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PubMedDate not supplied

Anti-AIDS agents, 2: Inhibitory effects of tannins on HIV reverse transcriptase and HIV replication in H9 lymphocyte cells.

Nine tannins, including gallo- and ellagitannins, were evaluated as potential inhibitors of HIV replication. 1,3,4-Tri-O-galloylquinic acid [1], 3,5-di-O-galloyl-shikimic acid [2], 3,4,5-tri-O-galloylshikimic acid [3], punicalin [6], and punicalagin [7] inhibited HIV replication in infected H9 lymphocytes with little cytotoxicity. Two compounds, punicalin and punicacortein C [8], inhibited purified HIV reverse transcriptase with ID50 of 8 and 5 microM, respectively. Further studies with H9 lymphocytes indicated that chebulagic acid [5] and punicalin did not inactivate virus directly. However, 1,3,4-tri-O-galloylquinic acid and 3,5-di-O-galloylshikimic acid were more effective inhibitors under those conditions. All tannins appear to inhibit virus-cell interactions. Thus, inspite of their anti-RT activity, the mechanism by which tannins inhibit HIV may not be associated with this enzyme.

Antiviral Agents↗

PubMed1987

Immunogenicity of human immunodeficiency virus (HIV) reverse transcriptase: detection of high levels of antibodies to HIV reverse transcriptase in sera of homosexual men.

Immunoglobulin isolated from sera of homosexual men infected with human immunodeficiency virus (HIV) inhibited the reverse transcriptase (RT) activity of HIV. The inhibitory activity was specifically directed against HIV RT, and not against other mammalian retrovirus RT, including human T-lymphotropic virus type I. The relative titer of anti-RT antibody was significantly higher in asymptomatic men than in patients with lymphadenopathy or acquired immune deficiency syndrome (AIDS)-related complex. There was no correlation between the relative titer of anti-RT antibody and the relative titers of antibodies to major virion structural protein as determined by the enzyme-linked immunosorbent assay (ELISA) technique. These data suggest that antibodies to HIV RT may be related to the clinical status and possibly to the different degree of HIV replication in HIV-infected homosexual men.

AIDS-Related Complex↗

PubMed1992

Structure-activity analyses of HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase is a dimeric enzyme which can exist in both homodimeric (p66/p66) and heterodimeric (p66/p51) forms. The monomeric subunits are catalytically inert. However, during DNA synthesis by the dimeric enzyme, only one subunit (p66) appears to carry out the catalysis, while the second subunit serves only a supportive role. In the case of the p66/p66 homodimers, we find that both the subunits are catalytically competent as judged by the observation that a) primer binding occurs to both subunits and b) catalytically inert dimers can be partially activated by replacement of one of the two inactive p66 subunits.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Resolution of microheterogeneity associated with recombinant HIV-1 heterodimeric reverse transcriptase.

HIV-1 reverse transcriptase (RT) has been successfully expressed as a biologically active recombinant protein in Escherichia coli and purified to homogeneity. After partial purification, RT was obtained primarily in a heterodimeric form represented by two subunits of 66 and 51 kDa, but the preparation also included several forms distinguishable in size and charge by chromatography on ionic-exchange and gel-filtration columns. We have developed a purification method that yields a single heterodimeric form of RT. Our strategy involves the selection of RT molecules exhibiting uniformity in elution from QAE Sepharose anion-exchange columns and Superose 12 gel-filtration columns. In the former, RT is resolved into multiple peaks on the basis of enzymatic activity, one of which represents highly active and pure p66:p51 heterodimeric RT. This highly active RT fraction, after gel-filtration chromatography, yields a compositionally pure protein product free of observable microheterogeneity by 1D and 2D polyacrylamide gel electrophoresis under a variety of conditions. Furthermore, the RNAse H enzymatic activity associated with HIV-1 RT has been demonstrated to coelute with the purified polymerase activity during gel filtration at a size (120 kDa) consistent with its location on the heterodimeric protein molecule.

Amino Acid Sequence↗

PubMed1991

HIV reverse transcriptase structure-function relationships.

