SUMMARYMicrococcal nuclease digestion was used to probe the structures in which herpes simplex virus type I (HSV-0 DNA is found during virus replication. Parental DNA, progeny DNA and DNA in nucleocapsids were analysed. Parental DNA was examined after infection of Vero cells with 32p. or 3H-thymidinelabelled HSV-L Progeny DNA was examined after HSV-I-infected Vero cells were pulse-labelled with 3H-thymidine during HSV-I DNA synthesis. In both cases, nuclei were isolated and digested with micrococcal nuclease. Digestion products were analysed by agarose or polyacrylamide gel electrophoresis (PAGE). Most parental DNA remained as intact molecules. However, a small amount was degraded into fragments which were heterogeneous in size or the size of nucleosomal cell DNA. These two classes of fragments were also produced upon digestion of progeny DNA. The heterogeneous fragments and nucleosomal fragments comprised major and minor fractions, respectively, of digested progeny DNA. When digested DNA from HSV-1-infected cells was transferred from composite polyacrylamide-agarose gels to diazobenzyloxymethyl paper, nucleosomal fragments hybridized to 32P-labelled HSV-l DNA as well as to 32P-labelled Vero cell DNA. Therefore, nucleosomal fragments contained HSV-I DNA sequences. HSV-I DNA in nucleocapsids was analysed by micrococcal nuclease digestion after nucleocapsids were disrupted with pH 9"3 buffer, pyridine, Sarkosyl or NaC1/urea. Only fragments of heterogeneous size were produced. Thus, HSV-~ DNA is found predominantly in structures other than nucleosomes during virus replication.
Phosphonoacetate was an effective inhibitor of both the Marek's disease herpesvirus- and the herpesvirus of turkey-induced DNA polymerase. Using the herpesvirus of turkey-induced DNA polymerase, phosphonoacetate inhibition studies for the DNA polymerization reaction and for the deoxyribonucleoside triphosphate-pyrophosphate exchange reaction were carried out. The results demonstrated that phosphonoacetate inhibited the polymerase by interacting with it at the pyrophosphate binding site to create an alternate reaction pathway. A detailed mechanism and rate equation for the inhibition were developed. For comparison to phosphonoacetate, pyrophosphate inhibition patterns and apparent inhibition constants were determined. Twelve analogues of phosphonoacetate were tested as inhibitors of the herpesvirus of turkey-induced DNA polymerase. At the concentrations tested, only one, 2-phosphonopropionate, was an inhibitor. The apparent inhibition constant for it was about 50 times greater than the corresponding apparent inhibition constant for phosphonoacetate. DNA polymerase alpha of duck embryo fibroblasts, the host cell for the herpesviruses, was inhibited by phosphonoacetate. The apparent inhibition constants for the alpha polymerase were about 10-20 times greater than the corresponding inhibition constants for the herpesvirus-induced DNA polymerase. Duck DNA polymerase beta, Escherichia coli DNA polymerase I, and avian myeloblastosis virus reverse transcriptase were not inhibited by phosphonoacetate.
Phosphonoacetate (PA), but not any of its analogues tested, effectively inhibited avian herpesvirus replication and viral DNA synthesis in cell cultures. At 100 mug/ml culture medium, PA completely inhibited the replication of Marek's disease virus (MDV), herpesvirus of turkeys, and owl herpesvirus, but had no measurable effect on normal cell growth. PA also inhibited DNA polymerases induced by these avian viruses. Enzyme inhibition was 50% at a PA concentration of 0.2 mug/ml. At a concentration of 3-6 mug/ml, the compound also effected a 50% inhibition of alpha (maxi) enzyme of the host DNA polymerase. It had no effect on the host beta (mini) enzyme. When administered to chickens, PA did not inhibit the replication of MDV, nor did it prevent the development of lymphoma.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.