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PubMed1989

Comparative studies of the inhibitory properties of antibiotics on human immunodeficiency virus and avian myeloblastosis virus reverse transcriptases and cellular DNA polymerases.

The inhibition of human immunodeficiency virus (HIV) reverse transcriptase by certain antibiotics and related compounds was studied in comparison with that of avian myeloblastosis virus (AMV) reverse transcriptase and cellular DNA polymerases alpha and beta. In general, compounds that inhibited HIV reverse transcriptase also inhibited AMV reverse transcriptase. For example, 10 micrograms/ml of the isoquinoline quinones used in this study inhibited approximately 80% of the activity of reverse transcriptases of HIV and AMV, but did not inhibit the activity of DNA polymerases alpha and beta even at 50 micrograms/ml. AMV enzyme was more sensitive than HIV enzyme to colistin, enduracidins A and B, janiemycin, glysperin A, and thielavins A and B. The streptonigrin alkyl esters, however, inhibited HIV reverse transcriptase only. Sakyomicin A, luzopeptins, ellagic acid and suramine inhibited the activities of reverse transcriptases and cellular DNA polymerases.

Anti-Bacterial Agents↗
PubMed1987

Human immunodeficiency virus reverse transcriptase. General properties and its interactions with nucleoside triphosphate analogs.

Using affinity purified human immunodeficiency virus (HIV) reverse transcriptase the reaction assay conditions were determined. The optimum incorporation of dTMP into the (rA)n(dT)10 template with HIV reverse transcriptase required 6 mM MgCl2 and 80 mM KCl. The template specificity of HIV reverse transcriptase is quite different from those of the human gamma-polymerase-associated reverse transcriptase or avian virus reverse transcriptase. The preferential inhibition of HIV reverse transcriptase as compared to human gamma-reverse transcriptase was observed with several nucleoside analog triphosphates. The Ki values for thymidine triphosphate analogs with HIV reverse transcriptase ranged from 5 to 13 nM with decreasing effectiveness for 3'-fluoro greater than 3'-amino greater than 2',3'-dideoxy greater than 3'-azido groups. This study provides information on the structure activity relationships of the triphosphate analogs inhibitory effects on HIV reverse transcriptase versus human gamma-polymerase-associated reverse transcriptase, and the possible mechanisms of action of 3' azido thymidine and the 2',3'-dideoxynucleosides, and also identifies other nucleoside analogs for possible development as inhibitors of HIV.

Deoxyguanine Nucleotides↗
PubMed1992

Antibodies to reverse transcriptase in HIV infection and progression to AIDS.

Serum antibodies to the reverse transcriptase (ART) of human immunodeficiency virus 1 (HIV-1) were sequentially determined by ELISA in a group of 41 HIV-seropositive male homosexuals and 101 matched healthy controls, over 1.5-6 years (mean follow-up 3.25 years). Mean ART levels were significantly higher in the patient group as compared to the controls (195 +/- 75 vs. 75 +/- 45 absorbance (A) units; P less than 0.05). When analyzed in parallel with clinical evaluation and T-cell subset determinations, a "surge" in ART activity was associated with a more favourable course: eleven patients whose ART profile showed an increase greater than 100 A units (mean delta A 159.6 units) showed an attenuated decrease of CD4+ (T helper) lymphocytes with a mean time of 42.5 months to reach a CD4+ number of 400 cells/mm3. In contrast, 25 matched seropositive patients whose ART remained constant became CD4+ less than 400 cells/mm3 within a mean time of 10.8 months (P less than 0.05). These results as well as individual patients' data support a surge in serum ART as a favourable prognostic indicator, and may indicate a protective role for this antibody which should be followed up and possibly utilized in the treatment or in the design of a vaccine against HIV-1.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Observations on the inhibition of HIV-1 reverse transcriptase by catechins.

The sensitivity and specificity of the inhibition of HIV-1 reverse transcriptase by various catechins have been examined. As previously reported, (-)epicatechin 3-gallate inhibits the viral polymerase. However, it is noted here that this inhibition is not observed in the presence of either serum albumin or Triton X-100. Other catechins behave similarly to (-)epicatechin 3-gallate in that they inhibit polymerase activity only in the absence of these reagents. Additionally, other DNA polymerases are inhibited to a similar degree by (-)epicatechin 3-gallate. Taken cumulatively, these results suggest that these catechins, and in particular (-)epicatechin 3-gallate, bind with no apparent selectivity and that the observed inhibition of HIV-1 reverse transcriptase is non-specific in nature.

