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PubMed1992

[The inhibitory effects of catechin derivatives on the activities of human immunodeficiency virus reverse transcriptase and DNA polymerases].

Catechin derivatives including (-)-epicatechin gallate (ECG), (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and green tea extract (GTE) were found to inhibit the activities of cloned human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT), duck hepatitis B virus replication complexes reverse transcriptase (DHBV RCs RT), herpes simplex virus 1 DNA polymerase (HSV-1 DNAP) and cow thymus DNA polymerase alpha (CT DNAP alpha). EGCG and ECG were shown to be very potent inhibitors of HIV-1 RT. According to the IC50 values for HIV-1 RT, these compounds can be ordered as EGCG 0.0066 mumol/L > ECG 0.084 mumol/L > GTE 0.1 microgram/ml > EGC 7.2 mumol/L. DHBV RCs RT was the least sensitive to these compounds. Kinetic study showed that EGCG exerts a mixed inhibition with respect to external template inducer poly (rA).oligo (dT) 12-18 and a noncompetitive inhibition with respect to substrate dTTP for HIV-1 RT. Bovine serum albumin significantly reduced the inhibitory effects of catechin analogues and GTE on HIV-1 RT. In tissue culture GTE inhibited the cytopathic effect of coxsackie B3 virus, but did not inhibit the cytopathic effects of HSV-1, HSV-2, influenza A or influenza B viruses.

Animals↗
PubMed1989

Detection of multiple, novel reverse transcriptase coding sequences in human nucleic acids: relation to primate retroviruses.

A variety of chemically synthesized oligonucleotides designed on the basis of amino acid and/or nucleotide sequence data were used to detect a large number of novel reverse transcriptase coding sequences in human and mouse DNAs. Procedures involving Southern blotting, library screening, and the polymerase chain reaction were all used to detect such sequences; the polymerase chain reaction was the most rapid and productive approach. In the polymerase chain reaction, oligonucleotide mixtures based on consensus sequence homologies to reverse transcriptase coding sequences and unique oligonucleotides containing perfect homology to the coding sequences of human T-cell leukemia virus types I and II were both effective in amplifying reverse transcriptase-related DNA. It is shown that human DNA contains a wide spectrum of retrovirus-related reverse transcriptase coding sequences, including some that are clearly related to human T-cell leukemia virus types I and II, some that are related to the L-1 family of long interspersed nucleotide sequences, and others that are related to previously described human endogenous proviral DNAs. In addition, human T-cell leukemia virus type I-related sequences appear to be transcribed in both normal human T cells and in a cell line derived from a human teratocarcinoma.

Amino Acid Sequence↗
PubMed2003

Response to efavirenz plus two nucleoside reverse-transcriptase inhibitors in patients with advanced stage human immunodeficiency virus-1 infection in Taiwan.

From July 1, 1999 to April 30, 2002, 111 consecutive human immunodeficiency virus-infected, antiretroviral-naïve Taiwan patients initiated highly active antiretroviral therapy with efavirenz plus 2 nucleoside reverse-transcriptase inhibitors. Their median baseline CD4+ count was 50 x 10(6)/L (0-559 x 10(6)/L) and plasma viral load was 5.51 log10 copies/mL (3.09 to > 5.88 log10) as assessed by reverse-transcriptase polymerase chain reaction. Of the patients, 52.3% had a CD4+ count of < or = 50 x 10(6)/L, 74.8% had plasma viral load over 5 log10 copies/mL, and 58.5% had active AIDS-defining opportunistic illnesses. The median observation duration of antiretroviral therapy was 350 days (range, 28-991 days). At week 48 to 52 following the initiation of highly active antiretroviral therapy, 81. 8% (45/55) and 91.8% (45/49) of the patients achieved undetectable plasma viral load by intent-to-treat and ontreat analysis, respectively. At week 80 to 84, these percentage decreased to 69.7% (23/33) and 85.2% (23/27), respectively. Median CD4+ count increased from baseline to week 48 to 52 by 147 x 10(6)/L and to week 80 to 84 by 227 x 10(6)/L. The virologic and immunologic responses at each time period by intention-to-treat or on-treat analysis were similar between patients with baseline plasma viral load over or < or = 5 log10, CD4+ count over or < or = 50 x 10(6)/L, and with or without active AIDS-defining opportunistic illnesses. After initiation of highly active antiretroviral therapy for a median duration of 57 days (range, 2-638 days), 11 episodes of AIDS-defining and 11 non-AIDS opportunistic illnesses occurred. The results of this study suggest that efavirenz plus 2 nucleoside reverse-transcriptase inhibitors is a potent antiretroviral combination regardless of whether the patient has a high baseline plasma viral load, low CD4+ count, or AIDS-defining opportunistic illnesses.

Adult↗
PubMed2003

A randomized trial comparing initial HAART regimens of nelfinavir/nevirapine and ritonavir/saquinavir in combination with two nucleoside reverse transcriptase inhibitors.

BACKGROUND: A triple-class HAART regimen may be associated with a better virological effect than conventional regimens, but may also lead to toxicity and more profound resistance. METHODS: Randomized, controlled, open-label trial of 233 protease inhibitor- and non-nucleoside reverse transcriptase inhibitor-naive HIV-infected patients allocated to a regimen of nelfinavir and nevirapine (1250/200 mg twice daily; n = 118) or ritonavir and saquinavir (400/400 mg twice daily; n = 115), both in combination with two nucleoside reverse transcriptase inhibitors. The primary end-point was HIV RNA < or = 20 copies/ml after 48 weeks (missing value = failure). Patients remained under follow-up also in case of switch from the randomized therapy. RESULTS: At baseline, the median CD4 cell counts were 126 (range: 0-942) (nelfinavir/nevirapine) and 150 (0-642) (ritonavir/saquinavir) cells/mm3, and HIV RNA measurements 5.0 copies/ml (1.3-6.4) in both groups. A total of 102 (86%) and 101 (88%) were antiretroviral-naive. 44% discontinued randomized therapy; P = 0.13. Of these, 80 and 73% switched therapy due to adverse events; P = 0.99. At week 48, 69 and 56%, respectively, had a HIV RNA < or = 20 copies/ml; P = 0.037. CONCLUSION: A regimen of nelfinavir/nevirapine had a favourable virological effect and tolerability over a 48-week period compared with ritonavir/saquinavir, when administered in combination with two nucleoside reverse transcriptase inhibitors. However, more extensive follow-up is required to determine the long-term consequences of triple class HAART regimens, including the development of broad drug resistance.

Adult↗
PubMed1996

Significance of amino acid variation at human immunodeficiency virus type 1 reverse transcriptase residue 210 for zidovudine susceptibility.

Amino acid variation at reverse transcriptase (RT) codon 210 (generally Leu-210 to Trp [L210W], TTG-->TGG) is occasionally detected after the initiation of azidothymidine (AZT) therapy. The impact of this variation on AZT resistance and viral replication was addressed by four different approaches. The frequency and genetic background of the L210W mutation in vivo were assessed by analyzing sera of AZT-naive and AZT-experienced patients by RT-PCR and DNA sequencing. The degree of AZT resistance (50% infective concentration [IC50]) of recombinant viruses constructed by using the RT of 21 clinical isolates was stratified by the presence or absence of the 210 mutation. The AZT IC50S of a panel of mutant viruses (with or without W-210) constructed by site-directed mutagenesis in an HXB2 background were assayed by using a HeLa CD4 plaque reduction assay. Finally, the effect of the 210 mutation on viral replication was assessed by replication competition of an AZT-resistant virus, RTMN (L-41/Y-215), and RTMN with the W-210 mutation in the presence and in the absence of AZT. In AZT-naive patients, tryptophan at RT residue 210 was rare. After AZT exposure, W-210 appeared in a minority of those patients, most commonly in association with L-41 and Y-215. The presence of W-210 increased the AZTIC50 by two- to fourfold, as determined by both the recombinant virus assay and site-directed mutagenesis. A significant replication advantage in favor of the wild-type L-210 over W-210 was observed, although the selection against the 210 mutant was two- to threefold lower when the viruses were grown in the presence of 5 microM AZT. In summary, the L210W mutation appears to be of marginal significance, conferring approximately two- to fourfold-reduced sensitivity to AZT compared with similar AZT-resistant genomes with L-210. The selection pressure against W-210 may account for the modest proportion of patients in which W-210 appears in vivo.

