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PubMed2002

Simultaneous quantitative assay of six HIV protease inhibitors, one metabolite, and two non-nucleoside reverse transcriptase inhibitors in human plasma by isocratic reversed-phase liquid chromatography.

A rapid (less than 30 min), sensitive, and specific liquid chromatography method for simultaneous assay of nine antiretroviral drugs in human plasma is described. This technique allows therapeutic drug monitoring of six approved protease inhibitors (amprenavir, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir) and two approved non-nucleoside reverse transcriptase inhibitors (efavirenz and nevirapine). Assays were performed after diethyl ether liquid-liquid extraction from 250-microL plasma samples. Chromatographic separation was achieved on an X-TERRA (Waters; Saint Quentin, France) column using a 58% water (with 3 mmol/L pyrrolidine) and 42% acetonitrile mobile phase. Three ultraviolet wavelengths were used for detection with a diode array detector. This method allowed quantitative assay of all nine antiretroviral drugs within a concentration range of 25 ng/mL to 9000 ng/mL. The method has been validated extensively and has been in routine use in our laboratory for several months for drug monitoring in plasma samples from patients treated with antiretroviral drugs.

Chromatography, High Pressure Liquid↗

PubMed1995

tRNAs as primer of reverse transcriptases.

Genetic elements coding for proteins that present amino acid identity with the conserved motifs of retroviral reverse transcriptases constitute the retroid family. With the exception of reverse transcriptases encoded by mitochondrial plasmids of Neurospora, all reverse transcriptases have an absolute requirement for a primer to initiate DNA synthesis. In retroviruses, plant pararetroviruses, and retrotransposons (transposons containing long terminal repeats), DNA synthesis is primed by specific tRNAs. All these retroelements contain a primer binding site presenting a Watson-Crick complementarity with the primer tRNA. The tRNAs most widely used as primers are tRNA(Trp), tRNA(Pro), tRNA(1,2Lys), tRNA(3Lys), tRNA(iMet). Other tRNAs such as tRNA(Gln), tRNA(Leu), tRNA(Ser), tRNA(Asn) and tRNA(Arg) are also occasionally used as primers. In the retroviruses and plant pararetroviruses, the primer binding site is complementary to the 3' end of the primer tRNA. In the case of retrotransposons, the primer binding site is either complementary to the 3' end or to an internal region of the primer tRNA. Additional interactions taking place between the primer tRNA and the retro-RNA outside of the primer binding site have been evidenced in the case of Rous sarcoma virus, human immunodeficiency virus type I, and yeast retrotransposon Ty1. A selective encapsidation of the primer tRNA, probably promoted by interactions with reverse transcriptase, occurs during the formation of virus or virus-like particles. Annealing of the primer tRNA to the primer binding site appears to be mediated by reverse transcriptase and/or the nucleocapsid protein. Modified nucleosides of the primer tRNA have been shown to be important for replication of the primer binding site, encapsidation of the primer (in the case of Rous sarcoma virus), and interaction with the genomic RNA (in the case of human immunodeficiency virus type I).

Binding Sites↗

PubMed1992

Inhibitory effects of flavonoids on Moloney murine leukemia virus reverse transcriptase activity.

Several flavonoids were tested for their effects on Moloney murine leukemia virus reverse transcriptase activity. Four groups of flavonoids, namely flavones, flavanones, flavonols, and flavanonols, were studied, and it was found that flavonols and flavanonols were very active in this regard while flavones and flavanones displayed very low activity. Among the flavonoids tested, fisetin, quercetin, myricetin, kaempferol, morin, (+/-)-taxifolin, (+)-catechin, and (-)-epicatechin were shown to be highly effective in inhibiting the reverse transcriptase activity. Structure-activity relationship analysis of these flavonoids revealed that the simultaneous presence of free hydroxyl groups at positions 3 and 4' enhanced the reverse transcriptase inhibitory activity. Replacement of the 3-hydroxyl group with a monosaccharide or of the 4'-hydroxyl group with a methyl group reduced inhibitory activity. The double bond at position 2 and 3 of the flavonoid's pyrone ring is not essential for inhibiting reverse transcriptase activity. The flavonoids studied demonstrated ability to inhibit the reverse transcriptase activity using either (rA)n(dT)12-18 or (rC)n(dG)12-18 as template-primers.

