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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Approximately 80 percent of all human sera that react with antigens of HTLV-III, the etiologic agent of the acquired immune deficiency syndrome (AIDS), recognize protein bands at 66 and 51 kilodaltons. A mouse hybridoma was produced that was specific to these proteins. Repeated cloning of the hybridoma did not separate the two reactivities. The p66/p51 was purified from HTLV-III lysates by immunoaffinity chromatography and subjected to NH2-terminal Edman degradation. Single amino acid residues were obtained in 17 successive degradation cycles. The sequence determined was a perfect translation of the nucleotide sequence of a portion of the HTLV-III pol gene. The purified p66/51 had reverse transcriptase activity and the monoclonal immunoglobulin G specifically removed the enzyme activity from crude viral extract as well as purified enzyme.
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PMID:Characterization of highly immunogenic p66/p51 as the reverse transcriptase of HTLV-III/LAV. 241 4

The ability to express the genes of pathogenic human viruses, such as the acquired immune deficiency syndrome (AIDS) virus (also called human immunodeficiency virus) in bacterial cells affords the opportunity to study proteins that are ordinarily difficult or inconvenient to obtain in amounts sufficient for detailed analysis. A segment of the AIDS virus pol gene was expressed in Escherichia coli. Expression resulted in the appearance of reverse transcriptase activity in the bacterial cell extracts. The extracts contained two virus-related polypeptides that have the same apparent molecular weights as the two processed forms of virion-derived reverse transcriptase (p66 and p51). The formation of these two polypeptides depended on the coexpression of sequences located near the 5' end of the pol gene, a region that is thought to encode a viral protease. This bacterial system appears to generate mature forms of the AIDS virus reverse transcriptase by a proteolytic pathway equivalent to that which occurs during virus infection of human cells.
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PMID:Expression and processing of the AIDS virus reverse transcriptase in Escherichia coli. 243 98

An activity gel analysis was performed in order to examine the catalytically active component of human immunodeficiency virus (HIV) reverse transcriptase in purified enzyme preparations and HIV-infected cell extracts. Immunoaffinity purified HIV reverse transcriptase contains two proteins with molecular weights 66,000 and 51,000 in approximately equal proportions. After denaturing polyacrylamide gel electrophoresis and removal of sodium dodecyl sulfate, the p66 component of reverse transcriptase was sufficient for both DNA- and RNA-directed DNA synthesis. No DNA synthetic activity of p51 was observed. Recovery of p66 catalytic activity was approximately 10% that of DNA polymerase beta, and the density of the autoradiographic band corresponding to p66 was linear with enzyme concentration. No additional HIV-specific DNA polymerases besides p66 were observed in HIV-infected H9 cell extracts.
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PMID:Enzyme activity gel analysis of human immunodeficiency virus reverse transcriptase. 245 63

Human immune deficiency virus (HIV) replicates by conversion of the RNA genome into the double-stranded DNA provirus. The reverse transcriptase is not the only enzymatic function crucial in DNA-provirus synthesis. A viral-coded RNase H activity which specifically degrades RNA in RNA-DNA hybrids has been shown to be essential as well. Here we demonstrate that the HIV-reverse transcriptase which consists of a two-polypeptide complex, p66 and p51, copurifies with an RNase H activity which exhibits properties of a processive exonuclease. Only the p66 molecule, not p51, is active as polymerase as evidenced by activated gel analysis. p66 exhibits RNase H activity when precipitated as immune complex by a monoclonal antibody raised against a bacterially expressed carboxy-terminal portion of p66. The monoclonal antibody which does not interfere with enzyme activity also precipitates a second population of molecules with RNase H activity which is of low mol. wt, p15. This RNase H appears therefore to be derived from the carboxy terminus of p66 during processing to the p51 polypeptide. It exhibits low template-binding ability and is of a non-processing mode of action which may be due to the absence of the reverse transcriptase domain. These results lend experimental support to the hypothesis that the RNase H gene maps at the carboxy terminus of the reverse transcriptase. Since both RNase H populations are virus-coded they may be essential for retrovirus replication in general and useful targets for chemotherapeutic agents.
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PMID:Identification and characterization of HIV-specific RNase H by monoclonal antibody. 245 83

The HIV immunoblot profiles of 700 HIV-antibody-positive sera were examined to determine the frequency of antibody reactivity with p66/p51, the reverse transcriptase of HIV. We report a remarkably high seroprevalence of antibodies to p66/p51, detected in 79% of the sera. Only gp41 is recognized more frequently in these assays. The level of anti-p66/p51 seroreactivity varies only slightly among the clinical stages of HIV infection.
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PMID:High prevalence of serum antibodies to reverse transcriptase in HIV-1-infected individuals. 245 48

