NgR1 binding to reovirus reveals an unusual bivalent interaction and a new viral attachment protein.

Nogo-66 receptor 1 (NgR1) binds a variety of structurally dissimilar ligands in the adult central nervous system to inhibit axon extension. Disruption of ligand binding to NgR1 and subsequent signaling can improve neuron outgrowth, making NgR1 an important therapeutic target for diverse neurological conditions such as spinal crush injuries and Alzheimer's disease. Human NgR1 serves as a receptor for mammalian orthoreovirus (reovirus), but the mechanism of virus-receptor engagement is unknown. To elucidate how NgR1 mediates cell binding and entry of reovirus, we defined the affinity of interaction between virus and receptor, determined the structure of the virus-receptor complex, and identified residues in the receptor required for virus binding and infection. These studies revealed that central NgR1 surfaces form a bridge between two copies of viral capsid protein σ3, establishing that σ3 serves as a receptor ligand for reovirus. This unusual binding interface produces high-avidity interactions between virus and receptor to prime early entry steps. These studies refine models of reovirus cell-attachment and highlight the evolution of viruses to engage multiple receptors using distinct capsid components.

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.

The Reovirus σ3 Protein Inhibits NF-κB-Dependent Antiviral Signaling.

Viral antagonism of innate immune pathways is a common mechanism by which viruses evade immune surveillance. Infection of host cells with reovirus leads to the blockade of NF-κB, a key transcriptional regulator of the hosts' innate immune response. One mechanism by which reovirus infection results in inhibition of NF-κB is through a diminishment in levels of upstream activators, IKKß and NEMO. Here, we demonstrate a second, distinct mechanism by which reovirus blocks NF-κB. We report that expression of a single viral protein, σ3, is sufficient to inhibit expression of NF-κB target genes. Further, σ3-mediated blockade of NF-κB occurs without changes to IκB kinase (IKK) levels or activity. Among NF-κB targets, the expression of type I interferon is significantly diminished by σ3 expression. Expression of NF-κB target genes varies following infection with closely related reovirus strains. Our genetic analysis identifies that these differences are controlled by polymorphisms in the amino acid sequence of σ3. This work identifies a new role for reovirus σ3 as a viral antagonist of NF-κB-dependent antiviral pathways. IMPORTANCE Host cells mount a response to curb virus replication in infected cells and prevent spread of virus to neighboring, as yet uninfected, cells. The NF-κB family of proteins is important for the cell to mediate this response. In this study, we show that a single protein, σ3, produced by mammalian reovirus, impairs the function of NF-κB. We demonstrate that by blocking NF-κB, σ3 diminishes the hosts' response to infection to promote viral replication. This work identifies a second, previously unknown, mechanism by which reovirus blocks this aspect of the host cell response.

Asymmetric reconstruction of mammalian reovirus reveals interactions among RNA, transcriptional factor µ2 and capsid proteins.

Mammalian reovirus (MRV) is the prototypical member of genus Orthoreovirus of family Reoviridae. However, lacking high-resolution structures of its RNA polymerase cofactor µ2 and infectious particle, limits understanding of molecular interactions among proteins and RNA, and their contributions to virion assembly and RNA transcription. Here, we report the 3.3 Å-resolution asymmetric reconstruction of transcribing MRV and in situ atomic models of its capsid proteins, the asymmetrically attached RNA-dependent RNA polymerase (RdRp) λ3, and RdRp-bound nucleoside triphosphatase µ2 with a unique RNA-binding domain. We reveal molecular interactions among virion proteins and genomic and messenger RNA. Polymerase complexes in three Spinoreovirinae subfamily members are organized with different pseudo-D3d symmetries to engage their highly diversified genomes. The above interactions and those between symmetry-mismatched receptor-binding σ1 trimers and RNA-capping λ2 pentamers balance competing needs of capsid assembly, external protein removal, and allosteric triggering of endogenous RNA transcription, before, during and after infection, respectively.

Generation of Genetically RGD σ1-Modified Oncolytic Reovirus That Enhances JAM-A-Independent Infection of Tumor Cells.

Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.

Z-FA-FMK demonstrates differential inhibition of aquatic orthoreovirus (PRV), aquareovirus (CSRV), and rhabdovirus (IHNV) replication.

