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

We have constructed a series of plasmids which, when introduced into Escherichia coli, induce the overexpression of soluble wild-type and mutated forms of the reverse transcriptases (RTs) from human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). These proteins were analyzed previously for their RNA-dependent DNA polymerase (RDDP) and ribonuclease H (RNase H) activities. In the present study we assayed the different mutant RTs for their DNA-dependent DNA polymerase (DDDP) activity, employing an in situ polyacrylamide gel activity assay. The results indicate that both the RDDP and DDDP catalytic functions of HIV-1 RT mutants are affected similarly by mutations suggesting a high degree of overlap between the catalytic domains involved in both activities. Contrariwise, many of the HIV-2 RT mutants display no correlation between these two DNA polymerase activities, that is, the DDDP activity was not affected by the mutations introduced in the native enzyme in contrast to the RDDP activity. We were thus able to generate mutants of HIV-2 RT that unlike the wild-type RT, are capable of transcribing only DNA and not RNA. The disparity in mutational-catalytic relations between the two HIV-related RTs may reflect a possible difference in the structure and folding properties of the two proteins.
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PMID:The DNA-dependent and RNA-dependent DNA polymerase activities of the reverse transcriptases of human immunodeficiency viruses types 1 and 2. 172 5

Using a computer-assisted molecular modeling protocol, we have completed the three-dimensional structures of HIV-1 reverse transcriptase and the Klenow fragment of DNA polymerase I based on the C alpha crystal coordinates of the individual enzymes. The two model-built structures were then used to compare the electrostatic potential contours and analyze the spatial positions of residues conserved in the catalytic domains of the two enzymes. In spite of rather weak sequence similarity and different folding patterns between the DNA-dependent DNA polymerase (pol I) and the RNA-dependent DNA polymerases (RT), we have noted the occurrence of identical or similar residues at common spatial positions in pol I and RT in a three-dimensional context. The homologous residues present at equivalent spatial position in the Klenow fragment and the p66 subunit of HIV-1 RT may therefore imply their functional similarity. Furthermore, these conserved residues may represent a similar structure-function feature in all polymerases.
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PMID:A computer-assisted analysis of conserved residues in the three-dimensional structures of the polymerase domains of Escherichia coli DNA polymerase I and HIV-1 reverse transcriptase. 750 63

Activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) in the organic extract of the Red Sea sponge Toxiclona toxius was traced by us to five novel natural compounds, namely toxiusol [1], shaagrockol B [3], shaagrockol C [4], toxicol A [6], all of which are sulfated hexaprenoid hydroquinones, and toxicol B [7], the p-hydroquinone derivative of compound 6. The hydrolysis of the two sulfated compounds 1 and 4 yielded the corresponding hydroquinones designated as compounds 2 and 5, and further oxidation of compound 7 afforded the corresponding p-quinone derivative, compound 8. All compounds exhibited inhibitory activity of both DNA polymerizing functions of HIV-1 RT but failed to inhibit the RT-associated ribonuclease H activity. Toxiusol [1] was found to be the most potent inhibitor of the RNA-dependent DNA polymerase function (with 50% inhibition obtained at 1.5 microM and 95% inhibition at 4.6 microM), whereas the DNA-dependent DNA polymerase was significantly less sensitive to the inhibitor (with 50% inhibition achieved at 6.6 microM and 95% inhibition only at 41.6 microM). The fact that compound 1 discriminates between the two DNA polymerase activities of the RT offers new prospects for developing potent and highly specific anti-RT compounds, since the RNA-dependent DNA polymerase activity of RT is the only unique function that is not expressed at significant levels in uninfected mammalian cells.
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PMID:Hexaprenoid hydroquinones, novel inhibitors of the reverse transcriptase of human immunodeficiency virus type 1. 751 Jul 86

The natural product of the Red Sea sponge Verongia sp., identified as 3,5,8-trihydroxy-4-quinolone, was found to be a potent inhibitor of the RNA-directed DNA synthesis of the reverse transcriptases (RTs) of human immunodeficiency viruses type 1 and type 2 (HIV-1 and HIV-2, respectively). This inhibition was unaffected by the nature of the primer template used for DNA synthesis. The DNA-dependent DNA polymerase activity was inhibited to a lesser extent, whereas the ribonuclease H (RNase H) function associated with both HIV RTs was only slightly inhibited. The inhibition by the trihydroxyquinolone is reversible and noncompetitive with respect to both substrates--dTTP and the template primer poly(rA)n.oligo(dT)12-18. The inhibitor binds HIV-1 RT with a high affinity (Ki = 0.46 microM). This compound was shown also to inhibit the catalytic activities of the RT of murine leukemia virus, establishing the general inhibitory effect on retroviral RTs. Introductions of acetyl or methoxy moieties at positions with potential activity have generated three synthetic analogs of the natural compound. Only one analog, 5,8-dimethoxy-4-quinolone, exhibited an inhibition potency similar to that of the unmodified compound. Analysis of the three analogs has led us to the conclusion that the hydroxyl group at the ortho position to the carbonyl group in the pyridinone ring is a key structural element for the inhibitory activity. Thus, it could well be that the inhibitor interacts with the enzyme through a hydrogen bond of this hydroxyl group. We hope that the identification of the inhibitory site of the compound might be an important step toward the rational design of new potent anti-HIV RT drugs.
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PMID:3,5,8-Trihydroxy-4-quinolone, a novel natural inhibitor of the reverse transcriptases of human immunodeficiency viruses type 1 and type 2. 751 Sep 44

