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

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*. 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. 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. The internal class III PBM of HPV16E6*I required for Dlg degradation is identified. This study highlights that a novel class III PBM as the domain responsible for Dlg degradation activity in high-risk HPVE6*. •Localization and Dlg degradation activity of E6* proteins from high-risk HPV58, HPV59 and HPV66 have been first evaluated.•Using HPV16E6* as a model, the internal class III PBM required for Dlg degradation activity have been first identified.•Insertion of 9 amino acids containing this PBM can convert HPV66E6* from non-degradable into degradable form.•HPV16E6*- induced Dlg degradation appeared independent of E6AP.