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J. Virol. doi:10.1128/JVI.01818-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The E6 oncoproteins from beta-HPVs differentially activate telomerase through an E6AP dependent mechanism and prolong the lifespan of primary keratinocytes

Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA; Department of Otolaryngology, University of Washington, Seattle, WA

^* To whom correspondence should be addressed. Email: dgallowa{at}fhcrc.org.

	Abstract

Human papillomaviruses (HPVs) belonging to the beta-genus have recently been implicated in squamous cell carcinomas of the skin, though the mechanisms by which they initiate carcinogenesis are unclear. We show that human foreskin keratinocytes (HFKs) expressing several beta-E6 proteins display lifespan extension, but not to the extent seen in HFKs expressing HPV16 E6. Additionally, we demonstrate that beta-E6 proteins can differentially activate telomerase. HFKs expressing 38E6 exhibit significant telomerase activity but to a lesser degree than that observed with 16E6; however, other beta-E6 proteins including 5E6, 8E6, 20E6 and 22E6 exhibit low or background levels of telomerase activity. Utilizing GST pull-down and coimmunoprecipitation experiments, the beta-E6 proteins were shown to interact with the cellular proteins E6AP and NFX1-91, two proteins known to be important for telomerase activation by 16E6. Interestingly, the relative strength of the interaction between the E6 and E6AP or NFX1-91 was proportionate to the activation of telomerase by each beta-E6 protein. To address the requirement for E6AP in telomerase activation by beta-E6 proteins, we utilized a shRNA to knockdown endogenous levels of E6AP. Lysates with decreased levels of E6AP showed a reduced ability to activate telomerase, suggesting that E6AP is a necessary component. This data suggests that complex formation between E6, E6AP, and NFX1-91 is a critical step in mediating telomerase activation, which may be one contributing factor to cellular lifespan extension during beta-HPV infection.