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: UMLS:C0021051 (immunodeficiency)
71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human immunodeficiency virus (HIV) reverse transcriptase (RT) is a heterodimeric enzyme composed of a 66 kDa (p66) and a 51 kDa (p51) subunit. Recently we showed that p51 plays an important role in the conformation of p66 within the HIV-1 RT heterodimer and hence appears to influence its catalytic activities [Amacker, M., and H ubscher, U. (1998) J. Mol. Biol. 278, 757-765]. This was further investigated here via construction of three intramolecular chimeras of HIV-1 and FIV RTs. The first 25 and 112 amino acids of the N terminus, respectively, as well as the last 22 amino acids of the C terminus in the p51 subunit of HIV-1 RT were exchanged with the corresponding regions of the FIV RT and combined with the wild-type HIV-1 p66. Characterization of these chimeric RT heterodimers demonstrated significant biochemical differences in (i) DNA-dependent DNA synthesis, (ii) strand displacement DNA synthesis, and (iii) RNase H activity. Our results indicate that both the N and C termini of HIV-1 RT p51 appear to be important in stabilizing the RT heterodimer for enzymatic functions.
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PMID:Intramolecular chimeras of the p51 subunit between HIV-1 and FIV reverse transcriptases suggest a stabilizing function for the p66 subunit in the heterodimeric enzyme. 993 Oct 31

The human immunodeficiency virus (HIV-1) nucleocapsid protein NCp7 containing two CX2CX4HX4C-type zinc fingers was proposed to be involved in reverse transcriptase (RT)-catalyzed proviral DNA synthesis through promotion of tRNA3Lys annealing to the RNA primer binding site, improvement of DNA strand transfers, and enhancement of RT processivity. The NCp7 structural characteristics are crucial because mutations altering the finger domain conformation led to noninfectious viruses characterized by defects in provirus integration. These findings prompted us to study a putative RT/NCp7 protein-protein interaction. Binding assays using far Western analysis or RT immobilized on beads clearly showed the formation of a complex between NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 microM with a 1:1 stoechiometry. This interaction was confirmed by chemical cross-linking and co-immunoprecipitation of the two proteins in a viral environment. Competition experiments using different NCp7 mutants showed that alteration of the finger structure disrupted RT recognition, giving insights into the loss of infectivity of corresponding HIV-1 mutants. Together with structural data on RT, these results suggest that the role of NCp7 could be to enhance RT processivity through stabilization of a p51-induced active form of the p66 subunit and open the way for designing new antiviral agents.
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PMID:Evidence of interactions between the nucleocapsid protein NCp7 and the reverse transcriptase of HIV-1. 1019 17

The biologically relevant and active form of human immunodeficiency virus reverse transcriptase is a heterodimer produced in a two-step dimerization process. Dimerization involves first the rapid association of the two subunits, followed by a slow conformational change yielding a fully active form. In the present study, we demonstrate that the interaction between the thumb domain of p51 and the RNase-H domain of p66 plays a major role in an essential conformational change required for proper folding of the primer/template and the tRNA-binding site, for maturation and for activation of heterodimeric reverse transcriptase. A synthetic peptide derived from the sequence within the thumb domain of p51, which forms the interface with the RNase-H domains of p66, binds heterodimeric reverse transcriptase with an apparent dissociation constant in the nanomolar range and selectively inhibits activation of heterodimeric reverse transcriptase with an inhibition constant of 1.2 microM. A detailed study of the mechanism of inhibition reveals that this peptide does not require dissociation of heterodimeric RT for efficient inhibition and does not affect subunit association, but interferes with the conformational change required for activation of heterodimeric reverse transcriptase, resulting in a decrease in the affinity of reverse transcriptase for the tRNA and an increase in the stability of the primer/template/reverse transcriptase complex. We have previously proposed that the dimeric nature of reverse transcriptase represents an interesting target for the design of antiviral agents. On the basis of this work, we propose that the conformational changes involved in the activation of reverse transcriptase similarly represent an important target for the design of novel antiviral compounds.
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PMID:The thumb domain of the P51-subunit is essential for activation of HIV reverse transcriptase. 1056 92