Benzyloxycarbonyl-phenylalanyl-alanyl-fluoromethyl ketone (Z-FA-FMK) is a protease inhibitor that has been shown to strongly inhibit mammalian orthoreovirus replication. Here we explore the ability of Z-FA-FMK to inhibit three important yet genetically discrete aquatic fish viruses: chum salmon aquareovirus (CSRV), piscine orthoreovirus (PRV), and the rhabdovirus infectious hematopoietic necrosis virus (IHNV). Z-FA-FMK significantly attenuated CSRV in vitro transcription and infectious yield following low-dose (2-20µM) exposure, yet a relatively high dose (200µM) was required to completely block CSRV replication. For PRV and IHNV, no significant attenuation of in vitro viral transcription was observed following low-dose (2-20µM) exposure; and although high dose (200µM) exposure significantly attenuated both PRV and IHNV transcription, neither was completely inhibited. These transcriptional results were similarly reflected in IHNV infectious titre observed at 7days post exposure. PRV titre is currently undeterminable in vitro; however, in vivo intra-peritoneal injection of PRV into juvenile Atlantic salmon (Salmo salar) in conjunction with 1.5mg/kg Z-FA-FMK did not affect PRV replication as measured by blood associated viral transcripts at 14days post challenge. These results indicate that aquatic ortho- and aqua-reoviruses appear to possess resilience to Z-FA-FMK relative to mammalian orthoreoviruses and suggest that environmental parameters or alternative mechanisms for viral replication may affect the efficacy of Z-FA-FMK as an antireoviral compound. Further, as Z-FA-FMK has been shown to irreversibly inhibit cysteine proteases such as cathepsins B and L in vitro at concentrations of ≤100µM, continued replication of IHNV (and possibly PRV) at 200µM Z-FA-FMK suggests that replication of these viruses can occur in a cathepsin-independent manner whereas CSRV likely requires cathepsins or similar cysteine proteases for successful replication.

Aging, mortality, and the fast growth trade-off of Schizosaccharomyces pombe.

Replicative aging has been demonstrated in asymmetrically dividing unicellular organisms, seemingly caused by unequal damage partitioning. Although asymmetric segregation and inheritance of potential aging factors also occur in symmetrically dividing species, it nevertheless remains controversial whether this results in aging. Based on large-scale single-cell lineage data obtained by time-lapse microscopy with a microfluidic device, in this report, we demonstrate the absence of replicative aging in old-pole cell lineages of Schizosaccharomyces pombe cultured under constant favorable conditions. By monitoring more than 1,500 cell lineages in 7 different culture conditions, we showed that both cell division and death rates are remarkably constant for at least 50-80 generations. Our measurements revealed that the death rate per cellular generation increases with the division rate, pointing to a physiological trade-off with fast growth under balanced growth conditions. We also observed the formation and inheritance of Hsp104-associated protein aggregates, which are a potential aging factor in old-pole cell lineages, and found that these aggregates exhibited a tendency to preferentially remain at the old poles for several generations. However, the aggregates were eventually segregated from old-pole cells upon cell division and probabilistically allocated to new-pole cells. We found that cell deaths were typically preceded by sudden acceleration of protein aggregation; thus, a relatively large amount of protein aggregates existed at the very ends of the dead cell lineages. Our lineage tracking analyses, however, revealed that the quantity and inheritance of protein aggregates increased neither cellular generation time nor cell death initiation rates. Furthermore, our results demonstrated that unusually large amounts of protein aggregates induced by oxidative stress exposure did not result in aging; old-pole cells resumed normal growth upon stress removal, despite the fact that most of them inherited significant quantities of aggregates. These results collectively indicate that protein aggregates are not a major determinant of triggering cell death in S. pombe and thus cannot be an appropriate molecular marker or index for replicative aging under both favorable and stressful environmental conditions.

Entirely plasmid-based reverse genetics system for rotaviruses.

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein-tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Orthoreovirus outer-fiber proteins are substrates for SUMO-conjugating enzyme Ubc9.

