Stimulation of Human Papillomavirus Type 1a DNA Replication by a Multimerized AT-Rich Palindromic Sequence

Replication of most papillomaviruses requires the viral E1 and E2 proteins and an origin of replication containing the E1 and E2 binding sites. In the case of human papillomavirus type 1a (HPV-1a), the E1 protein alone is sufficient for DNA replication although the E2 protein significantly stimulates replication. We have further analyzed the role of cis-acting sequences and the E1 and E2 proteins in HPV-1a replication. Previous studies have shown that a 60-bp region lacking the E2 binding sites but containing an imperfect 16-bp AT-rich palindrome corresponding to the putative E1 binding site contains the minimal origin of replication (ori). Using a transient replication assay, we demonstrate that duplication of this 60-bp region causes a severalfold increase in replication. Synthetic oligonucleotides containing a 39-bp region centered around the above palindromic sequence supported only low-level replication in the presence of either E1 alone or both E1 and E2, but replication was significantly increased in the presence of multiple copies of this sequence. Plasmids containing a 19-bp sequence which includes the AT-rich palindrome failed to replicate, but multiple copies of this region supported replication in the presence of both E1 and E2 to significant levels. The results presented indicate that the HPV-1a E1 protein is capable of recruiting all the cellular factors required for replication. Our results also suggest that multimerization of the AT-rich palindromic sequence may result in a significant increase in the recruitment of the E1 protein to the origin, thereby stimulating replication. This increased targeting of the E1 protein to the origin containing multiple copies of the putative E1 binding site may be functionally similar to the targeting of E1 to the origin by E2.