The papillomaviruses are epitheliotropic viruses which induce benign and malignant lesions in a variety of squamous epithelia such as skin and the cervix. The papillomavirus life cycle is intimately linked with the differentiation state of the squamous epithelium which it infects. Regulation of papillomavirus gene expression takes place at both transcriptional and posttranscriptional levels. The late pre-mRNAs of the bovine papillomavirus (BPV-1) are alternatively spliced and alternatively polyadenylated in a differentiation dependent manner. Changes in both alternative splicing and alternative polyadenylation are essential components of the early to late switch in viral gene expression. We have identified several cis-elements that regulate splice site choice. Immediately downstream of the first of two alternative 3 splice sites (the early 3 splice site) is a bipartite splicing regulatory element consisting of a purine-rich exonic splicing enhancer (SE1) and a pyrimidine-rich exonic splicing suppressor (ESS1) with an essential GGCUCCCCC motif. A second enhancer element (SE2) is located a short distance upstream of the second alternative 3 splice site (the late 3 splice site). We have used both in vitro and in vivo splicing assays to demonstrate that all three elements are essential for proper regulation of alternative splicing. All three elements require suboptimal 3 splice sites for proper function. SE2 is required to overcome the inhibitory effects of ESS1 at early stages of the life cycle and selects use of the early 3 splice site. This function of SE2 is due to its ability to act as a strong exonic splicing enhancer, even at a distance of more than 250 nt from the 3 splice site. SE2 does not appear to block use of the downstream late 3 splice site. Both ESE elements bind the same set of SR splicing factors. The early to late switch in splicing during keratinocyte differentiation could be due to a change in the activity of either SR proteins or as yet unknown proteins that bind ESS1. We are currently developing RNA affinity purification techniques to identify the factors that bind to the papillomavirus RNA regulatory elements and will use these techniques to determine how these factors change during keratinocyte differentiation.We have begun to investigate cis splicing elements in the exon downstream of the late 3 splice site and have identified a second bipartite splicing regulatory element consisting of an AC-rich exonic splicing enhancer (SE4) and a novel exonic splicing suppressor (ESS2). The role of these elements in the early to late switch in splicing is currently being investigated.We have generated replication- defective recombinant adenovirus vectors that express BPV-1 late pre- mRNAs from a CMV promoter. These viruses efficiently infect primary keratinocytes and will be used for studies of differentiation-dependent RNA processing. We have also become interested in parallels between the regulation of adenovirus and BPV-1 RNA processing. The early to late shift in adenovirus RNA processing also involves the switch from a promoter-proximal to promoter-distal 3 splice site and from a promoter- proximal to promoter-distal poly(A) sites. This switch in processing is due at least in part to virus-induced changes in the phosphorylation of SR proteins and changes in the activity of the general polyadenylation factor CstF. The chimeric BPV-1/adenovirus vectors can go through a complete adenovirus life cycle in 293 and 911 cells that complement the replication defect. Interestingly, the BPV-1 late pre-mRNA undergoes the correct early to late shift in both splicing and polyadenylation in parallel with similar changes in adenovirus splicing and polyadenylation. This suggests that both viruses regulate their RNA processing through the same splicing and polyadenylation factors. - Papillomaviruses, polyadenylation, post-transcriptional regulation, splicing, adenovirus vectors, Differentiation, Keratinocyte, - Human Tissues, Fluids, Cells, etc. & Neither Human Subjects nor Human Tissues
