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Antimicrobial Agents and Chemotherapy Jan 1978The phosphonacetic acid (PAA)-susceptible deoxyribonucleic acid (DNA) polymerase of herpes simplex virus type 1 was partially purified and isolated in sucrose gradients...
The phosphonacetic acid (PAA)-susceptible deoxyribonucleic acid (DNA) polymerase of herpes simplex virus type 1 was partially purified and isolated in sucrose gradients and on double-strand DNA cellulose columns. The DNA polymerase isolated from cells infected with the PAA-resistant mutant had the same molecular weight as the wild-type enzyme (140,000 to 149,000) but was consistently more resistant to PAA.
Topics: DNA-Directed DNA Polymerase; Mutation; Organophosphorus Compounds; Phosphonoacetic Acid; Simplexvirus
PubMed: 204249
DOI: 10.1128/AAC.13.1.124 -
Antimicrobial Agents and Chemotherapy Sep 1978Phosphonoacetic acid inhibited replication of simian varicella virus (Delta herpesvirus) in tissue culture. The drug was tested in patas monkeys 40 h after infection...
Phosphonoacetic acid inhibited replication of simian varicella virus (Delta herpesvirus) in tissue culture. The drug was tested in patas monkeys 40 h after infection with Delta herpesvirus. A total of 200 mg/kg per day was given intramuscularly, divided into two doses every day for a total of 10 days. The treated monkeys were protected from clinical illness, and Delta herpesvirus was not recovered from their lymphocytes. Complement-fixing and neutralizing antibody titers were significantly lower in phosphonoacetic acid-treated monkeys than in the untreated controls. In animals given the drug alone, there was dermatitis and blackening of the skin and hair, serum glutamic oxalacetic transaminase and serum glutamic pyruvic transaminase enzymes were significantly increased, and liver biopsy revealed diffuse cytoplasmic swelling and granulation of the hepatocytes. The therapeutic range of this drug should be studied carefully before considering its use in severe varicella-zoster infection in humans.
Topics: Animals; Erythrocebus patas; Haplorhini; Herpesviridae Infections; Herpesvirus 3, Human; Organophosphorus Compounds; Phosphonoacetic Acid
PubMed: 213014
DOI: 10.1128/AAC.14.3.331 -
Journal of Virology Sep 1981The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and...
The genome structures of herpes simplex virus type 1 (HSV-1)/HSV-2 intertypic recombinants have been previously determined by restriction endonuclease analysis, and these recombinants and their parental strains have been employed to demonstrate that mutations within the HSV DNA polymerase locus induce an altered HSV DNA polymerase activity, exhibiting resistance to three inhibitors of DNA polymerase. The viral DNA polymerases induced by two recombinants and their parental strains were purified and shown to possess similar molecular weights (142,000 to 144,000) and similar sensitivity to compounds which distinguish viral and cellular DNA polymerases. The HSV DNA polymerases induced by the resistant recombinant and the resistant parental strain were resistant to inhibition by phosphonoacetic acid, acycloguanosine triphosphate, and the 2',3'-dideoxynucleoside triphosphates. The resistant recombinant (R6-34) induced as much acycloguanosine triphosphate as did the sensitive recombinant (R6-26), but viral DNA synthesis in infected cells and the viral DNA polymerase activity were not inhibited. The 2',3'-dideoxynucleoside-triphosphates were effective competitive inhibitors for the HSV DNA polymerase, and the Ki values for the four 2',3'-dideoxynucleoside triphosphates were determined for the four viral DNA polymerases. The polymerases of the resistant recombinant and the resistant parent possessed a much higher Ki for the 2',3'-dideoxynucleoside triphosphates and for phosphonoacetic acid than did the sensitive strains. A 1.3-kilobase-pair region of HSV-1 DNA within the HSV DNA polymerase locus contained mutations which conferred resistance to three DNA polymerase inhibitors. This region of DNA sequences encoded for an amino acid sequence of 42,000 molecular weight and defined an active center of the HSV DNA polymerase enzyme.
Topics: Acyclovir; Antiviral Agents; Binding Sites; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Genes, Viral; Guanine; Mutation; Nucleotides; Phosphonoacetic Acid; Simplexvirus
PubMed: 6270349
DOI: 10.1128/JVI.39.3.746-757.1981 -
Journal of Virology Apr 1982A 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...
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.
Topics: DNA; DNA-Directed DNA Polymerase; Drug Resistance, Microbial; Mutation; Organophosphorus Compounds; Phosphonoacetic Acid; Simplexvirus; Temperature; Virus Replication
PubMed: 6283139
DOI: 10.1128/JVI.42.1.20-29.1982 -
American Journal of Physiology. Cell... Jan 2007During bone resorption, a large amount of inorganic phosphate (P(i)) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this P(i)...
