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PubMed1990

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure.

HIV-1 virions contain two reverse transcriptase polypeptides that have apparent molecular weights of 66 and 51 kDa. The 51-kDa form lacks the carboxy-terminal sequences found in the 66-kDa form, and is believed to be a proteolytic digestion product. We have treated purified 66-kDa reverse transcriptase with viral and nonviral proteases. The digestion products were characterized by their ability to react with monoclonal antibodies known to recognize particular segments of the HIV-1 reverse transcriptase. The approximate location of the segments recognized by the monoclonal antibodies was determined by testing the ability of the antibodies to recognize a series of amino- and carboxy-terminal-deleted forms of HIV-1 reverse transcriptase. The segments recognized are not uniformly distributed along the primary amino acid sequence of HIV-1 reverse transcriptase. We suggest that these segments are probably on the surface of the properly folded form of reverse transcriptase. Of the tested proteases, only the viral protease was able to cleave the 66-kDa form to the 51-kDa form without producing additional cleavage products, suggesting that the viral protease cleaves the 66-kDa protein to the 51-kDa form in virions.

Animals↗
PubMed1992

HIV inhibitors targeted at the reverse transcriptase.

HIV inhibitors targeted at the virus-associated reverse transcriptase (RT) can be divided into two groups, depending on whether they are targeted at the substrate or nonsubstrate binding site. To the first group belong the 2',3'-dideoxynucleosides (i.e., DDC, DDI), 3'-azido-2',3'-dideoxynucleosides (i.e., AZT), 3'-fluoro-2',3'-dideoxynucleosides (i.e., FLT), 2',3'-didehydro-2',3'-dideoxynucleosides (i.e., D4C, D4T) and carbocyclic derivatives thereof (i.e., carbovir), 2'-fluoro-ara-2',3'-dideoxynucleosides, 1,3-dioxolane derivatives (i.e., 2',3'-dideoxyl-3'-thiacytidine), oxetanocin analogues and carbocyclic derivatives thereof (i.e., cyclobut-G) and the 9-(2-phosphonylmethoxyethyl)adenine (PMEA) and 9-(3-fluoro-2-phosphonylmethoxypropyl)adenine (FPMPA) derivatives. These compounds need to be phosphorylated intracellularly to their triphosphate forms before they act as competitive inhibitors or alternate substrates (chain terminators) of HIV RT. The second group includes the tetrahydro-imidazo[4,5,l-jk][1,4]-benzodiazepin-2(1H)one (TIBO), 1-[(2-hydroxyethoxy)-methyl]-6-(phenylthio)thymine (HEPT), dipyrido[3,2-b:2',3'-e]-[1,4]diazepin-6-one (nevirapine) and pyridin-2(1H)one derivatives, which interact as such, noncompetitively, with a specific allosteric binding site of HIV-1 RT. Compounds belonging to the two different groups may give rise to synergism which combined, and, likewise, viral resistance to the compounds may arise through different mutations, depending on the nature of the compounds and the group to which they belong.

Adenine↗
PubMed1987

Inhibition of the reverse transcriptase from HIV by 3'-azido-3'-deoxythymidine triphosphate and its threo analogue.

3'-Azido-3'-deoxythymidine triphosphate (erythro) and its threo isomer were synthesized and investigated for their inhibition of HIV reverse transcriptase from virus isolate U 937/HTLV-III. The erythro isomer was a competitive inhibitor of the reaction directed by (rA)n(dT)12-18 and the Ki value was 0.0022 microM. The threo isomer was at least 100-fold less active. The inhibition was specific for dTMP incorporation, and dGMP incorporation using (rC)n(dG)12-18 as template/primer was not affected. The Km value for dTTP varied between 0.7 microM and 1.7 microM. Reverse transcriptase from nine HIV isolates were tested for inhibition by the erythro isomer and only slight differences in sensitivity were observed.

Antiviral Agents↗
PubMedDate not supplied

Evaluation of natural products as inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase.

