Conserved structure/function of the orthoreovirus major core proteins.
ABSTRACT Orthoreoviruses are infectious agents with genomes of 10 segments of double-stranded RNA. Detailed molecular information is available for all 10 segments of several mammalian orthoreoviruses, and for most segments of several avian orthoreoviruses (ARV). We, and others, have reported sequences of the L2, all S-class, and all M-class genome segments of two different avian reoviruses, strains ARV138 and ARV176. We here determined L1 and L3 genome segment nucleotide sequences for both strains to complete full genome characterization of this orthoreovirus subgroup. ARV L1 segments were 3958 nucleotides long and encode lambda A major core shell proteins of 1293 residues. L3 segments were 3907 nucleotides long and encode lambda C core turret proteins of 1285 residues. These newly determined ARV segments were aligned with all currently available homologous mammalian reovirus (MRV) and aquareovirus (AqRV) genome segments. Identical and conserved amino acid residues amongst these diverse groups were mapped into known mammalian reovirus lambda 1 core shell and lambda 2 core turret proteins to predict conserved structure/function domains. Most identical and conserved residues were located near predicted catalytic domains in the lambda-class guanylyltransferase, and forming patches that traverse the lambda-class core shell, which may contribute to the unusual RNA transcription processes in this group of viruses.
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ABSTRACT: The double-shelled grass carp reovirus (GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene segment 2 (S2), is the putative RNA-dependent RNA polymerase (RdRp). In previous work, we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity (denoted as rVP2(390-900)) in E. coli and have prepared a polyclonal antibody against VP2. To characterize the GCRV RNA polymerase, a recombinant full-length VP2 (rVP2) was first constructed and expressed in a baculovirus system, as a fusion protein with an attached His-tag. Immunofluorescence (IF) assays, together with immunoblot (IB) analyses from both expressed cell extracts and purifi ed Histagged rVP2, showed that rVP2 was successfully expressed in Sf9 cells. Further characterization of the replicase activity showed that purifi ed rVP2 and GCRV particles exhibited poly(C)-dependent poly(G) polymerase activity. The RNA enzymatic activity required the divalent cation Mg(2+), and was optimal at 28 °C. The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.Virologica Sinica 03/2014;
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ABSTRACT: Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.PLoS ONE 01/2013; 8(7):e70075. · 3.53 Impact Factor