Identification of domains of the HPV11 E1 protein required for DNA replication in vitro.

The HPV E1 and E2 proteins along with cellular factors, are required for replication of the viral genome. In this study we show that in vitro synthesized HPV11 E1 can support DNA replication in a cell-free system and is able to cooperate with E2 to recruit the host polymerase alpha primase to the HPV origin in vitro. Deletion analysis revealed that the N-terminal 166 amino acids of E1, which encompass a nuclear localization signal and a cyclin E-binding motif, are dispensable for E1-dependent DNA replication and for recruitment of pol alpha primase to the origin in vitro. A shorter E1 protein lacking the N-terminal 190 amino acids supported cell-free DNA replication at less than 25% the efficiency of wild-type E1 and was active in the pol alpha primase recruitment assay. An even shorter E1 protein lacking a functional DNA-binding domain due to a truncation of the N-terminal 352 amino acids was inactive in both assays despite the fact that it retains the ability to associate with E2 or pol alpha primase in the absence of ori DNA. We provide additional functional evidence that E1 interacts with pol alpha primase through the p70 subunit of the complex by showing that p70 can be recruited to the HPV origin by E1 and E2 in vitro, that the domain of E1 (amino acids 353-649) that binds to pol alpha primase in vitro is the same as that needed for interaction with p70 in the yeast two-hybrid system, and that exogenously added p70 competes with the interaction between E1 and pol alpha primase and inhibits E1-dependent cell-free DNA replication. On the basis of these results and the observation that pol alpha primase competes with the interaction between E1 and E2 in solution, we propose that these three proteins assemble at the origin in a stepwise process during which E1, following its interaction with E2, must bind to DNA prior to interacting with pol alpha primase.