Human papillomaviruses (HPVs) are a group of non-enveloped DNA viruses that have evolved to exploit a specialized biological niche in epithelial tissues such as the skin or the genital mucosa. Although most HPV types are associated with clinically inapparent or benign symptoms, such as common skin warts, about a dozen sexually-transmitted HPV types cause nearly all cases of cervical cancer, as well as a substantial fraction of anal, genital and throat cancers. HPV-induced cervical cancer kills nearly 300,000 women per year worldwide, mostly in developing countries where access to cervical cancer screening is limited. A recently-licensed vaccine targeting two cancer-causing HPV types has helped focus public attention on the substantial risk these viruses pose to public health. Work in the Tumor Virus Molecular Biology Section is focused on the HPV virion. The virions of non-enveloped viruses are dynamic structures that must undergo a range of conformational changes during different stages of the virus life cycle. Virions must be flexible enough to allow encapsidation of the viral genome, yet stable enough to withstand environmental insults encountered during transmission between hosts. Virions must also become pliable enough to release the viral genome upon infection of a host cell. Although each of these steps in the virus life cycle requires discrete structural motifs that are typically well-conserved among members of a virus family, immunological pressure tends to select for virions in which conserved functional motifs are occluded from immunological recognition. Using HPV vector technologies and a range of proteomics tools, we seek to characterize conserved functional motifs of the virion, as well as their cellular binding targets. Work in the lab is aimed at elucidating the mechanics of virion assembly and infectious entry, as well as the basic cell biology that underpins these phases of the viral life cycle. However, our work also has translational goals. High-titer HPV-based gene transfer vectors (also known as HPV pseudoviruses) are beginning to show promise as genetic vaccine vehicles. Improved understanding of the assembly, infectivity and in vivo tropism of HPV vectors should improve their practicality for in vivo gene transfer applications. Antibodies targeting conserved virion structures have the potential to confer sterilizing immunity against a wide variety of HPV types. Thus, another translational goal of our work is to facilitate the ongoing development of improved vaccines that might confer protection against a broader range of tumorigenic HPV types than the currently available vaccine. During FY09 we completed our goal of using tandem affinity purification (TAP) strategy for identifying cellular proteins that the HPV virion contacts during the infectious entry process. In brief, the TAP strategy involved purification of cell-bound HPV virions baited with chemical crosslinkers. We utilized mass spectrometric analysis to identify cellular proteins crosslinked to the virion. The approach revealed two classes of host protein that facilitate the infectious entry of HPVs into host cells. Once candidate factor is a cellular protease. In FY10 we will work toward understanding what protein clients the protease acts on to facilitate HPV infectious entry. Our TAP analysis also revealed that a chaperone protein that is a member of the cyclophilin family is required for HPV infectious entry. Cyclophilin-inhibitory drugs, such as cyclosporin A, were found to inhibit HPV infectivity in vitro. Although the initial candidate protein identified in our TAP screen was cyclophilin B, further analysis suggests that a cyclophilin other than cyclophilin B is required to support HPV infectious entry. The unidentified cyclophilin appears not to be produced endogenously by the cell, but is rather provided by serum in the cell culture medium. In a separate line of work, we completed our goal of elucidating the pattern of disulfide bonds that stabilize the HPV virion. This line of work led to improved methods for production of fully mature HPV virions.
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