The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the viral E1 helicase.

Abstract

Persistent infection by papillomaviruses involves the maintenance of viral DNA as a nuclear plasmid, the replication of which requires host DNA polymerases. The role of the cellular DNA polymerase a-primase holoenzyme was probed by using soluble extracts from rodent cells that replicate bovine papilloma virus 1 and human papilloma virus 6b DNA in the presence of the viral El helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase a inhibit DNA synthesis in this system. Addition of purified human polymerase a-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids ofEl forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against El that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase a antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine El can interface with the murine and human replication apparatus. Although the large tumor antigens encoded by simian virus 40 and polyoma share limited primary sequence homology with the papillomavirus El proteins, the organization of functional motifs at the level of primary protein structure is remarkably similar. In addition to their origin-specific DNA-binding activity, each of these helicases may function to help recruit the cellular polymerase primase complex to the viral replication origin.A central event in the initiation of DNA synthesis is the localization of one or more DNA polymerases (pols) to the origin of replication (ori). This localization appears to be accomplished through a series ofprotein-protein interactions in a variety of biological systems (for review, see ref. 1). Although these interactions have been well documented at the genetic and biochemical level in bacteria and their phages, little is known about how the DNA pols of higher eukaryotes recognize cellular oris. Although oris of higher eukaryotes, along with the presumed initiator proteins that recognize these elements, are ill defined, it is possible to examine how cellular DNA pols are targeted to the wellcharacterized oris of DNA tumor viruses (2-4).The papovaviruses constitute a family of small, transforming DNA viruses that exhibit a complete dependence on host-cell functions to replicate (5). Each virus encodes an initiator helicase capable of assembling a specific nucleoprotein structure on the viral ori and unwinding it (for review, see refs. 5 and 6). In the case of simian virus 40 (SV40) and polyoma, the viral large tumor antigen (T) associates with the cellular pol a-primase, and this interaction may aid in localizing pol a to the ori (2-4, 7, 8). DNA pol a is responsible for initiating bidirectional DNA synthesis from the SV40 ori in vitro (9).Papillomaviruses (PVs) behave as regulated replicons. Rodent cells transformed by bovine papilloma virus (BPV) 1 maintain the viral DNA as a mult...

Rolling circle replication of human papillomavirus type 16 DNA in epithelial cell extracts

“…The resultant E1 hexamer is an active replicative helicase that can induce melting of the DNA at the origin as well as subsequent unwinding of the double helical DNA during replication fork progression. With the exception of continuous DNA unwinding by E1, HPV uses host replication proteins, such as DNA polymerases, proliferating cell nuclear antigen and replication protein A, to accomplish its genome replication (Park et al 1994;Melendy et al 1995).…”

“…The resultant E1 hexamer is an active replicative helicase that can induce melting of the DNA at the origin as well as subsequent unwinding of the double helical DNA during replication fork progression. With the exception of continuous DNA unwinding by E1, HPV uses host replication proteins, such as DNA polymerases, proliferating cell nuclear antigen and replication protein A, to accomplish its genome replication (Park et al 1994;Melendy et al 1995).As two replication forks containing the E1 hexamers progress in opposite directions from the viral origin, bidirectional replication has been considered as a basic mode of HPV DNA replication (Stenlund 2003). Bidirectional replication was clearly demonstrated with an HPV origin-containing plasmid in a crude extract from human embryonic kidney 293 …”

Control of HPV 18 DNA replication by cellular and viral transcription factors

“…The BPV1 El protein contains activities known to be involved in DNA synthesis, such as ATP-dependent DNA helicase, and DNA-dependent ATPase (6)(7)(8). In addition, El interacts with the cellular DNA polymerase a-primase (9,10). It specifically binds with low affinity to a palindromic DNA sequence localised within the AT-rich sequence of the replication origin ( 1,11).…”

The human DnaJ protein, hTid-1, enhances binding of a multimer of the herpes simplex virus type 1 UL9 protein to ori s , an origin of viral DNA replication

“…The UL9 protein shares a number of properties with the SV40 Tag and the HPV E1 protein, including origin-specific binding, DNA-dependent ATPase and helicase activities (7)(8)(9). Like the Tag and the E1 protein, the UL9 protein binds to the cellular DNA polymerase-␣ primase (21,23,24). During SV40 DNA replication, the interaction between Tag, the single strand DNA binding protein (RP-A), topoisomerase I, and DNA polymerase ␣-primase are essential for the formation of the replication initiation complex (25)(26)(27)(28).…”