The papillomaviruses are small DNA tumor viruses which induce benign and malignant lesions in squamous epithelia of higher vertebrates. The complete life cycle of these viruses occurs only in the differentiated keratinocytes of a squamous epithelium. This project currently focuses on posttranscriptional regulatory mechanisms which may be important in regulating papillomavirus gene expression during keratinocyte differentiation, with particular emphasis on the regulation of bovine papillomavirus type 1 (BPV-1) translation and polyadenylation. Four short open reading frames (uORFs) in the 5' untranslated region (UTR) of the major capsid protein (L1) mRNA have been shown to block translation of this mRNA. Mutation of each AUG singly and in combination showed that mutation of the second and third AUGs gave the greatest increase in translation in transfection assays. It is not known whether the mechanism of translational inhibition is a block to ribosome scanning or a direct effect of one of the peptides. However, the second uORF encodes a short arginine-rich peptide with similarities to the RNA-binding domain of the human immunodeficiency virus (HIV-1) tat protein, suggesting that it may have an RNA target. We have also carried out a yeast two hybrid screen with this peptide to identify potential protein targets. This screen gave two unknown proteins, one of which also interacts with HIV-1 tat. The 3'UTR of the L1 mRNA also contains a negative posttranscriptional regulatory element. This element has been mapped to a 5' splice site which binds at least one splicing factor, the U1 snRNP, but is not used for splicing. In vivo studies from the LTVB as well as in vitro studies from other labs have shown that this element inhibits polyadenylation. We have also shown that the HIV-1 Rev protein can block the effect of this element. We are currently using mutant Rev proteins to investigate whether this function of Rev is due to its ability to facilitate nucleocytoplasmic transport or to interact with the splicing machinery. We are also testing other elements, such as retroviral constitutive transport elements and viral elements which confer splicing independent expression, for their ability to counteract the effect of a regulatory 5' splice site. Preliminary data indicates that the hepatitis B Virus (HBV) PRE element does not have this ability, indicating that it does not function in the same way as the HIV Rev protein.
