The role of cellular RNA processing functions in Human papillomavirus type 16 gene regulation

Abstract: AbstractInfections with Human papillomavirus type 16 (HPV16) is the most the most common, sexually transferred and is responsible for genital warts, cervical cancers and a growing number of head and neck cancers. Knowledge about how HPV16 interacts with the infected cell to regulate its gene expression is essential for therapeutic development. Here, we show that the viral E5 protein which may contribute to carcinogenesis can only be expressed efficiently during the early infection. E5 is the last open reading frame (ORF) on the early pre-mRNAs. Therefore, its expression requires removal of upstream, inhibitory E7 and E1 ORFs by alternative splicing. Alternative splicing is also required for HPV16 later gene expression together with an inhibition of the early polyadenylation signal. We report that induction of the DNA damage response (DDR) by alkylating agent melphalan caused both an activation of late gene splicing and inhibition of the early polyadenylation signal. The connection between induction of DDR and HPV16 late gene expression by melphalan was dependent on ATM signaling and caused an accumulation of phosphorylated BRCA1 on HPV16 DNA. BRAC1 also interacted with splice factors U2AF65 and hnRNPC that were recruited to HPV16 mRNAs to generate a expression of late, L1 and L2 HPV16 mRNAs. The ATM-branch of the DDR is hijacked by HPV for productive viral replication prior to late gene expression. Therefore, we suggest that HPV16 also has evolved to utilize the connection between DDR and RNA-processing for late gene activation. Finally, we investigated the presence of methylated N6-adenosine (m6A) on HPV16 mRNAs in connection to regulation of alternative splicing. Here, we report that HPV16 mRNAs undergoes methylation and that the proteins associated with m6A affects viral splicing. In particular, splicing of mRNAs that express the viral oncoproteins E6 and E7 were affected in a mutually exclusive manner. The same pattern was also observed for mRNAs expressing viral replication proteins E1 and E2. Further investigations will reveal the true regulatory importance of m6A for splicing of these mRNAs. In conclusion, this thesis identifies the main E5 mRNA, connects HPV16 late gene expression to the DDR and suggests a regulatory function of m6A in HPV16 alternative splicing.