This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Dr. Angeletti?s research investigates the mechanism of encapsidation of the persistent infecting virus, human papillomavirus (HPV) by utilizing a novel yeast-based system. The genetic analysis of early steps in HPV infection has been hampered by the lack of easily manipulatable systems in which to generate useful amounts of viral particles. Dr. Angeletti has recently developed an HPV-packaging system in which expression of two late HPV proteins, L1 and L2 proteins can generate intact virions in yeast. This system is not only useful for analysis of regulatory elements required for efficient packaging, but potentially in production of infectious HPV in yeast for further study. The long-term goal of this project is to understand what cis and trans acting signals are important for HPV assembly and how these relate to infectivity of virions. We hypothesize that an undefined packaging element exists in HPV DNA that is either partially or entirely independent of E2. Therefore, we plan to: (1) define the minimal cis-acting packaging signal in the HPV genome, (2) determine the role of trans-acting factors E2 and L2 in efficient DNA packaging, (3) determine the effect of L1 and L2 genotype-specific differences on infectivity of HPV pseudovirus created in yeast. Different HPV genotypes; lab strains and natural isolates derived from patient samples from Zambia Africa will be used as a source of capsid genes. These studies will provide a detailed analysis of the requirements for HPV encapsidation and infectivity that can be related to the known pathogenicity of HPV strains. Future analyses will allow detailed structure-function studies of HPV virions as well as analysis of viral attachment, entry and establishment in human keratinocytes. The yeast-based approach is potentially adaptable to other viral systems and therefore, provides a great opportunity for collaboration with NCV colleagues. Dr. Clinton Jones will act as a mentor for Dr. Angeletti. Dr. Jones has a breadth of experience in studies of persistent DNA viruses and will provide advice on the strategies employed in these studies.??
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