DNA Polymerase I↗

PubMed2001

Conformational analysis of nevirapine, a non-nucleoside HIV-1 reverse transcriptase inhibitor, based on quantum mechanical calculations.

The structure and the conformational behavior of the HIV-1 reverse transcriptase inhibitor, 11-cyclopropyl-5,11dihydro-4-methyl-6H-dipyrido[3,2-b2',3'-e][1,4]diazepin-6-one (nevirapine), is investigated by semiempirical (MNDO, AMI and PM3) method, ab initio at the HF/3-21G and HF/6-31G** levels and density functional theory at the B3LYP/6-31G** level. The fully optimized structure and rotational potential of the nitrogen and carbon bond in the cyclopropyl ring were examined in detail. A similar geometrical minimum is obtained from all methods which shows an almost identical structure to the geometry of the molecule in the complex structure with HIV-1 reverse transcriptase. To get some information on the structure in solution, NMR chemical shift calculations were also performed by a density functional theory at the B3LYP/6-31G** level, using GIAO approximation. The calculated 1H-NMR and 13C-NMR spectra for the energy minimum geometry agree well with the experimental results, which indicated that the geometry of nevirapine in solution is very similar to that of the molecule in the inhibition complex. Furthermore, the obtained results are compared to the conformational studies of other non-nucleoside reverse transcriptase inhibitors and reveal a common agreement of the non-nucleoside reverse transcriptase inhibitors. The specific butterfly-like shape and conformational flexibility within the side chain of the non-nucleoside reverse transcriptase inhibitors play an important role inducing conformational change of HIV-1 reverse transcriptase structure and are essential for the association at the inhibition pocket.

Anti-HIV Agents↗

PubMed1988

Mechanism of inhibition of reverse transcriptase by quinone antibiotics. II. Dependence on putative quinone pocket on the enzyme molecule.

Inhibition of avian myeloblastosis virus (AMV) reverse transcriptase by natural and synthetic quinones including antibiotics could be accounted for by an oxidation-reduction reaction. The quinones were shown to function as electron acceptors as revealed by the catalytic oxidation of NADH by Clostridium kluyveri diaphorase which was in excellent agreement with enzyme inhibition activity. The kinetics of inhibition of AMV reverse transcriptase by three synthetic quinones with different core structures, i.e., 6-methoxy-5,8-dihydroquinoline-5,8- dione, 5,8-dihydroisoquinoline-5,8-dione and 1,4-naphthoquinone, were studied. These quinones inhibited reverse transcriptase in the same manner as streptonigrin (STN) and were shown to act at a single class of reaction site(s) on the enzyme molecule. In contrast, the quinones with bulky substituents, i.e., 7-(2-nitrophenethylamino)-5,8-dihydroisoquinoline-5,8-dione and 7-methoxy-6-methyl-3-piperidino-5,8-dihydroisoquinoline-5,8-dione, were inactive as inhibitors of reverse transcriptase, whereas they retained competent catalytic activities in the oxidation of NADH by C. kluyveri diaphorase. Based on these observations, the existence of a specific site of interaction on the enzyme molecule, referred to as a quinone pocket, was proposed. The quinone pocket might play a crucial role in the early sequence of events leading to the inhibition of reverse transcriptase by quinones including STN and sakyomicin A (SKM). Access of SKM to a quinone pocket might be restricted due to its bulky structure in the vicinity of the quinone group. This is inferred from unsuccessful inhibition of reverse transcriptase by the quinones with bulky substituents, resulting in much poorer inhibition of reverse transcriptase in spite of more potent electron acceptor activity in the oxidation-reduction system as compared with those of STN.

Animals↗

PubMed2006

The contribution of HIV fitness to the evolution pattern of reverse transcriptase inhibitor resistance.

