John A. Boezi

Treatment of experimental herpesvirus infections with phosphonoformate and some comparisons with phosphonoacetate.

Phosphonoformate (PF) at a concentration of 5 to 10 μg/ml inhibited the growth of type 1 strains of herpes simplex virus (HSV) in tissue culture, whereas 20 to 30 μg/ml was required for inhibition of type 2 strains and about 50 μg/ml was required for murine cytomegalovirus. In mice inoculated intraperitoneally or intracerebrally with HSV or intraperitoneally with murine cytomegalovirus, treatment with 250 to 400 mg of PF per kg twice daily for 5 days had only minimal effectiveness. When mice were inoculated intravaginally (i.vg.) with HSV type 2 and treated i.vg. with 10% PF beginning 3 h after viral inoculation, treatment was effective in completely inhibiting viral replication in the genital tract. If i.vg. therapy was initiated 24 h after infection, when the mice had a mean virus titer of 105 plaque-forming units in vaginal secretions, a significant reduction in the mean virus titer was observed on days 3, 5, and 7 after infection as compared with control animals. In guinea pigs treated i.vg. with 10% PF beginning 6 h after i.vg. inoculation with HSV type 2 there was also complete inhibition of viral replication in the genital tract, and no extenal lesions developed. When therapy was initiated 24 h after infection there was a 4 to 5-log decrease in viral titers on days 3, 5, and 7 of the infection and a slight delay in the development of external lesions.

Inhibition of Herpesvirus Replication and Herpesvirus-Induced Deoxyribonucleic Acid Polymerase by Phosphonoformate

Phosphonoformate was found to be an inhibitor of the deoxyribonucleic acid polymerase induced by the herpesvirus of turkeys. The apparent inhibition constants were 1 to 3 μM. Phosphonoformate was also able to block the replication in cell culture of Mareks disease herpesvirus, the herpesvirus of turkeys, and herpes simplex virus. It was as effective as phosphonoacetate. Phosphonoformate was not an effective inhibitor of a phosphonoacetate-resistant mutant of the herpesvirus of turkeys nor of its induced deoxyribonucleic acid polymerase.

Isolation and characterization of a UV-sensitive hypermutable aphidicolin-resistant Chinese hamster cell line

Aphidicolin is a specific inhibitor of DNA polymerase α and blocks DNA synthesis in vivo. The inhibition of purified α-polymerase has been shown to be competitive with dCTP but not with the other three deoxynucleoside triphosphates (dNTPs). In order to study the various roles that the α-polymerase might play in DNA replication and/or repair, we have attempted to isolate Chinese hamster V79 cells that are resistant to aphidicolin. Four resistant mutants were isolated from BrdU-black light- and UV-mutagenized cells. None of the mutants isolated contains an α-polymerase that is resistant, in crude extract measurements, to aphidicolin. Three mutants isolated, however, were found to be resistant to araC. Two mutants tested were found to be sensitive to cytidine and have elevated levels of dCTP or all 4 dNTPs. These results indicate that they are nucleotide pool mutants instead of α-polymerase mutants. One mutant, aphr-4, is characterized by the following: (1) high level of dCTP; (2) thymidine (or CdR, UdR, auxotrophic; (3) sensitive to thymidine (and AdR, GdR); (4) slow-growing; (5) cytidine sensitive; (6) UV sensitive and hypermutable at the ouabain-resistant locus; and (7) a ninefold increase in frequency of chromatid gaps and breaks when cells are exposed to BrdU- containing medium. Revertants of aphr-4 which are partially aphidicolin-resistant and retain the first three characteristics listed above, but not the others, have been isolated. The appearance of this type of revertant indicates that either aphr- 4 or its “revertant” is a double mutant.

An enzymic method for determination of inorganic pyrophosphate and its use as an assay for RNA polymerase

1. 1. An enzymic method for the determination of PP1 has been developed. PP1 can be quantitatively estimated from the amount of NADPH formed via the action of UDP-glucose pyrophosphorylase, phosphoglucomutase, and glucose-6-phosphate dehydrogenase. Neither P1 nor any of the organic phosphate compounds tested interfered in the PP1 determination. 2. 2. The method has been used for the assay of DNA-dependent RNA polymerase—by coupling the generation of PP1 by RNA polymerase with NADPH formation. The RNA polymerase catalyzed syntheses of RNA and polyriboadenylate have been assayed by this procedure.

Inhibition of eucaryotic DNA polymerases by phosphonoacetate and phosphonoformate.