During bone resorption, a large amount of inorganic phosphate (P(i)) is generated within the osteoclast hemivacuole. The mechanisms involved in the disposal of this P(i) are not clear. In the present study, we investigated the efflux of P(i) from osteoclast-like cells. P(i) efflux was activated by acidic conditions in osteoclast-like cells derived by the treatment of RAW264.7 cells with receptor activator of nuclear factor-kappaB ligand. Acid-induced P(i) influx was not observed in renal proximal tubule-like opossum kidney cells, osteoblast-like MC3T3-E1 cells, or untreated RAW264.7 cells. Furthermore, P(i) efflux was stimulated by extracellular P(i) and several P(i) analogs [phosphonoformic acid (PFA), phosphonoacetic acid, arsenate, and pyrophosphate]. P(i) efflux was time dependent, with 50% released into the medium after 10 min. The efflux of P(i) was increased by various inhibitors that block P(i) uptake, and extracellular P(i) did not affect the transport of [(14)C]PFA into the osteoclast-like cells. Preloading of cells with P(i) did not stimulate P(i) efflux by PFA, indicating that the effect of P(i) was not due to transstimulation of P(i) transport. P(i) uptake was also enhanced under acidic conditions. Agents that prevent increases in cytosolic free Ca(2+) concentration, including acetoxymethyl ester of 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, 2-aminoethoxydiphenyl borate, and bongkrekic acid, significantly inhibited P(i) uptake in the osteoclast-like cells, suggesting that P(i) uptake is regulated by Ca(2+) signaling in the endoplasmic reticulum and mitochondria of osteoclast-like cells. These results suggest that osteoclast-like cells have a unique P(i) uptake/efflux system and can prevent P(i) accumulation within osteoclast hemivacuoles.
Topics: Animals; Antiviral Agents; Calcium; Calcium Signaling; Cell Line; Dose-Response Relationship, Drug; Extracellular Fluid; Foscarnet; Hydrogen-Ion Concentration; Kidney Tubules, Proximal; Opossums; Osmolar Concentration; Osteoblasts; Osteoclasts; Phosphates; RANK Ligand; Sodium; Substrate Specificity
PubMed: 16971494
DOI: 10.1152/ajpcell.00357.2006 -
Annals of Medicine Oct 1993The introduction of virus-specific anti-herpes virus agents such as acyclovir, ganciclovir and Foscarnet has had a significant impact on the management of herpes virus... (Review)
Review
The introduction of virus-specific anti-herpes virus agents such as acyclovir, ganciclovir and Foscarnet has had a significant impact on the management of herpes virus infections. The use of specifically acting antimicrobial agents, however, raises the question of drug resistance. Exposure in cell culture of herpes virus to these agents results in the selection of drug-resistant variants, with resistance being due to alterations in the genes encoding the target enzymes involved in the mechanism of action of the drugs concerned, e.g. in the case of acyclovir resistance occurs as a result of deletions or alterations in the thymidine kinase (TK) or alterations in DNA polymerase genes. Pathogenicity studies reveal that drug-resistant variants are disadvantaged, in particular the TK deletion variants which are less pathogenic and unable to reactivate from latent infections. Extensive studies in cell culture and animal models with herpes viruses have provided an understanding of the mechanisms of resistance and more recently these findings have been correlated with the clinical experience. The incidence of virus resistance in immunocompetent patients is extremely rare, whereas resistance has infrequently been reported in immunocompromised individuals where exposure to drug is prolonged. However, the understanding of the mechanisms and consequences of virus resistance gained in cell culture and in animal models has led to the successful management of resistant episodes in the clinic, either by temporary removal of the selection pressure or by providing alternative therapies.
Topics: Acyclovir; Animals; Antiviral Agents; Drug Resistance, Microbial; Foscarnet; Ganciclovir; Herpesviridae; Herpesviridae Infections; Humans
PubMed: 8251142
DOI: 10.3109/07853899309147309 -
Anais Brasileiros de Dermatologia 2014Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually...
BACKGROUND
Herpes simplex virus (HSV) infection is an endemic disease and it is estimated that 6095% of the adult population are infected with symptoms that are usually self-limiting, though they can be serious, extensive and prolonged in immunocompromised individuals, highlighted by the emergence of drug-resistant strains. The study of the wild-type HSV strains based on the cytopathogenic features and its antiviral sensitivity are important in the establishment of an antivirogram for controlling the infection.
OBJECTIVE
This study sought to isolate and examine the cytopathological characteristics of circulating strains of the Herpes simplex virus, from clinical specimens and their sensitivity to commercially available antiherpesvirus drugs, acyclovir, phosphonophormic acid and trifluridine.
METHODS
Herpes simplex virus isolation, cytopathological features and antiviral sensitivity assays were performed in cell culture by tissue culture infectious dose or plaque forming unit assay.
RESULTS
From twenty-two clinical specimens, we isolated and adapted nine strains. Overall, the cytopathic effect was detected 24 h post-infection (p.i.) and the presence of syncytia was remarkable 48 h p.i., observed after cell staining. Out of eight isolates, four developed plaques of varying sizes. All the isolates were sensitive to acyclovir, phosphonophormic and trifluridine, with the percentage of virus inhibition (%VI) ranging from 49.7-100%.
CONCLUSIONS
The methodology for HSV isolation and characterization is a straightforward approach, but the drug sensitivity test, regarded as being of great practical importance, needs to be better understood.