Recombinant vaccinia viruses containing either the entire gag/pol gene or the reverse transcriptase (RT) domain of the human immunodeficiency virus (HIV) were constructed. In mammalian cells infected with the recombinant vaccinia virus containing the gag/pol gene, major and minor polypeptides of 55 and 41 kDa were made, but processed gag products (p24/p17/p15) were not detected. In addition, none of the products of the pol open-reading frame were seen. Both the 55- and 41-kDa gag proteins were post-translationally modified by addition of myristic acid residues in recombinant vaccinia-infected cells, and were immunoprecipitated by antiserum to p24 gag, as well as by antisera from HIV-infected patients. These results indicate that neither proteolytic processing nor other HIV proteins are required for myristilation, and suggest that the 55- and 41-kDa gag precursors share the same amino terminus as p17. Cells infected with a separate vaccinia recombinant containing a truncated piece of the gag/pol gene with added start and stop codons at the 5' and 3' ends of the RT reading frame synthesized a major 61-kDa and a minor 51-kDa protein product which reacted immunologically with both a monoclonal antibody to native HIV p66/51 and antisera from HIV-infected patients. These proteins were purified from recombinant vaccinia-infected mammalian cells, and their enzyme activity was found to be similar to that of authentic HIV RT. Cells infected with the vaccinia/RT vector contained approximately 200-fold more RT per milligram of protein than cells infected with HIV. Recombinant RT was inhibited by dideoxynucleoside triphosphates and should be useful in screening for specific inhibitors of this enzyme. Mice inoculated intradermally with 10(8) plaque-forming units of the vaccinia/RT vector developed specific antibodies to the p66/51 proteins of HIV, but anti-HIV antibodies were not detected in mice inoculated with the vaccinia/gag vector.
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PMID:Characterization of human immunodeficiency virus gag/pol gene products expressed by recombinant vaccinia viruses. 245 42

The reverse transcriptase of HIV-1 (AIDS virus) is characterized by the presence of two highly immunogenic proteins of 66 and 51 kD known to be enzymatically active as a complex p66/51. Using an activity gel procedure that allows identification of catalytic polypeptides in situ after PAGE in denaturing conditions, we visualized two major active bands of 66 and 51 kD of reverse transcriptase from highly purified preparations of HIV-1. We show that both p66 and p51 are enzymatically active. An additional active band was also associated with a 165 kD polypeptide, representing about 2-4% of total activity and possibly corresponding to the putative gag-pol precursor. In H9-infected cells the 66 kD active band became visible 70 hours after infection. These studies show that the two major forms of reverse transcriptase (66 and 51 kD) of HIV-1 are independently active and that a higher Mr form of 165 kD is also enzymatically active.
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PMID:Enzymatically active forms of reverse transcriptase of the human immunodeficiency virus. 246 25

Biochemical characteristics of the RNase H activity associated with immunoaffinity purified human immunodeficiency virus (HIV) reverse transcriptase (RT) were examined. Glycerol gradient centrifugation of HIV RT resulted in a single peak of RNase H, associated with RT activity, with an apparent molecular weight of 110,000. HIV RNase H exhibited a marked substrate preference for poly(dC).[3H]poly(rG) compared to poly(dT).[3H]poly(rA). It did not hydrolyze single-stranded RNA or the DNA component of DNA.RNA hybrids. Products of the HIV RT-associated RNase H reaction consisted primarily of monomers, dimers, and trimers with 3' OH groups. This reaction was Mg2+ dependent, with greater than 90% of maximum activity at MgCl2 concentrations between 4 and 12 mM. The optimum KCl concentration for HIV RT catalyzed polymerization with a poly(rA).(dT)10 template. The optimum pH for HIV RNase H activity was between 8.0 and 8.5, in contrast to an optimum pH of 7.5 to 8.0 for HIV RT activity. The association of RNase H activity with the p66 component of HIV RT was demonstrated by activity gel analysis. These results indicate that HIV RT has an integral RNase H activity; however, some of its properties are different from those of RNase H associated with other retroviral RT's, and optimal assay conditions are different than those for HIV RT catalyzed DNA polymerization.
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PMID:Human immunodeficiency virus reverse transcriptase-associated RNase H activity. 246 65

A plasmid construct expressing the p66 version of the human immunodeficiency virus reverse transcriptase as a bacterial fusion protein was subjected to in vitro mutagenesis, and the resulting variant proteins were assayed to define the locations of the two major enzymatic activities. The DNA polymerase activity was localized to the N-terminal portion of the protein; mutations altering or eliminating the C-terminal portion had little or no effect on that activity. The results suggest that, in contrast with previous reports, the p51 subunit found in virions should exhibit DNA polymerase activity. Mutations in many parts of the protein eliminated RNase H activity, suggesting that several areas are needed for proper folding and generation of that activity.
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PMID:Linker insertion mutagenesis of the human immunodeficiency virus reverse transcriptase expressed in bacteria: definition of the minimal polymerase domain. 247 90

Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M. M. Lightfoote et al. (1986) J. Virol. 60, 771-775; F. diMarzo Veronese et al. (1986) Science 231, 1289-1291). The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor. Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51. RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenous E. coli RNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.
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PMID:Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities. 247 69


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