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Altered Rous sarcoma virus Gag polyprotein processing and its effects on particle formation
Y Xiang1, T W Ridky, N K Krishna
1Department of Biochemistry, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, USA.
Journal of Virology
|March 1, 1997
Summary
Altering Rous sarcoma virus (RSV) Gag polyprotein processing affects virus-like particle release. Mutations impacting protease (PR) activity or cleavage sites influence budding, suggesting maturation occurs post-protease release.
Area of Science:
- Virology
- Molecular Biology
- Protein Biochemistry
Background:
- Rous sarcoma virus (RSV) Gag polyprotein undergoes proteolytic processing for viral maturation.
- The viral protease (PR) is crucial for cleaving the Gag polyprotein into functional subunits.
- Understanding Gag processing and PR activity is key to viral replication and assembly.
Purpose of the Study:
- To investigate the in vivo effects of altering Rous sarcoma virus (RSV) Gag polyprotein cleavage junctions and protease (PR) coding sequences.
- To determine how specific amino acid substitutions impact Gag processing efficiency and virus-like particle (VLP) budding.
- To elucidate the relationship between PR activity, Gag maturation, and VLP release.
Main Methods:
- Introduction of specific amino acid substitutions into the RSV Gag polyprotein cleavage sites and PR coding sequence in vivo.
- Analysis of proteolytic processing of the Gag precursor in COS cells producing VLPs.
- Assessment of VLP release efficiency and analysis of viral protein content in released particles.
Main Results:
- Amino acid substitutions predicted to decrease PR release rate did not affect VLP budding but resulted in uncleaved Pr76gag.
- Substitutions increasing PR release rate also did not affect budding.
- A significant budding defect was observed with mutations increasing PR catalytic activity or linking PR subunits, suggesting premature processing or dimerization limitations.
Conclusions:
- Viral protein maturation, mediated by protease (PR) activity, appears to occur after PR subunits are released from the Gag polyprotein.
- Specific mutations in cleavage sites and PR can lead to budding defects, highlighting the delicate balance of proteolytic processing.
- PR dimerization may be a rate-limiting step in the viral maturation process.