Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the C viral cell attachment protein initiates over 600 nucleotides distal from the 5 end of the S1 mRNA and is preceded by two predicted small nonoverlapping ORFs. To more clearly define the translational properties of this unusual polycistronic RNA, we pursued a comparative analysis of the S1 genome segment of the related Nelson Bay reovirus (NBV). Sequence analysis indicated that the 3-proximal ORF present on the NBV S1 genome segment also encodes a C homolog, as evidenced by the presence of an extended N-terminal heptad repeat characteristic of the coiled-coil region common to the cell attachment proteins of reoviruses. Most importantly, the NBV S1 genome segment contains two conserved ORFs upstream of the C coding region that are extended relative to the predicted ORFs of ARV-S1133 and are arranged in a sequential, partially overlapping fashion. Sequence analysis of the S1 genome segments of two additional strains of ARV indicated a similar overlapping tricistronic gene arrangement as predicted for the NBV S1 genome segment. Expression analysis of the ARV S1 genome segment indicated that all three ORFs are functional in vitro and in virus-infected cells. In addition to the previously described p10 and C gene products, the S1 genome segment encodes from the central ORF a 17-kDa basic protein (p17) of no known function. Optimizing the translation start site of the ARV p10 ORF lead to an approximately 15-fold increase in p10 expression with little or no effect on translation of the downstream C ORF. These results suggest that translation initiation complexes can bypass over 600 nucleotides and two functional overlapping upstream ORFs in order to access the distal C start site.The orthoreoviruses consist of a number of diverse virus species, all of which have a similar capsid structure and a genome composed of 10 double-stranded RNA (dsRNA) segments (38). For the most part, the genome segments of reoviruses are monocistronic, each encoding a single unique polypeptide. The mammalian reovirus (MRV) S1 genome segment, however, is bicistronic and encodes two unrelated polypeptides from overlapping open reading frames (ORFs). The 1 viral cell attachment protein is encoded by a large ORF that originates near the 5Ј end of the mRNA and spans almost the complete length of the S1 mRNA (12, 37). A second small ORF, beginning at nucleotide position 71, is nested within the 1 ORF in a ϩ1 reading frame and encodes the small nonstructural 1NS protein of MRV (14,23).Since the translation start site for 1 is in a nonpreferred context (a pyrimidine at position Ϫ3), leaky scanning by the preinitiation complex most likely accounts for translation of the downstream 1NS ORF from the MRV S1 mRNA (28). In addition to leaky scanning, there is evidence suggesting that translation of the 1 protein from the bicistronic S1 mRNA is modulated by ribosomal pausing at the downstream 1NS start site (7, 15).As wit...

Among members of the genus Orthoreovirus, family Reoviridae, a group of non-enveloped viruses with genomes comprising ten segments of double-stranded RNA, only the "non-fusogenic" mammalian orthoreoviruses (MRVs) have been studied to date by electron cryomicroscopy and three-dimensional image reconstruction. In addition to MRVs, this genus comprises other species that induce syncytium formation in cultured cells, a property shared with members of the related genus Aquareovirus. To augment studies of these "fusogenic" orthoreoviruses, we used electron cryomicroscopy and image reconstruction to analyze the virions of a fusogenic avian orthoreovirus (ARV). The structure of the ARV virion, determined from data at an effective resolution of 14.6 A, showed strong similarities to that of MRVs. Of particular note, the ARV virion has its pentameric lambda-class core turret protein in a closed conformation as in MRVs, not in a more open conformation as reported for aquareovirus. Similarly, the ARV virion contains 150 copies of its monomeric sigma-class core-nodule protein as in MRVs, not 120 copies as reported for aquareovirus. On the other hand, unlike that of MRVs, the ARV virion lacks "hub-and-spokes" complexes within the solvent channels at sites of local sixfold symmetry in the incomplete T=13l outer capsid. In MRVs, these complexes are formed by C-terminal sequences in the trimeric mu-class outer-capsid protein, sequences that are genetically missing from the homologous protein of ARVs. The channel structures and C-terminal sequences of the homologous outer-capsid protein are also genetically missing from aquareoviruses. Overall, the results place ARVs between MRVs and aquareoviruses with respect to the highlighted features.

We determined that the highly pathogenic avian reovirus strain 176 (ARV-176) possesses an enhanced ability to establish productive infections in HD-11 avian macrophages compared to avian fibroblasts. Conversely, the weakly pathogenic strain ARV-138 shows no such macrophagotropic tendency. The macrophage infection capability of the two viruses did not reflect differences in the ability to either induce or inhibit nitric oxide production. Moderate increases in the ARV-138 multiplicity of infection resulted in a concomitant increase in macrophage infection, and under such conditions the kinetics and extent of the ARV-138 replication cycle were equivalent to those of the highly infectious ARV-176 strain. These results indicated that both viruses are apparently equally capable of replicating in an infected macrophage, but they differ in the ability to establish productive infections in these cells. Using a genetic reassortant approach, we determined that the macrophagotropic property of ARV-176 reflects a post-receptor-binding step in the virus replication cycle and that the ARV-176 M2 genome segment is required for efficient infection of HD-11 cells. The M2 genome segment encodes the major -class outer capsid protein (B) of the virus, which is involved in virus entry and transcriptase activation, suggesting that a host-specific influence on ARV entry and/or uncoating may affect the likelihood of the virus establishing a productive infection in a macrophage cell.The avian reoviruses (ARV) differ from the prototypical mammalian reoviruses (MRV) based on several biological properties other than just their distinct host ranges. Unlike MRV, ARV is naturally pathogenic in its avian host, lacks hemagglutinating ability, and is one of the few nonenveloped viruses capable of inducing syncytium formation in infected cell cultures and in vivo (14,18,24,28). Although ARV pathogenesis has been extensively described (5,6,15,34), the viral factors that influence ARV-host cell interactions and pathogenesis remain poorly understood.We have been investigating two ARV strains that possess distinct pathogenic and syncytium-inducing potentials. Previous results demonstrated that ARV-176 is highly pathogenic relative to ARV-138 in an embryonated egg model of virus pathogenesis, an attribute that correlates with the relative fusogenic capability of the virus (8). Both viruses infect and replicate with equal efficiency in cultured fibroblast cells, they display 94 to 98% amino acid sequence identity in the three sequenced S-class genome segment-encoded proteins (7a), and all 10 of their individual genome segments can be resolved by electrophoretic analysis (8); these properties make these two ARV strains ideal parental virus candidates for genetic and molecular approaches to identify viral determinants of host interaction and pathogenicity. We previously used a genetic reassortant approach to reveal that the S1 genome segment of ARV-176 is solely responsible for the syncytium-inducing property of the virus (8). Subsequent molecular and bio...