Purification and characterization of equine herpesvirus-induced DNA.
Abstract: Infection of cells with equine herpesvirus type 1 (EHV-1) or type 3 (EHV-3) resulted in the induction of a DNA polymerase activity distinguishable from host cell DNA polymerases by its high salt requirement for maximal activity. By column chromatography on DEAE-cellulose, DNA-cellulose, phosphocellulose, and hydroxyapatite, the EHV-1-induced polymerase was purified 500-fold with 1–2% recovery of total activity from the nuclei of infected hamster livers. The final enzyme preparation was homogeneous as judged by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. Calculations based on Stokes radius, sedimentation coefficient, and electrophoretic mobility indicated that the native enzyme is composed of a single subunit having a molecular weight of 160,000. The purified enzyme exhibited anomalous gel filtration behavior indicating molecular asymmetry. It required Mg2+, dithiothreitol, alkaline pH (8–9), all four deoxyribonucleoside triphosphates, and 150 mM salt [K2SO4, (NH4)2SO4, or K2HPO4] for maximal activity, and utilized templates in the following order of preference: activated DNA (100%), poly(dA)·oligo(dT) (40%), poly(dC)·oligo(dG) (21%), native DNA (7%), denatured DNA (4%), and poly(rA)·oligo(dT) (3%). N-Ethylmaleimide, Zn2+, and phosphonoacetate acted as inhibitors of the EHV-1-induced DNA polymerase. Antiserum elicited against the EHV-1 DNA polymerase induced in hamsters inactivated the viral enzyme from infected mouse and equine cell cultures. However, the DNA polymerase induced by EHV-3 and DNA polymerases present in uninfected cells were not inhibited by the antiserum. These results support the hypothesis that a new, virus-coded DNA polymerase is induced after equine herpesvirus infection.
Publication Date: 1977-01-01 PubMed ID: 13532DOI: 10.1016/0042-6822(77)90311-7Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
- Journal Article
- Research Support
- U.S. Gov\'t
- P.H.S.
Summary
This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.
This study investigates the induction of a novel DNA polymerase activity following infection with equine herpesvirus type 1 (EHV-1) or type 3 (EHV-3), distinguishing it from regular host cell DNA polymerases. The enzyme was isolated and associated characteristics including its composition, activity preferences and inhibitors were detailed, as well as the implications of this for the virus’ effects on the host.
DNA Polymerase Induction
- The research examines the phenomenon of EHV-1 or EHV-3 infection inducing DNA polymerase activity different from host cell DNA polymerases. This was notably visible in the high salt requirement for maximal enzyme activity.
- The induced polymerase was purified, allowing a closer examination of its characteristics.
Enzyme Characteristics
- The isolated enzyme, after purification, showed homogeneity when judged by electrophoresis processes.
- Calculations using Stokes radius, sedimentation coefficient, and electrophoretic mobility showed the enzyme to consist of a single subunit with a molecular weight of 160,000.
- The enzyme showed ‘anomalous gel filtration behavior’, indicative of molecular asymmetry.
Activity Preferences and Inhibitors
- The purified enzyme’s activity required all four deoxyribonucleoside triphosphates, Mg²⁺, dithiothreitol, an alkaline pH, and high salt concentrations.
- The enzyme’s preference for templates was most to least in the following order: activated DNA, poly(dA)·oligo(dT), poly(dC)·oligo(dG), native DNA, denatured DNA, and poly(rA)·oligo(dT).
- Three specific inhibitors of the EHV-1 induced DNA polymerase were identified: N-Ethylmaleimide, Zn²⁺, and phosphonoacetate.
Antiserum Experiments
- The antiserum made against EHV-1 DNA polymerase in hamsters effectively inactivated the viral enzyme from infected mouse and equine cell cultures.
- In contrast, the DNA polymerase induced by EHV-3 and DNA polymerases present in uninfected cells were not affected by the hamster antiserum.
Implications and Conclusion
- The study’s results bolster a hypothesis that a new, virus-coded DNA polymerase is generated following an equine herpesvirus infection, providing a new perspective on the mechanisms of EHV-1 and EHV-3 infections.
Cite This Article
APA
Allen GP, O'Callaghan DJ, Randall CC.
(1977).
Purification and characterization of equine herpesvirus-induced DNA.
Virology, 76(1), 395-408.
https://doi.org/10.1016/0042-6822(77)90311-7 Publication
Researcher Affiliations
MeSH Terms
- Cell Line
- Chromatography, Ion Exchange
- DNA / biosynthesis
- DNA / metabolism
- DNA-Directed DNA Polymerase / analysis
- DNA-Directed DNA Polymerase / isolation & purification
- DNA-Directed DNA Polymerase / metabolism
- Electrophoresis, Polyacrylamide Gel
- Enzyme Induction
- Ethylmaleimide / pharmacology
- Herpesviridae / enzymology
- Hydrogen-Ion Concentration
- Molecular Weight
- Organophosphonates / pharmacology
- Polyribonucleotides / metabolism
- Zinc / pharmacology
Citations
This article has been cited 9 times.- Kim S, Ahn BC, O'Callaghan DJ, Kim SK. The early UL31 gene of equine herpesvirus 1 encodes a single-stranded DNA-binding protein that has a nuclear localization signal sequence at the C-terminus. Virology 2012 Oct 25;432(2):306-15.
- Lidin BI, Lamon EW. Effects of DNA synthesis inhibitors on early antigen expression following primary infection or superinfection by Epstein-Barr virus. Arch Virol 1983;77(1):13-25.
- Grossberger D, Clough W. Incorporation into DNA of the base analog 2-aminopurine by the Epstein-Barr virus-induced DNA polymerase in vivo and in vitro. Proc Natl Acad Sci U S A 1981 Dec;78(12):7271-5.
- McGowan JJ, Wagner RR. Inhibition of cellular DNA synthesis by vesicular stomatitis virus. J Virol 1981 Apr;38(1):356-67.
- Kallin B, Sternås L, Saemundssen AK, Luka J, Jörnvall H, Eriksson B, Tao PZ, Nilsson MT, Klein G. Purification of Epstein-Barr virus DNA polymerase from P3HR-1 cells. J Virol 1985 May;54(2):561-8.
- Holmes AM, Wietstock SM, Ruyechan WT. Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells. J Virol 1988 Mar;62(3):1038-45.
- Ooka T, Lenoir G, Daillie J. Characterization of an Epstein-Barr virus-induced DNA polymerase. J Virol 1979 Jan;29(1):1-10.
- Allen GP, O'Callaghan DJ, Randall CC. Genetic relatedness of equine herpesvirus types 1 and 3. J Virol 1977 Dec;24(3):761-7.
- Powell KL, Purifoy DJ. Nonstructural proteins of herpes simplex virus. I. Purification of the induced DNA polymerase. J Virol 1977 Nov;24(2):618-26.
Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists
