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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of several forms (p51, p66, and p66/p51) of recombinant human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) with a synthetic derivative of its cognate replication primer, tRNA(Lys-3), has been determined by gel-mobility shift analysis. While p66/p51 RT is proficient in tRNA binding, preparations of p66 and p51 display only weak binding at elevated protein:tRNA ratios, despite the former containing both RNA-dependent DNA polymerase and ribonuclease H (RNase H) activity. Gel permeation analysis of purified p66 RT indicate this to be predominantly monomeric, suggesting that dimerization may be a prerequisite for efficient tRNA binding. Prolonged incubation of a mixture of the 66- and 51-kDa polypeptides results in heterodimer reconstitution, restoration of tRNA binding, and recovery of appreciable levels of RNA-dependent DNA polymerase activity. Under the same conditions, both the tRNA binding and RNA-dependent DNA polymerase activities of the 66- and 51-kDa polypeptides are unaffected, suggesting that they remain in the monomeric conformation.
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PMID:Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase. 137 42

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).
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PMID:Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase. 137 51

An in situ gel assay was applied to the study of double stranded RNA dependent RNase activity associated with reverse transcriptase (RT) of HIV-1 and murine leukemia virus. Polyacrylamide gels containing [32P] RNA/RNA substrate were used for electrophoresis of proteins under denaturing conditions. The proteins were renatured and in situ enzymatic degradation of 32P-RNA/RNA was followed. E. coli RNaseIII, but not E. coli RNaseH, was active in this in situ gel assay, indicating specificity of the assay to RNA/RNA dependent nucleases. Analysis of purified preparations of HIV-1 RT p66/p51 expressed in E. coli demonstrated an RNA/RNA dependent RNase activity comigrating with the large subunit (p66) of the enzyme. In addition, this activity of the RT was often accompanied by a contaminating RNA/RNA dependent RNase, with a molecular weight approximately 30,000 dalton identical to that of E. coli RNaseIII. As the p51 small subunit of HIV-1 RT and a mutant of RT p66/p51, at Glutamic acid #478, did not exhibit RNA/RNA dependent RNase activity, at least part of the active site of the RNA/RNA dependent RNase activity appeared to reside at the carboxy end of the molecule. As these RT proteins are also deficient of RNaseH, our results suggest overlapping or identical catalytic sites for degradation of the substrates RNA/DNA and RNA/RNA.
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PMID:Characterization of the double stranded RNA dependent RNase activity associated with recombinant reverse transcriptases. 138 38

HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule.
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PMID:Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase. 138 59

In the HIV Seroprevalence Survey among Childbearing Women (SCBW), antibodies to human immunodeficiency virus type 1 are detected using enzyme immunoassays (EIA) and Western blot (WB) methods modified to accommodate samples of blood dried on special collection paper. Dried blood spot (DBS) eluates positive by EIA are tested by one of two WB methods, the miniblot technique using equipment from Immunetics Corporation and the PBS Integra assay (pageblot) from Genetic Systems. In this report we compared the performance of the two WB methods. The identity and position of the viral proteins on the WB were identified using monoclonal antibodies and monospecific antisera. The blots differed substantially in their composition and concentration of viral glycoproteins. Performance of the WB assays with DBS elution buffers from different EIA kits was equivalent except for samples eluted in the Abbott buffer, which reduced detection of antibodies to the p31, p51, p55, and p66 viral proteins. Case classification of DBS, positive sera, dilution curve samples, and seroconversion panels was equivalent by both tests in the presence of all elution buffers. Proficiency evaluation panels sent to SCBW participating laboratories over a 3-year period were used to note the differences between the two WB methods in detection of antibodies to the viral glycoproteins.
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PMID:Factors influencing HIV-1 banding patterns in miniaturized western blot testing of dried blood spot specimens. 140 56

Introduction of a reactive 5-mercapto group into some of the cytosine and/or uracil bases of various oligo- and polynucleotides by partial thiolation resulted in several potent inhibitors of the replication of human immunodeficiency virus type 1 (HIV-1) in primary human lymphocytes. These compounds exhibited little if any toxicity against uninfected peripheral blood mononuclear cells and showed 15 to 75 times higher antitemplate activity against a p66/p51 HIV-1 recombinant reverse transcriptase (RT) than against the DNA polymerase alpha from human lymphocytes. In contrast, the unthiolated oligo- and polynucleotides are void of antitemplate activity, and their apparent inhibitory effect on HIV-1 closely paralleled their toxicity for the cells. Partially thiolated poly(dC) (MPdC) was the most potent of all the compounds tested against HIV-1 in peripheral blood mononuclear cells (50% effective concentration, 1.8 micrograms/ml or 0.019 microM), while showing low cytotoxicity (greater than 100 micrograms/ml). The corresponding unmodified poly(dC) showed no anti-HIV-1 activity at 50 micrograms/ml but had pronounced cytotoxicity. MPdC was also a potent inhibitor of HIV-1 RT (50% inhibitory concentration, 0.30 micrograms/ml). The inhibitory activities of thiolated homooligo(dCs) against both HIV-1 replication and HIV-1 RT increased with increasing chain length. The heterooligonucleotides included in this study were designed as structural analogs of portions of the natural primer of HIV-1 RT, i.e., tRNA(3Lys). An 18-mer analog of the 3' terminus, complementary (antisense) to the primer-binding site of the HIV-1 genome, was attached to an oligo(dC) tail and 5-thiolated; this increased its activity and decreased its toxicity. This compound will serve as a new lead in the development of more effective antitemplates against HIV-1.
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PMID:Structure-activity relationships and mode of action of 5-mercapto-substituted oligo- and polynucleotides as antitemplates inhibiting replication of human immunodeficiency virus type 1. 159 Jun 75

