Pull-down of human papillomavirus 16 E6 variants for interactome analysis: a methods development and analysis

Abstract

Human Papillomavirus 16 (HPV16) is a double-stranded DNA virus known as a causative agent in almost all cervical cancers and an increasing number of oropharyngeal cancers. Variants of HPV16, such as the Asian-American (AA) and L83V, have been found to have increased abilities to promote carcinogenesis. Even though previous interactome studies identified which proteins interact with HPV16 E6, few have looked at interactions between variants of E6 in particular AAE6 and the European Prototype (EPE6). This thesis had two objectives: develop a method to co-immunoprecipitate host cellular proteins that interact with E6 variants; and identify potential differences between cellular host proteins and variant E6. We were successful in developing a method that not only could pull-down and identify E6 variant interacting proteins but the E6 variant proteins themselves. Using liquid chromatography-mass spectrometry (LC MS/MS), we identified 13 proteins that interact with both AAE6 and EPE6, along with six unique AAE6 interacting proteins and six unique EPE6 interacting proteins. Of the interactors we found, seven were of particular interest: TRIP12, GNL2, INO80B, CHMP4B, MX2, RPSK6K4A, and PROK2. These proteins affect a variety of cellular functions, including DNA replication and repair, telomere maintenance, cellular proliferation, ERK1/2 signaling, signal transduction, and immune response. Identification of different proteins using different bioinformatic analyses further provide evidence that AAE6 and EPE6 may have unique interactions with their host cells resulting in varied abilities to promote carcinogenesis. The identification of these proteins has furthered our understanding of potential mechanisms that allow AAE6 to promote carcinogenesis more than EPE6. More wet lab work is still required to confirm these interactions and determine their exact effects on the host cell.