Evidence for the multimeric nature and cell binding ability of avian reovirus 3 protein

, p53, p21 cip1/waf1 , Cdc2, cyclin B1, Chk1, Chk2, and Cdc25C, suggesting that p17 induces a G 2 /M cell cycle arrest through activation of the ATM/p53/p21 cip1/waf1 /Cdc2/cyclin B1 and ATM/Chk1/Chk2/Cdc25C pathways. The G 2 /M cell cycle arrest resulted in increased virus replication. In the present study, we also provide evidence demonstrating that p17 protein is responsible for ARV-induced host cellular protein translation shutoff. Increased phosphorylation levels of the eukaryotic translation elongation factor 2 (eEF2) and initiation factor eIF2␣ and reduced phosphorylation levels of the eukaryotic translation initiation factors eIF4E, eIF4B, and eIF4G, as well as 4E-BP1 and Mnk-1 in p17-transfected cells, demonstrated that ARV p17 suppresses translation initiation factors and translation elongation factors to induce host cellular protein translation shutoff. Inhibition of mTOR by rapamycin resulted in a decrease in the levels of phosphorylated 4E-BP1, eIF4B, and eIF4G and an increase in the levels eEF2 but did not affect ARV replication, suggesting that ARV replication was not hindered by inhibition of cap-dependent translation. Taken together, our data indicate that ARV p17-induced G 2 /M arrest and host cellular translation shutoff resulted in increased ARV replication.

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confidence: 99%

Avian Reovirus Nonstructural Protein p17-Induced G ₂ /M Cell Cycle Arrest and Host Cellular Protein Translation Shutoff Involve Activation of p53-Dependent Pathways

Chulu

Huang

Wang

et al. 2010

“…The second ORF encodes p17, a nonstructural protein of unknown function. Finally, the third ORF expresses C, an elongated trimeric structural protein responsible for the initial attachment of the virus to cell receptors (30,47). When we initiated this study, nothing was known about the activity or properties of the avian reovirus nonstructural p17 protein.…”

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confidence: 99%

The Second Open Reading Frame of the Avian Reovirus S1 Gene Encodes a Transcription-Dependent and CRM1-Independent Nucleocytoplasmic Shuttling Protein

Costas

Martı́nez-Costas

Bodelón

et al. 2005

It was previously shown that the second open reading frame of the avian reovirus S1 gene encodes a 146-amino-acid nonstructural protein, designated p17, which has no known function and no sequence similarity to other known proteins. The results presented in this report demonstrate that p17 accumulates in the nucleoplasm of infected and transfected cells. An examination of the deduced amino acid sequence of p17 revealed the presence of a putative monopartite nuclear localization signal (NLS) between residues 119 and 128. Mutagenesis analysis revealed both that this sequence is indeed a functional NLS and that two of its basic residues are critical for the normal nuclear distribution of p17. An interspecies heterokaryon assay further showed that p17 shuttles continuously between the nucleus and the cytoplasm and that this activity is restricted to its NLS-containing C-terminal tail. Finally, an analysis of the intracellular distribution of p17 in the presence of inhibitors of both RNA polymerase II and CRM1 further revealed that the nucleocytoplasmic distribution of p17 is coupled to transcriptional activity and that the viral protein exits the nucleus via a CRM1-independent pathway.

“…A heptad repeat motif present in the MRV 1 cell attachment protein is responsible for the formation of a coiled-coil structure which imparts stability to the functional homotrimeric form of the protein (31,49). A similar heptad repeat was previously identified in the ARV-S1133 C cell attachment protein that presumably contributes to the formation of C multimers (33,46,47). The presence of this characteristic motif in the predicted NBV ORF3 gene product indicates that this ORF encodes the NBV C viral cell attachment protein that also likely functions as a coiled-coil-stabilized trimer.…”

Section: Resultsmentioning

confidence: 98%

“…As with MRV, the avian reovirus (ARV) cell attachment protein, termed C, is encoded by the S1 genome segment (33,46,47). However, the initiation codon for the ARV strain S1133 (ARV-S1133) C ORF occurs over 600 nucleotides distal from the 5Ј end of the RNA, and this ORF is preceded by two small nonoverlapping ORFs that could potentially encode 9.8-kDa and 3.8-kDa polypeptides (46).…”

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confidence: 99%

Sequential Partially Overlapping Gene Arrangement in the Tricistronic S1 Genome Segments of Avian Reovirus and Nelson Bay Reovirus: Implications for Translation Initiation

Shmulevitz

Yameen

Dawe

et al. 2002

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...