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PubMed1998

A novel mutation (F227L) arises in the reverse transcriptase of human immunodeficiency virus type 1 on dose-escalating treatment of HIV type 1-infected cell cultures with the nonnucleoside reverse transcriptase inhibitor thiocarboxanilide UC-781.

Treatment of wild-type human immunodeficiency virus [HIV-1(IIIB)]-infected cell cultures with the thiocarboxanilide UC-781 under low selective pressure (i.e., 0.01 microg/ml) resulted in the emergence of V106A RT mutant virus. On increasing drug concentrations (stepwise up to 30 microg/ml) the virus retained the V106A RT mutation but acquired the novel F227L mutation in the RT genome in addition to the L100I, K1O1I, and Y181C mutations. This multiple-mutant virus proved highly resistant to virtually all nonnucleoside RT inhibitors (NNRTIs) (e.g., nevirapine, delavirdine, and loviride), but retained full sensitivity to nucleoside analogs such as AZT, ddI, (-)FTC, and 3TC. The F227 amino acid is highly conserved in HIV-1 strains and forms part of the NNRTI-binding pocket. Our model suggests a hydrophobic interaction between F227 and the chloro atom of UC-781.

Anilides↗

PubMed2004

Structural and biochemical effects of human immunodeficiency virus mutants resistant to non-nucleoside reverse transcriptase inhibitors.

Non-nucleoside reverse transcriptase inhibitors are potent, highly specific, noncompetitive inhibitors of the Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase. They are used commonly as part of potent antiretroviral regimens for the treatment of HIV-1 infection, either in combination with nucleoside analogs, protease inhibitors, or both. A major limitation to the success of non-nucleoside inhibitors is the rapid emergence of HIV-1 variants resistant to these drugs. These drug-resistant variants contain one or more mutations in the non-nucleoside inhibitor binding pocket of reverse transcriptase. This review summarizes the effects that these mutations have on non-nucleoside inhibitor binding, reverse transcriptase structure, HIV-1 replication, and the ability of reverse transcriptase to catalyze DNA polymerization and RNase H cleavage. In addition, studies are summarized evaluating important interactions between mutations conferring resistance to non-nucleoside inhibitors and those conferring resistance to nucleoside analogs. The studies summarized in this review provide important insights into potentially useful approaches to minimize the development of HIV-1 resistant to non-nucleoside inhibitors.

Codon↗

PubMed1995

Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is the major target for antiretroviral therapy of the acquired immunodeficiency syndrome (AIDS). While some inhibitors exhibit activity against most retroviral RTs, others are specific for the HIV-1 enzyme. To develop an animal model for the therapy of the HIV-1 infection with RT inhibitors, the RT of the simian immunodeficiency virus (SIV) was replaced by the RT of HIV-1. Macaques infected with this SIV/HIV-1 hybrid virus developed AIDS-like symptoms and pathology. The HIV-1-specific RT inhibitor LY300046.HCl, but not zidovudine [3'-azido-3'-deoxythymidine (AZT)] delayed the appearance of plasma antigenemia in macaques infected with a high dose of the chimeric virus. Infection of macaques with the chimeric virus seems to be a valuable model to study the in vivo efficacy of new RT inhibitors, the emergence and reversal of drug resistance, the therapy of infections with drug-resistant viruses, and the efficacy of combination therapy.

Acquired Immunodeficiency Syndrome↗

PubMed2005

Effect of a bound non-nucleoside RT inhibitor on the dynamics of wild-type and mutant HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) is an important target for drugs used in the treatment of AIDS. Drugs known as non-nucleoside RT inhibitors (NNRTI) appear to alter the structural and dynamical properties of RT which in turn inhibit RT's ability to transcribe. Molecular dynamics (MD), principal component analysis (PCA), and binding free energy simulations are employed to explore the dynamics of RT and its interaction with the bound NNRTI nevirapine, for both wild-type and mutant (V106A, Y181C, Y188C) RT. These three mutations commonly arise in the presence of nevirapine and result in resistance to the drug. We show that a bound NNRTI hinders the motion of almost all RT amino acids. The mutations, located in the non-nucleoside RT inhibitor binding pocket, partially restore RT flexibility. The binding affinities calculated by molecular mechanics/Poisson-Boltzmann surface accessibility (MM-PBSA) show that nevirapine interacts stronger with wild-type RT than with mutant RT. The mutations cause a loss of van der Waals interactions between the drug and the binding pocket. The results from this study suggest that a good inhibitor should efficiently enter and maximally occupy the binding pocket, thereby interacting effectively with the amino acids around the binding pocket.

