Regulation of human papillomavirus E6 oncoprotein function via a novel ubiquitin ligase FBXO4

ABSTRACT Previous studies have shown that E6 interacts with the E6-associated protein (E6AP) ubiquitin-protein ligase and directs its ubiquitylation activity toward several specific cellular proteins, one of the most important of which is p53. Interestingly, E6AP not only aids in the E6-directed degradation of cellular substrates but also stabilizes the E6 protein by protecting it from proteasome-mediated degradation. However, there is no information available about the ubiquitin ligases that regulate the stability and activity of the human papillomavirus (HPV) E6 oncoprotein in the absence of E6AP. Therefore, to identify these novel ubiquitin ligases, we performed high-throughput human siRNA library screen against ubiquitin ligases in clustered regularly interspaced palindromic repeat (CRISPR)-edited E6AP-knockout human embryonic kidney (HEK) 293 cells, stably expressing green fluorescent protein (GFP)-tagged HPV-18E6. We found a number of ubiquitin ligases that increase the expression of GFP-tagged 18E6 upon their knockdown in the absence of E6AP. Upon validation of the interaction of 18E6 with these ubiquitin ligases in cervical cancer-derived cell lines, we found that the knockdown of ubiquitin ligase F-box protein 4 (FBXO4), together with E6AP knockdown, leads to a dramatic increase in the levels of endogenous HPV-18E6 oncoprotein. Furthermore, our data demonstrate that the combined knockdown of FBXO4 and E6AP not only rescues the protein levels of E6 but also induces high levels of cell death in a p53-dependent manner in the HPV-positive cervical cancer cell line, HeLa. These results indicate a close interplay between FBXO4, E6AP, and p53 in the regulation of cell survival in HPV-positive cervical tumor-derived cells.IMPORTANCEE6-associated protein (E6AP)-mediated stabilization of human papillomavirus (HPV) E6 plays a crucial role in the development and progression of cervical and other HPV-associated cancers. This study, for the first time, identifies a novel ubiquitin ligase, FBXO4 that targets the degradation of HPV E6 oncoprotein in the absence of E6AP in cervical cancer-derived cell lines. This may have significant implications for our understanding of HPV-associated cancers by providing deeper insights into the intricate interplay between viral proteins and host cellular machinery and the development of targeted therapies.