The papillomaviruses are epitheliotropic small DNA tumor viruses that cause both benign and malignant lesions in humans and animals. These viruses productively infect only squamous epithelia and progression of the viral life cycle is dependent on differentiation of the host cell, the keratinocyte. An oncogenic subset of the human papillomaviruses (including HPV-16, 18, 31, and 33) is associated with the vast majority of cervical cancers as well as squamous cell carcinomas in other locations. The E6 and E7 viral oncogenes interfere with the functions of p53 and pRb, respectively, and thus disrupt regulation of the cell cycle. The role of a third viral oncogene, E5, in the viral life cycle is less well understood. Expression of papillomavirus genes is regulated at both transcriptional and posttranscriptional levels. We are currently exploiting HPV alternative splicing to develop novel RNA-based antiviral and anticancer therapies. In addition we have been involved in several collaborations on HPV transcriptional and posttranscriptional regulation. Some of the techniques we have developed, such as isoform-specific real time quantitative RT-PCR (QRT-PCR), are also being used in other collaborations both within and outside the papillomavirus field. Targeting HPV infected cells using specific trans-splicing: The ideal cancer therapy would kill cancer cells and have no effect on normal cells. The expression of spliced human papillomavirus E6/E7 pre-mRNAs by the vast majority of cervical cancers provides an absolute difference between cancer cells and normal cells that can be exploited for the specific targeting of these cells. Through a CRADA between NCI and Intronn Inc., we are investigating the use of Intronn's Spliceosome Mediated RNA Trans-splicing (SMaRT) Technology to develop a novel RNA-based suicide gene therapy for cervical cancer. Trans-splicing refers to the joining together of exons from two different pre-mRNAs to form a novel chimeric mRNA. Trans-splicing is thought to be very rare in mammalian cells. However, pre-trans-splicing molecules (PTMs) can efficiently and specifically trans-splice to a target pre-mRNA if they contain an antisense binding domain that tethers the PTM to the target pre-mRNA. We have designed PTMs that target HPV-16 E6 and E7 pre-mRNAs and have used these to study the factors that are important for trans-splicing efficacy. Expression of the chimeric mRNA, and presumably also the expression of the therapeutic molecule encoded by the PTM exon, was directly proportional to the amount of target pre-mRNA over at least a 1000-fold range. Very efficient trans-splicing was achieved, with as much as 80% of an exogenous pre-mRNA and 17 % of an endogenous pre-mRNA being trans-spliced instead of cis-spliced. Trans-splicing efficiency was dependent on PTM expression level, so efficient delivery and PTM expression are also important. We are currently in the process of evaluating the specificity of trans-splicing using 5' RACE (rapid amplification of cDNA ends) and are also redesigning PTM binding domains as well as other aspects of PTM structure to optimize efficiency and specificity. Identification of additional HPV-16 promoters: A single promoter has so far been found in the long control region (LCRs) of human papillomavirus type 16 (HPV-16). Multiple promoters exist in the LCRs of several other papillomaviruses, which are spliced to become mRNAs for late and some early genes. In a collaboration between my lab and Uli Bernard (Institute of Molecular and Cell Biology, Singapore), we have investigated whether such promoters exist in the LCR of HPV-16. In in vitro transcription experiments, a strong transcriptional start site was identified 280 bp downstream from the 3' end of the L1 gene between a nuclear matrix attachment region and the epithelial-specific enhancer. Promoter activity coincides with a GCCATTTT motif, which binds the transcription factor YY1 (YY1-7436). The A of this motif is the first nucleotide of the transcripts and identifies YY1-7436 as an initiator. Genomic segments with YY1-7436 initiate expression of a luciferase reporter gene in transfection experiments. Mutational analysis of YY1-7436 suggests, however, that promoter function originates from another factor but YY1, which can contact overlapping sequences. Promoter activity of YY1-7436 is modulated by upstream A-T-rich sequences, which bind the basal transcription factor TFIID, and it is stimulated by the viral E2 protein binding to a downstream E2 binding site. Analysis of RNA from differentiating W12 cells, which contain episomal HPV-16 copies, revealed transcripts which included LCR sequences downstream of YY1-7436 and which were differentially spliced to early and late genes. Although 5' ends mapping to YY1-7436 were not detected, these studies did provide evidence for two novel HPV-16 promoters within the L1 gene. Conservation of the arrangement of the YY1 and E2 binding sites suggests a role in important biological functions, which, however, is difficult to confirm in every type of cell culture. The study of W12 cells complements the examination of YY1-7436 and points to yet undetected promoters upstream of the LCR. Codon optimization of the HPV-16 E5 gene enhances protein expression: The human papillomavirus type 16 (HPV-16) E5 protein is an 83-amino-acid, hydrophobic polypeptide that has been localized to intracellular membranes when overexpressed in COS-1 cells. While the HPV-16 E5 protein appears to modulate endosomal pH and signal transduction pathways, genetic analysis of its biological activities has been hampered by low (usually nondetectable) levels of expression in stable cell lines. Sequence analysis of the native HPV-16 E5 gene revealed that infrequent-use codons are used for 33 of its 83 amino acids and, in an effort to optimize E5 expression, these codons were converted to those more common in mammalian genes. The modified gene, 16E5*, generated protein levels that were six- to ninefold higher than those of wild-type HPV-16 E5, whereas the levels of mRNA were unchanged. 16E5* protein was detectable in keratinocytes by immunoblotting, immunoprecipitation, and immunofluorescence techniques and formed disulfide-dependent dimers and higher-order oligomers. Unlike the bovine papillomavirus E5 protein, which is present in the Golgi, 16E5* was localized primarily to the endoplasmic reticulum and its expression reduced the in vitro life span of keratinocytes. This work was done as a collaboration with Richard Schlegel (Georgetown University Medical School).
