Regulation of HPV16 Early Gene Expression

Sammanfattning: Human papillomavirus type 16 (HPV16) is the most common high-risk type and isassociated with more than 50% cervical cancer cases as well as a number of headand neck cancer cases. The life cycle of HPV16 is strictly linked to celldifferentiation in the squamous epithelium with expression of the HPV16 earlygenes. The oncoproteins E6 and E7 are essential for preventing apoptosis andinducing proliferation of HPV16 infected cells. As cell differentiation proceeds,viral protein E2 suppresses early promoter p97 and results in downregulation ofE6/E7 proteins and activation of HPV16 late gene expression. It is not surprisingthat the HPV16 E2 gene is inactivated in many HPV16-driven cancer cells.Therefore, the study of HPV16 early gene regulation especially regulation of E1,E2, E6 and E7 expression is important to understand HPV16 infection. Thesestudies may contribute to development of antiviral drugs to HPV16.The HPV16 E6 protein is translated from unspliced mRNAs, whereas the E7 proteinis translated from the mRNAs spliced from HPV16 5’-splice site SD226 to 3’-splicesite SA409. The balanced expression of unspliced and spliced mRNAs is importantto produce the E6 and E7 proteins that affect the HPV16 life cycle progression andpathogenesis. We have investigated the regulation of splicing from SD226 toSA409. We determined that cellular splicing factors hnRNP A1 and hnRNP A2B1function as inhibitors of HPV16 E7 expression. Both hnRNP A1 and hnRNP A2inhibited splicing to SA409 through direct and specific interaction with a C-lessRNA element located between HPV16 nucleotide position 594 and 604. Eventhough inhibition SA409 exerted by hnRNP A1 and hnRNP A2 had different effects.Overexpression of hnRNP A1 inhibited SA409 and resulted in enhanced expressionof unspliced E6 mRNAs at the expense of the E7 mRNAs. Overexpression ofhnRNP A2 inhibited SA409 but resulted in alternative splicing to 3’-splice siteSA742. The splice site SA742 is used for the production of the HPV16 E6^E7, E1,and E4 mRNAs.Since HPV16 E2 is a transcriptional regulator indirectly downregulates HPV16 E6and E7 expression via suppressing of HPV16 early promoter p97. HPV16 producesvarious E2 mRNAs that initiate at either early promoter p97 or late promoter p670and several splice sites are used. Our study demonstrated that the most efficientlytranslated E2 mRNA initiates at late promoter p670 spliced from 5’-splice siteSD880 to 3’-splice site SA2709. An alternative 3’-splice site named SA2582 andlocated upstream of the E2 ATG could also produce E2 mRNAs, but it is lessefficiently to be translated into E2 protein. These results suggest that a splicingenhancer may be located downstream of SA2709 to regulate E2 mRNA expression.To further understand how HPV16 regulates 3’-splice site SA2709, we introduceda number of deletions and mutations to HPV16 subgenomic plasmids. We identifieda 19-nucleotide RNA element consisting of three “AC(A/G)AGG” repeats locateddownstream of SA2709.This RNA element acted as a splicing enhancer. Thissplicing enhancer sequence interacted with cellular RNA binding protein hnRNP Gto enhance splicing to SA2709 and interacted with hnRNP A1 and A2B1 to inhibitsplicing to SA2709. Deletion of the splicing enhancer reduced mRNA splicing toSA2709, and redirected splicing to the upstream 3’-splice site SA2582. The latermRNA has poor ability to produce E2 protein, but it may produce a small proteincalled E1C. We also wish to establish a bio-assay that can monitor expression of thevarious E1 mRNAs and the alternatively spliced E2 mRNAs using either SA2582or SA2709. We constructed a number of subgenomic HPV16 plasmids containinggreen fluorescent protein, secreted luciferase, neomycin resistance gene, orthymidine kinase reporter genes. These reporter plasmids produce either E1-fusionproteins or E2-fusion proteins. We observed that the balance between unspliced E1mRNAs and spliced E2 mRNAs was affected by overexpression of RNA bindingproteins SRp30c, Tra2b, hnRNP A1, and hnRNP G. However, further study isrequired to optimize these reporter plasmids for quantitative analysis of HPV16 E1and E2 mRNA splicing. This bioassay could be used to enhance our understandingof HPV16 gene regulation and to identify small molecules that interfere withHPV16 mRNA splicing. Such substances could potentially be used as antiviraldrugs to HPV16 infections and HPV16-driven cancers.