Reoviruses are potential anticancer agents due to their ability to induce cell death in tumor cells. Grass carp reovirus (GCRV) is one of the best characterized models on reovirus pathogenesis in vitro. However, there is little known about how SUMOylation affects reovirus pathogenesis. The SUMO conjugating enzyme 9 (Ubc9) determines the targets of SUMOylation. Here, the protein interactions between reovirus outer fiber proteins, specifically GCRV-104 VP55, and Ubc9 were probed using a yeast two-hybrid system. The N-terminal coiled-coil domain of VP55, containing a single lysine residue, was responsible for the interaction between VP55 and Ubc9 in yeast. In solid phase binding assays, a single amino acid mutation (K87R) prevented Ubc9 from binding to VP55. Overexpression of Ubc9 enhanced GCRV-104 infection efficiency, and knockdown of Ubc9 in CIK cells inhibited viral replication, which suggested that Ubc9 was a proviral factor. Furthermore, Ubc9 was shown to bind outer fiber proteins from type II GCRV, avian reovirus and mammalian reovirus in yeast. To our knowledge, this is the first study to show that Ubc9 binds to reovirus outer-fiber proteins and likely contributes to efficient orthoreovirus replication. These results suggest that SUMOylation modifications could be targeted to improve the therapeutic efficacy of oncolytic reovirus.

The non-structural protein µNS of piscine orthoreovirus (PRV) forms viral factory-like structures.

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation in farmed Atlantic salmon. The virus is ubiquitous and found in both farmed and wild salmonid fish. It belongs to the family Reoviridae, closely related to the genus Orthoreovirus. The PRV genome comprises ten double-stranded RNA segments encoding at least eight structural and two non-structural proteins. Erythrocytes are the major target cells for PRV. Infected erythrocytes contain globular inclusions resembling viral factories; the putative site of viral replication. For the mammalian reovirus (MRV), the non-structural protein µNS is the primary organizer in factory formation. The analogous PRV protein was the focus of the present study. The subcellular location of PRV µNS and its co-localization with the PRV σNS, µ2 and λ1 proteins was investigated. We demonstrated that PRV µNS forms dense globular cytoplasmic inclusions in transfected fish cells, resembling the viral factories of MRV. In co-transfection experiments with µNS, the σNS, µ2 and λ1 proteins were recruited to the globular structures. The ability of µNS to recruit other PRV proteins into globular inclusions indicates that it is the main viral protein involved in viral factory formation and pivotal in early steps of viral assembly.

Reovirus FAST proteins: virus-encoded cellular fusogens.

Reovirus fusion-associated small transmembrane (FAST) proteins are the only known nonenveloped virus fusogens and are dedicated to inducing cell-to-cell, not virus-cell, membrane fusion. Numerous structural and functional attributes distinguish this novel family of viral fusogens from all enveloped virus membrane fusion proteins. Both families of viral fusogens play key roles in virus dissemination and pathogenicity, but employ different mechanisms to mediate membrane apposition and merger. However, convergence of these distinct families of viral membrane fusion proteins on common pathways needed for pore expansion and syncytium formation suggests syncytiogenesis represents a cellular response to the presence of cell-cell fusion pores. Together, FAST proteins and enveloped virus fusion proteins provide exceptional insights into the ubiquitous process of cell-cell membrane fusion and syncytium formation.

Efficient reovirus- and measles virus-mediated pore expansion during syncytium formation is dependent on annexin A1 and intracellular calcium.

UNLABELLED: Orthoreovirus fusion-associated small transmembrane (FAST) proteins are dedicated cell-cell fusogens responsible for multinucleated syncytium formation and are virulence determinants of the fusogenic reoviruses. While numerous studies on the FAST proteins and enveloped-virus fusogens have delineated steps involved in membrane fusion and pore formation, little is known about the mechanics of pore expansion needed for syncytiogenesis. We now report that RNA interference (RNAi) knockdown of annexin A1 (AX1) expression dramatically reduced both reptilian reovirus p14 and measles virus F and H protein-mediated pore expansion during syncytiogenesis but had no effect on pore formation. A similar effect was obtained by chelating intracellular calcium, which dramatically decreased syncytiogenesis in the absence of detectable effects on p14-induced pore formation. Coimmunoprecipitation revealed calcium-dependent interaction between AX1 and p14 or measles virus F and H proteins, and fluorescence resonance energy transfer (FRET) demonstrated calcium-dependent p14-AX1 interactions in cellulo. Furthermore, antibody inhibition of extracellular AX1 had no effect on p14-induced syncytium formation but did impair cell-cell fusion mediated by the endogenous muscle cell fusion machinery in C2C12 mouse myoblasts. AX1 can therefore exert diverse, fusogen-specific effects on cell-cell fusion, functioning as an extracellular mediator of differentiation-dependent membrane fusion or as an intracellular promoter of postfusion pore expansion and syncytium formation following virus-mediated cell-cell fusion. IMPORTANCE: Numerous enveloped viruses and nonenveloped fusogenic orthoreoviruses encode membrane fusion proteins that induce syncytium formation, which has been linked to viral pathogenicity. Considerable insights into the mechanisms of membrane fusion have been obtained, but processes that drive postfusion expansion of fusion pores to generate syncytia are poorly understood. This study identifies intracellular calcium and annexin A1 (AX1) as key factors required for efficient pore expansion during syncytium formation mediated by the reptilian reovirus p14 and measles virus F and H fusion protein complexes. Involvement of intracellular AX1 in syncytiogenesis directly correlates with a requirement for intracellular calcium in p14-AX1 interactions and pore expansion but not membrane fusion and pore formation. This is the first demonstration that intracellular AX1 is involved in pore expansion, which suggests that the AX1 pathway may be a common host cell response needed to resolve virus-induced cell-cell fusion pores.