All currently recommended anti-retroviral therapy protocols employ reverse transcriptase inhibitors (RTIs). However, mutations within the reverse transcriptase (RT) domain can lead to resistance to these agents and treatment failure. The contribution of the fitness of drug-resistant species to the evolution of RTI resistance has not been elucidated despite its potential implications for therapeutic strategies. In this study we utilized a competitive fitness assay to assess the relative fitness of 13 drug-resistant HIV-1 mutants in the presence and absence of inhibitor. Among these mutants were thymidine analog mutations (TAMs) such as 41L/210W/215Y and 67N/70R/219Q, as well as single mutants such as 103N and 181C that confer high-level resistance to non-nucleoside reverse transcriptase inhibitors (NNRTIs) such as nevirapine. These studies revealed that 67N/70R and 67N/70R/219Q were fitter than the 70R progenitor species, and the acquisition of 41L by 215Y substantially increased its fitness in the absence of drug. We also observed that 215Y was more fit than 70R and 67N/70R, and that 41L/215Y and 41L/210W/215Y were the most-fit species in the presence of zidovudine. Moreover, 103N was fitter than 181C without nevirapine but less fit with nevirapine. From these studies we conclude that viral fitness contributes substantially to the evolutionary pattern of TAMs suggesting that, as for protease inhibitor resistance, mutations can act in primary (increasing resistance) and secondary (increasing fitness) capacities. We also surmise that drug resistance and fitness are competing forces underlying the emergence of nevirapine resistant mutants 103N and 181C.

Anti-HIV Agents↗

PubMed1974

Separation of ribonuclease H and RNA directed DNA polymerase (reverse transcriptase) of murine type-C RNA tumor viruses.

Ribonuclease H (RNA.DNA-hybrid ribonucleotidohydrolase, EC 3.1.4.34) has been reported to copurify with reverse transcriptase (RNA directed DNA polymerase) of RNA tumor viruses. In addition, viral specific ribonuclease H and reverse transcriptase of avian type-C viruses are thought to be part of the same polypeptide. In this report we show that a fraction of the ribonuclease H activity from Rauscher murine leukemia and Kirsten murine sarcoma viruses was separated from reverse transcriptase by anion exchange chromatography while the remaining portion co-purified with the viral polymerase. The amount of this co-purified nuclease activity was about 4- to 8-fold lower than the activity found in avian myeloblastosis virus (with respect to the ratio of ribonuclease H to reverse transcriptase) and this nuclease activity can only be detected by using labeled substrate of high specific radioactivity. However, a complete separation of ribonuclease H activity from reverse transcriptase was obtained by purifying core structures of the virus by sucrose density gradient centrifugation. While reverse transcriptase was present in the cores, there was no detectable ribonuclease H. Furthermore, a specific antibody against Rauscher leukemia virus reverse transcriptase did not inhibit any virion associated ribonuclease H activity. Our results suggest that in these virions these two enzyme activities reside in two separate molecules and probably in two different compartments of the virus. These findings emphasize a basic difference between the avian and murine type-C virus DNA polymerases.

Antibodies, Viral↗

PubMed2006

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

Alkynes↗

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↗

PubMed1994

Expression of reverse transcriptase from feline immunodeficiency virus in Escherichia coli.

Reverse transcriptase from feline immunodeficiency virus (FIV) has been cloned and expressed in Escherichia coli. We have purified this recombinant enzyme and shown that it is a 66-kDa protein that is indistinguishable from virion-derived FIV reverse transcriptase in sensitivity to the 5'-triphosphates of 3'-azido-3'-deoxythymidine and the four 2',3'-dideoxynucleosides. The availability of large quantities of the FIV reverse transcriptase will allow more detailed physical and pharmacological studies.

Amino Acid Sequence↗

PubMed2000

Long-term exposure of HIV type 1-infected cell cultures to combinations of the novel quinoxaline GW420867X with lamivudine, abacavir, and a variety of nonnucleoside reverse transcriptase inhibitors.

