Posttranscriptional Regulation of Human Papillomavirus Type 16 Late mRNAs

Sammanfattning: The lifecycle of the human papillomavirus (HPV) is strictly linked to the programmed differentiation of the host cell it infects. Production of late proteins, which are used for the production of the major and minor capsid proteins, is only seen upon terminal differentiation of the epithelium.The regulation of late gene expression appears to be a multi-faceted process in which transcription, splicing, polyadenylation, mRNA stability and translation seem to play functional roles.In this study, we have identified and examined the roles of cis-acting negative elements present on the late mRNAs and trans-acting factors, which interact specifically with these sequences.The HPV-16 L2 mRNA has been shown to contain negative elements in both the 5’ and 3’ halves of the gene. The 5’ negative element had a destabilizing effect on the mRNA, while the element in the 3’end affected the level of protein production. The 3’ element was used in RNA-protein binding studies to identify proteins, which specifically interact. We identified three proteins, hnRNP K and the Poly(rC) binding proteins 1 and 2, interacting with the 3’ negative element. The binding of these proteins was further shown to inhibit the translation of HPV-16 L2 mRNA in vitro.The HPV-16 L1 mRNA also contains multiple negative elements, which appear to be restricted to the first 514 nucleotides (nts) of the L1 gene. The strongest negative element was located in the first 129 nucleotides and inhibition appears to be localised to the nucleus. Mutation of the negative elements in the first 514 nts, while retaining an open reading frame (ORF) that has the ability to produce L1 protein, resulted in loss of inhibition and production of L1 mRNA and protein. Furthermore, negative elements were also located in the ORFs of HPV 5, 6b, 18, 45, and 56.The usage of rare codons in certain genes has been suggested to affect translational efficiency.We wished to demonstrate that codon usage did not contribute to the lack of L1 protein production. Alterations of rare codons in the entire L1 ORF increased the translational efficiency of L1 protein 2-fold. Interestingly, RNA analysis of an L1 ORF with mutations in the first 514nts showed the appearance of an approximate 700 bp RNA species. RT-PCR analysis and sequencing demonstrated that this was an mRNA polyadenylated at an internal poly (A) site within the L1 ORF. Mutation of this poly (A) site, results in all L1 mRNA being polyadenylated at the late poly (A) site, downstream of L1.In conclusion, this study demonstrates that HPV-16 late gene expression is regulated at many levels, in particular translational inhibition mediated by RNA binding protein and mRNA stability. It remains to be seen if the use of the alternative poly (A) site within the L1 ORF also plays a role in regulating L1 expression.