Catechin↗
PubMed1992

High-performance liquid chromatographic procedure for the determination of a non-nucleoside HIV-1 reverse transcriptase inhibitor in human plasma.

A method for the determination of a non-nucleoside HIV-1 reverse transcriptase inhibitor in human plasma is described. Plasma samples are extracted using phenyl solid-phase extraction columns. The extract is analyzed by high-performance liquid chromatography with a polybutadiene-coated alumina column and a mobile phase of methanol-0.025 M pH 8 dibasic sodium phosphate buffer (1:1, v/v). Detection is based on ultraviolet absorbance at 326 nm. The assay was validated in the concentration range 10-500 ng/ml when 1-ml aliquots of plasma are extracted. The assay has been utilized to support human pharmacokinetic studies.

Administration, Oral↗
PubMed1992

Measurement of HIV-1 reverse transcriptase by a nonradioactive assay system.

Reverse transcriptase activity was measured by incorporation of dUMP linked to digoxigenin into a suitable template-primer molecule. Incorporation was monitored by using peroxidase-conjugated Fab fragments directed against digoxigenin. The standard assay measuring incorporation of radiolabeled nucleotides into acid-precipitable material was compared with this new immunochemical assay with regard to its usefulness for testing inhibitors of reverse transcriptase.

Digoxigenin↗
PubMed1989

Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities.

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.

Amino Acids↗
PubMed1990

Site-directed mutagenesis of the conserved Asp-443 and Asp-498 carboxy-terminal residues of HIV-1 reverse transcriptase.

Substitution of the conserved Asp-443 residue of HIV-1 reverse transcriptase by asparagine specifically suppressed the ribonuclease H activity of the enzyme without affecting the reverse transcriptase activity, suggesting involvement of this ionizable residue at the ribonuclease H active site. An analogous asparagine substitution of the Asp-498 residue yielded an unstable enzyme that was difficult to enzymatically characterize. However, the instability caused by the Asn-498 mutation was relieved by the introduction of a second distal Asn-443 substitution, yielding an enzyme with wild type reverse transcriptase activity, but lacking ribonuclease H activity.

Amino Acid Sequence↗
PubMed1992

Epitope mapping of HIV-1 reverse transcriptase with monoclonal antibodies that inhibit polymerase and RNase H activities.

Lysates from E. coli expressing HIV-1 reverse transcriptase (RT) as a TrpE fusion protein were used for immunization of BALB/c mice. Twenty hybridomas producing monoclonal antibodies (MAbs) recognizing the RT part of the TrpE-RT fusion protein by Western blot analysis were isolated. Of these, 18 were reactive in immunofluorescence assays when tested on HIV-infected cells. Twelve MAbs were reactive with both the p66 and p51 fragments of RT, while 6 of the MAbs were reactive only with the p66 band, indicating specificity for the C-terminal (RNase H) region of RT. Mapping of the monoclonal antibody binding sites was performed using deletion and insertion mutants of recombinant RT. The antibodies bound to five distinct regions within amino acid sequences 190-560 of RT. In order to map functionally important regions of the RT molecule, the MAbs were tested for their ability to interfere with the polymerase and RNase H activities of the polypeptide. MAbs binding to two different epitopes in the polymerase domain were found to inhibit the polymerase activity. Of these, three MAbs also inhibited the RNase H activity. Two MAbs binding to the same epitope in the RNase H region inhibited RNase H activity and further mediated an effect on the polymerase activity.

Amino Acid Sequence↗
PubMed2025

Kinetic Investigation of Resistance to Islatravir Conferred by Mutations in HIV-1 Reverse Transcriptase.

