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Published on: May 5, 2012
Control of Rous sarcoma virus RNA translation and packaging by the 5' and 3' untranslated sequences
Abstract:
A cytopathic mutant of Rous sarcoma virus-PrB was isolated and shown to have two large deletions, which result in the junction of Gag sequences in P27 to the 5' end of Env, and in the loss of the Src gene. This replication-defective (rd) and transformation-defective (td) mutant can replicate in the presence of its helper, which is also td, but the viral particles produced are poorly infectious. Most of the virions do not contain viral RNA, and the mutant RNA accumulates in infected cells, where it is poorly translated and packaged. Molecular clones of the mutant, of its helper and of a PrBtd strain were obtained in lambda-EMBL3, characterized, shown to be biologically active by transfection assays and sequenced. Nucleotide sequence comparisons indicate that the strong ribosome-binding site of Rous sarcoma virus RNA, responsible for the efficient RNA translation in vivo and in vitro, is mutated in PrB-(HM) mutant RNA; this causes the inhibition of RNA translation, as demonstrated by translation competition experiments using virus RNA made in vitro that carries the original or the mutated ribosome-binding site. In addition, an insertion present at the 3' end of both the mutant and the helper RNA, but absent in PrBtd RNA, is probably responsible for the inhibition of RNA packaging. Finally, these data are discussed in the light of a model of a 5'----3' Rous sarcoma virus RNA structure leading to a circular RNA molecule, which has implications in RNA translation, packaging and reverse transcription.
Insights
A cytopathic mutant of Rous sarcoma virus (RSV) was identified with deletions affecting Gag, Env, and Src genes. This mutant shows impaired RNA translation and packaging, impacting viral infectivity.
Area of Science:
- Molecular Virology
- Retroviral Genetics
- Cell Biology
Background:
- Rous sarcoma virus (RSV) is a retrovirus known for its ability to cause tumors.
- Understanding viral mutants is crucial for deciphering viral replication and pathogenesis.
- Previous studies have characterized various RSV strains, but specific cytopathic mutants offer unique insights.
Purpose of the Study:
- To characterize a novel cytopathic mutant of Rous sarcoma virus-PrB.
- To elucidate the genetic basis of the mutant's replication-defective and transformation-defective phenotype.
- To investigate the molecular mechanisms underlying impaired viral RNA translation and packaging.
Main Methods:
- Isolation and characterization of a cytopathic RSV-PrB mutant.
- Molecular cloning using lambda-EMBL3 and transfection assays.
- Nucleotide sequencing and sequence comparisons.
- In vitro RNA synthesis and translation competition experiments.
Main Results:
- The mutant possesses two large deletions, fusing Gag sequences to Env and losing the Src gene.
- The mutant is replication-defective and transformation-defective, requiring a helper virus for replication.
- Mutations in the ribosome-binding site inhibit viral RNA translation.
- An insertion at the 3' end of the RNA likely inhibits packaging.
- Viral particles produced are poorly infectious, with reduced viral RNA content.
Conclusions:
- The identified mutations directly correlate with the observed defects in RNA translation and packaging.
- A model of RSV RNA structure involving circularization is proposed to explain these findings.
- This study provides a detailed molecular understanding of RSV mutant pathogenesis and RNA processing.
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