Piscine reovirus encodes a cytotoxic, non-fusogenic, integral membrane protein and previously unrecognized virion outer-capsid proteins.

Piscine reovirus (PRV) is a tentative new member of the family Reoviridae and has been linked to heart and skeletal muscle inflammation in farmed Atlantic salmon (Salmo salar L.). Recent sequence-based evidence suggests that PRV is about equally related to members of the genera Orthoreovirus and Aquareovirus. Sequence similarities have also suggested that PRV might encode a fusion-associated small transmembrane (FAST) protein, which in turn suggests that PRV might be the prototype of a new genus with syncytium-inducing potential. In previous support of this designation has been the absence of identifiable PRV-encoded homologues of either the virion outer-clamp protein of ortho- and aquareoviruses or the virion outer-fibre protein of most orthoreoviruses. In the current report, we have provided experimental evidence that the putative p13 FAST protein of PRV lacks the defining feature of the FAST protein family - the ability to induce syncytium formation. Instead, p13 is the first example of a cytosolic, integral membrane protein encoded by ortho- or aquareoviruses, and induces cytotoxicity in the absence of cell-cell fusion. Sequence analysis also identified signature motifs of the outer-clamp and outer-fibre proteins of other reoviruses in two of the predicted PRV gene products. Based on these findings, we conclude that PRV does not encode a FAST protein and is therefore unlikely to be a new fusogenic reovirus. The presence of a novel integral membrane protein and two previously unrecognized, essential outer-capsid proteins has important implications for the biology, evolution and taxonomic classification of this virus.

Reovirus binding determinants in junctional adhesion molecule-A.

Junctional adhesion molecule-A (JAM-A) serves as a serotype-independent receptor for mammalian orthoreoviruses (reoviruses). The membrane-distal immunoglobulin-like D1 domain of JAM-A is required for homodimerization and binding to reovirus attachment protein sigma1. We employed a structure-guided mutational analysis of the JAM-A dimer interface to identify determinants of reovirus binding. We purified mutant JAM-A ectodomains for solution-phase and surface plasmon resonance binding studies and expressed mutant forms of full-length JAM-A in Chinese hamster ovary cells to assess reovirus binding and infectivity. Mutation of residues in the JAM-A dimer interface that participate in salt-bridge or hydrogen-bond interactions with apposing JAM-A monomers abolishes the capacity of JAM-A to form dimers. JAM-A mutants incapable of dimer formation form complexes with the sigma1 head that are indistinguishable from wild-type JAM-A-sigma1 head complexes, indicating that sigma1 binds to JAM-A monomers. Residues Glu(61) and Lys(63) of beta-strand C and Leu(72) of beta-strand C' in the dimer interface are required for efficient JAM-A engagement of strain type 3 Dearing sigma1. Mutation of neighboring residues alters the kinetics of the sigma1-JAM-A binding interaction. Prototype reovirus strains type 1 Lang and type 2 Jones share similar, although not identical, binding requirements with type 3 Dearing. These results indicate that reovirus engages JAM-A monomers via residues found mainly on beta-strands C and C' of the dimer interface and raise the possibility that the distinct disease phenotypes produced in mice following infection with different strains of reovirus are in part attributable to differences in contacts with JAM-A.

Atypical fusion peptide of Nelson Bay virus fusion-associated small transmembrane protein.

