INFECTION OF PRIMARY-CELLS BY ADENOASSOCIATED VIRUS TYPE-2 RESULTS INA MODULATION OF CELL CYCLE-REGULATING PROTEINS

It has been demonstrated that infection of primary human cells with ad eno-associated viruses (AAV) leads to a decrease in cellular prolifera tion and to growth arrest, We analyzed the molecular basis of this phe nomenon and observed that infection with AAV type 2 (AAV2) had an effe ct on several factors engaged in the control of the mammalian cell cyc le, In particular, all of the pRB family members, pRB, p107, and p130, which are involved in G(1) cell cycle checkpoint control, were affect ed. After infection, a shift from hyper- to hypophosphorylated forms w as observed, Cyclins A and B1, which are required for G(1)/S transitio n and progression into mitosis, respectively, were downregulated at th e transcriptional level as well as at the protein level, whereas the G (1) cyclins D1 and E remained unaffected, In addition, the steady-stat e levels of cyclin-dependent kinases CDK1 and CDK2 and of transcriptio n factor E2F-1 were diminished, Of all the factors known to be involve d in phosphorylation of pRB family proteins, only the CDK inhibitor p2 1(WAF1) exhibited a response to AAV2 infection, p21(WAF1) mRNA was qui ckly and progressively upregulated in a p53-independent manner over at least 72 h, Consistent with the increased p21(WAF1) protein levels, c yclin E- and cyclin A-dependent kinase activities declined to low leve ls and E2F-p130-cyclin-CDK2 complexes were disrupted, From these data, we conclude that the major effect of AAV2 infection on primary human fibroblasts appears to be upregulation of p21(WAF1) gene expression an d thus cell cycle arrest by the suppression of pRB family protein phos phorylation.