Inhibition of human immunodeficiency virus reverse transcriptase is currently considered a useful approach in the prophylaxis and intervention of acquired immunodeficiency syndrome (AIDS), and natural products have not been extensively explored as inhibitors of this enzyme. We currently report that the reverse transcriptase assay developed for the detection of the enzyme in virions involving polyadenylic acid.oligodeoxythymidylic acid (poly rA.oligo dT) and radiolabeled thymidine 5'-triphosphate (TTP), can be applied as a simple method for screening the human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) inhibitory potential of natural products. As reported herein, 156 pure natural products have been examined in this system. Benzophenanthridine alkaloids such as faragaronine chloride [1] and nitidine chloride, which are known inhibitors of avian myeloblastosis virus reverse transcriptase, demonstrated potent activity in the HIV-1 RT system, and 1 (IC50 10 micrograms/ml) was adopted as a positive-control substance. Additional inhibitors found were columbamine iodide [2] and other protoberberine alkaloids, the isoquinoline alkaloid O-methylpsychotrine sulfate [3], and the iridoid fulvoplumierin [4]. A number of indolizidine, pyrrolizidine, quinolizidine, indole, and other alkaloids, as well as compounds of many other structural classes, were tested and found to be inactive. A total of 100 plant extracts have also been evaluated, and 15 of these extracts showed significant inhibitory activity. Because tannins and other polyphenolic compounds are potent reverse transcriptase inhibitors, methods were evaluated for the removal of these from plant extracts prior to testing. Polyphenolic compounds were found to be responsible for the activity demonstrated by the majority of plant extracts. After appropriate tannin removal procedures were established, the bioassay system was shown to be generally applicable to both pure natural products and plant extracts. The method also proved useful in directing an isolation procedure with Plumeria rubra to yield fulvoplumierin [4] as an active compound (IC50 45 micrograms/ml).

Antiviral Agents↗
PubMed1992

Interaction of tRNALys with the p66/p66 form of HIV-1 reverse transcriptase stimulates DNA polymerase and ribonuclease H activities.

The precursor homodimeric p66/p66 form of human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) possesses the DNA polymerase and RNase H activities involved in the synthesis of the double-stranded provirus DNA. Reverse transcription is initiated from tRNALys in the case of HIV-1. The present study confirmed that interactions between HIV-1 RT and tRNALys induce protein conformational changes and demonstrated that these interactions stimulate the enzymatic activities associated with the p66 subunit. Thus, the p66/p66 form of the enzyme is strongly stimulated in both DNA polymerase and RNase H activities. Preincubation of the enzyme with tRNA is an obligatory step to obtain the stimulatory effect. The affinity of template, primer, or substrate for RT p66/p66 did not change when the enzyme was preincubated with tRNALys at stimulatory concentrations; the interaction of tRNA with p66/p66 has an effect only on the maximal rate of polymerization. It is further shown that the RNase H domain of RT is much more accessible to protease attack than the DNA polymerase active site.

Chymotrypsin↗
PubMed1992

Kinetics of inhibition of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase by the novel HIV-1-specific nucleoside analogue [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5 "- (4"-amino-1",2"-oxathiole-2",2"-dioxide)thymine (TSAO-T).

[2',5'-Bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5"-(4"-amino-1",2"-oxathiole-2", 2"-dioxide)thymine (TSAO-T) is a representative of a novel class of nucleoside analogues that are endowed with a potent and specific activity against human immunodeficiency virus (HIV) type 1 and are targeted at the HIV-1 reverse transcriptase (RT). Inhibition of HIV-1 RT by TSAO-T was reversible and noncompetitive with respect to dGTP as the substrate and poly(C).oligo(dG) as the template/primer. In contrast with the nonnucleoside derivatives tetrahydroimidazo-[4,5,1-jk][1,4]- benzodiazepin-2(1H)-thione (TIBO) (R-82150), nevirapine (BI-RG-587) and the HEPT derivative I-HEPU-SdM, TSAO-T was not inhibitory to HIV-1 RT in the presence of other homopolymeric template/primers. It did not interfere with the DNA-dependent DNA polymerase function of HIV-1 RT, HIV-2 RT, herpes simplex virus type 1 DNA polymerase, or Taq polymerase. However, TSAO-T proved inhibitory to the HIV-1 RT reaction primed by Escherichia coli 16S/23S rRNA, irrespective of the nature of the radiolabeled 2'-deoxynucleotide 5'-triphosphate (dNTP) used. TSAO-T does not act as a DNA chain terminator. It interacts with HIV-1 RT at a nonsubstrate (dNTP)-binding site.