A 10-kDa nonstructural transmembrane protein (p10) encoded by a reovirus, Nelson Bay virus, has been shown to induce syncytium formation (34). Sequence analysis and structural studies identified p10 as a type I membrane protein with a central transmembrane domain, a cytoplasmic basic region, and an N-terminal hydrophobic domain (HD) that was hypothesized to function as a fusion peptide. We performed mutational analysis on this slightly hydrophobic motif to identify possible structural requirements for fusion activity. Bulky aliphatic residues were found to be essential for optimal fusion, and an aromatic or highly hydrophobic side chain was found to be required at position 12. The requirement for hydrophilic residues within the HD was also examined: substitution of 10-Ser or 14-Ser with hydrophobic residues was found to reduce cell surface expression of p10 and delayed the onset of syncytium formation. Nonconservative substitutions of charged residues in the HD did not have an effect on fusion activity. Taken together, our results suggest that the HD is involved in both syncytium formation and in determining p10 transport and surface expression.

Increased ubiquitination and other covariant phenotypes attributed to a strain- and temperature-dependent defect of reovirus core protein mu2.

Reovirus replication and assembly are thought to occur within cytoplasmic inclusion bodies, which we call viral factories. A strain-dependent difference in the morphology of these structures reflects more effective microtubule association by the mu2 core proteins of some viral strains, which form filamentous factories, than by those of others, which form globular factories. For this report, we identified and characterized another strain-dependent attribute of the factories, namely, the extent to which they colocalized with conjugated ubiquitin (cUb). Among 16 laboratory strains and field isolates, the extent of factory costaining for cUb paralleled factory morphology, with globular strains exhibiting higher levels by far. In reassortant viruses, factory costaining for cUb mapped primarily to the mu2-encoding M1 genome segment, although contributions by the lambda3- and lambda2-encoding L1 and L2 genome segments were also evident. Immunoprecipitations revealed that cells infected with globular strains contained higher levels of ubiquitinated mu2 (Ub-mu2). In M1-transfected cells, cUb commonly colocalized with aggregates formed by mu2 from globular strains but not with microtubules coated by mu2 from filamentous strains, and immunoprecipitations revealed that mu2 from globular strains displayed higher levels of Ub-mu2. Allelic changes at mu2 residue 208 determined these differences. Nocodazole treatment of cells infected with filamentous strains resulted in globular factories that still showed low levels of costaining for cUb, indicating that higher levels of costaining were not a direct result of decreased microtubule association. The factories of globular strains, or their mu2 proteins expressed in transfected cells, were furthermore shown to gain microtubule association and to lose colocalization with cUb when cells were grown at reduced temperature. From the sum of these findings, we propose that mu2 from globular strains is more prone to temperature-dependent misfolding and as a result displays increased aggregation, increased levels of Ub-mu2, and decreased association with microtubules. Because so few of the viral strains formed factories that were regularly associated with ubiquitinated proteins, we conclude that reovirus factories are generally distinct from cellular aggresomes.

Orthoreovirus and Aquareovirus core proteins: conserved enzymatic surfaces, but not protein-protein interfaces.

Orthoreoviruses and Aquareoviruses constitute two respective genera in the family Reoviridae of double-stranded RNA viruses. Orthoreoviruses infect mammals, birds, and reptiles and have a genome comprising 10 RNA segments. Aquareoviruses infect fish and have a genome comprising 11 RNA segments. Despite these differences, recent structural and nucleotide sequence evidence indicate that the proteins of Orthoreoviruses and Aquareoviruses share many similarities. The focus of this review is on the structure and function of the Orthoreovirus core proteins lambda1, lambda2, lambda3, and sigma2, for which X-ray crystal structures have been recently reported. The homologous core proteins in Aquareoviruses are VP3, VP1, VP2, and VP6, respectively. By mapping the locations of conserved residues onto the Orthoreovirus crystal structures, we have found that enzymatic surfaces involved in mRNA synthesis are well conserved between these two groups of viruses, whereas several surfaces involved in protein-protein interactions are not well conserved. Other evidence indicates that the Orthoreovirus mu2 and Aquareovirus VP5 proteins are homologous, suggesting that VP5 is a core protein as mu2 is known to be. These findings provide further evidence that Orthoreoviruses and Aquareoviruses have diverged from a common ancestor and contribute to a growing understanding of the functions of the core proteins in viral mRNA synthesis.