Antiviral Agents↗
PubMed1977

A joint produce of the genes gag and pol of avian sarcoma virus: a possible precursor of reverse transcriptase.

A virus-specific protein of approximately 180,000 daltons has been identified in cells transformed by avian sarcoma virus. The protein, designated P180, includes immunological determinants of both viral core proteins and reverse transcriptase. Its tryptic peptides represent essentially the sum of those of the precursor of the core proteins (Pr76gag) and reverse transcriptase. Thus P180 must arise from the uninterrupted translation of gag and pol. The kinetics of its formation and decay suggest that P180 is the precursor of reverse transcriptase.

Alpharetrovirus↗
PubMed1988

A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity.

We have cloned the entire pol gene of human immunodeficiency virus type 2 into a high-level Escherichia coli expression system. Induction of cultures containing the recombinant plasmid, p2RTL1, leads to rapid accumulation of polypeptides of 66, 54, and 34 kilodaltons. We have designated the larger polypeptides reverse transcriptase, and we have designated the smaller polypeptide endonuclease. Purification of reverse transcriptase via ion-exchange and affinity chromatography yields the 66-kilodalton polypeptide, with which reverse transcriptase activity is associated. Purified enzyme furthermore displays a higher apparent molecular weight than its counterpart from human immunodeficiency virus type 1.

Escherichia coli↗
PubMed1998

Quantification of poliovirus in seawater and sewage by competitive reverse transcriptase--polymerase chain reaction.

Reverse transcriptase-polymerase chain reaction (RT-PCR) has been used extensively to detect enteric viruses in environmental samples. Advantages of RT-PCR include its high detection sensitivity and rapid turn-around time. However, unlike traditional cell culture, RT-PCR has not provided quantitation and infectivity information. In this study, we have developed a quantitative RT-PCR method that can be used to determine the amount of poliovirus RNA in environmental samples. An RNA internal standard for poliovirus RT-PCR was designed and obtained through genetic engineering. Serial dilutions of RNA internal standard templates were amplified with a 5'-carboxyfluorescein-labeled poliovirus downstream primer and a nonlabeled poliovirus upstream primer in the RT-PCR. The fluorescent light intensity of labeled RT-PCR products was quantified using an ABI DNA sequencer with GeneScan software. The internal standard was coamplified with poliovirus in the RT-PCR, allowing for enumeration of the poliovirus RNA present in the seawater and sewage samples. This method, using a cloned internal standard and specified primers in the PCR, may be applied to quantify other microorganisms in environmental samples. Although quantitative RT-PCR has begun to be used more extensively for detecting pathogens in clinical samples, the complex nature of many environmental samples has limited the sample range of the effectiveness of quantitative RT-PCR.

Environmental Microbiology↗
PubMed1996

(Alkylamino) piperidine bis(heteroaryl)piperizine analogs are potent, broad-spectrum nonnucleoside reverse transcriptase inhibitors of drug-resistant isolates of human immunodeficiency virus type 1 (HIV-1) and select for drug-resistant variants of HIV-1IIIB with reduced replication phenotypes.

The (alkylamino)piperidine bis(heteroaryl)piperizines (AAP-BHAPs) are a new class of human immunodeficiency virus type 1 (HIV-1)-specific inhibitors which were identified by targeted screening of recombinant reverse transcriptase (RT) enzymes carrying key nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-conferring mutations and NNRTI-resistant variants of HIV-1. Phenotypic profiling of the two most potent AAP-BHAPs, U-95133 and U-104489, against in vitro-selected drug-resistant HIV-1 variants carrying the NNRTI resistance-conferring mutation (Tyr->Cys) at position 181 of the HIV-1 RT revealed submicromolar 90% inhibitory concentration estimates for these compounds. Moreover, U-104489 demonstrated potent activity against BHA-P-resistant HIV-1MF harboring the Pro-236->Leu RT substitution and significantly suppressed the replication of clinical isolates of HIV-1 resistant to both delavirdine (BHAP U-90152T) and zidovudine. Biochemical and phenotypic characterization of AAP-BHAPresistant HIV-1IIIB variants revealed that high-level resistance to the AAP-BHAPs was mediated by a Gly-190->Glu substitution in RT, which had a deleterious effect on the integrity and enzymatic activity of virion-associated RT heterodimers, as well as the replication capacity of these resistant viruses.

