ABSTRACTWe show that E6 proteins from benign human papillomavirus type 1 (HPV1) and oncogenic HPV16 have the ability to alter the regulation of the G1/S transition of the cell cycle in primary human fibroblasts. Overexpression of both viral proteins induces cellular proliferation, retinoblastoma (pRb) phosphorylation, and accumulation of products of genes that are negatively regulated by pRb, such as p16INK4a, CDC2, E2F-1, and cyclin A. Hyperphosphorylated forms of pRb are present in E6-expressing cells even in the presence of ectopic levels of p16INK4a. The E6 proteins strongly increased the cyclin A/cyclin-dependent kinase 2 (CDK2) activity, which is involved in pRb phosphorylation. In addition, mRNA and protein levels of the CDK2 inhibitor p21WAF1/CIP1were strongly down-regulated in cells expressing E6 proteins. The down-regulation of the p21WAF1/CIP1gene appears to be independent of p53 inactivation, since HPV1 E6 and an HPV16 E6 mutant unable to target p53 were fully competent in decreasing p21WAF1/CIP1levels. E6 from HPV1 and HPV16 also enabled cells to overcome the G1arrest imposed by oncogenicras. Immunofluorescence staining of cells coexpressingrasand E6 from either HPV16 or HPV1 revealed that antiproliferative (p16INK4a) and proliferative (Ki67) markers were coexpressed in the same cells. Together, these data underline a novel activity of E6 that is not mediated by inactivation of p53.