The Role of E6 Domains and Promoter Regulation in the HPV 31 Life CyclePublic Deposited
Tonia René Holverson The HPV genome contains a bipartite promoter system that regulates the expression of early and late gene transcripts. The early proteins include the replication proteins E1 and E2, the oncoproteins E6 and E7, and the late functioning proteins E4 and E5. The late proteins include the capsid structural proteins L1 and L2. This body of work focuses on three main areas of the HPV31 life cycle: 1) the functional roles of the alpha-helical binding domain and the nuclear localization sequences of E6, 2) the regulation of the early and late promoters by histone modifications and transcription factor binding, and 3) the activation of late gene expression. E6 interacts with several proteins through the alpha-helical protein binding domain at its C-terminus. Previous studies in other HPV subtypes determined that residue 128 in this domain was responsible for the binding of these proteins to E6. Mutagenesis studies demonstrate this residue is necessary in HPV 31E6 for the binding and degradation of E6TP1, but not the other alpha-helical proteins. This interaction was shown to be necessary for cell proliferation and episomal maintenance. E6 also interacts with nuclear proteins that have roles in the regulation of transcription, DNA replication, and repair complexes. The nuclear localization of E6 has been demonstrated to occur through three nuclear localization sequences (NLSs) of E6, with the third NLS providing the major contribution to this activity. My work has demonstrated this sequence also contributes to the nuclear localization of HPV 31 E6 and this nuclear localization was required for cell proliferation and episomal maintenance. The transcription of E6 and the other early genes occurs through activation of the early promoter, while the differentiation-dependent late genes are initiated from the late promoter. Using chromatin immunoprecipitation, I showed that activated histones bind both promoter regions throughout the viral life cycle. I further observed differential binding of transcription factors to the promoters, suggesting these factors in the regulation of late gene expression. Additional reporter studies showed sequences within the URR are necessary for the activation of late expression.