Antiviral Agents↗
PubMed2002

Genotypic and phenotypic analysis of a novel 15-base insertion occurring between codons 69 and 70 of HIV type 1 reverse transcriptase.

An HIV-1 isolate possessing a 15-base insertion between codons 69 and 70 of the reverse transcriptase (RT) gene was derived from a patient plasma sample. Investigation of the insertion sequence revealed that this mutation is an ectopic duplication of the first 15 bases of the HIV-1 envelope gene. Phenotypic analysis yielded the following increases in resistance: 371-fold to zidovudine, 84-fold to lamivudine, 32-fold to abacavir, 15-fold to stavudine, 12-fold to didanosine, and 4-fold to zalcitabine. Phenotypic studies suggested that this change does not detract from the overall fitness of the virus. Together, data from this investigation support two conclusions. First, a previously unreported mechanism exists for generating diversity in HIV-1, namely long-distance duplication of genetic material from one portion of the genome to another. Second, large insertions in this region of RT are well tolerated and can confer high levels of resistance to multiple nucleoside analogue reverse transcriptase inhibitors.

Codon↗
PubMed2005

PCR inhibition by reverse transcriptase leads to an overestimation of amplification efficiency.

This study addresses the problem of PCR inhibition by reverse transcriptase. It has been shown that the inhibition occurs mostly when a small amount of RNA is taken for RT reaction, and it is more visible for rarely expressed transcripts. We show here that the inhibition takes place regardless of what amount of template is utilized for RT. The inhibition possesses a global nature, i.e. the amplification of any given transcript may be compromised with different levels of inhibition. The process of inhibition also explains wrongfully derived PCR amplification efficiencies, sometimes more than 100%, when the sequential dilutions of unpurified RT sample are utilized to build the calibration curve. The RT influences PCR not only by inhibiting it. When microgram(s) of RNA are taken for RT reaction, reverse transcriptase may cause overamplification of some transcripts under certain PCR conditions. The possible mechanism of RT influence on PCR is presented, and a purification method is implemented to remove the effects of RT on PCR.

Cell Line, Tumor↗
PubMed1991

Human immunodeficiency virus reverse transcriptase. Effect of primer length on template-primer binding.

Poly(rA).oligo(dT)n binding to human immunodeficiency virus type-1 reverse transcriptase heterodimer (p66-p51) was primer length-dependent. The estimated Kd for (n = 10-14) was 20-30 nM and for (n = 16-20) was 0.11-0.14 nM. Gel electrophoretic analysis of the patterns of primer extension was consistent with an abrupt change in the Kd between a primer length of 14 and 16 nucleotides. Further, the rate constant for dissociation of the reverse transcriptase-template-primer complex was determined from steady state kinetics and enzyme-template-primer trapping experiments to be independent of primer length. Thus, the abrupt change in Kd was most likely due to a change in the rate constant for formation of the reverse transcriptase-template-primer complex. A similar shift in the Kd for template-primer binding was observed with poly(dA).oligo(dT)n. Reverse transcriptase homodimer (p66) catalyzed the incorporation of dTMP into poly(rA).oligo(dT)n with the same primer length dependence observed for the heterodimer. In contrast, binding of the p51 homodimer to poly(rA).oligo(dT)n was independent of primer length. Thus, the RNase H domain may contribute to reverse transcriptase heterodimer or p66 homodimer binding to template-primers in which the primer length is greater than 14 nucleotides.

HIV-1↗
PubMedDate not supplied

Biotransformation of 5-chloro-3-phenylthioindole-2-carboxamide (L-734,005) in rhesus monkeys and rat liver microsomes to a potent HIV-1 reverse transcriptase inhibitor.

