Human cytomegalovirus. IV. Specific inhibition of virus-induced DNA polymerase activity and viral DNA replication by phosphonoacetic acid.

Phosphonoacetic acid specifically inhibited human cytomegalovirus DNA synthesis in virus-infected human fibroblasts as detected by virus-specific nucleic acid hybridization. Inhibition was reversible; viral DNA synthesis resumed upon the removal of the drug. The compound partially inhibited DNA synthesis of host cells in the log phase of growth but had little effect on confluent cells. Studies of partially purified enzymes indicated that phosphonoacetic acid specifically inhibited virus-induced DNA polymerase and had only a slight effect on normal host cell enzymes. The drug was shown to interact directly with virus-induced enzyme but not with the template-primers.     

Genetic evidence for vaccinia virus-encoded DNA polymerase: isolation of phosphonoacetate-resistant enzyme from the cytoplasm of cells infected with mutant virus.

Phosphonoacetate (PAA), at concentrations of 200 micrograms/ml or more, prevented growth of vaccinia virus in HeLa and BSC-1 cells. Spontaneous vaccinia virus mutants, selected at high PAA levels, were resistant to the antiviral effects of the drug. The action of PAA was directed toward an early viral function, since the drug was inhibitory only during the first 4 h of the approximately 15-h growth cycle. Conversely, significant reversal of the antiviral effects was obtained only when the drug was removed at or before the fourth hour of infection. Incorporation of [3H]thymidine into cytoplasmic viral DNA was severely inhibited in cells infected with wild-type virus but not in cells infected with mutant virus. Virus-induced DNA polymerase isolated from the cytoplasm of cells infected with wild-type or mutant virus had indistinguishable chromatographic properties on DEAE-cellulose and phosphocellulose columns. However, the wild-type enzyme was inhibited by relatively low concentrations of PAA, whereas 10-fold higher concentrations were needed for equivalent inhibition of the mutant enzyme. Kinetic analysis indicated that PAA inhibition was noncompetitive with deoxyribonucleoside triphosphates; Ki values for wild-type and mutant DNA polymerases were approximately 25 and 300 microM, respectively. Inhibition of wild-type DNA polymerase was immediate and complete even when PAA was added after initiation of DNA synthesis in vitro, suggesting that chain elongation was affected. These results established that the DNA polymerase is a target of the antiviral action of PAA and provided genetic evidence that this enzyme is virus encoded.     

Taxol inhibits stimulation of cell DNA synthesis by human cytomegalovirus

The microtubule (MT)-stabilizing drug, taxol, inhibited human cytomegalovirus (CMV)-initiated cell DNA synthesis by up to 100% in serum-arrested mouse embryo (ME) fibroblasts that were abortively infected by CMV. Taxol concentrations known to increase MT polymerization and to stabilize existing MTs (10 to 20 micrograms/ml) blocked CMV-stimulated cell DNA synthesis, while taxol concentrations of 2.5 micrograms/ml, or less, did not. Taxol maximally inhibited CMV initiation of cell DNA synthesis when added 3 h after virus infection and inhibited this initiation by greater than 50% when added up to 12 h after CMV infection. Control experiments suggest that taxol specifically inhibited CMV-stimulated cell DNA synthesis. Pretreatment of CMV stock with taxol did not reduce the stimulatory effect of CMV on cell DNA synthesis and taxol had no detectable effect on CMV-specific early protein synthesis. Moreover, taxol did not appear to alter thymidine pool sizes, affect cell viability, or compromise the DNA synthetic machinery in CMV-infected cells. Since taxol increases tubulin polymerization and inhibits MT disassembly, these results suggest that dynamic changes in MTs or in the pool of free tubulin subunits are necessary for CMV to stimulate cell entry into a proliferative cycle.     

Inhibition of herpes simplex virus-induced DNA polymerase activity and viral DNA replication by 9-(2-hydroxyethoxymethyl)guanine and its triphosphate.

The effect of the nucleoside analog 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine) on herpes simplex virus type 1 DNA synthesis was examined. Acycloguanosine inhibited herpesvirus DNA synthesis in virus-infected cells. The synthesis of host cell DNA was only partially inhibited in actively growing cells at acycloguanosine concentrations several hundred-fold greater than the 50% effective dose for herpes simplex virus type 1. Studies using partially purified enzymes revealed that the triphosphate of this compound inhibited the virus-induced DNA polymerases (DNA nucleotidyltransferases) to a greater degree than the DNA polymerase of the host cell, that the inhibition was dependent upon the base composition of the template, and that the triphosphate was a better substrate for the virus-induced polymerases than for the alpha cellular DNA polymerases.     

