Interaction between interferon-inducible IFI16 protein and human papillomavirus protein E6 and E7

Objective: To study the molecular mechanism underlying that interferon-inducible IFI16 protein overcomes the tumorgenesis activities of HPV16E6 and E7, the authors investigated whether HPV16E6 and E7 could associate with IFI16. Methods: GST-Pulldown was performed to examine the interaction between IFI16 and HPV16E6/E7 in vitro. Bacteria expression plasmids expressing GST-E6 and GST-E7 were generated and GST fusion proteins purified. The purified GST-E6 and GST-E7 were incubated with the whole cell extracts prepared from BJAB cells that expressed endogenous IFI16, and the IFI16 detained by GST fusion proteins were detected using Western Blotting assay with anti-IFI16 antibodies. In vivo association of IFI16 and HPV16E6 was tested via co-immunoprecipitation (Co-IP) using transfected mammalian cells. Briefly, an expression pcDNA4HisMax/HPV16E6 plasmid expressing His-HPV16 E6 protein was constructed and used to transfect 293T cells together with another expression pRcCMV/IFI16 plasmid expressing IFI16 protein. The whole cell lysate prepared from these transfected cells were incubated with anti-His serum followed by protein A agarose and the presence of IFI16 in the immunoprecipitated protein was visualized by immunoblotting with anti-IFI16 antibodies. Furthermore, in vivo interaction between IFI16 and HPV16E7 was also revealed via a Co-IP assay with cell extracts prepared from both SiHa and Caski cells in which the endogenous IFI16 and HPV16 E7 proteins were expressed. The cell lysates were immunoprecipitated with anti-HPV16 E7 serum and protein complex was detected with anti-IFI16 antibodies. Results: The plasmids expressing GST-HPV16E7 and His-HPV16E6 were successfully generated and the fusion proteins of interests were verified to be correct. In vitro pulldown assay clearly demonstrated that both GST-HPV16E6 and GST-HPV16E7 were capable of binding to IFI16, and co-immunoprecipitation assays revealed that HPV16E6/E7 and IFI16 were present in the same protein complexes and also associated with each other in vivo. Conclusion: IFI16 associates with HPV16E6 and E7 proteins both in vitro and in vivo. These foundings could provide potential molecular targets for designing compounds to treat human cervical cancer.