Over 25 selected naphthalenesulfonic acid derivatives were evaluated for their inhibitory effect on two different functional domains of the HIV-1 reverse transcriptase (RT), namely the ribonuclease H and DNA polymerase activities. Most of the analogues were found to be either specific toward the DNA polymerase activity or showed nonselective inhibition of both catalytic functions. The most active compounds are either symmetrical derivatives or nonsymmetrical derivatives containing a lipophilic appendage consisting of a palmitoyl or cholesteryl moiety. The six most active compounds in the preliminary screen, derivatives 6, 16, 17, 23, 26, and 27, were subjected to experiments to determine their 50% inhibitory concentration (IC50) values in the assays that measure RNA-dependent DNA polymerase (RDDP), DNA-dependent DNA polymerase (DDDP), and ribonuclease H (RNase H) functions of HIV-1 RT. The most potent derivative was a nonsymmetric cholesterol-linked 4-amino-5-hydroxy-2,7-naphthalenedisulfonic acid analogue, compound 23, which demonstrated an IC50 value of 0.06 microM for inhibiting RDDP activity. Inhibition of DDDP and RNase H activity for this compound was demonstrated at concentrations that were over 100-fold of that for inhibiting RDDP activity. However, the potency of this active compound does not correlate in the whole virus assay, probably due to a lack of cellular entry. The cholesterol derivative, 23, also possesses HIV-1 protease inhibitory activity and belongs to a unique class of multifunctional HIV-1 inhibitors.
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PMID:Synthesis of naphthalenesulfonic acid small molecules as selective inhibitors of the DNA polymerase and ribonuclease H activities of HIV-1 reverse transcriptase. 752 80

We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 and between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting RNase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the RNase H functions.
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PMID:Mutational analysis of the fingers and palm subdomains of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase. 752 67

Novel 3,4-dihydro-6-benzyl-4-oxopyrimidines (DABOs), variously substituted at both the C-2 and C-5 positions of the pyrimidine ring, proved to be specific inhibitors of the human immunodeficiency virus type 1 (HIV-1) in vitro. Some compounds showed potency at micromolar doses, no cytotoxicity at the maximum testable doses and selectivity indexes comparable to that of 2'-3'-dideoxyinosine (ddI). Mode of action studies suggested that DABOs interfered with a step of the virus multiplication cycle following adsorption and preceding integration. Enzyme assays indicated that DABOs targeted HIV-1 reverse transcriptase: they inhibited the RNA-dependent DNA polymerase activity in a template-dependent manner and, to a lesser extent, the DNA-dependent DNA polymerase activity. No inhibition of the RNase-H associated activity was observed. When DABOs were assayed in combination with 3'-azido-3'-dideoxythymidine (AZT) or ddI against HIV-1 in cell cultures, a slightly synergistic inhibitory effect was observed. The combination of DABO 546 and AZTTP in enzyme assays showed that the two compounds were kinetically mutually exclusive.
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PMID:Characterization of the anti-HIV-1 activity of 3,4-dihydro-2-alkoxy-6-benzyl-4-oxopyrimidines (DABOs), new non-nucleoside reverse transcriptase inhibitors. 753 70

The carboxanilides UC84 and UC38 are nonnucleoside inhibitors of both the RNA-dependent and DNA-dependent DNA polymerase activities of HIV-1 reverse transcriptase (RT). We have previously shown that UC84 and UC38 bind to the same site as nevirapine but interact with different RT mechanistic forms, with UC84 preferentially binding to the RT-primer/template complex and UC38 binding only to the RT-primer/template-dNTP ternary complex [Fletcher, R. S., et al. (1995) Biochemistry 34, 4346-4353]. Here we demonstrate that combinations of UC84 and UC38 inhibit RT DNA polymerase activity in vitro in a synergistic manner. This synergy was noted primarily in reactions containing high concentrations of primer/template and Km levels of dNTP substrate and was independent of both primer/template identity and the molar ratio of UC84:UC38. Combination indices were in the range of 0.4-0.6, indicating substantial synergy in the inhibition of RT activity. More importantly, combinations of UC84 and UC38 also showed a high degree of synergy in inhibiting HIV-1 replication in both MT-4 and cord blood mononuclear cells. We believe this to be the first example of synergistic inhibition of HIV-1 RT by combinations of structurally related nonnucleoside inhibitors.
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PMID:Synergistic inhibition of HIV-1 reverse transcriptase DNA polymerase activity and virus replication in vitro by combinations of carboxanilide nonnucleoside compounds. 754 75

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.
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PMID:Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase. 769 3

In the search for effective antiviral agents, we have found 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate (RD4-2025) to be a highly potent and selective inhibitor of human immunodeficiency virus type 1 (HIV-1) in vitro. The 50% effective concentration of RD4-2025 for HIV-1-induced cytopathic effect in MT-4 cells was 37 nM, yet no antiviral activity was observed against HIV-2. In HIV-1 reverse transcriptase (RT) assays, RD4-2025 inhibited both RNA-dependent and DNA-dependent DNA polymerase activities of a recombinant HIV-1 RT with 50% inhibitory concentrations of 0.11 and 3.5 microM, respectively. However, the compound did not affect the activity of human DNA polymerase alpha. Kinetic studies revealed that the inhibition was noncompetitive with respect to dGTP as the substrate and poly(C)/(dG) 12-18 as the template/primer. These results were in accordance with those of nonnucleoside RT inhibitors (NNRTIs), such as R89439 (an alpha-anilinophenylacetamide derivative) and nevirapine, indicating that RD4-2025 also belongs to the family of NNRTIs.
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PMID:Inhibitory effect of 4-(2, 6-dichlorophenyl)-1, 2, 5-thiadiazol-3-yl-N-methyl, N-ethylcarbamate on replication of human immunodeficiency virus type 1 and the mechanism of action. 879 26


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