HIV reverse transcriptase (RT) is the target of the most widely used treatments for AIDS. Biochemical and mutagenesis studies performed on HIV-1 RT are reviewed in light of the enzyme's structure and functions. Features described include domain arrangement, dimerization, proteolytic processing, and specific recognition of the priming tRNA. Possible regions of functional importance as determined by comparative amino acid sequence analysis and by site-directed mutagenesis are identified. Among the conclusions of the analysis is the unexpected realization that the substrate for proteolytic maturation of the HIV-1 RT p66/p66 homodimer to the p66/p51 heterodimer is most likely an unfolded RNase H domain. In addition, the current progress in crystallization and structure determination of HIV-1 RT is described. Finally, a functional-model of the active reverse transcription complex is presented.

Amino Acid Sequence↗

PubMedDate not supplied

[A micromethod for assaying HIV reverse transcriptase].

A micromethod for assaying HIV reverse transcriptase was developed. Accuracy, and sensitivity of the micromethod were comparable to those of the traditional macromethod. The micromethod is easy to perform and can be used in non specialised laboratories.

AIDS Serodiagnosis↗

PubMed1991

Functional characterization of RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase.

The DNA polymerase and RNase H activities of HIV reverse transcriptase are both essential for HIV replication. Although the two activities are both catalyzed by a single polypeptide, they are physically separate; i.e., the DNA polymerase resides in the N-terminal domain whereas the RNase H is localized in the C-terminal domain. The present study was undertaken to characterize the enzymatic properties of these two activities and to determine whether the two catalytic sites are also functionally distinct. We have observed that EGTA specifically stimulates, whereas CaCl2 selectively inhibits, the RNA-dependent DNA polymerase activity but that neither compound has any effect on the RNase H activity of a recombinant HIV reverse transcriptase. The stimulation of the DNA polymerase activity by EGTA is dependent on the Mg2+ concentration; the greatest stimulation is observed at low Mg2+ concentrations. Similarly, the inhibition of DNA polymerase activity by Ca2+ is influenced by Mg2+ concentration. Ca2+ inhibition can be reversed by increasing Mg2+ concentrations, suggesting the possibility that CaCl2 inhibits the reverse transcriptase activity by competing for a metal-binding site on the enzyme. The pyrophosphate analogue phosphonoformate selectively inhibits the polymerase activity but not the RNase H activity of HIV reverse transcriptase. In contrast, the RNase H activity can be selectively inhibited by deoxyadenosine 5'-monophosphate, whereas the DNA polymerase activity is not inhibited. These results suggest that the DNA polymerase and RNase activities are not only physically separate but that they are also functionally distinct.

Blotting, Western↗

PubMed1992

3'-Mercapto-2',3'-dideoxynucleotides are high effective terminators of DNA synthesis catalyzed by HIV reverse transcriptase.

Four 3'-mercapto-2',3'-dideoxynucleoside 5'-triphosphates (A, G, C and T) were tested as DNA chain terminator substrates for calf thymus alpha-DNA polymerase, E. coli DNA polymerase I Klenow fragment, terminal deoxynucleotidyl transferase and reverse transcriptases of AMV, HIV and MLV viruses. It was shown that the analogues selectively and irreversibly terminated DNA chain elongation by AMV and HIV reverse transcriptases and the terminal transferase. Other DNA polymerases tested did not use the nucleotide analogues as chain terminator substrate.

Base Sequence↗

PubMed1989

Stepwise mechanism of HIV reverse transcriptase: primer function of phosphorothioate oligodeoxynucleotide.