We have modified an Escherichia coli vector expressing 66-kDa HIV-1 reverse transcriptase (p66) so that it simultaneously expresses this and the pol-coded protease. The twin expression cassette yields high quantities of both reverse transcriptase and protease; however, under these conditions, 50% of the over-expressed p66 reverse transcriptase is processed, resulting in accumulation of large quantities of p66/p51 enzyme. Furthermore, addition of a poly(histidine) affinity label at the amino terminus of the reverse-transcriptase-coding sequence (His-p66) permits a simple, rapid purification of milligram quantities of either p66 or p66/p51 enzyme from a crude lysate by metal chelate affinity chromatography. Purified His-p66 and His-p66/His-p51 reverse transcriptase exhibit both reverse transcriptase and RNase H activity. Purification by metal chelate chromatography of a p66/p51 enzyme wherein only the p66 component is labelled strengthens the argument for the existence of a heterodimer.
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PMID:Rapid purification of homodimer and heterodimer HIV-1 reverse transcriptase by metal chelate affinity chromatography. 168 98

We have analysed the mechanism of ribonuclease H (RNaseH) induced cleavage of a defined RNA-DNA hybrid by human immuno-deficiency virus (HIV-1) reverse transcriptase (RT). An in vitro transcribed RNA labelled at the 3' end was hybridized to a pentadecameric DNA oligonucleotide complementary to an internal region of the RNA. Upon incubation of this RNA-DNA hybrid with recombinant p66 or p66/p51 HIV-1 reverse transcriptase, RT-RNaseH mediated cleavage is observed at most nucleotides within the short hybridized stretch, resulting in a spectrum of RNA fragments extending from the 3' label to this region and differing in length by one nucleotide. The same RNA, this time labelled at the 5' end, yields only one or two major cleavage products corresponding to RNA species extending from the 5' label to the middle of the hybridized region. Such a result can be explained by the action of both endonuclease and 3'----5' exonuclease activities inherent to the C-terminal domain of p66 RT. To investigate how RNaseH cleavage is coupled to reverse transcription, a combination of deoxynucleoside triphosphates was used which allowed controlled extension of the primer DNA. Concomitantly with the elongation of the oligonucleotide primer, RNaseH cleavage proceeds towards the 5' end of the RNA with identical increments, suggesting a simultaneous action of both activities.
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PMID:HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity. 169 Oct 93

Overexpression of the reverse transcriptase was designed in E. coli. For a high level of expression, HIV protein was expressed as a protein fusion with beta-galactosidase. When the proviral DNA fragment covering the 3' half of the gag gene and the entire pol gene was ligated to the 3' end of the lacZ gene to fuse the truncated gag to lacZ in frame, a small quantity of reverse transcriptase was produced, indicating that frameshifting and post-translational processing have occurred. Much more reverse transcriptase was produced when the entire pol region was directly fused to the lacZ gene. From a one liter culture of bacteria, 1 mg of highly purified reverse transcriptase consisting of approximately equimolar amounts of two species (p64 and p51) was obtained. These proteins had identical N-termini consistent with the deduced amino acid sequence and therefore, might be correctly processed from the fusion protein in E. coli by the protease encoded by the pol region. The purified reverse transcriptase was enzymatically as active as the enzyme purified from the virus particles, and immunoreactive to the sera of HIV carriers with high sensitivity and specificity.
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PMID:Overproduction of human immunodeficiency virus type I reverse transcriptase in Escherichia coli and purification of the enzyme. 169 13

The mode of action of the RNase H activity from HIV-1 was analyzed with a purified recombinant p66/p51 reverse transcriptase RT/RNase H protein and RNA-DNA hybrid consisting of RNA harboring the polypurine tract (ppt) and three complementary synthetic DNA oligonucleotides. Upon incubation of this preformed RNA-DNA hybrid with the p66/p51 RT/RNase H, a cleavage pattern is observed that indicates endonucleolytic RNase H activity with some sequence specificity for the next to last nucleotide of the 3'-end of the ppt RNA and one cut within the ppt. The RNase H avoids cleavage of G or A stretches. During RNA-directed DNA synthesis the RNA is hydrolyzed in a concerted action of RT and RNase H whereby the RNase H exhibits endonuclease as well as 3'-5'-exonuclease activity. The distance between the active centers of the RT and RNase H corresponds to 18 base pairs of the RNA-DNA hybrid. Plus-strand DNA-directed DNA synthesis initiates exactly at the next to last nucleotide of the 3'-end of the ppt RNA by means of the RNase H activity.
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PMID:Interaction of HIV-1 ribonuclease H with polypurine tract containing RNA-DNA hybrids. 170 2


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