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Mutations affecting cleavage at the p10-capsid protease cleavage site block Rous sarcoma virus replication

Marcy L Vana1, Aiping Chen, Peter Boross

  • 1Department of Microbiology and Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. mvana@stanford.edu

Retrovirology
|September 29, 2005
PubMed

Insights

Mutations in the p10-CA protease cleavage site significantly impair Rous sarcoma virus (RSV) replication and virus particle formation. Altering this site blocks viral processing and can lead to abnormal virus core structures.

Area of Science:

  • Virology
  • Molecular Biology
  • Structural Biology

Background:

  • The p10-CA protease cleavage site in Rous sarcoma virus (RSV) is crucial for viral polyprotein processing.
  • Understanding the kinetics of this cleavage site is essential for viral replication.

Purpose of the Study:

  • To investigate the impact of amino acid substitutions in the p10-CA protease cleavage site on RSV processing and replication.
  • To analyze the effects of these mutations on Gag polyprotein processing and virus particle morphology.

Main Methods:

  • Site-directed mutagenesis of the p10-CA cleavage site (VVAM*PVVI) with substitutions M239F, M239G, P240F, V241G.
  • Kinetic analysis of p10-CA cleavage using model peptide substrates.
  • Metabolic labeling and immunoprecipitation to assess Gag polyprotein processing in transfected cells.
  • Subcloning mutations into an RSV proviral vector (RCAN) for replication studies in DF-1 cells.
  • Electron microscopy to examine the morphology of released virus particles.

Main Results:

  • Most substitutions (M239G, P240F, V241G) decreased Gag polyprotein processing and release, and impaired production of CA proteins.
  • All mutations except M239F blocked RSV replication in DF-1 cells.
  • M239G substitution resulted in large, misshapen viral cores, unlike wild-type or M239F particles.

Conclusions:

  • Cleavage at the p10-CA site is critical for efficient RSV replication.
  • Mutations affecting this cleavage site can lead to defects in viral polyprotein processing and abnormal virus particle morphology.
  • Targeting the p10-CA cleavage site offers a potential strategy for antiviral development against RSV.

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