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Related Experiment Videos

Overlapping genes in a yeast double-stranded RNA virus.

M E Diamond1, J J Dowhanick, M E Nemeroff

  • 1Department of Biological Sciences, State University of New York, Buffalo 14260.

Journal of Virology
|September 1, 1989
PubMed
Summary

Saccharomyces cerevisiae viruses express a capsid-polymerase fusion protein from overlapping genes on their large double-stranded RNA. This fusion protein is found in virions and confirms the viral RNA-dependent RNA polymerase gene

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Area of Science:

  • Virology
  • Molecular Biology
  • Yeast Genetics

Background:

  • Saccharomyces cerevisiae viruses possess a large double-stranded RNA genome.
  • This viral RNA encodes the major capsid polypeptide and a putative RNA-dependent RNA polymerase.
  • Viral genome expression resembles retroviral gag-pol gene expression.

Purpose of the Study:

  • To elucidate the complete sequence and organization of the L1 RNA from Saccharomyces cerevisiae viruses.
  • To investigate the expression of viral genes, specifically the capsid and RNA polymerase.
  • To determine if a capsid-polymerase fusion protein is produced and present in virions.

Main Methods:

  • Complete sequencing of the L1 double-stranded RNA.
  • In vitro translation of denatured viral RNA.

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  • Western blot analysis using antibodies against a synthetic polymerase peptide.

    • Main Results:

    • The L1 sequence revealed two overlapping large open reading frames (ORFs) on the plus strand.
    • The first ORF encodes the capsid polypeptide, and the second encodes the putative RNA polymerase.
    • A -1 frameshift in the overlapping region during in vitro translation produced a capsid-polymerase fusion polypeptide; this fusion protein was detected in virions.

    Conclusions:

    • The Saccharomyces cerevisiae viral L1 RNA encodes both capsid and RNA-dependent RNA polymerase proteins.
    • Expression of the RNA polymerase likely occurs via a frameshift mechanism, producing a fusion protein.
    • The presence of the fusion protein in virions validates the proposed gene organization and expression strategy.