FUNCTIONAL CONSEQUENCES OF DIRECTED MUTATIONS IN HUMAN PAPILLOMAVIRUS E6 PROTEINS - ABROGATION OF P53-MEDIATED CELL-CYCLE ARREST CORRELATES WITH P53 BINDING AND DEGRADATION IN-VITRO

Clinical and epidemiological studies have implicated the involvement of human papillomavirus (HPV) infection in cervical tumorigenesis. We have previously shown that expression of high-risk (HPV16) E6 can abrogate an important cell cycle checkpoint mediated by p53. Sublethal DNA damage causes p53 accumulation and G(1) arrest in normal cells, but not in cells with mutant or absent p53, or in cells that express HPV16-E6. To investigate the functional consequences of low-risk (HPV11) 56 expression and to evaluate regions of E6 believed to mediate interaction with p53, we generated several 56 expression constructs, including HPV11-E6, and four different E6 mutants. HPV16E6 Delta D and HPV16E6 Delta B had short deletions of nucleotides encoding amino acids previously implicated in p53 degradation and binding, respectively. HPV16E6HL and HPV11E6LH had the putative p53 binding domain exchanged between the high- and the low-risk types. Unlike HPV16-E6, HPV11-E6 and the mutant 56 proteins were not able to bind or degrade p53 in in vitro assays. When expressed in RKO cells, HPV11-E6 or the mutant E6 proteins did not prevent p53 accumulation or interfere with p53-dependent WAF1/CIP1 mRNA expression, allowing p53-mediated G(1) cell cycle arrest after DNA damage. These findings demonstrate that low-risk and high-risk 56 proteins differ in their effects on p53-mediated cell cycle control and that rather subtle mutations of highrisk 56 can alter its ability to abrogate this important cellular response. (C) 1995 Academic Press, Inc.