Amino Acid Substitution↗

PubMed2004

Mutations conferring drug resistance affect eukaryotic expression of HIV type 1 reverse transcriptase.

Mutations in reverse transcriptase (RT) confer high levels of HIV resistance to drugs. However, while conferring drug resistance, they can lower viral replication capacity (fitness). The molecular mechanisms behind remain largely unknown. The aim of the study was to characterize the effect of drug-resistance mutations on HIV RT expression. Genes encoding AZT-resistant RTs with single or combined mutations D67N, K70R, T215F, and K219Q, and RTs derived from drug-resistant HIV-1 strains were designed and expressed in a variety of eukaryotic cells. Expression in transiently transfected cells was assessed by Western blotting and immunofluorescent staining with RT-specific antibodies. To compare the levels of expression, mutated RT genes were microinjected into the nucleus of the oocytes of Xenopus laevis. Expression of RT was quantified by sandwich ELISA. Relative stability of RTs was assessed by pulse-chase experiments. Xenopus oocytes microinjected with the genes expressed 2-50 pg of RT mutants per cell. The level of RT expression decreased with accumulation of drug-resistance mutations. Pulse-chase experiments demonstrated that poor expression of DR-RTs was due to proteolytic instability. Instability could be attributed to additional cleavage sites predicted to appear in the vicinity of resistance mutations. Accumulation of drug-resistance mutations appears to affect the level of eukaryotic expression of HIV-1 RT by inducing proteolytic instability. Low RT levels might be one of the determinants of impaired replication fitness of drug-resistant HIV-1 strains.

Animals↗

PubMed2000

Analysis of plus-strand primer selection, removal, and reutilization by retroviral reverse transcriptases.

The ability of reverse transcriptase to generate, extend, and remove the primer derived from the polypurine tract (PPT) is vital for reverse transcription, since this process determines one of the ends required for integration of the viral DNA. Based on the ability of the RNase H activity of Moloney murine leukemia virus reverse transcriptase to cleave a long RNA/DNA hybrid containing the PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5'-end, and such cleavages may serve to minimize cleavage events within the PPT itself. If the PPT were to be cleaved inappropriately just upstream of the normal plus-strand origin site, the resulting 3'-ends would not be extended by reverse transcriptase. Extension of the PPT primer by at least 2 nucleotides is sufficient for recognition and correct cleavage by RNase H at the RNA-DNA junction to remove the primer. Specific removal of the PPT primer after polymerase extension deviates from the general observation that primer removal occurs by cleavage one nucleotide away from the RNA-DNA junction and suggests that the same PPT specificity determinants responsible for generation of the PPT primer also direct PPT primer removal. Once the PPT primer has been extended and removed from the nascent plus-strand DNA, reinitiation at the resulting plus-strand primer terminus does not occur, providing a mechanism to prevent the repeated initiation of plus strands.

Base Sequence↗

PubMed1984

Mechanism of the action of the inhibitor of reverse transcriptase.

An inhibitor of reverse transcriptase of Moloney leukemia virus was reported previously in the cytoplasm of the cultured cells (1). In this report, the mechanism of the inhibition was examined. The inhibitory activity was completely abrogated by treatment at 55 degrees C for 20 min. Destruction of the reaction products of reverse transcriptase was not observed. Strong inhibition was observed by preincubation of poly rA oligo dT used as a template-primer, together with the inhibitor, but was not by incubation of poly rA or oligo dT with the inhibitor separately. It is suggested that the inhibitor reacts with poly rA oligo dT complex and interferes with attachment of the reverse transcriptase to the template-primer complex.

Cell Line↗

PubMed1997

Functional differences between the human LINE retrotransposon and retroviral reverse transcriptases for in vivo mRNA reverse transcription.

