DNA polymerase in nuclei isolated from herpes simplex virus type-2-infected cells. Characterization of the reaction product and inhibition by substrate analogs

Abstract

Nuclei isolated from herpes simplex virus (HSV) type 2-infected KB cells were examined for their capacity to serve as an in situ source of herpes DNA polymerase. In contrast to purified enzymes with added template, approx. 80% of the DNA synthesized in isolated nuclei was viral. The average size of DNA fragments labeled in vitro was 3.2 X 10(6) Da. Based on an increase in DNA density when nuclei were incubated in the presence of BrdUTP rather than dTTP, 16% of the nucleotides were added during the in vitro reaction. Sucrose gradient analysis of DNA polymerase activity in extracts of isolated nuclei demonstrated the nearly exclusive presence of herpes DNA polymerase. Km concentrations for the four dNTPs were from 0.14 to 0.55 microM. DNA synthesis was inhibited competitively by the 5'-triphosphates of ara-A and ara-C (Ki = 0.03 and 0.22 microM, respectively) but not by the 5'-triphosphate of dideoxythymidine. aATP also served as a substrate (Km = 0.014 microM) for the reaction. We conclude that nuclei from HSV-infected cells have significant advantages for the detailed study of inhibitors of herpesvirus replication.

Inhibition of herpes simplex virus DNA replication by ara-tubercidin

“…The average ZCi concentration of ara-tubercidin-TP derived from two experiments was 16 p,M and was similar in magnitude to the value (5 p.M) obtained using the partially purified enzyme. In contrast, we previously reported that the S-triphosphate of vidarabine had a ZCi concentration of 0.03 FM in this system (Barnett et al, 1984). The >500-fold difference in potency is similar to that noted above in HSV-1 titer reduction experiments with these two compounds and further implicates the viral DNA polymerase as possessing a significant role in the antiviral activity of ara-tubercidin.…”

“…(2) Viral DNA synthesis also was measured in nuclei isolated from HSV-2 infected cells. We previously have shown that this system provides a convenient in situ source of herpes DNA polymerase under conditions in which the natural replication complex remains intact (Barnett et al, 1984). In brief, monolayer cultures of KB cells were infected with the X-79 strain of HSV-2 (obtained from Dr. E.R.…”

“…In brief, monolayer cultures of KB cells were infected with the X-79 strain of HSV-2 (obtained from Dr. E.R. Kern, University of Utah) at a multiplicity of infection of 2 PFU/cell and harvested 9 h post-infection (Barnett et al, 1984). Nuclei were isolated and DNA synthesis measured by quantitating the amount of [3H]dATP incorporated into acid-insoluble material.…”