Rhesus monkeys were dosed orally with 10 mg/kg 5-chloro-3-phenylthioindole-2-carboxamide (L-734,005), a nonnucleoside human immunodeficiency virus type 1 (HIV-1) reverse transcriptase inhibitor, in polyethylene glycol 300. Plasma samples from these monkeys demonstrated greater bioactivity in an HIV-1 reverse transcriptase inhibition assay than anticipated from the parent compound concentrations as determined by an HPLC-UV assay. One major and three minor metabolites, as well as the parent compound, were detected in the plasma. One of the minor metabolites was determined to be several-fold more active, and the major metabolite one-half as active as the parent compound in the inhibition assay. Identical metabolites were formed during an incubation of L-734,005 with rat liver microsomes. The most active minor metabolite was identified as a sulfone analog (L-737,126) of the parent compound by NMR and MS analyses. The less active major metabolite and two relatively inactive minor metabolites were similarly identified as the sulfoxide, 4-hydroxythiophenyl and 6-hydroxyindole analogs of L-734,005. The synthetic sulfone analog was highly potent against HIV-1, with a 95% inhibitory concentration of 3.0 nM for the spread of virus infection in a cell culture.

Animals↗
PubMed1997

Linear mitochondrial plasmids of Fusarium oxysporum contain genes with sequence similarity to genes encoding a reverse transcriptase from Neurospora spp.

Two linear mitochondrial plasmids called pFOXC1 and pFOXC2 from the fungus Fusarium oxysporum were previously described. DNA sequence comparisons indicated that the derived amino acid sequences of both plasmids exhibit similarity to the reverse transcriptase of the Mauriceville and Varkud plasmids of Neurospora spp. The derived amino acid sequence of pFOXC2 has 51% similarity and 32% identity to the Neurospora reverse transcriptase; sequence similarity was greatest for seven blocks of amino acids that are conserved in reverse transcriptases from a wide range of biological sources. Northern analysis suggests that full-length RNAs corresponding to the plasmids are found in representative isolates.

Amino Acid Sequence↗
PubMed2005

Structure based activity prediction of HIV-1 reverse transcriptase inhibitors.

We have developed a fast and robust computational method for prediction of antiviral activity in automated de novo design of HIV-1 reverse transcriptase inhibitors. This is a structure-based approach that uses a linear relation between activity and interaction energy with discrete orientation sampling and with localized interaction energy terms. The localization allows for the analysis of mutations of the protein target and for the separation of inhibition and a specific binding to the enzyme. We apply the method to the prediction of pIC(50) of HIV-1 reverse transcriptase inhibitors. The model predicts the activity of an arbitrary compound with a q(2) of 0.681 and an average absolute error of 0.66 log value, and it is fast enough to be used in high-throughput computational applications.

Anti-HIV Agents↗
PubMed2003

Single-tube fluorescent product-enhanced reverse transcriptase assay with Ampliwax (STF-PERT) for retrovirus quantitation.

A TaqMan fluorescent probe-based product enhanced reverse transcriptase (RT) assay is described in which the RT and polymerase chain reaction (PCR) steps are set-up in a single tube, in two compartments separated by Ampliwax (designated as single-tube fluorescent product-enhanced reverse transcriptase assay (STF-PERT)). This simplification of the two-step method resulted in increased assay reproducibility and handling efficiency while maintaining the sensitivity of the PERT assay (<10 virions). The STF-PERT assay can be used to quantitate low amounts of retrovirus in clinical and research materials and to evaluate retrovirus contamination in cell substrates and biological products in human use.

Animals↗
PubMed1991

Reverse transcriptase encoded by a human transposable element.

L1 elements are highly repeated mammalian DNA sequences whose structure suggests dispersal by retrotransposition. A consensus L1 element encodes a protein with sequence similarity to known reverse transcriptases. The second open reading frame from the human L1 element L1.2A was expressed as a fusion protein targeted to Ty1 virus-like particles in Saccharomyces cerevisiae and shown to have reverse transcriptase activity. This activity was eliminated by a missense mutation in the highly conserved amino acid motif Y/F-X-D-D. Thus, L1 represents a potential source of the reverse transcriptase activity necessary for dispersion of the many classes of mammalian retroelements.

Base Sequence↗
PubMedDate not supplied

Classification and regression trees--studies of HIV reverse transcriptase inhibitors.

In this paper, the application of Classification And Regression Trees (CART) is presented for the analysis of biological activity of Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). The data consist of the biological activities, expressed as pIC50, of 208 NNRTIs against wild-type HIV virus (HIV-1) and four mutant strains (181C, 103N, 100I, 188L) and the computed interaction energies with the Reverse Transcriptase (RT) binding pocket. CART explains the observed biological activity of NNRTIs in terms of interactions with individual amino acids in the RT binding pocket, i.e., the original data variables.

Algorithms↗