Inhibition of herpes simplex virus DNA polymerase by purine ribonucleoside monophosphates

Purine ribonucleoside monophosphates were found to inhibit chain elongation catalyzed by herpes simplex virus (HSV) DNA polymerase when DNA template-primer concentrations were rate-limiting. Inhibition was fully competitive with DNA template-primer during chain elongation; however, DNA polymerase-associated exonuclease activity was inhibited noncompetitively with respect to DNA. Combinations of 5'-GMP and phosphonoformate were kinetically mutually exclusive in dual inhibitor studies. Pyrimidine nucleoside monophosphates and deoxynucleoside monophosphates were less inhibitory than purine riboside monophosphates. The monophosphates of 9-beta-D-arabinofuranosyladenine, Virazole (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), 9-(2-hydroxyethoxymethyl)guanine, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine exerted little or no inhibition. In contrast to HSV DNA polymerase, human DNA polymerase alpha was not inhibited by purine ribonucleoside monophosphates. These studies suggest the possibility of a physiological role of purine ribonucleoside monophosphates as regulators of herpesvirus DNA synthesis and a new approach to developing selective anti-herpesvirus compounds.     

Pyrophosphate analogues as inhibitors of DNA polymerases of cytomegalovirus, herpes simplex virus and cellular origin

Several pyrophosphate analogues have been compared for their ability to inhibit the activities of isolated cytomegalovirus (CMV) DNA polymerase, herpes simplex virus type 1 (HSV 1) DNA polymerase and calf thymus DNA polymerase alpha. The most effective inhibitors were phosphonoformate and phosphonoacetate. Although not identical, the structural requirements for compounds inhibitory to CMV and HSV-1 DNA polymerase were specific, with two negatively charged groups in close vicinity. The CMV DNA polymerase was more susceptible to certain phosphonoacetates containing bulky hydrophobic alpha-substituents than was the HSV-1 DNA polymerase. No example of the converse preference was found. The inhibition of CMV DNA polymerase by phosphonoformate, hypophosphate, alpha-hydroxyphosphonoacetate and alpha-nonylphosphonoacetate was linear non-competitive with the deoxyribonucleoside triphosphates as variable substrates. Phosphonoformate, phosphonoacetate, and to a lesser extent alpha-hydroxyphosphonoacetate, carbonyldiphosphonate and alpha-nonylphosphonoacetate also inhibited the focus formation by CMV in cell-culture.     

Cytofluorometry of lymphocytes infected with Epstein-Barr virus: effect of phosphonoacetic acid on nucleic acid.

DNA synthesis in Epstein-Barr virus (EBV)-infected lymphocytes was inhibited by phosphonoacetic acid (PAA) as measured by [3H]thymidine incorporation. PAA, at a concentration of 200 microgram/ml, inhibited [3H]thymidine incorporation by human umbilical cord lymphocytes infected with EBV strain P94 but had little effect on DNA synthesis in mitogen-stimulated cells. Transformed cell lines did not develop from infected cord cell cultures treated with 100 microgram of PAA per ml. Cytofluorometric analysis showed marked increases in cellular nucleic acid content (RNA plus DNA) as early as 9 days after infection of cord cells in the absence of PAA and before significant enhancement of [3H]thymidine incorporation became apparent. Moreover, EBV led to increases in cellular nucleic acid even when 200 microgram of PAA per ml was added to cell cultures before infection. The apparent discrepancy between results obtained by [3H]thymidine incorporation and cytofluorometry is explained either by significant inhibition of cellular DNA polymerases by PAA or by a block at the G2 + M phase of the cell cycle. The data suggest that EBV initiates alterations in cellular nucleic acid synthesis or cell division without prior replication of viral DNA by virus-induced DNA polymerases.     

Involvement of DNA polymerase delta in DNA repair synthesis in human fibroblasts at late times after ultraviolet irradiation

DNA repair synthesis following UV irradiation of confluent human fibroblasts has a biphasic time course with an early phase of rapid nucleotide incorporation and a late phase of much slower nucleotide incorporation. The biphasic nature of this curve suggests that two distinct DNA repair systems may be operative. Previous studies have specifically implicated DNA polymerase delta as the enzyme involved in DNA repair synthesis occurring immediately after UV damage. In this paper, we describe studies of DNA polymerase involvement in DNA repair synthesis in confluent human fibroblasts at late times after UV irradiation. Late UV-induced DNA repair synthesis in both intact and permeable cells was found to be inhibited by aphidicolin, indicating the involvement of one of the aphidicolin-sensitive DNA polymerases, alpha or delta. In permeable cells, the process was further analyzed by using the nucleotide analogue (butylphenyl)-2'-deoxyguanosine 5'-triphosphate, which inhibits DNA polymerase alpha several hundred times more strongly than it inhibits DNA polymerase delta. The (butylphenyl)-2'-deoxyguanosine 5'-triphosphate inhibition curve for late UV-induced repair synthesis was very similar to that for polymerase delta. It appears that repair synthesis at late times after UV irradiation, like repair synthesis at early times, is mediated by DNA polymerase delta.     

