Inhibition of S-phase progression by adeno-associated virus Rep78 protein is mediated by hypophosphorylated pRb

Adeno-associated virus (AAV) has an antiproliferative action on cells. We i nvestigated the effect of the AAV replication proteins (Rep) on the cell di vision 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 activ ity. Rep78-expressing cells were also impaired in S-phase progression and a ccumulated almost exclusively with hypophosphorylated retinoblastoma protei n (pRb). The differences between Rep78 and Rep68 were mapped to the C-termi nal zinc finger domain of Rep78, Rep78-induced S-phase arrest could be bypa ssed by adenoviral E1A or papillomaviral E7 proteins but not by E1A or E7 m utants unable to bind pRb. Rb-/- primary mouse embryonic fibroblasts displa yed a strongly reduced S-phase arrest when challenged with Rep78, compared with matched Rb+/+ controls. These results suggest that physiological level s 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 involv ing the ectopic accumulation of active pRb.