Purification and characterization of equine herpesvirus-induced DNA.

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.