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The cellular DNA polymerase alpha-primase is required for papillomavirus DNA replication and associates with the
1Department of Molecular and Cell Biology, University of California, Berkeley 94720.
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 alpha-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 E1 helicase and the E2 transcription factor. Monoclonal antibodies directed against the catalytic 180-kDa subunit of polymerase alpha inhibit DNA synthesis in this system. Addition of purified human polymerase alpha-primase holoenzyme to neutralized extracts restores their DNA synthetic activity. The amino-terminal 424 amino acids of E1 forms a specific protein complex with the p180 polymerase subunit. Immune complexes can be isolated with antibodies directed against E1 that contain a DNA polymerase activity. Moreover, this polymerase activity can be neutralized by anti-polymerase alpha antibodies. Permissivity barriers were not encountered in this in vitro system, as bovine E1 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 E1 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 alpha-primase complex to the viral replication origin.
Insights
Papillomaviruses use host DNA polymerase alpha-primase for viral DNA replication. The viral E1 helicase specifically binds and recruits this polymerase to initiate replication.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Persistent papillomavirus infection relies on maintaining viral DNA as a nuclear plasmid.
- Replication of this viral DNA necessitates host cell DNA polymerases.
Purpose of the Study:
- To investigate the role of the cellular DNA polymerase alpha-primase holoenzyme in papillomavirus DNA replication.
- To elucidate the interaction between the viral E1 helicase and host DNA replication machinery.
Main Methods:
- Utilized soluble extracts from rodent cells capable of replicating bovine papillomavirus 1 and human papillomavirus 6b DNA.
- Employed monoclonal antibodies against the polymerase alpha catalytic subunit and purified human polymerase alpha-primase holoenzyme.
- Investigated protein complex formation between the viral E1 helicase and the polymerase p180 subunit.
Main Results:
- Monoclonal antibodies against polymerase alpha inhibited DNA synthesis, while purified holoenzyme restored activity.
- The amino-terminal 424 amino acids of E1 formed a complex with the p180 polymerase subunit.
- Immune complexes with anti-E1 antibodies contained and were neutralized by anti-polymerase alpha antibodies, indicating polymerase activity.
Conclusions:
- The study demonstrates that papillomavirus E1 helicase directly interacts with and recruits host DNA polymerase alpha-primase for viral DNA replication.
- This interaction is crucial for initiating the replication of the viral genome.
- Functional similarities exist between papillomavirus E1 and other viral helicases in recruiting cellular polymerases.