Antiviral Agents↗
PubMed1990

Expression, purification, and crystallization of the HIV-1 reverse transcriptase (RT).

The HIV-1 pol gene proteins (protease, reverse transcriptase, and endonuclease) were expressed in Escherichia coli N4830-1 by the use of the inducible expression vector pWS60 into which the pol gene was inserted. The p66/p51 heterodimer of reverse transcriptase (RT) was isolated in a highly pure and active form. Crystals of the p66/p51 heterodimer were obtained by the vapor diffusion hanging drop technique. The present crystal quality is still not adequate for high resolution X-ray investigation.

Crystallization↗
PubMed1992

On the engineering of rDNA proteins for purification by immobilized metal affinity chromatography: applications to alternating histidine-containing chimeric proteins from recombinant Escherichia coli.

Recently we reported (D. B. Evans, W. G. Tarpley, and S. K. Sharma, 1991, Protein Expression Purif. 2, 205-213) the cloning, expression, and characterization of recombinant chimeric proteins with an N-terminal metal-binding peptide (mbp), His-Asp-His-Asp-His, and a renin cleavage site. Using these chimerics as examples, we describe here the use of genetically engineered alternating histidines in the purification of these chimerics by immobilized metal affinity chromatography (IMAC). In these chimerics, an alternate histidine-containing peptide was fused to the N-termini of HIV reverse transcriptase (HIV RT) and beta-galactosidase. These chimerics were retarded on immobilized nickel very strongly and could be completely eluted only by the use of 100 mM imidazole, whereas the wildtype HIV RT and Escherichia coli contaminating proteins were eluted between 10 and 35 mM imidazole. When the DNA coding for the mbp was removed, the resulting chimerics were recovered from the IMAC column at 35 mM imidazole. The strong and specific interaction between the chimeric protein and the immobilized metal ion was also abolished when the mbp was specifically cleaved by human renin. It is concluded from these studies that tailoring recombinant proteins with three or more alternate histidines should result in the isolation of such chimeric proteins from crude mixtures in a single step. Since IMAC is amendable to scale up, the tailored specificity engineered into the protein of interest via an mbp should allow one to achieve large-scale isolation of recombinant proteins from bacterial and nonbacterial hosts in a highly predictable manner.

Amino Acid Sequence↗
PubMed1988

Fidelity of HIV-1 reverse transcriptase.

The human immunodeficiency virus type 1 (HIV-1) shows extensive genetic variation and undergoes rapid evolution. The fidelity of purified HIV-1 reverse transcriptase was measured during DNA polymerization in vitro by means of three different assays. Reverse transcriptase from HIV-1 introduced base-substitution errors in DNA from the bacteriophage phi X174 amber3 at estimated frequencies of 1/2000 to 1/4000. Analyses of misincorporation rates opposite a single template adenine residue showed that HIV-1 reverse transcriptase catalyzed nucleotide mismatches with a specificity of A:C much greater than A:G greater than A:A. The high error rate of HIV-1 reverse transcriptase in vitro translates to approximately five to ten errors per HIV-1 genome per round of replication in vivo. This high error rate suggests that misincorporation by HIV-1 reverse transcriptase is, at least in part, responsible for the hypermutability of the AIDS virus. The specificity of misincorporation may provide a basis for the systematic construction of antiviral nucleosides.

