An internal class III PDZ binding motif in HPV16 E6* protein is required for Dlg degradation activity.

BACKGROUND: A splice product of the E6 oncoprotein, E6*, is found in cells infected with HPV associated with a high-risk for cervical cancer. Both E6* and E6 promote Dlg degradation, considered a contributing factor for the tumorigenic potential of high-risk HPVs. The full-length E6 utilizes a conserved PDZ binding motif (PBM) at the extreme C-terminus to promote Dlg degradation. In contrast, this PBM is absent in E6*. METHODS: We performed western blot analysis, site-directed mutagenesis and co-immunoprecipitation to identify the key elements required for Dlg degradation activity of high-risk HPVE6*, using HPV16E6* as a model. RESULTS: Our data indicate that only one of the two internal putative class III PBMs, located between amino acids 24-27 (HDII) of HPV16E6*, was required to facilitate degradation of Dlg protein. Substitution of the two consensus residues in this region (D25 and I27) to glycine greatly diminished activity. Whereas substitution of the two conserved residues in the putative internal class I PBM (amino acids 16-19) or the second putative class III PBM (amino acids 28-31) was without effect. Interestingly, HPV66E6* which does not promote Dlg degradation can be converted into a form capable of facilitating Dlg degradation through the insertion of nine amino acids (20-28) containing the class III PBM from HPV16E6*. HPV16E6*-induced Dlg degradation appeared independent of E6AP. CONCLUSIONS: The internal class III PBM of HPV16E6*I required for Dlg degradation is identified. GENERAL SIGNIFICANCE: This study highlights that a novel class III PBM as the domain responsible for Dlg degradation activity in high-risk HPVE6*.

HPV16 oncoproteins promote cervical cancer invasiveness by upregulating specific matrix metalloproteinases.

Production of matrix metalloproteinases (MMPs) for degradation of extracellular matrix is a vital step in cancer metastasis. We investigated the effects of HPV16 oncoproteins (16E6, 16E6*I and 16E7), either individually or combined, on the transcription of 7 MMPs implicated in cervical cancer invasiveness. The levels of 7 MMPs reported to be increased in cervical cancer were determined in C33A stably expressing different HPV16 oncoproteins using quantitative RT-PCR and compared with invasion ability of cell lines using in vitro invasion and wound healing assays. Overexpression of MMP-2 and MT1-MMP was detected in HPV16E6E7 expressing cells which correlated with increased cell invasion. Combination of HPV oncoproteins always showed greater effects than its individual form. Inhibition of cell invasion using a specific MMP-2 inhibitor, OA-Hy, and anti-MT1-MMP antibody confirmed that invasion in these cells was dependent on both MMP-2 and MT1-MMP expression. Depletion of HPV16E6E7 by shRNA-mediated knock-down experiments resulted in decreased MMP-2 and MT1-MMP expression levels as well as reduced invasion ability which strongly suggested specific effects of HPV oncoproteins on both MMPs and on cell invasion. Immunohistochemistry study in invasive cervical cancers confirmed the enhanced in vivo expression of these two MMPs in HPV16-infected cells. In addition, possible sites required by HPV16E6E7 on the MMP-2 and MT1-MMP promoters were investigated and PEA3 (at -552/-540 for MMP-2, -303 for MT1-MMP) and Sp1 (at -91 for MMP-2, -102 for MT1-MMP) binding sites were shown to be essential for mediating their transactivation activity. In conclusion, our study demonstrated that HPV16E6 and E7 oncoproteins cooperate in promoting cervical cancer invasiveness by specifically upregulating MMP-2 and MT1-MMP transcription in a similar manner.