The novel quinoxaline GW420867X has been combined with a variety of nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) in HIV-1(IIIB)-infected CEM cell cultures. Whereas the antiviral efficacy of combinations of GW420867X with the NRTIs lamivudine (3TC) and abacavir (ABC) proved additive when administered to HIV-1-infected cells in a short-term (4-day) infection experiment, combination of GW420867X with the NRTIs 3TC and ABC resulted in a marked delay of virus breakthrough compared with the single drugs alone in a long-term (2-month) infection experiment. Delay of virus breakthrough was less pronounced for combinations of GW420867X with the NNRTIs. Combination of GW420867X with the NRTIs and NNRTIs resulted in additive inhibitory effects on recombinant HIV-1 reverse transcriptase as evident from isobolograms. Lamivudine plus GW420867X selected for the 3TC-specific M184I mutation and a number of NNRTI-characteristic mutations (i.e., V106A, V108I, and Y188H). Abacavir plus GW420867X selected only for NNRTI-specific mutations (i.e., K101E, K103R, V106A, and Y181C), including the novel L100V mutation. Combination of GW420867X with five different NNRTIs selected solely for NNRTI-specific mutations, and also for the L100V mutation in the combined presence of efavirenz, nevirapine, or emivirine, respectively. Five single-, two double-, and two triple-mutated HIV-1 strains that emerged from this study were evaluated for their sensitivity/resistance to AZT, lamivudine, and seven different NNRTIs. In all cases, efavirenz, GW420867X, and UC-781 retained pronounced antiviral potency. Our data suggest that combinations of GW420867X with 3TC, ABC, and NNRTIs (e.g., efavirenz) would be worth pursuing as therapeutic modalities against HIV-1 infections.

Alkynes↗

PubMed1987

Low prevalence of particle-associated reverse transcriptase activity in serum from patients with non A - non B hepatitis.

Sera from 367 patients presumed to have NANB hepatitis were screened for reverse transcriptase activity. In 29 cases significantly increased enzyme activities could be observed. In contrast, sera from 338 patients did not contain significant reverse transcriptase activities. 207 healthy individuals, 7 patients with hepatitis A and 6 patients with hepatitis B who served as controls were all negative for reverse transcriptase activity. The specificity of the enzyme assay was demonstrated by estimation of reverse transcriptase activity in sera from 10 "healthy" HIV-1-antibody positive individuals. In 3 out of 10 cases significant reverse transcriptase activity was observed associated with the human immunodeficiency virus. Our results indicate that the presence of particle-associated reverse transcriptase activity in serum from patients with NANB hepatitis is indicative of the presence of a retrovirus-like agent in these cases. However, the relatively low prevalence of reverse transcriptase positive cases associated with the NANB hepatitis makes it rather questionable whether this agent is a frequent and specific factor in the etiology of NANB hepatitis.

Acquired Immunodeficiency Syndrome↗

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↗

PubMed1975

Evolutionary nature of human reverse transcriptase and of viral-related DNA synthesized in vitro by human leukemic cells.

The reverse transcriptase and endogenous DNA product synthesized by virus-like particles in the cytoplasm of human leukemic cells have been studied for their genetic relatedness to homologous components obtained from several animal RNA tumor viruses. The human reverse transcriptase activity was inhibited by antibodies prepared against reverse transcriptase from some animal RNA tumor viruses. The DNA molecules synthesized endogenously by the human cytoplasmic particle in the presence of actinomycin D, using the reverse transcriptase enzyme and RNA template residing in the particle, hybridized to 70S RNA purified from certain animal RNA tumor viruses. Both the human reverse transcriptase and DNA product are closely related to homologues from primate type-C viruses, more distantly related to those from murine type-C viruses, and essentially unrelated to similar structures from feline or avian type-C viruses. They are not related to type-B RNA tumor viruses. The results demonstrate that the components from the human leukemic cells are viral (type-C) and primate in nature.

Cytoplasm↗

PubMed1983

Production and characterization of monoclonal antibodies against avian retrovirus reverse transcriptase.

Monoclonal antibodies were prepared against the avian myeloblastosis virus reverse transcriptase. These monoclonal antibodies specifically immunoprecipitated the alpha and beta subunits of the reverse transcriptase molecule, as well as the Pr180gag-pol precursor protein present in virus-infected cells. In addition, these monoclonal antibodies inhibited the DNA polymerase activity associated with the reverse transcriptase molecule but not the RNase H activity. The monoclonal antibody preparations were specific for the amino-terminal portion of the protein, as determined by the immunoprecipitation of a reverse transcriptase-beta-galactosidase fusion protein produced in Escherichia coli by molecular cloning procedures.

Animals↗

PubMed1994

Detection of poliovirus, hepatitis A virus, and rotavirus from sewage and ocean water by triplex reverse transcriptase PCR.

