Research Summary:
The papillomaviruses are epitheliotropic viruses that induce benign
and malignant lesions in a variety of squamous epithelia. The
human papillomaviruses are etiologic agents of several human cancers and
have been found in >95% of cervical cancers, >50% of other anogenital cancers,
and 20% of oral, laryngeal, and nasal cancers. One interesting feature
of these viruses is that their life cycle is intimately linked with the
differentiation state of the squamous epithelium that they infect. The
primary goals of our research are 1) to elucidate the regulatory mechanisms
that control papillomavirus gene expression during keratinocyte differentiation;
2) to study the cellular signaling pathways that regulate keratinocyte
growth and differentiation and to determine how these pathways are perturbed
by papillomavirus proteins; and 3) to study the genetic changes and
cellular and viral gene expression changes that occur during malignant
progression of papillomavirus lesions.
Regulation of papillomavirus gene expression occurs at both transcriptional and posttranscriptional levels. A major focus of the laboratory has been the post-transcriptional regulation of bovine papillomavirus (BPV-1) late gene expression. Previously we have used in situ hybridization to demonstrate that alternative splicing of BPV-1 late pre-mRNAs is regulated in a differentiation dependent manner; the distal 3' splice site at nt 3605 is used only in keratinocytes of the granular layer of the epidermis (Figure 1). Several cis-elements have been identified that regulate splice site choice. Immediately downstream of the first of two alternative 3' splice sites is a bipartite splicing regulatory element that regulates use of this site (Figure 2). This element consists of a purine-rich positive element known as an exonic splicing enhancer (ESE) and a pyrimidine-rich negative element known as an exonic splicing suppressor (ESS). Both the ESE and the ESS are capable of regulating splicing of heterologous pre-mRNAs containing suboptimal splice sites. Only two other bipartite exonic splicing regulatory elements are known, one in HIV-1 and the other in the human fibronectin gene (Figure 2). A third element similar to the ESE has been identified a short distance upstream of the second alternative 3' splice site (Figure 2). Although this element can function as a splicing enhancer when located near a 3' splice site in an exonic position, we speculate that it functions in its normal location as an intronic splicing suppressor (Figure 3). This arrangement potentially allows the coordinated regulation of two alternative splice sites by the same trans-acting factors. Combined UV crosslinking and immunoprecipitation experiments indicated that both splicing enhancers bind a subset of the SR family of splicing factors (ASF/SF2, SRp55, and SRp75). Mutational analysis indicated that this binding is functionally relevant. Over the past year we have also carried out a detailed analysis of the ESS. The ESS consists of three parts: a U-rich 5' end, a C-rich central part and an AG-rich 3' end (Figure 2). We have shown that the 5' region binds U2AF65 and PTB, the central region binds 30 and 55 kDa SR proteins, and the 3' region binds ASF/SF2. Finally, mutational analysis indicated that the central region is functionally the most important. We are currently investigating whether the activity of any of these splicing factors is regulated by differentiation of the epithelial cells.
Expression of the major capsid (L1) protein is also regulated by sequences in both the 5' and 3' UTR. Mutational analysis of the 5'UTR of the L1 mRNA indicated that four short upstream ORFs (uORFs) block translation. 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 HIV-1 tat protein, suggesting that it may have an RNA target.
The 3'UTR of the L1 mRNA also contains a negative post-transcriptional regulatory element which has been mapped to a nonfunctional 5' splice site. In vivo studies from the LTVB as well as in vitro studies from other labs have shown that this element inhibits polyadenylation through a U1 snRNP-dependent mechanism. 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 indicate that the HBV PRE element does not have this ability, indicating that it does not function in the same way as the HIV Rev protein.
A major new initiative over the last two years has been to set up in
vivo assays that can be used to investigate differentiation dependent changes
in gene expression. In one of these assays, keratinocytes from a
variety of sources are grafted onto the backs of nude mice. In this environment
these cells form a fully differentiated squamous epithelium that is capable
of supporting the full papillomavirus life cycle. Expression vectors as
well as whole viral genomes are being introduced into keratinocytes prior
to grafting. This system is being used to study virus-host interactions
in the context of a differentiating squamous epithelium.
Finally, we are initiating a new project to look at changes in gene
expression as a function of both differentiation and malignant progression
of HPV lesions of the cervix. Laser capture microdissection
will be used to collect clusters of similar cells in heterogeneous lesions.
Patterns of gene expression will be analyzed through both cDNA cloning
and high throughput assays such as hybridization to gene microarrays.
This project will hopefully identify cellular genes that regulate progression
of the viral life cycle as well as genes involved in malignant progression.
Collaborators on this research include Alison McBride, Ph.D., Laboratory of Viral Disease, National Institute of Allergy and Infectious Diseases; Andrzej Dlugosz, U. of Michigan Comprehensive Cancer Center; Lance Liotta, Laboratory of Pathology, NCI; Matthew Gonda, Ph.D., Greg Tobin, Ph.D., and Steve Fong, Ph.D., Lab of Cell and Molecular Structure, SAIC, NCI-Frederick Cancer Research and Development Center; and Alfred M. Del Vecchio, Ph.D., and Jane Tsai, Smith Kline Beecham Pharmaceuticals, King of Prussia, PA.
Laboratory Information and Reagents
INQUIRIES FOR POSITIONS:
The CRTS periodically has postdoctoral and technical positions open. Applicants must have molecular biology experience. Minorities are encouraged to apply. Please send CV and letters of recommendation to Carl C. Baker at the above address.
Return to the NCI Intramural
Research Home Page
Return to RNA Club Home Page