Regulation of Human Papillomavirus Type 16 Late L1 mRNA Splicing

Abstract: Human papillomaviruses (HPVs) cause almost half of the human cancers that are attributable to viruses. HPV type 16 is the most carcinogenic type among the HPVs and is detected in 50% of all cervical cancers. HPV-16 infects epithelial cells and HPV-16 gene expression is tightly linked to the differentiation stage of the infected cells. Early HPV-16 genes are expressed in basal layers of the epithelium whereas the late genes which encode highly immunogenic viral structural proteins are only expressed in the suprabasal layers. HPV-16 infections are normally cleared within 18-24 months, but HPV-16 can establish persistent infections that progress to cancer. Such HPV-infected cancer cells express early HPV-16 genes but never expressed the late genes. We speculate that inhibition of HPV-16 late gene expression is a prerequisite for viral persistence and progression to cervical cancer. HPV-16 uses alternative splicing to regulate expression of early and late genes. HPV RNA elements and cellular factors control the expression level of viral proteins by regulating alternative splicing. This project was carried out to enhance our understanding of the regulation of HPV-16 gene expression, in particular at the RNA splicing level. The goal of this thesis was to identify viral RNA elements and cellular factors that regulate the processing of HPV-16 early and late mRNA splicing. These studies may also contribute to the identification of diagnostic biomarkers for premalignant infections at risk of progressing to cervical cancer. We identified a splicing silencer that interacts with hnRNP D proteins and hnRNP A2/B1 to suppress HPV-16 late gene expression in mitotic cells, including cervical cancer cells. We also characterized a splicing enhancer that promotes HPV-16 early gene expression, thereby indirectly inhibiting late gene expression. Mutation in this enhancer reduced its binding to the ASF/SF2 splicing factor. This resulted in decreased expression of the viral oncogenes E6 and E7 and a reduced ability of HPV-16 to immortalize human epithelial cells, thereby, linking HPV-16 mRNA splicing regulation to its pathogenic prospects. We also identified the hnRNP G protein binding to this enhancer and has opposite effects to ASF/SF2 on splicing matched by antagonism in RNA binding.