Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
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.
 ...
PMID:Interaction of tRNA(Lys-3) with multiple forms of human immunodeficiency virus reverse transcriptase. 137 42

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.
 ...
PMID:HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity. 169 Oct 93

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.
 ...
PMID:Reconstitution and properties of homologous and chimeric HIV-1.HIV-2 p66.p51 reverse transcriptase. 172 Jul 76

The drug sensitivities of human immunodeficiency virus type 1 (HIV-1) isolates from a group of four untreated and seven TIBO R82913-treated patients were determined in a reverse transcriptase (RT) assay. Five of the treated patients harbored HIV-1 isolates with R82913 sensitivity comparable to that of the isolates of untreated patients, ranging from almost 2-fold higher sensitivity to 13-fold lower sensitivity than that of recombinant p66 RT. From one of the seven treated patients, an HIV-1 strain with a 20-fold reduced sensitivity to R82913 could be isolated; and from another patient, a strain with 100-fold reduced sensitivity (resistance) was isolated. The drug-resistant strain in this patient emerged after 3 weeks of treatment and was due to the Y188L mutation in its RT. On passaging the virus in cord blood lymphocytes, but not in CEM cells, the resistant virus was lost in favor of a different HIV-1 strain harboring the wild-type Y188 with a sensitivity to R82913 comparable to that of wild-type p66 RT. In several HIV-1 isolates (from treated and untreated patients), some HIV-2- and CIVgab-specific amino acids were found. One of these substitutions, that is, I/V179D (from an untreated patient), conferred a sevenfold reduced RT sensitivity to R82913.
 ...
PMID:Characterization of HIV-1 strains isolated from patients treated with TIBO R82913. 751 16

We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.
 ...
PMID:Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli. 768 63

We compared the inhibition of HIV-1 reverse transcriptase (RT) by 1-[2',5'-bis-O-(t-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'- spiro-5"-(4"-amino-1", 2"-oxathiole-2",2"-dioxide)-3-ethylthymine (TSAOe3T) and the nonnucleoside RT inhibitor (NNRTI) 9-aminonevirapine (9-NH2N). Both compounds were equally effective against p51/p66 heterodimeric RT RNA-dependent DNA polymerase activity, although TSAOe3T was a much better inhibitor of the p51/p51 and p66/p66 RT homodimers. Inhibition by TSAOe3T and 9-NH2N combinations was essentially additive. TSAOe3T did not protect either free RT or the RT-template/ primer-deoxynucleoside triphosphate ternary complex from irreversible inactivation by the photolabel 9-azidonevirapine. Slight protection of the RT-template/primer binary complex was noted, but only at high TSAOe3T/photolabel ratios. Analysis of RT polymerization product profiles under both continuous- and single-processive cycle conditions showed that 9-NH2N prevented the formation of full-length product with a corresponding accumulation of smaller polymerization products. In contrast, all products formed in the absence of inhibitor, including full-length product, were noted in TSAOe3T-inhibited reactions, albeit at reduced levels. TSAOe3T thus inhibits HIV-1 RT by a different mechanism than NNRTI such as nevirapine. Our data suggest that TSAOe3T and 9-NH2N interact differently with HIV-1 RT, perhaps by binding to distinct sites on the enzyme.
 ...
PMID:Differences in the inhibition of human immunodeficiency virus type 1 reverse transcriptase DNA polymerase activity by analogs of nevirapine and [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro-5"-(4"-amino-1", 2"-oxathiole-2",2"-dioxide] (TSAO). 891 35

Radiation target analysis is a powerful technique that can be used to determine both the structural and functional sizes of macromolecules. We have used this technique to probe the structure-function relationships of the recombinant forms of HIV-1 reverse transcriptase (RT). For the p66/p51 and p66/p66 dimeric forms of HIV-1 RT, both the structural and functional target sizes corresponded to that of the dimeric protein, indicating that a primary ionization in one subunit of the HIV-1 RT enzyme results in the concomitant polymer scission of both subunits. In contrast to p66/p51 and p66/p66 RT, the individually isolated p51 subunit of HIV-1 RT inactivated as a monomer. However, in the presence of a DNA template/primer substrate, the radiation inactivation analyses of p51 yielded a structural target size corresponding to that of a dimeric protein. This would indicate that the DNA substrate acted as a scaffold or template for p51 RT homodimer formation. In light of this observation, radiation inactivation studies can readily be applied to other DNA polymerase enzymes, such as the murine leukemia virus RT, for which the functional form of the enzyme has yet to be determined.
 ...
PMID:Structure-activity relationships in HIV-1 reverse transcriptase revealed by radiation target analysis. 1293 Oct 6