We have analysed the reverse transcriptase (RT) activity of the human LINE retrotransposon and that of two retroviruses, using an in vivo assay within mammalian (murine and human) cells. The assay relies on transfection of the cells with expression vectors for the RT of the corresponding elements and PCR analysis of the DNA extracted 2-4 days post-transfection using primers bracketing the intronic domains of co-transfected reporter genes or of cellular genes. This assay revealed high levels of reverse-transcribed cDNA molecules, with the intron spliced out, with expression vectors for the LINE. Generation of cDNA molecules requires LINE ORF2, whereas ORF1 is dispensable. Deletion derivatives within the 3.8 kb LINE ORF2 allowed further delineation of the RT domain: > 0.7 kb at the 5'-end of the LINE ORF2 is dispensable for reverse transcription, consistent with this domain being an endonuclease-like domain, as well as 1 kb at the 3'-end, a putative RNase H domain. Conversely, the RT of the two retroviruses tested, Moloney murine leukemia virus and human immunodeficiency virus, failed to produce similar reverse transcripts. These experiments demonstrate a specific and high efficiency reverse transcription activity for the LINE RT, which applies to RNA with no sequence specificity, including those from cellular genes, and which might therefore be responsible for the endogenous activity that we previously detected within mammalian cells through the formation of pseudogene-like structures.

3T3 Cells↗

PubMed1990

Characterization of reverse transcriptase from feline immunodeficiency virus.

Reverse transcriptase has been purified from feline immunodeficiency virus (FIV) by DEAE-cellulose and phosphocellulose chromatography. The purified enzyme consists of a single protein with a Mr of 67,000. When proteolysis is not minimized during purification, a fragment of Mr 54,000 is also observed. This is similar to the reverse transcriptase from human immunodeficiency virus type 1 (HIV), which consists of a polypeptide of Mr 66,000; when proteolysis is not minimized during purification, a fragment of Mr 51,000 is also observed. In direct comparisons, the FIV reverse transcriptase is very similar to the HIV reverse transcriptase in template specificity and requirements for Mg2+. In contrast to these similarities, the FIV and HIV reverse transcriptases are substantially different in primary sequence, as determined by peptide mapping.

Animals↗

PubMed2001

Mitochondrial Toxicity Associated with Nucleoside Reverse Transcriptase Inhibitor Therapy.

Nucleoside reverse transcriptase inhibitors result in a wide range of toxic side effects. These include lactic acidosis syndrome, myopathy, cardiomyopathy, pancreatitis, peripheral neuropathy, and possibly lipodystrophy. Despite the seemingly diverse nature of these side effects, all of these toxicities may be mediated by a common pathophysiologic mechanism, namely, mitochondrial toxicity resulting from nucleoside reverse transcriptase inhibitor-induced inhibition of DNA polymerase g. This article reviews the relevant mitochondrial biology and mechanism underlying nucleoside reverse transcriptase inhibitor-induced mitochondrial toxicity. Clinical manifestations of this toxicity are reviewed followed by a discussion of clinical management.

Journal Article↗

PubMed2001

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription.

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT). The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch). In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT. Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Anti-HIV Agents↗

PubMed2003

Bench-to-bedside review: severe lactic acidosis in HIV patients treated with nucleoside analogue reverse transcriptase inhibitors.

Nucleoside reverse transcriptase inhibitors (NRTIs) are effective antiretroviral therapy for the treatment of HIV-infected patients. NRTIs can induce mitochondrial impairment that leads to a number of adverse events, including symptomatic lactic acidosis. In the present review, we describe the underlying mechanism of NRTI-induced toxicity and the main clinical features of this infrequent, but severe, emerging complication. We also summarise experimental data and clinical observations that support the use of L-carnitine supplementation to reverse NRTI-induced mitochondrial impairment.

Acidosis, Lactic↗

PubMed1995

Enhancement of HIV-1 proteinase activity by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) was found to increase the activity of HIV-1 proteinase in vitro and in eukaryotic cells. The effect of RT on proteinase activity was dose-dependent and independent of pH or salt concentration. The cleavage of sequences corresponding to all the naturally occurring cleavage sites that could be tested in vitro was enhanced. The effect of RT on cleavage was greatest at the cleavage site between RT and integrase. The enhancement of viral proteinase activity by the virus RT may contribute to regulation of the order and/or efficiency of cleavage at different sites during virus replication and maturation.

Base Sequence↗

PubMed2001

Detection of acute bee paralysis virus and black queen cell virus from honeybees by reverse transcriptase pcr.

