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Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb
1Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland.
Abstract:
Adeno-associated virus (AAV) has an antiproliferative action on cells. We investigated the effect of the AAV replication proteins (Rep) on the cell division cycle using retroviral vectors. Rep78 and Rep68 inhibited the growth of primary, immortalized and transformed cells, while Rep52 and Rep40 did not. Rep68 induced cell cycle arrest in phases G(1) and G(2), with elevated CDK inhibitor p21 and reduced cyclin E-, A- and B1-associated kinase activity. Rep78-expressing cells were also impaired in S-phase progression and accumu lated almost exclusively with hypophosphorylated retinoblastoma protein (pRb). The differences between Rep78 and Rep68 were mapped to the C-terminal zinc finger domain of Rep78. Rep78-induced S-phase arrest could be bypassed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 mutants unable to bind pRb. Rb(-/-) primary mouse embryonic fibroblasts displayed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb(+/+) controls. These results suggest that physiological levels of active pRb can interfere with S-phase progression. We propose that the AAV Rep78 protein arrests cells within S-phase by a novel mechanism involving the ectopic accumulation of active pRb.
Insights
Adeno-associated virus (AAV) Rep78 protein halts cell division by accumulating active retinoblastoma protein (pRb). This novel S-phase arrest mechanism highlights pRb
Area of Science:
- Molecular Biology
- Cell Biology
- Virology
Background:
- Adeno-associated virus (AAV) exhibits antiproliferative effects on cells.
- AAV replication proteins (Rep) play a role in viral replication and potentially host cell manipulation.
Purpose of the Study:
- To investigate the impact of AAV Rep proteins on the cell division cycle.
- To elucidate the mechanism by which Rep proteins affect cell proliferation and cell cycle progression.
Main Methods:
- Retroviral vectors were used to express AAV Rep proteins (Rep78, Rep68, Rep52, Rep40) in various cell types.
- Cell cycle progression was analyzed using flow cytometry and Western blotting for cell cycle regulators.
- The role of retinoblastoma protein (pRb) in Rep78-mediated cell cycle arrest was assessed using Rb-deficient cells and viral oncoproteins.
Main Results:
- Rep78 and Rep68 significantly inhibited the growth of primary, immortalized, and transformed cells.
- Rep68 induced G1 and G2 cell cycle arrest, associated with increased p21 and decreased cyclin-dependent kinase activity.
- Rep78 caused S-phase arrest, characterized by hypophosphorylated pRb accumulation, and this arrest was dependent on pRb binding by viral proteins E1A and E7.
Conclusions:
- AAV Rep78 protein arrests cells in S-phase through a novel mechanism involving the accumulation of active pRb.
- Physiological levels of active pRb can impede S-phase progression, suggesting a broader role in cell cycle regulation.
- The C-terminal zinc finger domain of Rep78 is critical for its S-phase arresting activity.