The nonnucleoside inhibitor binding pocket is a well-defined region in the p66 palm domain of the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT). This binding pocket opens toward the interface of the p66/p51 heterodimer and we have investigated whether ligand binding at or near this site induces structural changes that have an impact on the dimeric structure of HIV-1 RT. 1-[2',5'-bis-O-(tert-butyldimethylsilyl]-3'-spiro-5' '-(4' '-amino-1' ',2' '-oxathiole-2' ',2' '-dioxide)-3-ethylthymine (TSAOe(3)T) was found to destabilize the subunit interactions of both the p66/p51 heterodimer and p66/p66 homodimer enzymes. The Gibbs free energy of dimer dissociation (DeltaG(D)(H)2(O)) is decreased with increasing concentrations of TSAOe(3)T, resulting in a loss in dimer stability of 4.0 and 3.2 kcal/mol for the p66/p51 and p66/p66 HIV-1 RT enzymes, respectively. This loss of energy is not sufficient to induce the dissociation of the subunits in the absence of denaturant. This destabilizing effect seems to be unique for TSAOe(3)T, since neither the tight-binding inhibitor UC781 nor nevirapine showed any effects on the stability of HIV-1 RT dimers. TSAOe(3)T was unable to destabilize the subunit interactions of the E138K mutant enzyme, which exhibits significant resistance to TSAOe(3)T inhibition. Molecular modeling of TSAOm(3)T into the nonnucleoside inhibitor binding pocket of wild-type RT suggests that it makes significant interactions with the p51 subunit of the enzyme, a feature that has not been observed with other types of nonnucleoside inhibitors. The observed destabilization of the dimeric HIV-1 RT may result from structural/conformational perturbations at the reverse transcriptase subunit interface.
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PMID:Human immunodeficiency virus type 1 reverse transcriptase dimer destabilization by 1-[Spiro[4"-amino-2",2" -dioxo-1",2" -oxathiole-5",3'-[2', 5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]]]-3-ethylthy mine. 1068 24

Cys(38) and Cys(280) of p66/p51 human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) can be converted to Ser without affecting enzyme function. We have exploited this feature to construct and purify "monocysteine" RT derivatives for site-specific modification with the photoactivable cross-linking agent, p-azidophenacyl bromide. Acylation of a unique cysteine residue introduced at the extreme C terminus of the p66 subunit (C(561)) with an azidophenacyl group allowed us to probe contacts between residues C-terminal to alpha-helix E' of the RNase H domain and structurally divergent nucleic acid duplexes. In a binary complex of RT and template-primer, we demonstrate efficient cross-linking to primer nucleotides -21 to -24/-25, and template nucleotides -18 to -21. Cross-linking specificity was confirmed by an analogous evaluation following limited primer extension, where the profile is displaced by the register of DNA synthesis. Finally, contact with a DNA primer hybridized to an isogenic RNA or DNA template indicates subtle alterations in cross-linking specificity, suggesting differences in nucleic acid geometry between duplex DNA and RNA/DNA hybrids at the RNase H domain. These data exemplify how site-specific acylation of HIV-1 RT can be used to provide high resolution structural data to complement crystallographic studies.
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PMID:Probing contacts between the ribonuclease H domain of HIV-1 reverse transcriptase and nucleic acid by site-specific photocross-linking. 1074 61

Crystallographic studies of the Mn(2+)-doped RNase H domain of human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) have revealed two bound Mn2+ separated by approximately 4A and surrounded by a cluster of four conserved carboxylates. Escherichia coli RNase H is structurally similar to the RNase H domain of HIV-1 RT, but requires one divalent metal cation for its activity, implying either that the HIV-1 RT RNase H domain contrasts in its ability to bind two divalent metal ions, or that the crystallographic data reflect specific use of Mn2+ and/ or the doping technique employed. Metal binding stoichiometry has been determined for Mn2+ and the biologically more relevant Mg2+ cation by solution calorimetric studies of native and recombinant p66/p51 HIV-1 RT. Three Mn2+ ions bind to HIV-1 RT apo-enzyme: one at the DNA polymerase and two at the RNase H catalytic center, the latter being consistent with crystallographic results. However, only one Mg2+ ion is bound in the RNase H catalytic center. Several mechanistic implications arise from these results, including the possibility of mutually exclusive Mg2+ binding sites that might be occupied according to the specific reaction being catalyzed by the multifunctional RNase H domain. The occurrence of distinct binding stoichiometries for Mg2+ and Mn2+ to multifunctional enzymes has previously been reported.
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PMID:Metal-ion stoichiometry of the HIV-1 RT ribonuclease H domain: evidence for two mutually exclusive sites leads to new mechanistic insights on metal-mediated hydrolysis in nucleic acid biochemistry. 1076 38

