Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antiviral drug resistance is an area of increasing clinical importance in treatment of a number of viruses including herpes simplex virus (HSV) and human cytomegalovirus (CMV). Work with these herpesviruses illustrates the value of studies of drug resistance. Novel aspects of drug mechanisms, such as a CMV gene product that contributes to ganciclovir phosphorylation, can be identified via drug resistance mutations. Drug targets such as the HSV DNA polymerase that are involved in drug recognition can be dissected by sequencing of drug-resistance mutations, which can point to alternate therapeutic strategies. Analysis of virus mutants in animal models and in patient populations can help assess the value of viral proteins such as the HSV thymidine kinase and ribonucleotide reductase as drug targets and the pathogenic potential of drug resistant mutants. Such studies reveal a broad spectrum of alterations conferring resistance and emphasize the importance of heterogeneous populations of virus in resistance and pathogenesis and the need to develop alternate therapies.
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PMID:The implications of resistance to antiviral agents for herpesvirus drug targets and drug therapy. 165 11

A herpes simplex virus ribonucleotide reductase (RR) null mutant, ICP6 delta, exhibited hypersensitivity to hydroxyurea, and to the precursors of allosteric inhibitors of cellular RR. The mutant was also much more sensitive than the parental KOS to all of antiviral nucleoside analogs tested, including acyclovir (ACV), ganciclovir (DHPG) and BVaraU. Our data indicate that cellular RR is essential for the growth of ICP6 delta, and suggest that inhibitors of viral RR could act as potentiators of all of anti-herpetic nucleoside analogs whose targets are viral DNA polymerase.
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PMID:Susceptibility of a herpes simplex virus ribonucleotide reductase null mutant to deoxyribonucleosides and antiviral nucleoside analogs. 166 66

A single pair of oligonucleotide primers selected within a highly conserved region of the DNA polymerase gene of the herpesviruses was designed to amplify related viral genomes, i.e., herpes simplex virus type 1, herpes simplex virus type 2, Epstein-Barr virus, and cytomegalovirus, by the polymerase chain reaction. A simple restriction enzyme analysis of these amplified products allowed accurate characterization of the herpesvirus type. Ninety-nine cerebrospinal fluid samples from 36 patients (including newborns, children, and adults) with acute encephalitis were tested for the presence and identification of herpesvirus DNA by this approach. High levels of viral DNA, which were readily visualized by simple ethidium bromide staining, were found in all these patients from the first days of the disease and, in some cases, until the third week following the onset of acute encephalitis. The herpesvirus type was rapidly identified by enzymatic digestion in 33 patients' samples and by hybridization and direct sequencing in the last 3 patients' samples. Our results show that the polymerase chain reaction provides a highly sensitive and specific technique for the identification of herpesviruses DNA in cerebrospinal fluid that should be of value for early and rapid diagnosis, therapeutic decisions, prognostic evaluation, and epidemiological studies.
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PMID:Amplification and characterization of herpesvirus DNA in cerebrospinal fluid from patients with acute encephalitis. 166 8

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
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PMID:Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses. 169 11

Using as antigens fusion proteins expressed in bacteria, we have generated polyclonal antisera specific for the herpes simplex virus (HSV) type 1 DNA polymerase. A variety of immunologic, genetic, and biochemical assays were used to characterize these antisera and demonstrate their specificity for the HSV DNA polymerase. Using these antisera, measurements of the synthesis and accumulation of HSV DNA polymerase in infected Vero cells were made. Peak rates of polymerase synthesis were observed at 4 h postinfection, as much as 2 h before peak levels of polymerase mRNA accumulation. At all times examined, the HSV DNA polymerase polypeptide was found to be synthesized at a lower rate per mRNA than the viral thymidine kinase, with this difference being especially dramatic at later times. Infected-cell RNA isolated at 2 and 6 h postinfection directed the synthesis of similar amounts of polymerase polypeptide per polymerase transcript in rabbit reticulocyte lysates, indicating that polymerase transcripts are inherently as translatable at both times. An HSV mutant in which sequences including a short upstream open reading frame in the HSV DNA polymerase transcript were deleted specified polymerase mRNA whose translational efficiency was no more than marginally greater than that of the wild-type virus. These results demonstrate that polymerase expression is regulated by inefficient translation mediated by sequences other than the short upstream open reading frame and that this leads to an early shutoff of polymerase synthesis during HSV infection.
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PMID:Translational regulation of herpes simplex virus DNA polymerase. 169 12