Islatravir (EFdA) is a novel nucleoside reverse transcriptase translocation inhibitor (NRTTI) that potently blocks HIV-1 replication in vivo. Its unique structural features in contrast to nucleoside reverse transcriptase inhibitors (NRTIs), particularly the 4'-ethynyl and 3'-hydroxy groups, contribute to its high clinical potency. Once intracellularly activated to EFdA 5'-triphosphate (EFdA-TP), it competes with dATP for incorporation by HIV-1 reverse transcriptase (RT) during HIV-1 genomic replication. The 4'-ethynyl group of incorporated EFdA-MP interacts with a hydrophobic pocket of HIV-1 RT, hindering DNA translocation and terminating DNA synthesis. The M184V mutation, commonly associated with resistance to NRTIs such as lamivudine and emtricitabine, and the M184V/A114S mutations, both located within the hydrophobic pocket, were shown to reduce Islatravir susceptibility in cell-based viral resistance selection assays. To elucidate the mechanisms by which these mutations affect Islatravir inhibition, we employed pre-steady-state kinetics to investigate their impact on EFdA-TP incorporation by HIV-1 RT using both DNA and RNA templates. We found that M184V had a modest effect on EFdA-TP incorporation efficiency, increasing it 2-fold with the DNA template and decreasing it 3-fold with the RNA template. In contrast, M184V/A114S significantly inhibited EFdA-TP incorporation, reducing its incorporation efficiency 5.4-fold with the DNA template and 181-fold with the RNA template. These reductions were primarily attributable to corresponding decreases in EFdA-TP incorporation rate constants of 18-fold and 105-fold, respectively. These results suggest that, unlike FDA-approved NRTIs, the clinical efficacy of Islatravir, may not be substantially compromised by the M184V mutation alone but will be significantly reduced by the M184V/A114S mutations.

HIV Reverse Transcriptase↗
PubMed1989

Analysis of the ribonuclease H activity of HIV-1 reverse transcriptase using RNA.DNA hybrid substrates derived from the gag region of HIV-1.

The RNase H activity associated with recombinant p66/p51 HIV-1 reverse transcriptase (RT) has been analyzed in the absence of DNA synthesis by using homogeneous RNA.DNA substrates. The substrates consisted of SP6 runoff transcripts from a portion of the gag region of the HIV-1 genome hybridized to complementary single-stranded DNA from either an M13 subclone or a phagemid transcription vector subclone. The corresponding hybrids either carried a 5'-mismatch of seven nucleotides or were fully base-paired. Analysis of recombinant HIV-1 p66/p51 RT by an activated gel assay employing these substrates suggested that the RNase H activity was exclusively associated with the p66 polypeptide. Denaturing gel electrophoresis was used to analyze the oligonucleotide products generated by hydrolysis of the hybrids by HIV-1 RT, M-MuLV RT, and Escherichia coli RNase H. The significant difference in the time-dependent distribution of products of HIV-1 RT vs E. coli RNase H catalyzed cleavage of 5'-mismatched hybrids indicated that the preparation of recombinant HIV-1 RT was free of contaminating bacterial RNase H. Although the HIV-1 RT associated RNase H activity shares many of the general mechanistic features of other retroviral enzymes [Gerard, G. F. (1981) Biochemistry 20, 256-265], the appearance of unique intermediates and end products in the course of hydrolysis of 5'-mismatched and fully base-paired hybrids indicated a significant difference in the sequence dependence of the kinetics of RNase H cleavage by HIV-1 RT and M-MuLV RT.

Base Sequence↗
PubMed1990

Endoribonucleolytic cleavage of RNA: oligodeoxynucleotide hybrids by the ribonuclease H activity of HIV-1 reverse transcriptase.

Endoribonucleolytic cleavage by the ribonuclease H activity associated with HIV-1 reverse transcriptase was observed in vitro using substrates consisting of synthetic oligodeoxynucleotides hybridized to a 345 nucleotide T7 RNA polymerase transcript derived from the gag region of HIV-1. This observation suggests that a possible mechanism of action of antisense oligonucleotides in the inhibition of viral replication and expression may involve the selective "suicidal" ribonucleolytic cleavage of viral RNA by reverse transcriptase at the site of hybridization of the oligonucleotide.

Base Sequence↗
PubMed1989

Presence of HIV-1 reverse transcriptase inhibitory antibodies in the sera of some HTLV-1 carriers and absence of neutralizing antibodies against HIV-1 in the sera of HTLV-1 carriers.

