HPV-18 E6*I modulates HPV-18 full-length E6 functions in a cell cycle dependent manner.

The E6 ORFs of the high-risk Human Papillomavirus (HPV) Types 16 and 18 have been shown to encode (besides the full-length product) several truncated forms, termed E6*. We have reported previously that the HPV-18 E6*I protein interacts with the full-length E6 protein as well as with the ubiquitin ligase E6-AP and, as a result of this, E6* can inhibit E6-mediated degradation of p53. Moreover, ectopic expression of the HPV-18 E6*I protein has an antiproliferative effect in cervical cancer-derived cell lines. These results led us to investigate further the modulatory functions of E6*I on E6. Using epitope tagged versions of the 2 proteins we have analyzed the sub-cellular distribution of the full-length HPV18 E6 and HPV18 E6*I, as well as their respective cellular abundance during the cell cycle, and show specific upregulation of E6*I during G2/M. We also investigated the effect of E6*I overexpression in cell lines derived from cervical tumors, with respect to the expression levels of E6 target proteins, such as p53, hDlg and Scribble and find a corresponding increase in p53 expression also during G2/M. In addition we show that the overexpression of E6*I reduces the amount of E6 in the insoluble nuclear and membrane fractions of the cell. E6 levels can, however, be restored by the addition of a specific proteasome inhibitor, suggesting that the interaction between E6 and E6*I leads to the destabilization of a subset of the E6 protein. These results suggest that the E6*I protein can function as a fine regulator of the full-length E6 protein by direct interaction that leads both to changes in its cellular abundance as well as its distribution during particular phases of the cell cycle. Copyright 2004 Wiley-Liss, Inc.