Abstract Phosphonoacetate was found to be an inhibitor of the DNA polymerase α from three human cells, HeLa, Wi-38, and phytohemagglutinin-stimulated lymphocytes. The inhibition patterns were determined. The apparent inhibition constants (Kii) were about 30 μ m . Thus the DNA polymerase α is 15 to 30 times less sensitive to Phosphonoacetate than the herpesvirus-induced DNA polymerase. The DNA polymerase α from Chinese hamster ovary cells and calf thymus was also inhibited. The DNA polymerases β and γ from the eucaryotic cells were relatively insensitive to phosphonoacetate. The sensitivity of the DNA polymerase α and the relative insensitivity of the DNA polymerase β and γ appeared to be general characteristics of the vertebrate polymerases, DNA polymerases from two other eucaryotic cells, yeast DNA polymerase A and B and tobacco cell DNA polymerase, were inhibited by phosphonoacetate, and to about the same extent as the α-polymerases. Fourteen phosphonate analogs were examined for inhibition of the HeLa DNA polymerase α. Only one, phosphonoformate, was an inhibitor. The mechanism of inhibition for phosphonoformate was analogous to that for phosphonoacetate.

Selection of aphidicolin-resistant CHO cells with altered levels of ribonucleotide reductase.

Chinese hamster ovary cells were initially selected for resistance to aphidicolin at 0.3 μg/ml. Serial cultivation with aphidicolin at concentrations up to 5.0 μg/ml yielded a series of mutants with increasing resistance. The most resistant mutant isolated was 44 times more resistant to aphidicolin than the parental CHO. The α-polymerases, assayed in the cytoplasmic extracts of the mutants, did not increase in specific activity or differ from the parental CHO in their sensitivity to aphidicolin. When cultured in the presence of deoxythymidine, deoxyadenosine, and 1-β-d-arabinofuranosyl cytosine (araC) the mutants showed considerably more resistance to these inhibitors than did the parental CHO. The intracellular pools of all four deoxynucleoside triphosphates (dNTPs) in the mutants increased with increasing resistance to aphidicolin. The elevated dNTP pools in the mutant most resistant to aphidicolin appear to be the result of a 4- to 8-fold increase in the level of ribonucleotide reductase (2′-deoxyribonucleoside diphosphate: oxidized thioredoxin 2′- oxidoreductase, EC 1.17.4.1).

Studies of Escherichia coli ribonucleic acid-deoxyribonucleic acid complex

Abstract The reaction of Escherichia coli pulse-labeled [ 3 H]RNA and denatured DNA to form ribonuclease (EC 2.7.7.16) resistant RNA-DNA complex was studied. The reaction is optimal at 78° in 0.50 M KCl, 0.01 M Tris (pH 7.3). Native E. coli DNA or denatured DNA extracted from Bacillus subtilis or Pseudomonas fluorescens does not react with E. coli [ 3 H]RNA. RNA-DNA complex formation is dependent upon both RNA and denatured DNA concentrations. In 0.50 M KCl, 0.01 M Tris (pH 7.3) the complex melts with a denaturation temperature, t m , of 94–95°. Under the same conditions, native DNA melts with a t m of 99°. Approx. one-third of pulse-labeled [ 3 H]RNA reacts readily with denatured DNA. Competition between pulse-labeled RNA and unlabeled RNA for reaction with denatured DNA was used to compare RNAs from cells grown under different conditions. Cells grown on C medium with casamino acids and yeast extract and those treated with chloramphenicol contain all or most of the messenger RNA present in cells grown on glucose-C medium. Little or no messenger RNA is present in cells starved for glucose.

Template activity of 2′-O-methylpolyribonucleotides with Pseudomonas putida DNA-dependent RNA polymerase

Summary Pseudomonas putida RNA polymerase can use the single stranded 2′- O -methylpolyribonucleotides, poly(Um) and poly(Cm), as templates for the synthesis of poly(A) and poly(G), respectively. No template activity was detected with the purine-containing 2′- O -methylated homopolymers, poly(Am) and poly(Im). The poly(Am) strand of either poly(Am)·poly(U) or poly(Am)·poly(Um) was not a template for poly(U) synthesis, and did not prevent the poly(U) or poly(Um) strand of the duplex from serving as a template for poly(A) synthesis. The poly(Um) strand of the duplex poly(Am)·poly(Um) was an effective template for poly(A) synthesis, but the poly(Um) strand of poly(A)·poly(Um) was not.