Sequential partially overlapping gene arrangement in the tricistronic S1 genome segments of avian reovirus and Nelson Bay reovirus: implications for translation initiation.
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Previous studies of the avian reovirus strain S1133 (ARV-S1133) S1 genome segment revealed that the open reading frame (ORF) encoding the final sigmaC 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 final sigmaC 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 final sigmaC 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 final sigmaC 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 final sigmaC 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 final sigmaC start site. (+info)
Subunit composition and conformational stability of the oligomeric form of the avian reovirus cell-attachment protein sigmaC.
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Previous work has shown that the avian reovirus cell-attachment sigma C (sigmaC) protein is a multimer. In the first part of this study the oligomerization state of intracellularly synthesized sigmaC was analysed by different approaches, including SDS-PAGE, chemical cross-linking, sedimentation and gel filtration analysis. All these approaches indicated that protein sigmaC in its native state is a homotrimer. In the second part of the present work we investigated the effect of different factors and reagents on oligomer stability, in order to elucidate the nature of the forces that maintain the conformational stability of the homotrimer. Our results, based on the stabilizing effect conferred by reducing agents, demonstrate that the sigmaC subunits are not covalently bound via disulfide linkages. They further suggest that the formation of an intrachain disulfide bond between the two cysteine residues of the sigmaC polypeptide has a negative effect on oligomer stability. The susceptibility of the trimer to pH, temperature, ionic strength, chemical denaturants and detergents indicates that hydrophobic interactions contribute much more to oligomer stability than do ionic interactions and hydrogen bonding. Finally, our results also reveal that mammalian and avian reovirus cell attachment proteins follow different subunit dissociation pathways. (+info)
The avian reovirus genome segment S1 is a functionally tricistronic gene that expresses one structural and two nonstructural proteins in infected cells.
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The avian reovirus S1 gene contains three partially overlapping, out-of-phase open reading frames (ORFs) that the highly conserved in all avian reovirus strains examined to date. The three S1 ORFs of the avian reovirus strain S1133 were individually expressed in bacterial cells, and their purified translation products used as antigens to raise specific polyclonal antibodies. With these antibodies we were able to demonstrate that all three S1 ORFs from different avian reovirus strains are translatable in infected cells. Proteins p10 and p17, which are specified by ORF1 and ORF2, respectively, are nonstructural proteins which associate with cell membranes, whereas ORF3 directs the synthesis of protein sigma C, a structural oligomeric protein responsible for cell attachment. While intracellular synthesis of protein sigma C was demonstrated a long time ago and that of protein p10 was reported recently, this is the first time that expression of the S1 ORF2 has been demonstrated experimentally. Thus, the previously reported coding capacity of the avian reovirus genome is now expanded to 14 proteins, of which ten are structural (lambda A, lambda B, lambda C, microA, microB, microBC, microBN, sigma A, sigma B, and sigma C) and four are nonstructural (microNS, sigma NS, p17, and p10). Finally, protein p10, but not p17 or sigma C, induces cell-cell fusion when transiently expressed in mammalian cells, supporting a previously published observation that the polypeptide encoded by the S1 ORF1 plays an important role in the syncytial phenotype displayed by avian reoviruses. (+info)
Evidence of nucleotidyl phosphatase activity associated with core protein sigma A of avian reovirus S1133.
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Both avian reovirus core protein sigma A purified from virus-infected cell extracts and the purified bacterially expressed protein sigma A (e sigma A) were characterized for their nucleoside triphosphate (NTP) hydrolysis activity by thin-layer chromotography. Protein sigma A from both preparations has a nonspecific nucleotidyl phosphatase activity that hydrolyzes four types of NTP to their corresponding nucleoside di- and monophosphates and free phosphate. The divalent cation requirement for this activity of e sigma A was further examined by the addition of Mn(2+), Mg(2+), Ca(2+), and Zn(2+) ions. NTP hydrolysis by e sigma A was maximal when Mn(2+), Mg(2+), or Ca(2+) concentrations were 5, 4, or 1 mM, respectively. Addition of Mn(2+) or Mg(2+) stimulated the reactions up to 4- or 3-fold, respectively, higher than Ca(2+) (2.2-fold). However, Zn(2+) ion inhibited this activity of e sigma A. The results suggest that nucleotidyl phosphatase activity of e sigma A is absolutely dependent on the divalent cations Mn(2+), Mg(2+), or Ca(2+), but not Zn(2+). Similar results were obtained from the analysis of divalent cation requirements for the protein sigma A nucleotidyl phosphatase activity. Optimal pH for nucleotidyl phosphatase activity of protein sigma A from both preparations was determined using reaction mixtures buffered at different pH. The results show that the optimal activities of both proteins were similar and were achieved between pH 7.5 and 8.5. (+info)
Modification of late membrane permeability in avian reovirus-infected cells: viroporin activity of the S1-encoded nonstructural p10 protein.