Primer recognition by purified HIV reverse transcriptase has been investigated. Earlier we found that the reaction pathway for DNA synthesis is ordered, with template-primer and free enzyme combining to form the first complex in the reaction sequence (Majumdar et al., 1988). We now find that d(C)28 is a linear competitive inhibitor of DNA synthesis against poly[r(A)].oligo[d(T)] as template.primer, indicating that d(C)28 and the template.primer combine with the same form of the enzyme in the reaction scheme, i.e., the free enzyme. The phosphorothioate oligodeoxynucleotide Sd(C)28 also is a linear competitive inhibitor against template.primer. However, the Ki for inhibition (approximately 2.8 nM) is approximately 200-fold lower than the Ki for inhibition by d(C)28. Since the inhibition is linear competitive, the dissociation constant is equal to the Ki for inhibition. Filter binding assays confirmed high-affinity binding between Sd(C)28 and the enzyme and yielded a KD similar to the Ki for inhibition. Substrate kinetic studies of DNA synthesis using Sd(C)28 as primer, and poly[r(I)] as template, revealed that the Km for Sd(C)28 is 24 nM. The Km for this primer is, therefore, 8-fold higher than the KD for enzyme-primer binding (2.8 nM). These results enable calculation of real time rate values for the enzyme-primer association (kon = 5.7 x 10(8) M-1 s-1) and dissociation (koff = 1.6 s-1).

Binding, Competitive↗

PubMed1989

Structural characterization of HIV reverse transcriptase: a target for the design of specific virus inhibitors.

The reverse transcriptase (RT) of HIV is an important target for chemotherapy as demonstrated by the effective treatment of AIDS patients with zidovudine, a potent inhibitor of RT. Structural studies of HIV RT were therefore undertaken with a view to designing more effective inhibitors. To obtain sufficient quantities of enzyme for these studies the reverse transcriptase gene of HIV was cloned into a high level expression plasmid yielding reverse transcriptase at a level of 10% of the total Escherichia coli proteins. Monoclonal antibodies to RT were raised in mice and have been used to purify the enzyme by immunoaffinity chromatography. Crystallization of the enzyme has been achieved and studies are underway to determine its three-dimensional structure. In addition, carboxy-terminal truncated mutants were prepared by inserting stop codons into the gene at appropriate sites. The proteins expressed were analysed for RT and RNase H activity and used for mapping RT epitopes. This, together with previous data on site-directed mutagenesis of conserved regions of HIV RT has helped to map some of the structural and functional regions of the enzyme.

Animals↗

PubMed1991

HIV reverse transcriptase inhibiting antibodies detected by a new technique: relation to p24 and gp41 antibodies, HIV antigenemia and clinical variables.

A new assay for HIV reverse transcriptase activity inhibiting antibodies (RTI-ab) was used for the analysis of a large collection of sera sampled before and after confirmation of HIV infection. In this assay HIV-RT was preincubated with diluted serum, after which residual RT activity was determined by a technique using a template coupled to macrobeads and 125I-lodo-deoxyuridine-triphosphate as the tracer-substrate. Of the 936 sera analysed, 818 were found positive for RTI-ab, and 824 were positive in Western blot (Wb). The prevalence of RTI-ab compared to Wb was therefore 99.3%. The corresponding figure for 930 sera analysed for envelope-ab, i.e., gp41-ab, was 823 positive, and of these 930 sera 815 were Wb positive, giving a comparative prevalence of 101%. In contrast, only 678 samples of 993 analyzed for core ab, i.e., p24, were positive, giving a prevalence of 77.0% as 880 of these samples were Wb positive. Thus, RTI-ab was as prevalent as gp41-ab, and although the analyses of RTI-ab amounts in different stages showed decreasing levels in stage IV compared to stages II or III, all of the sera except 1 were found positive in stages III and IV. Further, it was found that both the few RTI-ab negative samples in stage II and the few RTI-ab positive samples among Wb negative sera were sampled in connection with seroconversion. The specificity of the RTI-ab assay was 100% in a test of 200 serum samples from HIV negative blood donors. It was concluded that RTI-ab analyses can be made highly sensitive and specific and useful for studies of HIV infection.

AIDS Serodiagnosis↗

PubMed1991

Inhibition of the RNase H activity of HIV reverse transcriptase by azidothymidylate.