We have detected inhibitory antibody against HIV-1 reverse transcriptase (RT) in the sera of HTLV-1 carriers, although the lack of immunological cross reaction between HIV-RT and other mammalian retroviruses, including HTLV-1, has been reported elsewhere (1). Sera from 72 HTLV-1 carriers in the southern part of Kyushu in Japan and 61 controls from the same district were studied and controlled for age and sex. One of the 72 HTLV-1 carriers was HTLV-1 antibody-positive, while the others were HIV-1 antibody-negative by ELISA and Western blot method. Sera from two HTLV-1 carriers and one hemophiliac with combined HIV-1 and HTLV-1 infections were found to inhibit HIV-1 RT activity. The inhibition ratios were 72, 65 and 72%, respectively. Protein A effluents of inhibitory sera retained no inhibitory capacity. These results might suggest that HTLV-1 and HIV-1 reverse transcriptase may share common or similar epitopes and that antibodies raised to the HTLV-1 RT react with that of HIV-1 RT in an inhibitory mode. However, the RT inhibitory antibody-positive sera had no neutralizing capacity against HIV-1 infection, supporting the idea that HIV-1 RT inhibitory antibody may not contribute to neutralization against HIV-1. The results also suggest that previous exposure to HTLV-1 does not contribute to neutralizing antibodies against HIV-1 in HIV-1 infected individuals.

Blotting, Western↗
PubMed1991

Crosslinking of substrates occurs exclusively to the p66 subunit of heterodimeric HIV-1 reverse transcriptase.

Photoaffinity labeling of the hetero- and homodimeric forms of HIV-1 reverse transcriptase has been carried out using [32P]rA12-18.dT10 as a representative template-primer and [alpha-32P]dTTP as a representative 2'-deoxynucleoside-5'-triphosphate. UV irradiation produces stable, covalent crosslinks between each of the reactants and both the hetero-(p66/p51) and homodimeric (p66/p66, p51/p51) forms of the enzyme. In the case of the p66/p51 heterodimer, the form of the enzyme believed to be involved in viral replication, crosslinking occurs exclusively to the p66 subunit. These results suggest that the polymerase activity of the heterodimer residues on p66.

Affinity Labels↗
PubMed1991

Inhibitory effect of N3-methyl derivative of 3'-azido-3'-deoxythymidine 5'-triphosphate on the activity of HIV-1 reverse transcriptase.

N3-Methyl derivative of 3'-azido-3'-deoxythymidine 5'-triphosphate (Me-AZTTP) showed a potent inhibitory effect on HIV-1 reverse transcriptase using MS2 phage RNA as the template. The inhibition mechanism of MeAZTTP was noncompetitive with respect to any of the template MS2 RNA, dATP and dCTP. On the other hand, MeAZTTP showed a mixed-type inhibition with respect to dGTP and dTTP. These results indicate that MeAZTTP competes not only with dTTP but also with dGTP.

Antiviral Agents↗
PubMed1991

Crystal structure of the ribonuclease H domain of HIV-1 reverse transcriptase.

The crystal structure of the ribonuclease (RNase) H domain of HIV-1 reverse transcriptase (RT) has been determined at a resolution of 2.4 A and refined to a crystallographic R factor of 0.20. The protein folds into a five-stranded mixed beta sheet flanked by an asymmetric distribution of four alpha helices. Two divalent metal cations bind in the active site surrounded by a cluster of four conserved acidic amino acid residues. The overall structure is similar in most respects to the RNase H from Escherichia coli. Structural features characteristic of the retroviral protein suggest how it may interface with the DNA polymerase domain of p66 in the mature RT heterodimer. These features also offer insights into why the isolated RNase H domain is catalytically inactive but when combined in vitro with the isolated p51 domain of RT RNase H activity can be reconstituted. Surprisingly, the peptide bond cleaved by HIV-1 protease near the polymerase-RNase H junction of p66 is completely inaccessible to solvent in the structure reported here. This suggests that the homodimeric p66-p66 precursor of mature RT is asymmetric with one of the two RNase H domains at least partially unfolded.

Amino Acid Sequence↗