High-risk anogenital papillomaviruses (HPVs) are associated with 95% of invasive cervical cancers worldwide. These viruses infect and replicate in stratified squamous epithelia, where they can persist for long periods. The double-stranded circular HPV genomic DNA is maintained as a low-copy-number nuclear plasmid in the mitotically active basal layer keratinocytes. Trans-acting viral factors E1 and E2 play an important role in replication; El, a helicase, recruits Polalpha to the origin; E2 acts cooperatively by binding E1 and the viral genome through its binding sites. Studies of bovine papillomavirus type 1 (BPV-1) suggest that E2 mediates segregation of newly synthesized viral genomes to daughter cells by linking them to mitotic chromosomes. We have recently developed a yeast-based system to study the cis and trans requirements for HPV16 replication and maintenance. Experiments performed in this system and in human cells indicate that E2 may not be strictly required for long-term maintenance of HPV16 genomes. This result suggests that the cis and trans requirements for extrachromosomal maintenance of HPV16 need to be further evaluated. The objectives of this proposal are: (1) To investigate the role of E2 in HPV16 maintenance; (2) To map cis-acting elements in the HPV16 genome that provide maintenance functions independently of E2; and (3) To identify cellular factors that contribute to genome maintenance. These studies will clarify the mechanism by which these oncogenic viruses maintain their genomes in the host.