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

Metal chelate affinity chromatography has been used to follow reconstitution of the 66- and 51-kDa human immunodeficiency (HIV)-1 and HIV-2 reverse transcriptase (RT) subunits into heterodimer, as well as chimeric enzymes comprised of heterologous subunits. By adding a small N-terminal polyhistidine extension to the 51-kDa subunit of either enzyme, reconstituted RT could be recovered from a cell lysate by chromatography on Ni(2+)-nitrilotriacetic acid-Sepharose. Homologous RT subunits rapidly associated to form the respective heterodimers (1-p66.1-p51 and 2-p66.2-p51) when bacterial lysates containing the individual components were mixed. Under the same conditions, association of p66 HIV-2 and p51 HIV-1 RT was inefficient and could be improved slightly by prolonged incubation of the respective p66 and p51 subunits. In contrast, HIV-1 p66 RT rapidly associated with the 51-kDa subunit of the HIV-2 enzyme. RNA-dependent DNA polymerase activity was associated with all reconstituted enzymes, and the response of each chimeric RT to an inhibitor selective for the HIV-1 enzyme indicated that sensitivity to inhibition was determined by the source of its 66-kDa subunit.
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PMID:Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase. 172 Jul 76

With the aid of monoclonal antibodies to the reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1), low-molecular-mass subunits (p29, p32, and p40) were identified in HIV-1 RT purified from HIV (HTLV-IIIB) virions by isoelectric focusing. Epitope mapping with synthetic polypeptides from various regions of the pol gene suggests that the low-molecular-mass subunits result from N-terminal cleavage of the p51 subunit. The subunits could be separated only by SDS-polyacrylamide gel electrophoresis and detected by immunoblotting. They could not be separated on chromatographic columns, suggesting that the subunits are complexed or conformationally arranged in such a way that their separation on the basis of molecular mass is not possible. The molecular mass of the active enzyme eluted from a chromatographic column (Sephacryl S-300) loaded with a mixture of the subunits was estimated to be 100 kDa.
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PMID:Epitope mapping of the low-molecular-mass subunits of reverse transcriptase in human immunodeficiency virus type 1 by monoclonal antibodies. 172 5

Simian immunodeficiency virus protease (SIV-PR) was produced in Escherichia coli with a recombinant expression system in which the mature enzyme autoprocessed from a precursor form. Recombinant SIV and HIV-1 (human immunodeficiency virus, type 1) proteases were purified from bacterial cell lysates by use of sequential steps of ammonium sulfate precipitation and size-exclusion and ion-exchange chromatography. The amino acid composition, amino-terminal sequence, and molecular weight (monomer) of the recombinant SIV-PR were in accord with that of the 99 amino acid polypeptide predicted from the SIVMac-PR nucleotide sequence. The active form of SIV-PR was shown to be dimeric by gel filtration chromatography. Inhibition by pepstatin A, time-dependent inactivation by 1,2-epoxy-3-(4-nitrophenoxy)propane, and pH rate profiles using oligopeptide substrates demonstrated that SIV-PR behaves as an aspartic protease. Recombinant HIV-1 Pr55gag precursor was processed in vitro by SIV-PR and HIV-1 PR with indistinguishable proteolytic patterns upon NaDodSO4-polyacrylamide gel electrophoresis. Oligopeptide substrates for HIV-1 PR were found to be suitable substrates for recombinant SIV-PR with the exception of a peptide containing the site identified for p66/p51 cleavage (Phe*Tyr) within HIV-1 reverse transcriptase (RT). Several synthetic peptide analogue inhibitors of HIV-1 PR were also potent inhibitors of SIV-PR, indicating that SIV infection in macaques and rhesus monkeys should be useful models for the preclinical evaluation of acquired immunodeficiency syndrome (AIDS) therapeutics targeted towards the virally encoded HIV-1 protease.
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PMID:Purification and biochemical characterization of recombinant simian immunodeficiency virus protease and comparison to human immunodeficiency virus type 1 protease. 188 29

Mutations were introduced into the P2 and P1 positions of the junctions, (a) linking reverse transcriptase (RT) and integrase (IN) (-Leu*Phe-) and (b) between the p51 and RNase H domain (-Phe*Tyr-) within p66 of RT in the HIV-1 pol polyprotein. Processing by HIV proteinase (PR) in cis was monitored upon expression of these constructs in E. coli. Whereas the presence of Leu or Phe in P1 permitted rapid cleavage at either junction, substitution of a beta-branched (Ile) hydrophobic residue essentially abolished hydrolysis. By contrast, placement of a beta-branched (Val) residue in the P2 position flanking such -Hydrophobic*Hydrophobic- junctions resulted in effective cleavage of the scissile peptide bond. Gly in P2, however, abrogated cleavage. The significance of these findings in terms of PR specificity, polyprotein processing and the generation of homodimeric (p51/p51) RT for crystallisation purposes is discussed.
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PMID:Mutating P2 and P1 residues at cleavage junctions in the HIV-1 pol polyprotein. Effects on hydrolysis by HIV-1 proteinase. 204 56

Western blot is the technique most frequently used to determine the presence of HIV-1 antibodies. However, some patients remain with an indeterminate diagnosis for HIV-1 according to the technique. We followed 8 such patients for 2 to 18 months. Immunologic studies included standard ELISA, immunofluorescence and Abbott anti-p24 ELISA. We found selective reactivity for certain viral proteins such as isolated p24, p24 plus p51 or p55, gp41 plus gp 120, p17 plus p24 and p25.
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PMID:[Patients with an indeterminate serological diagnosis of HIV-1 infection]. 215 49

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

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

To investigate a non-RI test which is equivalent to the reverse transcriptase inhibiting antibody test, a reverse transcriptase inhibiting antibody was compared to the absolute number of CD-4 or CD-8 cells or CD-4/-8 ratio and also to photodensitometric analysis for western blotting. There is no correlation of the reverse transcriptase with any test for cell numbers and their ratio. In photodensitometry, relative units of anti-p65 and anti-p51 were compared with reverse transcriptase inhibiting antibody. The reverse transcriptase inhibiting antibody showed a higher correlation to the relative unit of p65 antibodies than that of p51 antibodies. The photodensitometric analysis of western blotting for a serum test may be a possible method to find a prognostic marker in HIV-1 infection.
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PMID:[Comparative evaluation of markers with reverse transcriptase inhibiting antibody in human immunodeficiency virus type 1 infection]. 247 51

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

The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3.
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PMID:HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA. 247 43


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