Transcriptional Regulation Mediated by Human Papillomavirus E2 Protein

201 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 2003.Human papillomaviruses (HPVs) are one of the most common sexually transmitted pathogens in the world. The American Social Health Association reports that there are at least 20 million Americans already infected with HPVs and approximately 5.5 million new cases are reported annually. In HPVs, regulation of gene expression is mainly controlled by virus-encoded E2 proteins which act as sequence-specific DNA-binding proteins in concert with host cellular transcription factors. Using reconstituted cell-free transcription systems, cellular enhancer-binding factors and general cofactors, such as TAFIIs, TFIIA, Mediator, and PC4, were found not to be required for E2-mediated repression. HPV E2 is able to directly target components of the general transcription machinery to exert its repressor activity on the natural HPV E6 promoter. These results indicate that E2 does not simply work by displacing TBP or TFIID from binding to the adjacent TATA box. Instead, E2 appears to function as an active repressor that directly inhibits HPV transcription at steps after TATA recognition by TBP or TFIID. To investigate whether E2 proteins encoded by high-risk HPVs may function differentially from E2 proteins encoded by low-risk HPVs and animal papillomaviruses, comparative DNA-binding and transcription studies were conducted using E2 proteins encoded by HPV-16, HPV-18, HPV-11 and bovine papillomavirus type 1 (BPV-1). Although different types of E2 proteins all exhibited transactivation and repression activities, depending on the sequence context of the E2-binding sites, HPV-16 E2 shows stronger transcription activity and greater DNA-binding affinity than those displayed by the other E2 proteins. To identify cellular components that mediate E2 transcriptional function, stable human cell lines conditionally expressing epitope-tagged HPV-11 E2 protein were established. Silver-stained SDS-PAGE and Western blotting analysis of immunopurified E2-cellular complexes isolated from nuclear extract of induced cell lines revealed specific protein factors that were absent in the negative control and showed E2 interactions with SWI/SNF chromatin remodeling complex, GCN5 histone acetyltransferase, and components of TFIID. These results indicate that full-length E2 may function as a transcriptional activator not only by directly recruiting components of the general transcription machinery to the promoter, but also by recruiting multiple chromatin cofactors to facilitate HPV chromatin transcription.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD