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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Simian immunodeficiency viruses (SIV) are a family of primate lentiviruses similar to human immunodeficiency viruses (HIV) in their genetic sequence and pathogenesis. However, host-derived cofactors which may determine the extent of viral replication are not clearly defined for SIV or HIV infections. A HuT-78 cell line chronically infected with SIV/mac strain 251, was biologically cloned and characterized for the ability to produce infectious viral particles, viral structural protein profile, cellular antigen surface phenotype and tested to determine the effects of recombinant cytokines on SIV replication. Reverse transcriptase (RT) assay was used to measure the replication of SIV/mac in response to various concentrations of recombinant cytokines (1-1000 units/ml). We report that tumor necrosis factor-alpha (rTNF-alpha), gamma-interferon (rIFN-gamma), interleukin 2 (rIL-2), and granulocyte-macrophage colony stimulating factor (rGM-CSF) induced approximately a 2 to 3 fold increase in virus RT activity compared with untreated SIV-infected HuT-78 cells. In contrast, viral replication was not enhanced or minimally enhanced by interleukin 1 (rIL-1), interleukin 3 (rIL-3), or interleukin 4 (rIL-4) at similar dosages. Furthermore, SIV replication in response to rTNF-alpha and rIFN-gamma occurred in a dose dependent fashion. These data suggest that SIV-infected T-lymphocyte lines are responsive to particular cytokines resulting in increased virus production.
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PMID:Cytokine enhancement of simian immunodeficiency virus (SIV/mac) from a chronically infected cloned T-cell line (HuT-78). 172 91

Operator-induced biological contamination in cell cultures is a multifaceted problem involving the unexpected introduction of other animal cells, microbial and viral contaminants. Detailed studies on animal cell cross contaminations have been performed and published. The frequency of detection of problem cultures has been as high as 36% for one service performed in the USA, with interspecific cross contamination accounting for 25% and human intraspecific contamination representing 11%. Awareness of the potential of this problem plus the application of several characterizations are key factors for its control. For example, fluorescent antibody staining, isoenzyme analyses, cytogenetic evaluations and DNA fingerprinting using molecular probes are needed for quality assurance on master seed stocks. Detection of microbial contamination is relatively straightforward, but the prevalence of mycoplasmal infections in cell cultures used in general research is still a significant problem. Detection services report frequencies of infection varying from 10% upwards, depending upon the country and laboratory of origin. The utilization of prescreened reagents and antibiotic-free cultivation, plus the application of improved procedures, such as fluorescent dyes and molecular probes for detection, provide effective means of avoiding mycoplasma infection and facilitating control. For many viruses, the presence of mycoplasma reduces immunoreactivity, suppresses transcriptase and other enzyme activities, reverses viral neutralization etc. The introduction of viral contaminants into cell cultures is perhaps the most problematic, especially where no cytopathic effect is produced. Few cases are documented where technicians infected with specific viruses have introduced these unwittingly into cultures in their care. The potential exists, however, as reports have appeared documenting the considerable stability of rhinoviruses, respiratory syncytial virus, rotaviruses and others, in aerosols on workers' hands and safety hood surfaces. The infection of cell cultures via other contaminated cells or reagents such as sera is a related problem. In this regard, the infection of transplantable tumor cell lines with lymphocytic choriomeningitis virus from host animals led to an outbreak of the disease in medical center personnel. Similar infection of rat cell lines exposed to animals harboring hantaviruses has been reported. Technical staff in US government laboratories have been infected with human immunodeficiency virus produced in cultured cells. Such serious public health hazards warrant repeated emphasis. The use of multiple cell lines in a given laboratory, including cultures known to be virally infected, compounds the problems and necessitates application of preventive methods both to avoid cross-infections and to document freedom from contamination.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Operator-induced contamination in cell culture systems. 179 20

Reverse transcriptase activity of the human immunodeficiency virus (HIV) was blocked in vitro by immunoglobulin G (IgG) derived from certain individuals infected with this retrovirus. A heterogeneous immune response for inhibition of enzyme function was noted. Catalytic activity was depressed by 50% or more with the use of 10 micrograms of IgG from 11 of 16 HIV-seropositive asymptomatic carriers, but from 0 of 8 seronegative controls and 2 of 12 patients with acquired immune deficiency syndrome (AIDS) or the AIDS-related complex (ARC). The inhibitor was confined to the F(ab')2 fragment. It was not directed against the poly(rA) X oligo(dT) template, nor against major envelope or structural viral antigens, and did not cross-react with bacterial, avian, or other mammalian DNA polymerases. It did not correlate with recognition of polymerase antigens by radioimmunoprecipitation. Loss of this inhibitor may be associated with development of clinical disease. Ten asymptomatic HIV-seropositive carriers with high titers of IgG antibodies to reverse transcriptase were followed for a mean of 3 years. All of four lost inhibitory capability prior to development of AIDS or ARC, while titers persist in the six who remain clinically healthy.
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PMID:Characterization and clinical association of antibody inhibitory to HIV reverse transcriptase activity. 243 4

The ability of granulocyte-macrophage colony-stimulating factor (GM-CSF) and 3'-azido-3'-deoxythymidine (AZT) to inhibit human immunodeficiency virus (HIV) in the U-937 monocytic cell line was examined. Acutely HIV-infected U-937 cells were exposed to GM-CSF (0.03, 0.3, 3.0, or 30.0 U/ml) and AZT (0.1, 1.0, or 10.0 microM) alone and in combination for 14 to 17 days. Reverse transcriptase activity in the supernatant, the percentage of cells expressing viral antigens by indirect immunofluorescence, and the 50% tissue culture infectious dose per milliliter of supernatant were determined to assess the level of viral replication in treated and control cultures. By the fractional-product method of analysis, nearly all combinations of GM-CSF and AZT synergistically inhibited HIV replication by these three measurements. The most effective combinations were 30 U of GM-CSF per ml with 0.1, 1.0, or 10.0 microM AZT. These treatments resulted in no reverse transcriptase activity in the supernatants, less than 1% immunofluorescent positive cells, and less than 8 50% tissue culture infectious doses per ml in the absence of cytotoxicity. Despite this degree of suppression, productive viral replication returned in all cultures within 4 to 10 days after drug removal. Combined therapy with GM-CSF and AZT merits consideration in the approach to HIV-associated illnesses.
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PMID:Synergistic activity of granulocyte-macrophage colony-stimulating factor and 3'-azido-3'-deoxythymidine against human immunodeficiency virus in vitro. 244 55

Ribavirin was administered orally in escalating doses for 2 or 4 weeks to 15 symptom-free, human immunodeficiency virus seropositive homosexual men with generalized lymphadenopathy. Reverse transcriptase activity was inhibited during therapy when steady-state plasma concentrations were greater than 6 mumol/L. These concentrations were achieved with 1200 or 2400 mg/day for 2 weeks or a loading dose of 2400 mg/day for 3 days followed by 600 mg/day for 4 weeks. Drug accumulation occurred at all doses. The elimination half-life appeared to be approximately 2 weeks. Reversible adverse reactions, principally resulting in central nervous system symptoms and anemia, correlated with dose and duration of therapy. Immunologic enhancement of T-lymphocyte-mediated mitogen-induced responses was observed in the majority of patients who had reduction in reverse transcriptase activity. However, specific T4+ lymphocyte-mediated antigen-induced responses increased to within the normal range in only three patients. Significant enhancement appeared to correlate with the severity of baseline antigen-induced functional impairment. These data indicate that oral ribavirin can be given for at least 1 month with acceptable toxicity at doses that appear to inhibit human immunodeficiency virus replication.
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PMID:Ribavirin pharmacodynamics in high-risk patients for acquired immunodeficiency syndrome. 244 79

Inhibition of visna virus replication in vitro by several compounds previously reported to inhibit replication of human immunodeficiency virus (HIV) was examined. Ribavirin concentrations as high as 1 mM reduced virus production by less than 50% relative to controls. The concentration of phosphonoformate reducing virus replication by 50% was 80 microM. 2',3'-Dideoxynucleosides were potent inhibitors of visna virus replication. The 50% inhibitory concentrations for dideoxyguanosine, dideoxyadenosine, and dideoxycytidine were 0.1, 0.2, and 0.3 microM, respectively. In contrast, weak inhibition was produced by 100 microM dideoxythymidine. These results are consistent with the reported susceptibility of HIV replication to inhibition by these compounds in vitro. The interaction of visna virus reverse transcriptase with several inhibitors was also examined. Reverse transcriptase was inhibited by phosphonoformate, ribavirin 5'-triphosphate, ddATP, ddCTP, ddGTP, and ddTTP. The last four compounds inhibited incorporation of homologous 2'-deoxynucleoside 5'-triphosphates into polynucleotides by a competitive mechanism. In view of the biological similarities between visna virus and HIV and the similar in vitro susceptibility of visna virus replication to known inhibitors of HIV, visna virus may provide a good model for studying the inhibition of HIV replication in vitro. Because visna virus is not pathogenic to humans, this model may facilitate the identification of compounds for further investigation into the treatment of HIV-induced disease.
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PMID:Visna virus as an in vitro model for human immunodeficiency virus and inhibition by ribavirin, phosphonoformate, and 2',3'-dideoxynucleosides. 244 82

