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Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.

ABSTRACT: Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney (CK) cells and in chicken embryos was not affected. In chicken tracheal organ cultures (TOCs), the recombinant rIBVs reached titers similar to those of the wild-type virus, but in the case of viruses lacking the 3a protein, the titer declined reproducibly earlier. Translation of the IBV E protein is believed to be initiated by internal entry of ribosomes at a structure formed by the sequences corresponding to ORFs 3a and 3b. To assess the necessity of this mechanism, we deleted most of the sequence representing 3a and 3b to produce a gene in which ORF 3c (E) was adjacent to the gene 3 transcription-associated sequence. Western blot analysis revealed that the recombinant IBV produced fivefold less E protein. Nevertheless, titers produced in CK cells, embryos, and TOCs were similar to those of the wild-type virus, although they declined earlier in TOCs, probably due to the absence of the 3a protein. Thus, neither the tricistronic arrangement of gene 3, the internal initiation of translation of E protein, nor the 3a and 3b proteins are essential for replication per se, suggesting that these proteins are accessory proteins that may have roles in vivo.

SUBMITTER: Hodgson T

PROVIDER: S-EPMC1317528 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.

Hodgson Teri T Britton Paul P Cavanagh Dave D

Journal of virology 20060101 1

Gene 3 of infectious bronchitis virus is tricistronic; open reading frames (ORFs) 3a and 3b encode two small nonstructural (ns) proteins, 3a and 3b, of unknown function, and a third, structural protein E, is encoded by ORF 3c. To determine if either the 3a or the 3b protein is required for replication, we first modified their translation initiation codons to prevent translation of the 3a and 3b proteins from recombinant infectious bronchitis viruses (rIBVs). Replication in primary chick kidney ( ...[more]

PMID: 16352554

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Project description:The subgenomic RNA 3 of IBV has been shown to be a tricistronic mRNA, encoding three products in IBV-infected cells. To explore if the least expressed ORF, ORF 3b, which encodes a nonstructural protein, is evolutionarily conserved and functionally indispensable for viral propagation in cultured cells, the Beaudette strain of IBV was propagated in chicken embryonated eggs for three passages and then adapted to a monkey kidney cell line, Vero. The 3b gene of passage 3 in embryonated eggs and passages 7, 15, 20, 25, 30, 35 50, and 65 in Vero cells were amplified by reverse transcription-polymerase chain reaction and sequenced. The results showed that viral RNA extracted from passages 35, 50, and 65 contained a single A insertion in a 6A stretch of the 3b gene between nucleotides 24075 and 24080, whereas the early passages carried the normal 3b gene. This insertion resulted in a frameshift event and therefore, if expressed, a C-terminally truncated protein. We showed that the frameshifting product, cloned in a plasmid, was expressed in vitro and in cells transfected with the mutant construct. The normal product of the 3b gene is 64 amino acids long, whereas the frameshifting product is 34 amino acids long with only 17 homogeneous amino acid residues at the N-terminal half. Immunofluorescent studies revealed that the normal 3b protein was localized to the nucleus and the truncated product showed a "free" distribution pattern, indicating that the C-terminal portion of 3b was responsible for its nuclear localization. Comparison of the complete genome sequences (27.6 kb) of isolates p20c22 and p36c12 (from passages 20 and 36, respectively) revealed that p36c12 contains three amino acid substitutions, two in the 195-kDa protein (encoded by gene 1) and one in the S protein, in addition to the frameshifting 3b product. Further characterization of the two isolates demonstrated that p36c12 showed growth advantage over p20c22 in both Vero cells and chicken embryos and was more virulent in chicken embryos than p20c22. These results suggest that the 3b gene product is not essential for the replication of IBV.

Dataset Information

Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.

Publications

Neither the RNA nor the proteins of open reading frames 3a and 3b of the coronavirus infectious bronchitis virus are essential for replication.

Similar Datasets

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