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Infection of chicken embryo fibroblasts by avian reovirus induces an increase in the permeability of the host plasma membrane at late, but not early, infection times. The absence of permeability changes at early infection times, as well as the dependence of late membrane modification on both viral protein synthesis and an active exocytic route, suggest that a virus-encoded membrane protein is required for avian reovirus to permeabilize cells. Further studies revealed that expression of nonstructural p10 protein in bacterial cells arrested cell growth and enhanced membrane permeability. Membrane leakiness was also observed following transient expression of p10 in BSC-40 monkey cells. Both its permeabilizing effect and the fact that p10 shares several structural and physical characteristics with other membrane-active viral proteins indicate that p10 is an avian reovirus viroporin. Furthermore, the fusogenic extracellular NH(2)-terminal domain of p10 appears to be dispensable for permeabilizing activity, because its deletion entirely abolished the fusogenic activity of p10, without affecting its ability to associate with cell membranes and to enhance membrane permeability. Similar properties have reported previously for immunodeficiency virus type I transmembrane glycoprotein gp41. Thus, like gp41, p10 appears to be a multifunctional protein that plays key roles in virus-host interaction. (+info)
Muscovy duck reovirus sigmaC protein is atypically encoded by the smallest genome segment.
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Although muscovy duck reovirus (DRV) shares properties with the reovirus isolated from chicken, commonly named avian reovirus (ARV), the two virus species are antigenically different. Similar to the DRV sigmaB-encoded gene (1201 bp long) previously identified, the three other double-stranded RNA small genome segments of DRV have been cloned and sequenced. They were 1325, 1191 and 1124 bp long, respectively, and contained conserved terminal sequences common to ARVs. They coded for single expression products, except the smallest (S4), which contained two overlapping open reading frames (ORF1 and ORF2). BLAST analyses revealed that the proteins encoded by the 1325 and 1191 bp genes shared high identity levels with ARV sigmaA and sigmaNS, respectively, and to a lesser extent with other orthoreovirus counterparts. No homology was found for the S4 ORF1-encoded p10 protein. The 29.4 kDa product encoded by S4 ORF2 appeared to be 25% identical to ARV S1 ORF3-encoded sigmaC, a cell-attachment oligomer inducing type-specific neutralizing antibodies. Introduction of large gaps in the N-terminal part of the DRV protein was necessary to improve DRV and ARV sigmaC amino acid sequence alignments. However, a leucine zipper motif was conserved and secondary structure analyses predicted a three-stranded alpha-helical coiled-coil feature at this amino portion. Thus, despite extensive sequence divergence, DRV sigmaC was suggested to be structurally and probably functionally related to ARV sigmaC. This work provides evidence for the diversity of the polycistronic S class genes of reoviruses isolated from birds and raises the question of the relative classification of DRV in the Orthoreovirus genus. (+info)
Diarrhea-inducing activity of avian rotavirus NSP4 glycoproteins, which differ greatly from mammalian rotavirus NSP4 glycoproteins in deduced amino acid sequence in suckling mice.
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Avian rotavirus NSP4 glycoproteins expressed in Escherichia coli acted as enterotoxins in suckling mice, as did mammalian rotavirus NSP4 glycoproteins, despite great differences in the amino acid sequences. The enterotoxin domain of PO-13 NSP4 exists in amino acid residues 109 to 135, a region similar to that reported in SA11 NSP4. (+info)
Cloning, expression, and characterization of avian reovirus guanylyltransferase.
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We have cloned and sequenced the L3 genome segment of avian reovirus strain 1733, which specifies the viral guanylyltransferase protein, lambdaC. The L3 gene is 3907 nucleotides long and encodes, in a single large open-reading frame, a polypeptide of 1285 amino acid residues, with a calculated M(r) of 142.2 kDa. Expression of this gene in a baculovirus/insect cell system produced a recombinant protein that comigrated with reovirion lambdaC and reacted with anti-reovirus polyclonal serum in a Western blot assay. Incubation of recombinant lambdaC with GTP led to the formation GMP-lambdaC complex via a phosphoamide linkage. Interestingly, a 42-kDa amino-terminal proteolytic fragment of recombinant lambdaC protein also exhibited autoguanylylation activity, demonstrating both that this fragment is necessary and sufficient for autoguanylylation activity and that the 100-kDa complementary fragment is expendable for that activity. Comparison of the deduced amino acid sequence of protein lambdaC with those of the mammalian and grass carp reovirus guanylyltransferases revealed that only two of eight lysine residues within the amino-terminal 42-kDa region are conserved. Interestingly, these two lysines match with the lysine residues in the mammalian reovirus capping enzyme proposed to be essential for autoguanylylation activity. Our alignment analysis also showed that the S-adenosyl-l-methionine-binding pocket previously detected in the mammalian reovirus capping enzyme is fully conserved in its avian and grass carp reovirus counterparts, suggesting that all three enzymes have methylase activity. (+info)