Interactions between papillomavirus E1 and E2 proteins and cellular replication factors modulate each others functions
Along with the viral proteins E1 and E2, papillomavirus DNA replication is carried out by the cellular DNA replication machinery. Many of these cellular replication factors have been identified using a viral cell-free DNA replication system. PV E1 is an essential initiator protein with ATPase and helicase activity and has been shown to bind to several of these replication factors, such as DNA polymerase α-primase, RPA, and topoisomerase I. E1 actively regulates the functions of RPA and topoisomerase I through these interactions. We hypothesized those interactions also allow cellular factors to modulate the function of viral protein E1. During the initiation step of DNA replication, E1 binds to the viral origin and initiates DNA replication. However, PV E1 has low sequence specificity. Origin recognition is a challenging process, especially during the initial stage of viral infection when the levels E1 and E2 are low and host DNA is in a vast excess over viral DNA. We first tested whether cellular DNA replication proteins play any role in viral origin recognition. We demonstrated that topo I, but not other cellular E1 interacting cellular factors, specifically stimulated PV E1 binding to origin DNA, but not to non-origin DNA. This stimulation by topo I is not synergistic with the stimulation by viral E2. The results that topo I and E2 independently stimulate origin binding by E1 suggest that in vivo topo I may provide an alternate pathway to assist in origin recognition by E1. Topo I appears to stimulate origin binding by E1 through a protein-protein interaction. Interestingly, the binding of E1 to DNA disrupts the interaction between E1 and topo I. A model for the role of topo I in viral DNA replication is proposed. Also presented in this study is an investigation of the interaction between PV E2 and topo I. As part of our investigation of the role of topo I in PV DNA replication, we evaluated whether E2 and topo I interact. Using ELISA protein interaction and GST co precipitation assays, we demonstrated that E2 binds to topo I. This suggests that E2 may help to recruit cellular tops I to the PV origin of replication. E2 can stimulate the DNA relaxation activity of topo I. Conversely, topo I does not stimulate the DNA binding activity of E2. These results suggest that after stimulating the origin binding of E1, E2 may remain active at the replication fork, playing multiple roles during viral DNA replication.