HPV‐18 E6I 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. © 2004 Wiley‐Liss, Inc.