Avian Myeloblastosis Virus↗
PubMed1990

Inhibition of the p66/p51 form of human immunodeficiency virus reverse transcriptase by tRNA(Lys).

Human immunodeficiency virus (HIV) reverse transcriptase (RT) uses host tRNA(Lys) partially annealed to the primer binding site (PBS) as primer for the initiation of cDNA synthesis. When assaying cDNA synthesis with a template-primer complex formed by an RNA fragment carrying the PBS site and bovine tRNA(Lys) we noticed that an excess of primer tRNA inhibited strongly the DNA polymerase activity of a recombinant HIV RT (p66-p51 heterodimeric form) produced in transformed yeast cells. The same inhibitory effect was observed with animal DNA polymerase alpha, while avian retrovirus RT was neither affected by tRNA(Lys) nor by its specific primer tRNA(Trp). Although the strongest inhibition was observed with tRNA(Lys), other tRNas like tRNA(Phe) and tRNA(Trp) inhibited also the HIV RT, whereas tRNAs specific for valine, proline and glycine had no effect on enzyme activity. Digestion of tRNA(Lys) with pancreatic RNase abolished the inhibition; on the other hand T1 RNase digestion had no effect on the inhibition suggesting a role of the anticodon region in this effect. The 12- and 14-mers corresponding to the anticodon regions of the three bovine tRNA(Lys) isoacceptors inhibited RT activity, indicating that at least an important part of the inhibitory effect could be ascribed to this tRNA region. A strong stimulation of DNA polymerase activity was observed when the effect of tRNA(Lys) was assayed on a recombinant HIV reverse transcriptase produced in a protease deficient yeast strain, which leads to the production of an active p66 enzyme. The same tRNAs that inhibited strongly the heterodimeric form stimulated the p66 form of HIV reverse transcriptase. The results suggest that although both enzymatic forms are able to interact with tRNA(Lys) the topography, as well as the functional implications of the interaction between the precursor and the mature form of HIV reverse transcriptase with the tRNA(Lys) primer, are different.

Base Sequence↗
PubMed1992

The observed inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate for HIV-1 reverse transcriptase depends on the length of the poly(rA) region of the template.

The inhibitory potency of 3'-azido-3'-deoxythymidine 5'-triphosphate (AZTTP) against HIV-1 reverse transcriptase (HIV-1 RT) has been further evaluated. The results indicate that the previously reported low Ki values for AZTTP against HIV-1 RT (2.35 nM) are due neither to the to the direct tight binding of AZTTP to HIV-1 RT nor to the interaction of the enzyme with AZTMP moiety terminated primer-templates, but instead they are an artifact of the use of a homotemplate-primer [poly(rA).oligo(dT)]. With a set of RNAs of defined sequence as templates, we demonstrate that the observed Ki value for AZTTP depends on the length of the poly(rA) region following the primer in the RNA template. The more adenosyl residues in the RNA template that are available for processive incorporation of TMP moieties, the lower is the observed Ki value for AZTTP. Since the potencies of new inhibitors of HIV-1 RT are usually compared with that for AZTTP, these results have important consequences for the process of discovery of new HIV inhibitors that are of potential use in AIDS therapy.

Base Sequence↗
PubMed1992

Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease.

Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.

Amino Acid Sequence↗
PubMed1992

Anti-aids agents, 6. Salaspermic acid, an anti-HIV principle from Tripterygium wilfordii, and the structure-activity correlation with its related compounds.

Salaspermic acid [1], an inhibitor of HIV reverse transcriptase and HIV replication in H9 lymphocyte cells, was isolated from the roots of Tripterygium wilfordii for the first time. The structure of 1 derived from spectral data was established unequivocally by an X-ray analysis of crystals of the monohydrate. A structure-activity correlation of 1 with ten related compounds indicated that the acetal linkage in ring A and the carboxyl group in ring E of 1 may be required for the anti-HIV activity.

