FUNCTIONAL CONSEQUENCES OF DIRECTED MUTATIONS IN HUMAN PAPILLOMAVIRUSE6 PROTEINS - ABROGATION OF P53-MEDIATED CELL-CYCLE ARREST CORRELATESWITH P53 BINDING AND DEGRADATION IN-VITRO

Clinical and epidemiological studies have implicated the involvement o f human papillomavirus (HPV) infection in cervical tumorigenesis. We h ave previously shown that expression of high-risk (HPV16) E6 can abrog ate an important cell cycle checkpoint mediated by p53. Sublethal DNA damage causes p53 accumulation and G(1) arrest in normal cells, but no t in cells with mutant or absent p53, or in cells that express HPV16-E 6. To investigate the functional consequences of low-risk (HPV11) 56 e xpression and to evaluate regions of E6 believed to mediate interactio n with p53, we generated several 56 expression constructs, including H PV11-E6, and four different E6 mutants. HPV16E6 Delta D and HPV16E6 De lta B had short deletions of nucleotides encoding amino acids previous ly implicated in p53 degradation and binding, respectively. HPV16E6HL and HPV11E6LH had the putative p53 binding domain exchanged between th e high- and the low-risk types. Unlike HPV16-E6, HPV11-E6 and the muta nt 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 D NA damage. These findings demonstrate that low-risk and high-risk 56 p roteins 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, In c.