The two subunits of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (HIV-1 RT), p66 and p51, were coexpressed in Escherichia coli along with the E. coli chaperonin system GroEL/GroES. Coexpression increases the yield of heterodimeric HIV-1 RT by a factor of 4 to 5 and improves the nucleic acid binding affinity of HIV-1 RT by a factor of 1.6. We have analyzed the reasons for the improvements. The total increase in yield of HIV-1 RT can be attributed to an accumulation of RT subunits in the cells (factor of about 2.8) and an increased growth of the E. coli cells (factor of about 1.4). One reason for the accumulation in the cells is an improved stability of HIV-1 RT subunits toward bacterial proteases. In vitro studies showed that the nucleic acid binding affinity of HIV-1 RT purified from cells that did not coexpress GroELS was stimulated by adding purified GroELS (approx 1.5-fold), whereas HIV-1 RT stemming from cells coexpressing GroELS was stimulated only marginally (approx 1.1-fold). The in vivo as well as the in vitro studies suggest that the chaperonin interacts with HIV-1 RT and therefore affects the folding process of HIV-1 RT.
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PMID:Effect of Escherichia coli chaperonin GroELS on heterologously expressed human immunodeficiency virus type 1 reverse transcriptase in vivo and in vitro. 1094 91

An ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) has been developed using recombinant HIV-1 reverse transcriptase (rRT) as antigen. However, some disadvantages were noted in the use of rRT as antigen: rRT was produced only with low efficiency in widely used strains of Escherichia coli using a rather long DNA fragment (3,012 bp) of the whole HIV-1 pol gene, and it was impossible to produce fusion proteins of RT for simple purification, since rRT is a heterodimer of p66 and p51. In this study, recombinant HIV-1 p51 and p66 with Ser-Ser at the N termini (Ser-Ser-rp51 and Ser-Ser-rp66) were produced in E. coli as fusion proteins with maltose binding protein containing a factor Xa site between the two proteins and were purified after digestion with factor Xa. Ser-Ser-rp51 was produced in larger amounts and purified in higher yields with less polymerization than Ser-Ser-rp66. Polymerized Ser-Ser-rp66 tended to be precipitated on mercaptoacetylation for conjugation to beta-D-galactosidase (used as a label) and showed higher nonspecific and lower specific signals in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 than Ser-Ser-rp51. The signals for serum samples of HIV-1-seropositive subjects by immune complex transfer enzyme immunoassay of antibody IgG to HIV-1 using Ser-Ser-rp51 as antigen (Y) were well correlated to those obtained using rRT as antigen (X) (log Y = 0.99 log X + 0.23; r = 0.99). Thus, the use of rp51 as antigen was advantageous over that of rp66 and rRT in an immune complex transfer enzyme immunoassay of antibody IgG to HIV-1.
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PMID:Recombinant p51 as antigen in an immune complex transfer enzyme immunoassay of immunoglobulin G antibody to human immunodeficiency virus type 1. 1106 8

We have recently demonstrated that metalloporphyrins are potent inhibitors of both human immunodeficiency virus type 1 (HIV-1) and human immunodeficiency virus type 2 (HIV-2) reverse transcriptases (RTs) [Argyris, E.G., Vanderkooi, J.M., Venkateswaran, P.S., Kay, B.K., and Paterson, Y. (1999) J. Biol. Chem. 274, 1549-1556]. In addition, by screening a phage peptide library we discovered that a peptide with sequence similarity to residues 398-407 from the connection subdomain of HIV RTs binds heme. These findings suggested that this highly conserved region may be the binding site for metalloporphyrins and a novel site for inhibition of enzymatic activity. Our most recent data presented here confirm this suggestion. Screening of HIV-1 RT 398-407 peptide analogs by fluorescence assays demonstrates that Trp residues at positions 401 and 402 are important for heme binding. Furthermore, site-directed mutagenesis of these residues verified these findings and indicated that heme inhibits HIV-1 RT by binding on the connection subdomain of the p66 subunit of the enzyme but not on the p51 subunit. This was also confirmed by analyzing the binding affinities of heme for mutant HIV-1 RT heterodimers, using intrinsic fluorescence assays. The clear identification of the connection domain as a novel inhibition site is crucial in understanding the mechanism of heme binding and enzymatic inhibition and will facilitate the generation of novel porphyrin-based inhibitors of RT.
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PMID:Mutagenesis of key residues identifies the connection subdomain of HIV-1 reverse transcriptase as the site of inhibition by heme. 1117 58

We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations. To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication. Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods. To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA. Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages. Thus, the pol gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome. The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance.
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PMID:An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase. 1131 28


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