Human papillomaviruses (HPVs) are widespread and important pathogens that cause both benign and malignant epithelial lesions, most notably various types of cutaneous warts, genital warts, laryngeal papillomas, and cervical and penile dysplasias and carcinomas. Infections by the mucosotrophic HPVs are the most prevalent sexually transmitted diseases of viral etiology. Papillomaviruses have stringent species and tissue tropisms and cannot be propagated in conventional cell cultures. Recently we and others have succeeded in replicating HPV DNA in transiently transfected cells and also in recapitulating the viral productive program by culturing tissues or cells that harbor episomal HPV DNAs at the medium-air interface on a dermal equivalent of collagen containing fibroblasts (i.e., as raft cultures). Tremendous strides have also been made in many laboratories in understanding other aspects of the molecular and cellular biology of the viruses and their host cells on the basis of studies performed with cloned genes and regulatory sequences and with patient biopsies. However, there are still significant unanswered questions concerning viral DNA replication, mRNA transcriptional regulation, viral protein functions, virus-host interactions and pathogenesis. The overall goal of this application is to perform a genetic dissection the papillomavirus in the context of its productive program in differentiating epithelium. The roles of viral cis sequence elements and trans-acting E1 and E2 proteins in extra-chromosomal DNA replication will be further investigated in a cell-free system and in transiently transfected cells. The functions of the other E region proteins and the regulatory sequences will be studied in primary human keratinocytes (PHK) cultured by the collagen raft technique. To achieve these goals, efficient means for introducing wild type or mutant viral DNAs into PHK will be developed, including packaging DNA into virions in surrogate cells. The mechanisms by which viral proteins activate host genes essential for DNA replication, a necessity for viral propagation in differentiated cells that have long exited from the cell cycle, will continue to be examined. Finally, the epithelial raft culture system provides an opportunity to design and test specific antiviral therapeutic agents. We will explore the possibilities of using sequence-specific triplex-forming oligonucleotides targeted to the regulatory region to interfere with viral transcription and replication.