Our recent efforts have been directed at the development of selective inhibitors of different classes of viruses, including adeno, pox, and herpesviruses [herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster (VZV), cytomegalovirus (CMV), Epstein-Barr virus (EBV)], (+/-)RNA viruses (reo- and rotavirus), (-)RNA viruses (influenza, parainfluenza, measles, respiratory syncytial, vesicular stomatitis and rabies virus) and retroviruses [i.e. human immunodeficiency virus (HIV), the causative agent of AIDS]. In this search, the following molecular targets were envisaged: for DNA viruses in general, the viral DNA polymerase; for herpes simplex virus and varicella-zoster virus, the viral DNA polymerase via a specific phosphorylation by the viral 2'-deoxythymidine (dThd) kinase; for (+/-)RNA and (-)RNA viruses, S-adenosylhomocysteine (SAH) hydrolase, a key enzyme in transmethylation reactions required for the maturation of viral mRNA; for retroviruses, reverse transcriptase as initiator of virus replication and/or cell transformation; and for several enveloped viruses (i.e. retro-, herpes- and rhabdoviruses), virus adsorption to the outer cell membrane. Several new compounds have been developed that appear to act at these targets: i.e. (E)-5-(2-bromovinyl)-2'-deoxyuridine [bromovinyldeoxyuridine (BVDU)] and derivatives thereof [i.e. carbocyclic BVDU (C-BVDU)] as well as derivatives of acyclovir (i.e. 8-substituted acyclovir derivatives) as inhibitors of herpesviruses; (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine [(S)-HPMPA], 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and other phosphonylmethoxyalkylpurines and -pyrimidines as inhibitors of DNA viruses and retroviruses; acyclic and carbocyclic analogues of adenosine [such as (S)-9-(2,3-dihydroxypropyl)adenine [S)-DHPA), carbocyclic 3-deazaadenosine (C-c3Ado), (RS)-3-adenin-9-yl-2-hydroxypropanoic acid (AHPA) alkyl esters, neplanocin A, 3-deazaneplanocin A and the 5'-nor derivatives of neplanocin A and 3-deazaneplanocin A] as inhibitors of (+/-)RNA and (-)RNA viruses; 2',3'-dideoxynucleoside analogues as inhibitors of retroviruses; and sulfated polysaccharides (i.e. heparin, dextran sulfate, pentosan polysulfate, mannan sulfate), sulfated polyvinylalcohol and co-polymers of sulfated polyvinylalcohol with acrylic acid as inhibitors of retro-, herpes- and rhabdoviruses.
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PMID:Selective virus inhibitors. 169 49

A monoclonal antibody (mAb) designated 55H3 was produced by using chemically induced Epstein-Barr virus genome-positive B95-8 cells. mAb 55H3, which reacted with an 85- to 80-kDa polypeptide, neutralized Epstein-Barr virus-encoded DNA polymerase activity in crude extracts of chemically induced M-ABA, HR-1, and B95-8 cells, as well as the partially purified Epstein-Barr virus DNA polymerase in a dose-dependent manner. The mAb also neutralized the virus-encoded DNA polymerase activity from cells infected with human cytomegalovirus, human herpesvirus 6, and the purified bacteriophage T4 DNA polymerases. However, mAb 55H3 did not neutralize the DNA polymerase activities encoded for by herpes simplex virus types 1 and 2, the reverse transcriptase of avian myeloblastosis virus, or Escherichia coli DNA polymerase 1 (Klenow fragment). These results suggest that mAb 55H3 recognizes an epitope common to some herpesviruses and T4 DNA polymerases and further supports the hypothesis that these organisms are evolutionarily related.
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PMID:A monoclonal antibody that neutralizes Epstein-Barr virus, human cytomegalovirus, human herpesvirus 6, and bacteriophage T4 DNA polymerases. 170 Apr 22

