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Related Experiment Videos

Simian virus 40 host range/helper function mutations cause multiple defects in viral late gene expression.

T Stacy1, M Chamberlain, C N Cole

  • 1Molecular Genetics Center, Dartmouth Medical School, Hanover, New Hampshire 03756.

Journal of Virology
|December 1, 1989
PubMed
Summary
This summary is machine-generated.

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Simian virus 40 (SV40) host range/helper function (hr/hf) mutants with altered T antigens show defects in late viral gene expression. This impacts capsid protein production and agnoprotein synthesis, leading to reduced progeny yields.

Area of Science:

  • Virology
  • Molecular Biology
  • Cell Biology

Background:

  • Simian virus 40 (SV40) is a well-characterized DNA tumor virus used extensively in molecular biology research.
  • Host range/helper function (hr/hf) mutants of SV40, such as dlA2459 and dlA2475, possess deletions in the T antigen's carboxy terminus.
  • These hr/hf mutants exhibit altered growth properties on specific monkey kidney cell lines and a reduced ability to support adenovirus replication.

Purpose of the Study:

  • To investigate the molecular basis for the growth defect of SV40 hr/hf mutants.
  • To elucidate the role of the T antigen carboxy terminus in viral DNA replication, gene expression, and progeny production.
  • To understand the impact of these mutations on late viral macromolecular synthesis at different temperatures and cell lines.

Main Methods:

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  • Analysis of viral macromolecular synthesis (DNA replication, mRNA, and protein production) in infected cell lines (Vero, BSC-1, CV-1) at various temperatures (40, 37, 32°C).
  • Quantification of viral late mRNA levels using Northern blot or similar techniques.
  • Assessment of viral capsid protein (VP1) steady-state levels and agnoprotein synthesis.
  • Primer extension analysis to determine mRNA start sites and initiation codon usage.

Main Results:

  • SV40 hr/hf mutants replicated viral DNA similarly to wild-type SV40, indicating defects occur late in the lytic cycle.
  • Late mRNA levels were reduced in mutant-infected CV-1 and BSC-1 cells at permissive temperatures, and VP1 protein levels mirrored mRNA reduction.
  • Agnoprotein synthesis was undetectable in mutant-infected cells, and late mRNAs initiated from alternative start sites downstream of the wild-type cap site.

Conclusions:

  • Deletion of the T antigen carboxy-terminal domain critically affects both the quantity and quality of viral late mRNA production.
  • Reduced capsid protein synthesis and the absence of agnoprotein likely contribute to the decreased progeny yields observed in hr/hf mutants.
  • The T antigen carboxy terminus is essential for efficient late gene expression, virion assembly, or maturation in the SV40 lytic cycle.