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The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase
J Gottlieb1, A I Marcy, D M Coen
1Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Abstract:
Genetic experiments have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication, and a number of studies have suggested that these two proteins specifically interact. We have confirmed and extended these findings. The viral DNA polymerase from HSV-1-infected cells has been purified as a complex containing equimolar quantities of the UL30 (Pol, the catalytic subunit) and UL42 polypeptides. Sedimentation and gel filtration analyses of this complex are consistent with the idea that the complex consists of a heterodimer of Pol and UL42. A complex with identical physical and functional properties was also purified from insect cells coinfected with recombinant baculoviruses expressing the two polypeptides. Therefore, the formation of the Pol-UL42 complex does not require the participation of any other HSV-encoded protein. We have compared the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells. The specific activity of the catalytic subunit alone was nearly identical to that of the complex when assayed on activated DNA. When assayed on a defined template such as singly primed M13 DNA, however, the combination of Pol and UL42 utilized fewer primers and formed larger products than Pol alone. Template challenge experiments demonstrated that the Pol-UL42 complex was more highly processive than Pol alone. Our data are consistent with the idea that the UL42 polypeptide is an accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization.
Insights
Herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 proteins form a complex essential for viral DNA replication. This UL42 protein acts as an accessory subunit, enhancing the DNA polymerase
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Herpes simplex virus type 1 (HSV-1) DNA replication is crucial for viral propagation.
- Previous studies suggested an interaction between HSV-1 DNA polymerase (UL30) and the UL42 protein.
- Both UL30 and UL42 gene products are necessary for efficient viral DNA synthesis.
Purpose of the Study:
- To confirm and further investigate the interaction between HSV-1 DNA polymerase (Pol) and the UL42 protein.
- To characterize the physical and functional properties of the Pol-UL42 complex.
- To determine the role of the UL42 polypeptide in the catalytic activity and processivity of HSV-1 DNA polymerase.
Main Methods:
- Purification of the HSV-1 DNA polymerase complex from infected cells.
- Purification of the Pol-UL42 complex from insect cells coinfected with recombinant baculoviruses.
- Sedimentation and gel filtration analyses to determine complex composition.
- Enzymatic assays comparing the catalytic properties of the isolated UL30 subunit and the Pol-UL42 complex.
Main Results:
- The HSV-1 DNA polymerase purified from infected cells exists as a complex of equimolar UL30 (Pol) and UL42 subunits.
- An identical Pol-UL42 complex was formed in insect cells, indicating no requirement for other viral proteins.
- The Pol-UL42 complex exhibited significantly increased processivity compared to the isolated Pol subunit on a defined DNA template.
- UL42 enhanced the ability of Pol to utilize primers and synthesize longer DNA products.
Conclusions:
- The UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase.
- UL42 significantly enhances the processivity of the viral DNA polymerase.
- The Pol-UL42 complex is the functional form of the enzyme required for efficient viral DNA replication.