3'-Azido-2',3'-dideoxy-5-methylcytidine (CS-92, AzddMeC) is an antiviral nucleoside analogue structurally related to 3'-azido-3'-deoxythymidine (AZT). CS-92 is a potent and selective inhibitor of HIV-1 reverse transcriptase and HIV-1 replication in human lymphocytes and macrophages. The EC50 for CS-92 in HIV-1-infected human PBM cells was 0.09 microM. In HIV-1-infected human macrophages, the EC50 was 0.006 microM. This compound was also effective against human immunodeficiency virus type 2 in lymphocytes. The replication of Friend murine virus was only weakly inhibited, and no effect was observed against herpes simplex virus type 1 and type 2 and coxsackievirus B4. CS-92 was not toxic to PBM or Vero cells when tested up to 200 microM and was, furthermore, at least 40 times less toxic to granulocyte-macrophage and erythroid precursor cells in vitro than was AZT. The interaction of the 5'-triphosphate of CS-92 with HIV-1 reverse transcriptase indicated competitive inhibition (the inhibition constant, Kis, was 0.0093 microM) with a 30-fold greater affinity for CS-92-TP than for ddCTP. CS-92-TP inhibited HIV-1 reverse transcriptase by 50% at a concentration 6,000-fold lower than that which was required for a similar inhibition of DNA polymerase alpha. Pharmacokinetic studies showed that CS-92 was not deaminated to AZT in rats, but this compound was found to have a half-life of 2.7 hours. In rhesus monkeys, however, a compound with a retention time and ultraviolet spectra characteristics similar to AZT was detected. The mean half-life in rhesus monkeys for CS-92 was 1.52 and 1.74 h after intravenous and oral administration, respectively, and the oral bioavailability was about 21 percent. Additional preclinical studies with CS-92 will determine the ultimate utility of this antiviral agent for the treatment of HIV-1 infections.
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PMID:Antiretroviral activity, biochemistry, and pharmacokinetics of 3'-azido-2',3'-dideoxy-5-methylcytidine. 170 74

The conservation of the herpesvirus DNA polymerases has allowed cross-hybridization studies to be used for their identification and mapping on the viral genome. With the use of a DNA fragment containing the DNA polymerase gene of human cytomegalovirus (HCMV) as a hybridization probe, we were able to localize the DNA polymerase gene of murine cytomegalovirus (MCMV) to a region within MCMV EcoRI fragment B which spans the HindIII site separating HindIII fragments D and H. This site is colinear with the HCMV strain AD169 DNA polymerase gene. To confirm that this region encoded the MCMV DNA polymerase gene, we sequenced a 5131 nucleotide fragment from the PstI site in HindIII fragment D to a BglII site in HindIII fragment H. Initiating in HindIII fragment D and extending into HindIII fragment H was a long open reading frame (ORF) 1097 amino acids in length with extensive homology to the DNA polymerases of HCMV, herpes simplex virus, and Epstein-Barr virus. Upstream of the polymerase ORF was a reading frame with considerable homology to the carboxy terminal half of the glycoprotein B gene of human herpesviruses. At early times in the infection, we could detect with a probe representing part of the polymerase ORF two 3' coterminal transcripts, 3.9 kb and 1.7 kb in length. S1 nuclease and exonuclease VII analyses indicated that both transcripts were unspliced and initiated at independent sites in HindIII fragment D. By primer extension, we were able to map precisely the 5' end of the 3.9-kb RNA to a site 186 nucleotides upstream of the beginning of the DNA polymerase ORF.
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PMID:Transcription analysis and sequence of the putative murine cytomegalovirus DNA polymerase gene. 171 83

The acyclic nucleoside phosphonate analogues (HPMPA, HPMPC, PMEA, FPMPA) show great promise for the treatment of infections with such important human pathogens as adeno, pox (vaccinia) and hepadna (hepatitis B) viruses (HPMPA), herpes (herpes simplex, varicella-zoster, cytomegalo, Epstein-Barr) viruses (HPMPC), and retro (human immunodeficiency) viruses (PMEA, FPMPA). All these compounds seem to be targeted at the viral DNA polymerase, with which they interact, as either competitive inhibitors or alternative substrates (or chain terminators), following their intracellular phosphorylation to the diphosphoryl derivatives. Of particular interest is the prolonged anti-viral action, lasting for several days or even weeks, that has been noted both in vitro and in vivo after a single administration of the acyclic nucleoside phosphonates.
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PMID:Chemotherapy of the acquired immune deficiency syndrome (AIDS): acyclic nucleoside phosphonate analogues. 182 10


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