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.