Regulation of Human Papillomavirus Type 16 Early and Late Gene Expression

Abstract: Human papillomavirus type 16 (HPV16) is a cancer-causing virus that contributes to at least 70% of cervical cancer as well as to other anogenital cancers and head and neck cancer. Understanding HPV16 gene regulation is important to enhance our understanding of HPV16 infections and may contribute to development of antiviral drugs. In this study, we show that nucleosides/nucleoside analogues have the ability to regulate HPV16 gene expression and we identify cellular splicing factor that control HPV16 mRNA processing.Nucleosides play a significant role in cell differentiation and tumorigenesis and have been used in antiviral and anticancer treatments. We report that adenosine activates HPV16 late gene expression in a dose-and time- dependent manner, but only in the presence of guanosine. Induction of HPV16 late gene expression by adenosine and guanosine was mainly causing read-through at the HPV16 early polyadenylation signal to the HPV16 late region and resulted in HPV16 late L2 mRNA production. This effect was dependent on ENT1 nucleoside transporter that transports adenosine into cells where it is metabolized further by adenosine kinase (ADK) or adenosine deaminase (ADA). Adenosine and guanosine also increased the nuclear export of the cellular HuR protein, which suggested that HuR contributed to nucleoside-mediated induction of HPV16 late gene expression. In addition to adenosine and guanosine, we found that nucleoside analogue cordycepin could also induce HPV16 late gene expression. In this case by increasing the production of polyadenylation factor NUDT21/CPSF5, resulted in inhibition of the early HPV16 polyadenylation site and HPV16 late L2 mRNAs production. We conclude that nucleosides adenosine and guanosine as well as nucleoside analogue cordycepin can induce HPV16 late gene expression, suggesting that nucleosides may play an important role in the control of HPV16 gene expression.The levels of the cellular splicing factor heterogeneous nuclear ribonucleoprotein G (hnRNP G) was also affected by nucleosides and was studied further. Overexpression of hnRNP G caused inhibition of a small-exon between HPV16 splice sites SA3358 and SA3632 on the L1 mRNA by interacting with an 8-nucleotide enhancer located within this exon. This interaction resulted in increased HPV16 late L1i mRNA production. Furthermore, we found that overexpression of hnRNP G strongly inhibited splicing of the HPV16 E6/E7 oncogene mRNAs. This splicing inhibitory effect resulted in production of unspliced mRNAs encoding the E6 protein at the expense of HPV16 E7 (E6'I) mRNA, indicating that hnRNP G controls expression of both HPV16 E6 and E7 oncogenes. Since hnRNP G affected splicing of both E6/E7 oncogene and late gene L1 mRNAs, the levels of hnRNP G may contribute to the final outcome of the HPV16 infections. In summary, this thesis shows that nucleosides/nucleoside analogues can affect HPV16 gene expression at the level of RNA processing and identifies hnRNP G as a major regulator of expression of the HPV16 oncogenes E6 and E7, and the HPV16 late L1 gene.