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Evidence for a herpesvirus saimiri-specified DNA polymerase activity which is aphidicolin-resistant and
Abstract:
Phosphonoacetic acid (PAA) effectively inhibited herpesvirus saimiri (HVS) replication and the onset of virus DNA synthesis. A PAA-resistant (Pr) mutant of HVS was isolated which plaqued efficiently in the presence of concentrations of PAA sufficient to reduce the growth of wild-type virus to less than 0.02% of control values. In contrast, virus growth and DNA synthesis in cells infected with unselected strains of herpesvirus saimiri was highly resistant to concentrations of aphidicolin, an inhibitor of alpha-type polymerases, which completely inhibited the growth and DNA replication of uninfected cells. An increased level of DNA polymerase activity was induced in cells infected with herpesvirus saimiri. This HVS-induced DNA polymerase was more sensitive to PAA but more resistant to aphidicolin in vitro than the uninfected cell activity and could also be distinguished on the basis of its response to ionic strength (40 to 50 mM-ammonium sulphate for optimal activity versus 20 mM for the uninfected cell activity). Under suitable in vitro assay conditions, an increase in the PAA-resistance of the DNA polymerase induced by the HVS(Pr) mutant was demonstrated. Comparison of the effects of aphidicolin and PAA demonstrated that the former was a much more effective and rapid inhibitor of susceptible cell DNA synthesis in vivo. Taken together, these results demonstrate novel properties of a DNA polymerase activity in cells infected with herpesvirus saimiri and suggest that aphidicolin should provide a useful reagent to analyse the functions of this enzyme in productive and non-productive infections with the virus.
Insights
Phosphonoacetic acid (PAA) inhibits herpesvirus saimiri (HVS) replication. A PAA-resistant HVS mutant revealed a unique viral DNA polymerase, offering new tools to study HVS infections.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Herpesvirus saimiri (HVS) replication and DNA synthesis are sensitive to phosphonoacetic acid (PAA).
- Aphidicolin, an inhibitor of alpha-type polymerases, affects uninfected cells but not HVS-infected cells.
- HVS infection induces increased DNA polymerase activity in host cells.
Purpose of the Study:
- To characterize the properties of the HVS-induced DNA polymerase.
- To investigate the differential sensitivity of viral and cellular DNA polymerases to inhibitors.
- To explore the potential of aphidicolin as a tool for analyzing HVS DNA polymerase function.
Main Methods:
- Isolation and characterization of a PAA-resistant HVS mutant.
- In vitro assays of DNA polymerase activity using PAA and aphidicolin.
- Comparison of viral and cellular DNA polymerase sensitivity to inhibitors and ionic strength.
Main Results:
- A PAA-resistant HVS mutant was isolated, exhibiting resistance to PAA at concentrations inhibiting wild-type virus.
- The HVS-induced DNA polymerase showed increased sensitivity to PAA and resistance to aphidicolin compared to uninfected cell polymerase.
- Optimal activity for HVS DNA polymerase occurred at higher ionic strength than the cellular enzyme.
- Aphidicolin was a more potent inhibitor of cellular DNA synthesis in vivo than PAA.
Conclusions:
- HVS encodes a DNA polymerase with distinct biochemical properties from the host cell enzyme.
- The differential sensitivity of viral and cellular DNA polymerases to PAA and aphidicolin can be exploited for mechanistic studies.
- Aphidicolin is a valuable reagent for dissecting the roles of HVS DNA polymerase in productive and non-productive viral infections.