Herpes simplex virus resistance and sensitivity to phosphonoacetic acid.

Phosphonoacetic acid (PAA) inhibited the synthesis of herpes simplex virus DNA in infected cells and the activity of the virus-specific DNA polymerase in vitro. In the presence of concentrations of PAA sufficient to prevent virus growth and virus DNA synthesis, normal amounts of early virus proteins (alpha- and beta-groups) were made, but late virus proteins (gamma-group) were reduced to less than 15% of amounts made in untreated infected cells. This residual PAA-insensitive synthesis of gamma-polypeptides occurred early in the virus growth cycle when rates were identical in PAA-treated and untreated infected cells. Passage of virus in the presence of PAA resulted in selection of mutants resistant to the drug. Stable clones of mutant viruses with a range of drug sensitivities were isolated and the emergence of variants resistant to high concentrations of PAA involved the sequential selection of mutants progressively better adapted to growth in the presence of the drug. Increased drug resistance of virus yield or plaque formation was correlated with increased resistance of virus DNA synthesis, gamma-protein synthesis, and resistance of the virus DNA polymerase reaction in vitro to the inhibitory effects of the drug. PAA-resistant strains of herpes simplex virus type 1 (HSV-1) complemented the growth of sensitive strains of homologous and heterologous types in mixed infections in the presence of the drug. Complementation was markedly dependent upon the proportions of the resistant and sensitive partners participating in the mixed infection. Intratypic (HSV-1A X HSV-1B) recombination of the PAA resistance marker(s), Pr, occurred at high frequency relative to plaque morphology (syn) and bromodeoxyuridine resistance (Br, thymidine kinase-negative phenotype) markers, with the most likely order being syn-Br-Pr. Recombinant viruses were as resistant or sensitive to PAA as the parental viruses, and viruses recombinant for their PAA resistance phenotype were also recombinant for the PAA resistance character of the virus DNA polymerase. The results provide additional evidence that the herpesvirus DNA polymerase is the site of action of PAA and illustrate the potential usefulness of PAA-resistant mutants in genetic studies of herpesviruses.     

H_(18)细胞中Epstein-Barr病毒DNA复制的抑制和恢复

采用~(32)P标记的EBV DNA BamH Ⅰ-W片段为探针,通过细胞点杂交测出每个H_(18)细胞中EBV基因组拷贝数约207个。在PAA存在下,这些病毒基因组数快速下降。每毫升细胞培液中加75γPAA后第11天,每个细胞中EBV基因组平均拷贝数降到17个,只占未处理细胞的8%,而后既使继续处理也稳定地维持在该水平。然而EBV-VCA的合成被完全地抑制了,只是EBV-EA的合成不受影响。如去除PAA,第3天时细胞中EBV基因组数可恢复到对照组的29%,而第40天则可恢复到90%。但是在去除PAA处理的同时加n-BA处理则明显地抑制这种恢复。一般认为EBV附加体DNA的复制由宿主细胞DNA多聚酶催化,病毒线状DNA由病毒诱导的PAA敏感的DNA多聚酶复制。由于n-BA能有效地阻碍宿主的DNA复制功能,因而我们的实验结果提示,宿主细胞和病毒诱导的DNA多聚酶??可能都参与了去除PAA处理后的H_(18)细胞中EBV DNA的恢复。     

Specific inhibition of human DNA ligase adenylation by a distamycin derivative possessing antitumor activity.

