Regulation of HPV E7 Stability by E6-Associated Protein (E6AP).

High-risk human papillomaviruses (HPVs) are responsible for most human cervical cancers, and uncontrolled expression of the two key viral oncoproteins, E6 and E7, stimulates the induction of carcinogenesis. Previous studies have shown that both E6 and E7 are closely associated with different components of the ubiquitin proteasome pathway, including several ubiquitin ligases. Most often these are utilized to target cellular substrates for proteasome-mediated degradation, but in the case of E6, the E6AP ubiquitin ligase plays a critical role in controlling E6 stability. We now show that knockdown of E6AP in HPV-positive cervical cancer-derived cells causes a marked decrease in E7 protein levels. This is due to a decrease in the E7 half-life and occurs in a proteasome-dependent manner. In an attempt to define the underlying mechanism, we show that E7 can also associate with E6AP, albeit in a manner different from that of E6. In addition, we show that E6AP-dependent stabilization of E7 also leads to an increase in the degradation of E7's cellular target substrates. Interestingly, ectopic overexpression of E6 oncoprotein results in lower levels of E7 protein through sequestration of E6AP. We also show that increased E7 stability in the presence of E6AP increases the proliferation of the cervical cancer-derived cell lines. These results demonstrate a surprising interplay between E6 and E7, in a manner which is mediated by the E6AP ubiquitin ligase. IMPORTANCE This is the first demonstration that E6AP can directly help stabilize the HPV E7 oncoprotein, in a manner similar to that observed with HPV E6. This redefines how E6 and E7 can cooperate and potentially modulate each other's activity and further highlights the essential role played by E6AP in the viral life cycle and malignancy.