The effects of AZTMP and other nucleoside 5'-monophosphates on the RNA-dependent DNA polymerase and RNase H activities of a recombinant HIV reverse transcriptase have been investigated. Both activities are sensitive to inhibition by millimolar concentrations of AZTMP with MgCl2 as divalent cation activator. Substitution of Mn2+ for Mg2+ markedly potentiates the inhibition of RNase H activity by AZTMP, reducing the IC50 from 5 to 0.05 mM. In contrast, Mn2+ does not alter the sensitivity of the RNA-dependent DNA polymerase activity to inhibition by AZTMP. The inhibition of RNase H activity by AZTMP can be reversed by increasing concentrations of the substrate poly(A)/poly(dT), suggesting that AZTMP may compete with the substrate for binding at the active site of RNase H. Other nucleoside 5'-monophosphates do not inhibit RNase H in the presence of Mg2+. However, in the presence of Mn2+, deoxy- and dideoxynucleoside 5'-monophosphates that are complementary to the DNA strand of the heteroduplex substrate are somewhat inhibitory. The RNA-dependent DNA polymerase activity is a slightly inhibited by AZTMP and ddTMP in either Mg2+ or Mn2+, and substitution of Mn2+ for Mg2+ results in inhibition by ddAMP as well. Naturally occurring ribo- or deoxyribonucleoside 5'-monophosphates are not inhibitory at concentrations up to 5 mM. Since AZTTP inhibits the RNA-dependent DNA polymerase activity of HIV reverse transcriptase at nanomolar concentrations, it is unlikely that the inhibition of this activity by AZTMP plays a significant role in the antiviral effect of AZT. However, the inhibition of the RNase H activity by AZTMP, which can reach millimolar concentrations in vivo, may account for part of the sensitivity of the virus to AZT.

Antiviral Agents↗

PubMed1988

Conformation and sandwiching of bases by azido groups in the crystal structure of 3'-azido-3'-deoxy-thymidine (AZT), an antiviral agent that inhibits HIV reverse transcriptase.

The crystal structure of 3'-azido-3'-deoxy-thymidine (AZT), an antiviral agent that inhibits HIV reverse transcriptase, has been determined from three-dimensional x-ray diffractometer data. The crystal structure contains two independent molecules of AZT forming a hydrogen bonded dimer but exhibiting different conformations. These conformations are different from those theoretically calculated by molecular mechanics methods. The azido groups associate with each other and interrupt the base stacking, forming a sandwich of two stacked bases. The close conformational similarity of AZT to thymidine explains why AZT is a good substrate for thymidine kinase. The selective inhibition of reverse transcriptase by AZT is not due to any conformational restrictions imposed by the azido group but likely due to their stereoelectronic properties.

Azides↗

PubMed1990

Denaturation/refolding of purified recombinant HIV reverse transcriptase yields monomeric enzyme with high enzymatic activity.

We engineered a prokaryotic expression vector encoding the HIV reverse transcriptase (RT). We grew Escherichia coli JM109 carrying the vector in a 250-liter stirred tank fermentor and purified RT (p66) under native conditions to apparent homogeneity. Purified p66 (greater than or equal to 5 mg/ml) was not stable, and was rapidly processed to its 51 kD derivative (p51), until p66:p51 levels were approximately 1:1. These latter RT preparations were chromatographed as heterodimers and had approximately fivefold higher specific RT enzymatic activities compared with those containing predominantly p66. P66 purified under dilute concentrations (less than or equal to 0.5 mg/ml) was monomeric in solution, resistant to p51 processing for weeks at 4 degrees C, but also had low specific RT enzymatic activities. To attempt the preparation of homogeneous p66 with specific RT enzymatic activities equivalent to p66:p51 heterodimers, purified heterodimers were denatured and p66 was purified and refolded during extensive dialysis (refolded p66). Refolded p66 (less than or equal to 0.5 mg/ml) was monomeric in solution and had identical specific RT enzymatic activities, Km for dTTP, and inhibition by 3'-azido-3'-deoxythymidine triphosphate compared with heterodimeric p66:p51 RT. The data indicates that HIV RT obtained from recombinant E. coli under native conditions is extensively processed at concentrations promoting dimerization. Moreover, RT denaturation and refolding yields apparently homogeneous monomeric p66, with specific RT enzymatic activities equivalent to heterodimeric RT.