The antiviral distamycin A and its phenyl mustard derivative FCE24517 possessing antitumor activity were tested for their ability to inhibit macromolecular synthesis in three human and one murine cell line. While distamycin A was poorly active in these systems, FCE24517 inhibited DNA synthesis efficiently, RNA synthesis to a lower extent and had little effect on protein synthesis. These findings suggest that the in vivo activity of FCE24517 derives from the specific inhibition of DNA synthesis. When the two drugs were tested on several enzymes involved in human DNA metabolism a strikingly similar pattern of inhibition appeared, with distamycin A being the more potent. Both drugs showed: A), no inhibitory activity against thymidine kinase and DNA primase; B), low activity against DNA topoisomerases I and II and the 3'-5' exonuclease associated with the DNA polymerase epsilon; C), high activity against DNA polymerases alpha and epsilon, uracil-DNA glycosylase and the joining activity of the replicative DNA ligase; D), the highest inhibitory activity against the AMP-dependent DNA relaxing activity of DNA ligase. The strong in vitro inhibition of several DNA enzymatic activities, including DNA ligase, do not match with the in vivo activities of the two drugs. However a unique difference was observed: only FCE24517 inhibited the DNA-independent reaction of adenylation of human DNA ligase while the adenylation reaction of T4 and E. coli DNA ligase was unaffected by either drug. It is still unclear whether these properties are relevant for modulating the killing activity of FCE24517 against proliferating cells both in culture and in vivo. Nevertheless FCE24517 is the first known molecule capable of interacting directly and specifically with human DNA ligase.     

Aphidicolin inhibition of the production of replicative-form DNA during bovine parvovirus infection.

Since parvoviruses apparently do not possess a DNA polymerase activity, one or more of the host cell DNA polymerases must be responsible for replicating the single-stranded DNA genome. We have focused on determining which polymerase, alpha, beta, or gamma (pol alpha, pol beta, or pol gamma, respectively), is responsible for the first step in bovine parvoviral DNA replication: conversion of the single-stranded DNA genome to a parental replicative form (RF). In this study, we used aphidicolin, a specific inhibitor of DNA pol alpha, to assay for the requirement of pol alpha activity in parental RF formation in vivo. Synchronized cell cultures were infected with bovine parvovirus with or without aphidicolin, and the products of viral replication were separated on agarose gels and identified by Southern blot analysis. We found that complete inhibition of viral DNA synthesis resulted when 20 microM aphidicolin was present throughout the infection. In addition, viral DNA synthesis was inhibited by as little as 1 microM aphidicolin, whereas lower concentrations (0.1 and 0.01 microM) resulted in partial inhibition of the replication process. Using 32P-labeled bovine parvovirus as the input virus we differentiated parental RF from daughter RF and progeny DNA synthesis. We conclude that DNA pol alpha is required for the production of RF during bovine parvovirus replication in vivo and that this requirement is most likely for the conversion of bovine parvovirus input single-stranded DNA to parental RF. These results do not rule out a possible role for DNA pol gamma in the first step, nor do they rule out a role for pol alpha or pol gamma in later stages of the replication cycle.     

Replication of human cytomegalovirus DNA: lack of dependence on cell DNA synthesis.

Human cytomegalovirus (CMV) DNA synthesis was studied in 5-fluorouracil (FU)-treated and untreated human embryonic lung cells, which differ greatly with respect to the number of cells in the culture synthesizing cellular DNA. CMV DNA synthesis proceeded at the same rate in FU-treated and in untreated cells. CMV infection also reversed the inhibitory effects of FU and activated cellular DNA synthesis in some of the cells in the FU-treated culture. Autoradiographic studies showed that more than 20% of the cells in the infected FU-treated culture synthesized viral DNA when less than 1% had synthesized cellular DNA, indicating that the synthesis of viral macromolecules proceeds in cells that do not synthesize cellular DNA from the time of infection, and that viral DNA synthesis proceeds independently of the host cell DNA synthesis. Combined autoradiographic and immunofluorescence studies of both the FU-treated and untreated infected cells showed that, whereas 20% of the cells in the cultures synthesize viral DNA and viral antigens, only about 3 to 6% of those cells that synthesize cellular DNA also synthesize viral antigen. Thus, productive infection was delayed or inhibited in those cells that were stimulated by CMV infection to synthesize cellular DNA.     

Inhibition of purified human and herpes simplex virus-induced DNA polymerases by 9-(2-hydroxyethoxymethyl)guanine triphosphate. Effects on primer-template function