A triplex reverse transcriptase PCR (RT-PCR) was developed to simultaneously detect poliovirus, hepatitis A virus (HAV), and rotavirus in sewage and ocean water. Sewage and ocean water samples seeded with the three different viruses were concentrated by ultrafiltration. The unseeded ocean water and sewage samples were concentrated by vortex flow filtration and/or ultrafiltration. Random hexamers and a rotavirus downstream primer were used to initiate reverse transcription. Three different sets of primers specific for poliovirus, HAV, and rotavirus cDNAs were mixed in the PCR mixture to amplify the target DNA. Three distinct amplified DNA products representing poliovirus, HAV, and rotavirus were identified by gel electrophoresis as 394-, 192-, and 278-bp sequences, respectively. Dot blot and Southern analyses were used to confirm the amplified products for each virus present in the environmental samples. Except for poliovirus, the sensitivity of triplex RT-PCR for the detection of rotavirus and HAV was found to be similar to that of monoplex RT-PCR, which uses only one set of primers to amplify a single type of virus. The triplex RT-PCR has greater advantages over monoplex RT-PCR for virus detection, namely, the rapid turnaround time and cost effectiveness.

Base Sequence↗

PubMed1997

Two step synthesis of (-) strong-stop DNA by avian and murine reverse transcriptases in vitro.

Retroviral reverses transcriptases (RTs) are RNA- and DNA-dependent DNA polymerases that use a tRNA bound at the so-called primer binding site (PBS) located near the 5'end of the genomic RNA as primer. Thus, RTs must be able to accommodate both RNA and DNA in the primer strand. To test whether the natural primer confers some advantages to the priming process, we compared initiation of reverse transcription of avian and murine retroviral RNAs, using either their natural tRNA primer, tRNATrp and tRNAPro, respectively, or synthetic 18mer oligodeoxyribonucleotides (ODNs) and oligoribonucleotides (ORNs) complementary to their PBS. In both retroviral systems, the initial extension of ODNs was fast and processive. The initial extension of ORNs, tRNATrp and tRNAPro was much slower and distributive, giving rise to the transient accumulation of short pausing products. Synthesis of (-) strong-stop DNA was delayed when using ORNs and tRNAs, compared to ODNs. Even though ORNs and tRNAs were initially extended at the same rate, the short pausing products were more rapidly extended when using the tRNA primers. As a consequence, synthesis of (-) strong-stop DNA was much more efficient with tRNA primers, compared to ORNs. Taken together, these results suggest that the tRNA-primed synthesis of (-) strong-stop DNA is a two-step process, as already observed for HIV-1. The initiation mode corresponds to the initial non-processive nucleotide addition and extension of the short pausing products. It is more efficient with the natural primers than with ORNs. Initiation is followed by a more processive and unspecific elongation mode. Elongation is observed when the primer strand is DNA, i.e. when using the ODNs as primers or when the ORN and tRNA primers have been extended by a sufficient number (depending on the retroviral system) of deoxyribonucleotides.

Animals↗

PubMed1993

Generation and characterization of murine monoclonal antibodies reactive against N-terminal and other regions of HIV-1 reverse transcriptase.

We produced a series of monoclonal antibodies against the human immunodeficiency virus (HIV-1) reverse transcriptase by immunizing mice with either purified recombinant HIV-1 p66 protein or with recombinant vaccinia virus which expresses HIV-1 pol sequences. The antibodies generated were specific for the reverse transcriptase protein, and recognized only the p51 and p66 subunits of the enzyme in each of the HIV-1 viral lysates and lysates of HIV-1 infected cells. The antibodies did not cross-react with HIV-2 reverse transcriptase. Most important, several of the antibodies are unique, in that they are the first that can bind to sites close to the N-terminal. This latter region has been suggested to form part of the polymerase domain of the reverse transcriptase. None of the antibodies could neutralize either the RNA-dependent DNA polymerase or RNase H activities of either p66 or p51/66 proteins. The binding patterns of these various antibodies to p66 and p51/66 were dependent on each of three independent variables: the source of antigen amployed, the individual specificity of the antibody, and the method employed to detect reactivity. These monoclonal antibodies provide useful reagents for the study of reverse transcriptase native structure-function relationships.

Animals↗