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A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs.

ABSTRACT: Two murine hepatitis virus strain A59 defective interfering (DI) RNAs were generated by undiluted virus passages. The DI RNAs were encapsidated efficiently. The smallest DI particle, DI-a, contained a 5.5-kb RNA consisting of the following three noncontiguous regions from the MHV-A59 genome, which were joined in frame: the 5'-terminal 3.9 kb, a 798-nucleotide fragment from the 3' end of the polymerase gene, and the 3'-terminal 805 nucleotides. A full-length cDNA clone of the DI-a genome was constructed and cloned downstream of the bacteriophage T7 promoter. Transcripts derived from this clone, pMIDI, were used for transfection of MHV-A59-infected cells and found to be amplified and packaged. Deletion analysis of pMIDI allowed us to identify a 650-nucleotide region derived from the 3' end of the second open reading frame of the polymerase gene that was required for efficient encapsidation.

SUBMITTER: van der Most RG

PROVIDER: S-EPMC240979 | biostudies-other | 1991 Jun

REPOSITORIES: biostudies-other

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A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs.

van der Most R G RG Bredenbeek P J PJ Spaan W J WJ

Journal of virology 19910601 6

Two murine hepatitis virus strain A59 defective interfering (DI) RNAs were generated by undiluted virus passages. The DI RNAs were encapsidated efficiently. The smallest DI particle, DI-a, contained a 5.5-kb RNA consisting of the following three noncontiguous regions from the MHV-A59 genome, which were joined in frame: the 5'-terminal 3.9 kb, a 798-nucleotide fragment from the 3' end of the polymerase gene, and the 3'-terminal 805 nucleotides. A full-length cDNA clone of the DI-a genome was cons ...[more]

PMID: 2033672

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Project description:UnlabelledThe replication of plus-strand RNA virus genomes is mediated by virally encoded RNA-dependent RNA polymerases (RdRps). We have investigated the role of the C-proximal region in the RdRp of tomato bushy stunt virus (TBSV) in mediating viral RNA synthesis. TBSV is the prototype species in the genus Tombusvirus, family Tombusviridae, and its RdRp is responsible for replicating the viral genome, transcribing two subgenomic mRNAs, and supporting replication of defective interfering RNAs. Comparative sequence analysis of the RdRps of tombusvirids identified three highly conserved motifs in their C-proximal regions, and these sequences were subsequently targeted for mutational analysis in TBSV. The results revealed that these motifs are important for (i) synthesizing viral genomic RNA and subgenomic mRNAs, (ii) facilitating plus- and/or minus-strand synthesis, and (iii) modulating trans-replication of a defective interfering RNA. These motifs were also found to be conserved in other plant viruses as well as in a fungal and insect virus. The collective findings are discussed in relation to viral RNA synthesis and taxonomy.ImportanceLittle is currently known about the structure and function of the viral polymerases that replicate the genomes of RNA plant viruses. Tombusviruses, the prototype of the tombusvirids, have been used as model plus-strand RNA plant viruses for understanding many of the steps in the infectious process; however, their polymerases remain poorly characterized. To help address this issue, the function of the C-terminal region of the polymerase of a tombusvirus was investigated. Three conserved motifs were identified and targeted for mutational analysis. The results revealed that these polymerase motifs are important for determining what type of viral RNA is produced, facilitating different steps in viral RNA production, and amplifying subgenomic RNA replicons. Accordingly, the C-terminal region of the tombusvirus polymerase is needed for a variety of fundamental activities. Furthermore, as these motifs are also present in distantly related viruses, the significance of these results extends beyond tombusvirids.

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A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs.

Publications

A domain at the 3' end of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs.

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