Base Sequence↗

PubMed1992

Enzymatic kinetic studies with the non-nucleoside HIV reverse transcriptase inhibitor U-9843.

The polymer of ethylenesulfonic acid (U-9843) is a potent inhibitor of HIV-1 RT (reverse transcriptase) and the drug possesses excellent antiviral activity at nontoxic doses in HIV-infected lymphocytes grown in tissue culture. The drug also inhibits RTs isolated from other species such as AMV and MLV retroviruses. Enzymatic kinetic studies of the HIV-1 RT catalyzed RNA-directed DNA polymerase function, using synthetic template:primers, indicate that the drug acts generally noncompetitively with respect to the template:primer binding site but the specific inhibition patterns change somewhat depending on the drug concentration. The inhibitor acts noncompetitively with respect to the dNTP binding sites. Hence, the drug inhibits this RT polymerase function by interacting with a site distinct from the template:primer and dNTP binding sites. In addition, the inhibitor also impairs the DNA-dependent DNA polymerase activity of HIV-1 RT and the RNase H function. This indicates that the drug interacts with a target site essential for all three HIV RT functions addressed (RNA- and DNA-directed DNA polymerases, RNase H).

Antiviral Agents↗

PubMed1992

Non-nucleoside inhibitors of HIV reverse transcriptase: screening successes--clinical failures.

A little less than two years ago, the first report describing non-nucleoside inhibitors of HIV reverse transcriptase (RT) led to the high anticipation that a range of new drugs could soon be available for the treatment of AIDS. The intervening period has given rise to several such agents but recent clinical trial data has indicated this optimism to be premature. This short review seeks to trace the brief history of the drug discovery process and to assess whether there are lessons to be learnt from the episode.

Antiviral Agents↗

PubMed1991

Factors contributing to the inhibition of HIV reverse transcriptase by chain-terminating nucleotides in vitro and in vivo.

Arguments are presented leading to the conclusion that two major factors contribute to the potency of inhibition of DNA-polymerase activity by chain-terminating nucleotides. The relative significance of these factors varies with the reaction conditions, particularly with the length of the template and the concentration ratio of enzyme (reverse transcriptase or other DNA polymerase) to primer. It is concluded that potent inhibition of HIV-reverse transcriptase activity under typical in vitro and in vivo conditions arises from different features of the interaction of chain terminators with the enzyme. A new method of testing for the parameter important under in vivo conditions is suggested.

HIV↗

PubMed1991

Inhibition of HIV-reverse transcriptase activity by some phloroglucinol derivatives.

Four phloroglucinol derivatives, named mallotophenone (5-methylene-bis-2,6-dihydroxy-3-methyl-4-methoxyacetophenone), mallotochromene (8-acetyl-5,7-dihydroxy-6-(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-2,2-dimethylchromene), mallotojaponin (3-(3,3(dimethylallyl)5-(3(acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone) and mallotolerin (3-(3-methyl-2-hydroxybut-3-enyl)-5(3-acetyl-2,4- dihydroxy-5-methyl-6-methoxybenzyl)-phloracetophenone), have been tested for their ability to inhibit the activity of human immunodeficiency virus (HIV)-reverse transcriptase. Under the reaction conditions with (rA)n.(dT)12-18 as the template.primer, the enzyme activity was inhibited by approximately 70% in the presence of 10 micrograms/ml mallotochromene or mallotojaponin, whereas mallotophenone and mallotolerin were much less inhibitory to the enzyme. The enzyme activity was also inhibited, though to lesser extent, by these compounds under similar conditions with initiated MS-2 phage RNA as the template.primer. The mode of inhibition was, as analyzed with mallotojaponin, competivite with respect to the template.primer, (rA)n.(dT)12-18, and non-competitive with respect to the triphosphate substrate, dTTP. The Ki value of mallotojaponin for HIV-reverse transcriptase was determined to be 6.1 microM.

Base Sequence↗