The inhibition of highly purified herpes simplex virus (HSV)-induced and host cell DNA polymerases by the triphosphate form of 9-(2-hydroxyethoxymethyl)guanine (acyclovir; acycloguanosine) was examined. Acyclovir triphosphate (acyclo-GTP) competitively inhibited the incorporation of dGMP into DNA, catalyzed by HSV DNA polymerase; apparent Km and Ki values of dGTP and acyclo-GTP were 0.15 microM and 0.003 microM, respectively. HeLa DNA polymerase alpha was also competitively inhibited; Km and Ki values of dGTP and acyclo-GTP were 1.2 microM and 0.18 microM, respectively. In contrast, HeLa DNA polymerase beta was insensitive to the analogue. The "limited" DNA synthesis observed when dGTP was omitted from HSV or alpha DNA polymerase reactions was inhibited by acyclo-GTP in a concentration-dependent manner. Prior incubation of activated DNA, acyclo-GTP, and DNA polymerase (alpha or HSV resulted in a marked decrease in the utilization of the primer-template in subsequent DNA polymerase reactions. This decreased ability of preincubated primer-templates to support DNA synthesis was dependent on acyclo-GTP, enzyme concentration, and the time of prior incubation. Acyclo-GMP-terminated DNA was found to inhibit HSV DNA polymerase-catalyzed DNA synthesis. Kinetic experiments with variable concentrations of activated DNA and fixed concentrations of acyclo-GMP-terminated DNA revealed a noncompetitive inhibition of HSV-1 DNA polymerase. The apparent Km of 3'-hydroxyl termini was 1.1 X 10(-7) M, the Kii and Kis of acyclo-GMP termini in activated DNA were 8.8 X 10(-8) M and 2.1 X 10(-9) M, respectively. Finally, 14C-labeled acyclo-GMP residues incorporated into activated DNA by HSV-1 DNA polymerase could not be excised by the polymerase-associated 3',5'-exonuclease activity.     

Herpes simplex virus-specified DNA polymerase is the target for the antiviral action of 9-(2-phosphonylmethoxyethyl)adenine.

9-(2-Phosphonylmethoxyethyl)adenine (PMEA) is a new antiviral compound with activity against herpes simplex virus (HSV) and retroviruses including human immunodeficiency virus. Although it has been suggested that the anti-HSV action of PMEA is through inhibition of the viral DNA polymerase via the diphosphorylated metabolite of PMEA (PMEApp), no conclusive evidence for this has been presented. We report that in cross-resistance studies, a PMEA-resistant HSV variant (PMEAr-1) was resistant to phosphonoformic acid, a compound which directly inhibits the HSV DNA polymerase. In addition, phosphonoformic acid-resistant HSV variants with defined drug resistance mutations within the HSV DNA polymerase gene were resistant to PMEA. Furthermore, the HSV DNA polymerase purified from PMEAr-1 was resistant to PMEApp in comparison with the enzyme from the parental virus. Moreover, PMEA inhibited HSV DNA synthesis in cell culture. These results provide strong evidence that HSV DNA polymerase is the major target for the anti-viral action of PMEA. Further studies showed that HSV DNA polymerase incorporated PMEApp into DNA in vitro, while the HSV polymerase-associated 3'-5' exonuclease was able to remove the incorporated PMEA. Thus, the inhibition of HSV DNA polymerase by PMEApp appears to involve chain termination after its incorporation into DNA.     

c-myc protein and DNA replication: separation of c-myc antibodies from an inhibitor of DNA synthesis.

Antibodies against human c-myc protein have been reported to inhibit DNA polymerase activity and endogenous DNA synthesis in isolated nuclei, suggesting a role for c-myc in DNA replication. Using the same antibody preparations, we observed equivalent inhibition of simian virus 40 DNA replication and DNA polymerase alpha and delta activities in vitro, as well as inhibition of DNA synthesis in isolated nuclei. However, the c-myc antibodies could be completely separated from the DNA synthesis inhibition activity. c-myc antibodies prepared in other laboratories also did not interfere with initiation of simian virus 40 DNA replication, DNA synthesis at replication forks, or DNA polymerase alpha or delta activity. Therefore, the previously reported inhibition of DNA synthesis by some antibody preparations resulted from the presence of an unidentified inhibitor of DNA polymerases alpha and delta and not from the action of c-myc antibodies.     

Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and preliminary characterization of a phosphonoacetic acid-resistant and temperature-sensitive mutant of herpes simplex virus type 1.

A group of 43 phosphonoacetic acid (PAA)-resistant mutants of herpes simplex virus type 1 was isolated after the mutagenesis of infected cells with nitrosoguanidine. One of these mutants, designated PAA1rts1, was found to be temperature sensitive (ts), that is, unable to replicate at 39.5 degrees C, the nonpermissive temperature. Recombination analysis of PAA1rts1 indicated that the PAA1r mutation and the ts1 mutation are loosely linked and are located on two separate genes. PAA1rts1 showed a defect in viral DNA synthesis at 39.5 degrees C, which presumably can be attributed to the production of a PAA-resistant and thermolabile DNA polymerase. PAA1rts1 was also defective in the shutoff of host DNA synthesis at the restrictive temperature.