Antiviral Agents↗
PubMed1992

Purification, characterization and crystallization of recombinant HIV-1 reverse transcriptase.

The pol I gene from HIV-1 encoding the protease, reverse transcriptase (RT) and endonuclease has been expressed in Escherichia coli. By modifying the fermentation conditions and developing a new purification scheme, the yield of purified RT has been increased substantially compared with that obtained in an earlier procedure. The expressed RT was purified to homogeneity by ammonium sulphate fractionation followed by chromatography on DEAE Sepharose, Heparin Sepharose, S Sepharose and Poly(A)-Sepharose. The purified HIV-RT is a heterodimer (p66/p51) with an isoelectric point close to 8 and with a tendency to aggregate. The proteolytic product (p51), corresponding to the N-terminal end of the RT molecule, was isolated and identified, as were also some bacterial polypeptides that co-elute with HIV-RT during the early stages of the purification. The heterodimer was crystallized in several morphological forms using the vapour-diffusion hanging drop technique. To concentrate the protein and to change the buffer for crystallization, reverse-salt-gradient chromatography and micropreparative columns were used. The best crystals diffracted to 9 A resolution. The best crystals of native RT diffracted to 9 A resolution and in complex with nucleic acids to 4.5 A resolution (using a rotating anode X-ray source).

Amino Acid Sequence↗
PubMed1991

Interaction of fluorescently labeled dideoxynucleotides with HIV-1 reverse transcriptase.

Succinylfluorescein-labeled dideoxyTTP has been used as a substrate for reverse transcriptase from HIV-1. On addition to the 3'-end of a primer molecule, there is a reduction of fluorescence yield of a factor of ca. 4. Release of a fluorescent DNA/DNA primer/template duplex from its complex with reverse transcriptase results in a reduction of fluorescence by a further factor of 2. The fluorescent nucleotide is incorporated somewhat less efficiently than 3'-azidoTMP and TMP, which show similar incorporation kinetics. Fluorescent chain-terminated primers have been used to investigate the interaction of normal and chain-terminated primer/template complexes with reverse transcriptase. The dissociation constant of a 36/18-mer was 0.65 nM, whereas that of the same complex after the addition of the fluorescent chain-terminating nucleotide to the primer was 3 nM at 25 degrees C. The rate of dissociation of the latter complex from the enzyme was 0.04 s-1. This was decreased by a factor of ca. 10 at high concentrations (greater than 200 microM) of the nucleotide triphosphate complementary to the next position of the template. The results obtained suggest that potent inhibition of reverse transcriptase activity in in vitro assays results from formation of a slowly dissociating complex between the enzyme and chain-terminated primer/template complexes. However, arguments are presented that lead to the conclusion that this is not the mode of inhibition in cells invaded by HIV. At the prevailing relative concentrations in this situation, chain termination resulting in incomplete transcription is likely to be the major factor.

Binding, Competitive↗
PubMed1991

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase.

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.

Amino Acid Sequence↗
PubMed1992

HIV-1 and HIV-2 reverse transcriptases: a comparative study of sensitivity to inhibition by selected natural products.

One hundred and fifty six pure natural products, which had previously been tested against HIV-1 reverse transcriptase, were evaluated for HIV-2 reverse transcriptase inhibitory activity. Compounds that lacked effect in the HIV-1 reverse transcriptase system were found also to be inactive against HIV-2 reverse transcriptase. However, compounds belonging to the benzophenanthridine and protoberberine classes of alkaloids, certain flavonoids, the iridoid, fulvoplumierin, and the ansamycin antibiotic, daunomycin, exhibited similar potencies in both enzyme systems. In contrast, HIV-2 reverse transcriptase was observed to be four-fold more sensitive toward the inhibitory effects of the ipecac alkaloids, O-methylpsychotrine sulfate heptahydrate and psychotrine dihydrogen oxalate. Such differences in susceptibilities to inhibitors may indicate subtle dissimilarities in enzyme structure and function.

Alkaloids↗