Reverse transcriptase was purified from human immunodeficiency virus (HIV). It utilized the artificial primer-template poly(rA)-oligo(dT)12-18 more efficiently than activated calf thymus DNA, poly(rI)-oligo(dC)12-18, poly(rC)-oligo(dG)12-18, or poly(rCm)-oligo(dG)12-18. Maximum activity was observed at pH 7.0 to 7.6 in the presence of 5 mM MgCl2 and 100 mM KCl. 3'-Azido-3'-deoxythymidine triphosphate competed with dTTP for binding to HIV reverse transcriptase. Different kinetic constants were obtained with different primer-templates. Km and Ki values of 2.8 and 0.04 microM, respectively, were obtained with poly(rA)-oligo(dT)12-18. The corresponding values were 1.2 and 0.3 microM, respectively, with activated calf thymus DNA and 0.3 and 0.01 microM, respectively, with extracted virus and native template. Inhibition of the host cell DNA polymerases alpha and beta was considerably weaker. The Km and Ki values obtained with activated calf thymus DNA as the primer-template were 2.4 and 230 microM, respectively, for DNA polymerase alpha and 6.0 and 73 microM, respectively, for DNA polymerase beta. 3'-Azido-3'-deoxythymidine triphosphate could also serve as an alternate substrate for HIV reverse transcriptase. The resulting incorporation of 3'-azido-3'-deoxythymidine triphosphate into poly(rA)-oligo(dT)12-18 caused chain termination and premature deceleration of the reaction. The terminated primer could not be elongated when incubated with dTTP and HIV reverse transcriptase.
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PMID:3'-Azido-3'-deoxythymidine triphosphate as an inhibitor and substrate of purified human immunodeficiency virus reverse transcriptase. 244 66

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.
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PMID:Fidelity of HIV-1 reverse transcriptase. 246 Sep 24

Human immunodeficiency virus (HIV) was detected by assay of reverse transcriptase activity in a "virus pellet" obtained by differential sucrose density centrifugation of cell-free semen from three patients with the acquired immune deficiency syndrome (AIDS), one individual with AIDS-related complex (ARC), and in an asymptomatic homosexual male. Reverse transcriptase assays indicated virus concentrations in the range of 10(8) particles/ml of semen, an accumulation substantiated by electron microscopic visualization of cell-free virus. This is the first description of cell-free retrovirus in seminal fluid and at a greater concentration than reported for blood or other body fluids or tissues. These results suggest that the male reproductive tract of humans may be a reservoir of HIV expression, and raises the possibility that the cells lining the epididymal lumen could be chronically infected with HIV. These are important considerations in formulating treatment and preventive strategies.
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PMID:Detection of human immunodeficiency virus in cell-free seminal fluid. 263 53

Reverse transcriptase from the human immunodeficiency virus type I (HIV-1) was expressed in E. coli and purified to near homogeneity. The enzyme was shown to contain reverse transcriptase, DNA polymerase and ribonuclease H activities. The DNA polymerase activity converted singly-primed phi X174 (+) DNA into the double-stranded form. Two third of the replication product is ligatable to covalently closed circular DNA (RFIV-form DNA) indicating that DNA synthesis by HIV reverse transcriptase can proceed until the enzyme matches the 5'-end of a pre-existing primer molecule. The in vitro accuracy of HIV reverse transcriptase was measured with the phi X174am16 reversion assay to be 1/7,400. Reversion rates for the individual mispairs were determined from pool bias studies to be 1/8,000 for the dGMP:T template mismatch, 1/35,000 for the dGMP:A template mismatch, 1/45,000 for the dAMP:G template mismatch, 1/73,000 for the dCMP:T template mispair, 1/140,000 for the dCMP:A template mispair, and 1/180,000 for the dGMP:G template mismatch. The dTMP:T template mispair was below the detection limit of the assay indicating a reversion rate of less than 1/300,000 for this particular mispair.
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PMID:Fidelity of human immunodeficiency virus type I reverse transcriptase in copying natural DNA. 246 38


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