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Host range and cell cycle activation properties of polyomavirus large T-antigen mutants defective in pRB binding
R Freund1, P H Bauer, H A Crissman
1Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore 21201.
Abstract:
We have examined the growth properties of polyomavirus large T-antigen mutants that are unable to bind pRB, the product of the retinoblastoma tumor suppressor gene. These mutants grow poorly on primary mouse cells yet grow well on NIH 3T3 and other established mouse cell lines. Preinfection of primary baby mouse kidney (BMK) epithelial cells with wild-type simian virus 40 renders these cells permissive to growth of pRB-binding polyomavirus mutants. Conversely, NIH 3T3 cells transfected by and expressing wild-type human pRB become nonpermissive. Primary fibroblasts from mouse embryos that carry a homozygous knockout of the RB gene are permissive, while those from normal littermates are nonpermissive. The host range of polyomavirus pRB-binding mutants is thus determined by expression or lack of expression of functional pRB by the host. These results demonstrate the importance of pRB binding by large T antigen for productive viral infection in primary cells. Failure of pRB-binding mutants to grow well in BMK cells correlates with their failure to induce progression from G0 or G1 through the S phase of the cell cycle. Time course studies show delayed synthesis and lower levels of accumulation of large T antigen, viral DNA, and VP1 in mutant compared with wild-type virus-infected BMK cells. These results support a model in which productive infection by polyomavirus in normal mouse cells is tightly coupled to the induction and progression of the cell cycle.
Insights
Polyomavirus mutants unable to bind retinoblastoma protein (pRB) grow poorly in primary cells but well in established lines. This study shows pRB is crucial for polyomavirus infection in primary cells by regulating cell cycle progression.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Polyomavirus large T-antigen typically binds the retinoblastoma tumor suppressor protein (pRB).
- The role of pRB binding in polyomavirus host range and replication is not fully understood.
Purpose of the Study:
- To investigate the impact of pRB binding by polyomavirus large T-antigen on viral growth properties.
- To determine the influence of host cell pRB expression on polyomavirus infection.
Main Methods:
- Examined growth of polyomavirus large T-antigen mutants lacking pRB-binding ability in various mouse cell lines.
- Assessed viral growth in primary baby mouse kidney (BMK) cells pre-infected with simian virus 40.
- Analyzed viral replication in NIH 3T3 cells expressing human pRB and in mouse embryonic fibroblasts with and without RB gene knockout.
Main Results:
- Polyomavirus pRB-binding mutants exhibited poor growth on primary cells but replicated efficiently in established cell lines.
- Simian virus 40 pre-infection rendered primary cells permissive to mutant viruses, while pRB expression in NIH 3T3 cells caused non-permissiveness.
- RB gene knockout fibroblasts were permissive, confirming host pRB expression dictates viral host range.
- Mutant virus-infected cells showed delayed cell cycle progression and reduced synthesis of viral components.
Conclusions:
- Host cell pRB expression is a critical determinant of polyomavirus host range.
- pRB binding by large T-antigen is essential for productive polyomavirus infection in primary cells.
- Polyomavirus infection is tightly linked to the induction of cell cycle progression, mediated by pRB interaction.