DNA replication and genome maintenance of human papillomavirus type 16 in mammalian cells

Human papillomavirus (HPV) is a major causative agent of cervical cancer and a number of human cancers. The high-risk types can be detected in more than 90% of cervical cancer, of which HPV16 is the most common found. The virus establishes a latent infection in basal epithelial cells in which the viral genomes can be stably maintained as extrachromosomal DNA for decades before the development of cancer. Several attempts have been made in bovine papillomavirus (BPV) to understand how the virus can maintain its genome at constant copy numbers in dividing cells. The viral protein E2 has been proposed to serve as a molecular linker that tethers the viral genome to host chromosomes. However, there is a great deal of dispartities between BPV and HPV with respect to variations of E2 binding pattern, cellular targets, and number of E2 binding sites within their genomes. It is unclear whether HPV utilizes the same maintenance mechanism. Papillomaviruses utilize two viral factors, E1, a DNA helicase, and E2, a transcriptional activator and auxiliary replication factor for replication and DNA maintenance in host cells. However, previous studies in yeast and findings reported here demonstrated that HPV16 can replicate independently of the viral E1 and E2 proteins. It was also shown that HPV16 possibly contains alternate origin of replication outside the LCR region that relies entirely on cellular replication proteins. In this study, we further identified cis-elements and trans-acting factors that are required for HPV genome maintenance during persistent infection. Two distinct regions have been mapped as maintenance elements as they provided DNA stability in mammalian cells in the absence of any viral proteins. We found three nonamer sites (TTAGGGTTA) which resemble telomeric repeats on the cis-maintenance elements within the late region and observed altered expression of proteins associated with telomeric repeats in HPV16 immortalized cells. The telomere binding proteins such as telomeric repeat binding factors 1(TRF1),TRF2, TRF2–interacting protein hRAP, the telomere-associated poly (ADP-ribose) polymerase (tankyrase) were previously found on EBV latent origin of replication and contributed to episomal maintenance. These observations motivated us to hypothesize that HPV16 has evolved similar mechanism to maintain its genome in host cells. Using ChIP assay, we found that TRF2, protection of telomere 1 (POT1) and a RecQ helicase WRN bound to the putative binding sites within HPV16 genome. Deletion mutations of TRF binding sites altered the plasmid maintenance activity suggesting the implication of these binding sites as well as the neighboring sequences in HPV life cycle. These results imply that the telomere binding factors are novel cellular factors for HPV16 DNA maintenance. Furthermore, several binding sites for topoisomeraseII (TopoII), centromere binding protein CENP-B, and high mobility group HMG were also predicted in the late region suggesting that HPV maintenance is regulated by multiple cellular factors