Topics: Acyclovir; Antiviral Agents; Cells, Cultured; Foscarnet; Hematoxylin; Humans; Microbial Sensitivity Tests; Simplexvirus; Time Factors; Trifluridine; Viral Plaque Assay
PubMed: 24937819
DOI: 10.1590/abd1806-4841.20142574 -
Genetics Jun 2005A mutant allele (pol3-L612M) of the DNA polymerase delta gene in Saccharomyces cerevisiae that confers sensitivity to the antiviral drug phosphonoacetic acid (PAA) was...
A mutant allele (pol3-L612M) of the DNA polymerase delta gene in Saccharomyces cerevisiae that confers sensitivity to the antiviral drug phosphonoacetic acid (PAA) was constructed. We report that PAA-sensitivity tagging DNA polymerases is a useful method for selectively and reversibly inhibiting one type of DNA polymerase. Our initial studies reveal that replication by the L612M-DNA pol delta requires Rad27 flap endonuclease activity since the pol3-L612M strain is not viable in the absence of RAD27 function. The L612M-DNA pol delta also strongly depends on mismatch repair (MMR). Reduced viability is observed in the absence of any of the core MMR proteins-Msh2, Mlh1, or Pms1-and severe sensitivity to PAA is observed in the absence of the core proteins Msh6 or Exo1, but not Msh3. We propose that pol3-L612M cells need the Rad27 flap endonuclease and MMR complexes composed of Msh2/Msh6, Mlh1/Pms1, and Exo1 for correct processing of Okazaki fragments.
Topics: Adaptor Proteins, Signal Transducing; Alleles; Amino Acid Motifs; Base Pair Mismatch; Carrier Proteins; Cell Survival; Culture Media; DNA Mutational Analysis; DNA Polymerase III; DNA Repair; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Exodeoxyribonucleases; Flap Endonucleases; Fungal Proteins; Genetic Techniques; MutL Protein Homolog 1; MutL Proteins; MutS Homolog 2 Protein; Mutation; Phenotype; Phosphonoacetic Acid; Polymerase Chain Reaction; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Time Factors
PubMed: 15802517
DOI: 10.1534/genetics.104.040295 -
Scientific Reports Jul 2018Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of...
Aspartate carbamoyltransferase (ATCase) is a large dodecameric enzyme with six active sites that exhibits allostery: its catalytic rate is modulated by the binding of various substrates at distal points from the active sites. A recently developed method, bond-to-bond propensity analysis, has proven capable of predicting allosteric sites in a wide range of proteins using an energy-weighted atomistic graph obtained from the protein structure and given knowledge only of the location of the active site. Bond-to-bond propensity establishes if energy fluctuations at given bonds have significant effects on any other bond in the protein, by considering their propagation through the protein graph. In this work, we use bond-to-bond propensity analysis to study different aspects of ATCase activity using three different protein structures and sources of fluctuations. First, we predict key residues and bonds involved in the transition between inactive (T) and active (R) states of ATCase by analysing allosteric substrate binding as a source of energy perturbations in the protein graph. Our computational results also indicate that the effect of multiple allosteric binding is non linear: a switching effect is observed after a particular number and arrangement of substrates is bound suggesting a form of long range communication between the distantly arranged allosteric sites. Second, cooperativity is explored by considering a bisubstrate analogue as the source of energy fluctuations at the active site, also leading to the identification of highly significant residues to the T ↔ R transition that enhance cooperativity across active sites. Finally, the inactive (T) structure is shown to exhibit a strong, non linear communication between the allosteric sites and the interface between catalytic subunits, rather than the active site. Bond-to-bond propensity thus offers an alternative route to explain allosteric and cooperative effects in terms of detailed atomistic changes to individual bonds within the protein, rather than through phenomenological, global thermodynamic arguments.
Topics: Adenosine Triphosphate; Allosteric Regulation; Allosteric Site; Aspartate Carbamoyltransferase; Aspartic Acid; Catalytic Domain; Cytidine Triphosphate; Enzyme Stability; Models, Molecular; Phosphonoacetic Acid; Protein Multimerization; Protein Subunits; Substrate Specificity
PubMed: 30038211
DOI: 10.1038/s41598-018-27992-z -
The Yale Journal of Biology and Medicine Mar 1976In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and...
In tissue culture phosphonoacetic acid (PAA) specifically inhibited DNA synthesis of human cytomegalovirus (CMV), murine CMV, simian CMV, Epstein-Barr virus, and Herpesvirus saimiri. Fifty to one hundred micrograms per milliliter PAA completely inhibited viral DNA synthesis with no significant damage to host cell DNA synthesis. In vitro DNA polymerization assays showed that 10 μg/ml of PAA specifically inhibited partially purified human CMV-induced DNA polymerase, while little inhibition of host-cell DNA polymerase activity was found. The specific inhibition of herpes-group virus DNA synthesis with little toxicity to host cells suggests that PAA has great potential as an antiherpesvirus therapeutic agent.
Topics: Acetates; Culture Techniques; DNA Nucleotidyltransferases; DNA, Viral; Herpesviridae; Organophosphorus Compounds; Virus Replication
PubMed: 960726
DOI: No ID Found