Identification and characterization of a baboon reovirus-specific nonstructural protein encoded by the bicistronic s4 genome segment.

All characterized orthoreoviruses encode a characteristic spike-like protein on their polycistronic S1 genome segments that mediates virus cell attachment. In the case of baboon reovirus (BRV), the polycistronic S-class genome segment corresponds to the smallest S4 segment. We recently determined that the 5'-proximal open reading frame (ORF) of the bicistronic S4 segment encodes a nonstructural protein responsible for virus-induced syncytium formation. Current analysis indicates that the p16 protein encoded by the 3'-proximal ORF of the BRV S4 genome segment shows no sequence similarity to any other protein encoded by the orthoreoviruses, including the well-characterized sigma1/sigmaC reovirus cell attachment protein. Results indicate that p16 is a BRV-specific nonstructural protein that is not required for virus infection in cell culture and is not involved in viral cell attachment. In conjunction with previous studies of the BRV S1, S2, and S3 genome segments, the current results indicate that, unlike all other orthoreoviruses, BRV does not encode a cell attachment protein in its S-class genome segments. Furthermore, cell binding and infectivity studies suggested BRV may not utilize a functional homolog of the prototypical reovirus sigma1/sigmaC cell receptor-binding protein to mediate endocytic uptake by cells.

The S4 genome segment of baboon reovirus is bicistronic and encodes a novel fusion-associated small transmembrane protein.

We demonstrate that the S4 genome segment of baboon reovirus (BRV) contains two sequential partially overlapping open reading frames (ORFs), both of which are functional in vitro and in virus-infected cells. The 15-kDa gene product (p15) of the 5"-proximal ORF induces efficient cell-cell fusion when expressed by itself in transfected cells, suggesting that p15 is the only viral protein required for induction of syncytium formation by BRV. The p15 protein is a small, hydrophobic, basic, integral membrane protein, properties shared with the p10 fusion-associated small transmembrane (FAST) proteins encoded by avian reovirus and Nelson Bay reovirus. As with p10, the BRV p15 protein is also a nonstructural protein and, therefore, is not involved in virus entry. Sequence analysis indicates that p15 shares no significant sequence similarity with the p10 FAST proteins and contains a unique repertoire and arrangement of sequence-predicted structural and functional motifs. These motifs include a functional N-terminal myristylation consensus sequence, an N-proximal proline-rich motif, two potential transmembrane domains, and an intervening polybasic region. The unique structural properties of p15 suggest that this protein is a novel member of the new family of FAST proteins.

Identification of carbohydrate-binding domains in the attachment proteins of type 1 and type 3 reoviruses.

The reovirus attachment protein, sigma1, is responsible for strain-specific patterns of viral tropism in the murine central nervous system and receptor binding on cultured cells. The sigma1 protein consists of a fibrous tail domain proximal to the virion surface and a virion-distal globular head domain. To better understand mechanisms of reovirus attachment to cells, we conducted studies to identify the region of sigma1 that binds cell surface carbohydrate. Chimeric and truncated sigma1 proteins derived from prototype reovirus strains type 1 Lang (T1L) and type 3 Dearing (T3D) were expressed in insect cells by using a baculovirus vector. Assessment of expressed protein susceptibility to proteolytic cleavage, binding to anti-sigma1 antibodies, and oligomerization indicates that the chimeric and truncated sigma1 proteins are properly folded. To assess carbohydrate binding, recombinant sigma1 proteins were tested for the capacity to agglutinate mammalian erythrocytes and to bind sialic acid presented on glycophorin, the cell surface molecule bound by type 3 reovirus on human erythrocytes. Using a panel of two wild-type and ten chimeric and truncated sigma1 proteins, the sialic acid-binding domain of type 3 sigma1 was mapped to a region of sequence proposed to form the more amino terminal of two predicted beta-sheet structures in the tail. This unit corresponds to morphologic region T(iii) observed in computer-processed electron micrographs of sigma1 protein purified from virions. In contrast, the homologous region of T1L sigma1 sequence was not implicated in carbohydrate binding; rather, sequences in the distal portion of the tail known as the neck were required. Results of these studies demonstrate that a functional receptor-binding domain, which uses sialic acid as its ligand, is contained within morphologic region T(iii) of the type 3 sigma1 tail. Furthermore, our findings indicate that T1L and T3D sigma1 proteins contain different arrangements of receptor-binding domains.