IDRs). To determine the importance of σ1 length and flexibility at different stages of reovirus infection, we generated viruses with mutant σ1 molecules of altered length and flexibility and tested these viruses for the capacity to bind the cell surface, internalize, uncoat, induce protein synthesis, assemble, and replicate. We reduced the length of the α-helical σ1 tail to engineer mutants L1 and L2 and deleted midpoint and head-proximal σ1 IDRs to generate ΔIDR1 and ΔIDR2 mutant viruses, respectively. Decreasing length or flexibility of σ1 resulted in delayed reovirus infection and reduced viral titers. L1, L2, and ΔIDR1 viruses but not ΔIDR2 virus displayed reduced cell attachment, but altering σ1 length or flexibility did not diminish the efficiency of virion internalization. Replication of ΔIDR2 virus was hindered at a postdisassembly step. Differences between wild-type and σ1 mutant viruses were not attributable to alterations in σ1 folding, as determined by experiments assessing engagement of cell-surface carbohydrate and JAM-A by the length and IDR mutant viruses. However, ΔIDR1 virus harbored substantially less σ1 on the outer capsid. Taken together, these data suggest that σ1 length is required for reovirus binding to cells. In contrast, IDR1 is required for stable σ1 encapsidation, and IDR2 is required for a postuncoating replication step. Thus, the structural architecture of σ1 is required for efficient reovirus infection of host cells.

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.

Mammalian orthoreovirus can exit cells in extracellular vesicles.

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.