Related Experiment Videos
Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication
Abstract:
In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.
Insights
Mouse cell extracts can replicate polyoma virus DNA in vitro, requiring specific viral proteins and host DNA polymerase alpha. Different host cell polymerases influence viral DNA replication, explaining host range specificity.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- In vitro DNA replication systems are crucial for studying viral replication mechanisms.
- Polyoma virus (Py) and Simian virus 40 (SV40) are DNA viruses with distinct replication requirements.
- Host cell factors play a significant role in viral DNA replication.
Purpose of the Study:
- To investigate the in vitro DNA replication of polyoma virus (Py) using cell-free extracts.
- To identify the essential viral and host factors required for Py DNA replication.
- To understand the role of DNA polymerase alpha in Py DNA replication and its interaction with viral T antigens.
Main Methods:
- Utilized cell-free extracts from mouse FM3A cells for in vitro DNA replication assays.
- Investigated the requirement for Py virus large tumor (T) antigen, origin of replication DNA, and ATP.
- Assessed the effect of aphidicolin and different T antigens (Py vs. SV40) on replication efficiency.
- Examined the role of host DNA polymerase alpha-primase complex by cross-complementation between FM3A and HeLa cell extracts.
Main Results:
- Py DNA replication in vitro required Py T antigen and was dependent on DNA polymerase alpha.
- SV40 T antigen could not support Py DNA replication.
- HeLa cell extracts, which support SV40 replication, showed poor Py DNA replication.
- Supplementation with mouse FM3A DNA polymerase alpha-primase complex enhanced Py DNA replication in HeLa extracts.
- Conversely, FM3A extracts required HeLa DNA polymerase alpha-primase complex for efficient SV40 DNA replication.
Conclusions:
- The host-cell DNA polymerase alpha-primase complex is a critical determinant in discriminating between Py and SV40 T antigen-dependent DNA replication.
- Differences in DNA polymerase alpha-primase complex between cell types contribute to the host-range specificity observed for Py and SV40 viruses in vivo.