HIV-1 reverse transcriptase (RT) is a heterodimer comprising a 66 kDa subunit (p66) and a p66-derived 51 kDa subunit (p51). RT is translated as part of a larger gag-pol polyprotein and subsequently processed to the p66/p51 heterodimer by HIV-1 protease (PR) during viral maturation. The processing events involved in the formation of the RT p66 and p51 subunits and the pathway(s) of RT dimer formation are poorly characterized. Attempts to study the kinetics of PR-catalyzed formation of p66/p51 HIV-1 RT in isolated HIV virions produced in the presence of HIV PR inhibitors were unsuccessful due to difficulties in removal of the tight-binding inhibitor to initiate proteolytic processing. Accordingly, an inducible bacterial expression vector encoding a 90 kDa pol polyprotein fragment was constructed. Following expression in Escherichia coli, the pol polyprotein underwent time-dependent proteolytic processing to the RT p66/p51 heterodimer. This processing was catalyzed entirely by HIV-1 PR since mutations that inactivate PR prevented RT heterodimer formation. The kinetics of RT processing follow an ordered sequential pathway in which RT p66 is first excised from the pol polyprotein, followed by formation of the p51 subunit. Processing of the p66 subunit to form p51 apparently proceeds through a p66/p66 RT homodimer intermediate since the L234A mutation in RT, a mutation that prevents RT dimerization, resulted in the formation of RT p66 only. These results provide the first experimental data defining the pathway for the HIV-1 PR catalyzed formation of the p66/p51 HIV-1 RT heterodimer.
 ...
PMID:Proteolytic processing of an HIV-1 pol polyprotein precursor: insights into the mechanism of reverse transcriptase p66/p51 heterodimer formation. 1518 48

The highly conserved Asn136 is in close proximity to the nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-specific lipophilic pocket of human immunodeficiency virus type 1 (HIV-1) RT. Site-directed mutagenesis has revealed that the catalytic activity of HIV-1 RT mutated at position Asn136 is heavily compromised. Only 0.07 to 2.1% of wild-type activity is retained, depending on the nature of the amino acid change at position 136. The detrimental effect of the mutations at position 136 occurred when the mutated amino acid was present in the p51 subunit but not in the p66 subunit of the p51/p66 RT heterodimer. All mutant enzymes could be inhibited by second-generation NNRTIs such as efavirenz. They were also markedly more sensitive to the inactivating (denaturating) effect of urea than wild-type RT, and the degree of increased urea sensitivity was highly correlated with the degree of (lower) catalytic activity of the mutant enzymes. Replacing wild-type Asn136 in HIV-1 RT with other amino acids resulted in notably increased amounts of free p51 and p66 monomers. Our findings identify a structural/functional role for Asn136 in stabilization of the RT p66/p51 dimer and provide hints for the rational design of novel NNRTIs or drugs targeting either Asn136 in the beta7-beta8 loop of p51 or its anchoring point on p66 (the peptide backbone of His96) so as to interfere with the RT dimerization process and/or with the structural support that the p51 subunit provides to the p66 subunit and which is essential for the catalytic enzyme activity.
 ...
PMID:The amino acid Asn136 in HIV-1 reverse transcriptase (RT) maintains efficient association of both RT subunits and enables the rational design of novel RT inhibitors. 1583 34

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the proteins that currently available anti-HIV-1 drugs target. Inhibitors of HIV-1 PR have become available, and they have lowered the rate of mortality from acquired immune deficiency syndrome (AIDS) in advanced countries. However, the rate of emergence of drug-resistant HIV-1 variants is quite high because of their short retroviral life cycle and their high mutation rate. Serious drug-resistant mutations against HIV-1 PR inhibitors (PIs) frequently appear at the active site of PR. Exceptionally, some other mutations such as L90M cause drug resistance, although these appear at nonactive sites. The mechanism of resistance due to nonactive site mutations is difficult to explain. In this study, we carried out computational simulations of L90M PR in complex with each of three kinds of inhibitors and one typical substrate, and we clarified the mechanism of resistance. The L90M mutation causes changes in interaction between the side chain atoms of the 90th residue and the main chain atoms of the 25th residue, and a slight dislocation of the 25th residue causes rotation of the side chain at the 84th residue. The rotation of the 84th residue leads to displacement of the inhibitor from the appropriate binding location, resulting in a collision with the flap or loop region. The difference in levels of resistance to the three inhibitors has been explained from energetic and structural viewpoints, which provides the suggestion for promising drugs keeping its efficacy even for the L90M mutant.
 ...
PMID:Computational simulations of HIV-1 proteases--multi-drug resistance due to nonactive site mutation L90M. 1677 2


1 2 3 Next >>