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Cytoskeleton-associated Pr65gag and retrovirus assembly
Abstract:
Our studies have shown a rapid and specific association of Rauscher murine leukemia virus (R-MuLV) precursor polyprotein Pr65gag with cytoskeletal elements in infected mouse fibroblasts. The Pr65gag associated with Nonidet P-40 (NP-40)-insoluble cytoskeletal structures appears to be subphosphorylated in comparison to NP-40-soluble Pr65gag. The association of Pr65gag with skeletal elements can be disrupted by extraction of the cytoskeleton with sodium deoxycholate, an ionic detergent, or with buffers of high ionic strength. Both the skeleton-associated Pr65gag and its NP-40-soluble counterpart can be labeled with [3H]palmitate, indicating their probable association with lipids presumably in the plasma membrane. Pr65gag molecules bound to skeletal elements in the infected cell appear to be more stable to proteolytic processing than NP-40-soluble Pr65gag. While the association of Pr65gag with cytoskeleton elements in the cell is neither increased nor decreased by blocking virus assembly and release with interferon, Pr65gag appears to accumulate in the cytoskeleton-enriched fraction of cells chronically infected with a temperature sensitive mutant of R-MuLV (ts 17) when such cells are grown at the nonpermissive temperature. Based on these and other results, we have proposed a model for the active role of cytoskeleton associated Pr65gag in retrovirus assembly.
Insights
Rauscher murine leukemia virus (R-MuLV) Pr65gag protein binds to cell structures, influencing retrovirus assembly. This cytoskeleton association affects protein stability and lipid interactions during infection.
Area of Science:
- Molecular Biology
- Virology
- Cell Biology
Background:
- Rauscher murine leukemia virus (R-MuLV) is a retrovirus that infects mouse fibroblasts.
- The precursor polyprotein Pr65gag is essential for retrovirus assembly.
Purpose of the Study:
- To investigate the association of R-MuLV Pr65gag with cytoskeletal elements in infected cells.
- To elucidate the role of Pr65gag-cytoskeleton interaction in retrovirus assembly.
Main Methods:
- Utilized Nonidet P-40 (NP-40) and sodium deoxycholate for protein extraction.
- Employed [3H]palmitate labeling to assess lipid association.
- Investigated protein stability through proteolytic processing assays.
- Examined Pr65gag localization in cells infected with wild-type and temperature-sensitive R-MuLV mutants.
Main Results:
- R-MuLV Pr65gag rapidly and specifically associates with cytoskeletal elements in infected fibroblasts.
- Skeleton-associated Pr65gag is subphosphorylated and more stable to proteolysis compared to soluble Pr65gag.
- Pr65gag associates with lipids, suggesting plasma membrane involvement.
- Pr65gag accumulates in the cytoskeleton fraction of cells infected with a temperature-sensitive R-MuLV mutant at nonpermissive temperatures.
Conclusions:
- Cytoskeleton-associated Pr65gag plays an active role in Rauscher murine leukemia virus assembly.
- The interaction between Pr65gag and the cytoskeleton influences viral protein stability and localization.
- This association is a critical factor in the retrovirus life cycle.