HPV DIFFERENTIATION DEPENDENT REPLICATION

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Worldwide, cervical cancer is the second leading cause of death due to cancer in females. Human papillomaviruses (HPV) have been associated with over 90 percent of all cervical cancers examined. The working hypothesis is that all aspects of HPV's differentiation-dependent replication cycle are controlled by the temporal and spatial growth mechanisms of its natural host tissue, squamous epithelium. Therefore, temporal and spatial controls on gene expression, viral DNA replication, and virion morphogenesis are stipulated by the differentiation state of the host cell. Two areas of the HPV differentiation-dependent life that are the focus of this application include: first, describing the cis elements important for viral transcriptional control during different stages of the viral life cycle and second, defining viral protein expression in terminally differentiating host tissue. In the first specific aim, HPV mutants will be utilized to investigate transcriptional control using in vitro model systems for (i) the initial stage of the viral life cycle; (ii) the differentiation of the host epithelial tissue; (iii) the contribution of the viral transactivating protein, E2; and (iv) the complete HPV life cycle. The ability to study mutations incorporated into the HPV genome on viral transcription, viral DNA replication, and late gene expression represents a breakthrough advance for investigating the biology of these human oncogenic viruses. These studies represent the first proposed mutational analysis of a HPV using a permissive in vitro system that is capable of propagating infectious HPV particles. In the second specific aim, we will define the temporal and spatial expression of HPV proteins in differentiating epithelium during the complete viral life cycle. The use of the organotypic (raft) culture system permissive for the complete HPV life cycle, including the synthesis of infectious viral particles, will be an integral part of these studies. The ability of the raft culture system to reproduce the complete HPV life cycle is a technical advance capable of providing important and novel insights concerning the regulation of viral transcription and protein expression during the complete HPV life cycle. When the proposed studies are completed we expect to have defined the role of cis regulatory elements located upstream of the major HPV31b promoter in viral transcriptional control, viral DNA replication, and virion synthesis. Additionally, we expect to have defined the temporal and spatial expression patterns of HPV31b proteins and to correlate viral protein expression with transcript expression, promoter usage, expression of epithelial differentiation-specific proteins, and virion morphogenesis.