Replication and packaging of coronavirus infectious bronchitis virus defective RNAs lacking a long open reading frame
| Autor: | Paul Britton, Z. Penzes, Dave Cavanagh, C. Wroe, T. D. K. Brown |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
DNA
Complementary Infectious bronchitis virus Molecular Sequence Data Immunology Biology Virus Replication medicine.disease_cause Microbiology Defective virus Open Reading Frames Bacteriophage T7 Virology medicine Protein biosynthesis Animals Cloning Molecular Gene Sequence Deletion Coronavirus Base Sequence Virus Assembly Defective Viruses RNA Molecular biology Stop codon Open reading frame Viral replication Protein Biosynthesis Insect Science RNA Viral Gene Deletion Research Article |
| Zdroj: | Journal of Virology. 70:8660-8668 |
| ISSN: | 1098-5514 0022-538X |
| DOI: | 10.1128/jvi.70.12.8660-8668.1996 |
| Popis: | The construction of a full-length clone of the avian coronavirus infectious bronchitis virus (IBV) defective RNA (D-RNA), CD-91 (9,080 nucleotides [Z. Penzes et al., Virology 203:286-293]), downstream of the bacteriophage T7 promoter is described. Electroporation of in vitro T7-transcribed CD-91 RNA into IBV helper virus-infected primary chick kidney cells resulted in the production of CD-91 RNA as a replicating D-RNA in subsequent passages. Three CD-91 deletion mutants were constructed--CD-44, CD-58, and CD-61--in which 4,639, 3,236, and 2,953 nucleotides, respectively, were removed from CD-91, resulting in the truncation of the CD-91 long open reading frame (ORF) from 6,465 to 1,311, 1,263, or 2,997 nucleotides in CD-44, CD-58, or CD-61, respectively. Electroporation of in vitro T7-transcribed RNA from the three constructs into IBV helper virus-infected cells resulted in the replication and packaging of CD-58 and CD-61 but not CD-44 RNA. The ORF of CD-61 was further truncated by the insertion of stop codons into the CD-61 sequence by PCR mutagenesis, resulting in constructs CD-61T11 (ORF: nucleotides 996 to 1,058, encoding 20 amino acids), CD-61T22 (ORF: nucleotides 996 to 2,294, encoding 432 amino acids), and CD-61T24 (ORF: nucleotides 996 to 2,450, encoding 484 amino acids), all of which were replicated and packaged to the same levels as observed for either CD-61 or CD-91. Analysis of the D-RNAs showed that the CD-91- or CD-61-specific long ORFs had not been restored. Our data indicate that IBV D-RNAs based on the natural D-RNA, CD-91, do not require a long ORF for efficient replication. In addition, a 1.4-kb sequence, corresponding to IBV sequence at the 5' end of the 1b gene, may be involved in the packaging of IBV D-RNAs or form part of a cis-acting replication element. |
| Databáze: | OpenAIRE |
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