A reverse transcriptase PCR (RT-PCR) assay was developed for the detection of acute bee paralysis virus (ABPV) and black queen cell virus (BQCV), two honeybee viruses. Complete genome sequences were used to design unique PCR primers within a 1-kb region from the 3' end of both genomes to amplify a fragment of 900 bp from ABPV and 700 bp from BQCV. The combined guanidinium thiocyanate and silica membrane method was used to extract total RNA from samples of healthy and laboratory-infected bee pupae. In a blind test, RT-PCR successfully identified the samples containing ABPV and BQCV. Sensitivities were approximately 1,600 genome equivalents of purified ABPV and 130 genome equivalents of BQCV.

Animals↗

PubMed2002

Lack of hepatotoxicity associated with nonnucleoside reverse transcriptase inhibitors.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs), particularly nevirapine, have been associated with hepatotoxicity. We performed a retrospective study to determine the incidence of NNRTI hepatotoxicity in a group of HIV-infected patients from a New York City practice. These patients are predominantly homosexual white males. We also analyzed the effect of coinfection with hepatitis B (HBV) or hepatitis C (HCV) virus. In total, 272 patients received NNRTIS: 40 (15%) received delavirdine, 91 (33%) received efavirenz, and 141 (52%) received nevirapine. Of the patients with known hepatitis status, 18 of 190 (9%) were coinfected with HBV, and 24 of 205 were coinfected (12%) with HCV. The overall rate of grade 3 to 4 elevations in aspartate aminotransferase (AST) or alanine aminotransferase (ALT) was 3 of 272 (1.1%) and did not differ significantly among the three NNRTIs. HBV or HCV was not associated with a significant increase in AST or ALT elevations. We conclude that NNRTIs are relatively free from hepatotoxicity in this population, despite the presence of coinfection with HBV or HCV.

Alanine Transaminase↗

PubMed2000

Analysis of mutations and suppressors affecting interactions between the subunits of the HIV type 1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) catalyzes the conversion of genomic RNA into cDNA. The enzyme is a heterodimer of p66 and p51 subunits, and the dimerization of these subunits is required for optimal enzyme activity. To analyze this process at the genetic level, we developed constructs that permit the detection of the interaction between these subunits in the yeast two-hybrid system. Genetic analysis of RT subdomains required for heterodimerization revealed that the fingers and palm of p66 were dispensable for p51 interaction. However, as little as a 26-amino acid deletion at the C terminus of p51 prevented dimerization with p66. A primer grip mutation, L234A, previously shown to inhibit RT dimerization by biochemical assays, also prevented RT dimerization in the yeast two-hybrid system. Second-site mutations that restored RT dimerization in yeast to the L234A parent were recovered in the tryptophan repeat region at the dimer interface and at the polymerase active site, suggesting the involvement of these sites in RT dimerization. In vitro binding experiments confirmed the effects of the L234A mutation and the suppressor mutations on the interaction of the two subunits. The RT two-hybrid assay should facilitate the extensive genetic analysis of RT dimerization and should make possible the rapid screening of potential inhibitors of this essential process.

Dimerization↗

PubMed1999

Compression of the DNA minor groove is responsible for termination of DNA synthesis by HIV-1 reverse transcriptase.

HIV-1 reverse transcriptase (RT) generally terminates plus strand DNA synthesis at the centre of the viral genome. The central termination sequence (CTS) contains two termination sites which are located at the 3' end of AnTm motifs. These motifs generate a global curvature of the DNA helix which correlates with termination of DNA synthesis. Here, we have characterized HIV-1 RT termination sites on different DNA sequences. Again, they are located at the 3' end of A-tracts. Using hydroxyl radicals as a probe of the width of the DNA helix, we have shown that RT termination sites are always located a few base-pairs downstream of a compressed minor groove. Mutations which relieve these compressions also abolish the termination events. The replacement of the adenine tracts by 2,6-diaminopurine tracts has a similar effect. Moreover, no termination site is observed on DNA sequences containing phased GC-tracts which curve the DNA helix but compress the major groove. The compression of the DNA minor groove and not necessarily the curved trajectory of the DNA is, therefore, responsible for termination of DNA synthesis at the CTS by HIV-1 RT. This conclusion is consistent with interpretation of other biochemical data on the processivity of HIV-1 RT, based on the structure of a DNA-enzyme complex.

2-Aminopurine↗

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↗