Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The YXDD motif is highly conserved in the reverse transcriptase family. The variable X residue is occupied by valine and methionine in MuLV RT and HIV-1 RT, respectively. Previous studies have shown that Tyr 222, the Y residue of the YXDD motif in MuLV RT, constitutes a major component of the fidelity center of the enzyme [Kaushik, N., Singh, K., Alluru, I., and Modak, M. J. (1999) Biochemistry 38, 2617-2627]. In this work, we present evidence that reverse transcriptases containing valine in the "X" position of the YXDD motif generally catalyze DNA synthesis with greater fidelity than those containing methionine or alanine. In the MuLV RT system, the two mutants V223M and V223A exhibited an overall reduced fidelity of DNA synthesis, specifically for RNA-templated reactions. Further analysis revealed that these mutants exhibit a higher efficiency of misinsertion on MS2 RNA than the wild-type enzyme for every mispair tested. However, unlike HIV-1 RT, the insensitivity of the wild-type MuLV RT to all four ddNTPs remained unchanged by mutation of V223 to Met or Ala. A 3D molecular model of the ternary complex of MuLV RT, template primer, and dNTP suggests that Val 223 along with its neighboring Tyr 222 stabilizes the substrate binding pocket via hydrophobic interactions with the dNTP substrate and template-primer.
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PMID:Valine of the YVDD motif of moloney murine leukemia virus reverse transcriptase: role in the fidelity of DNA synthesis. 1081 83

Human immunodeficiency virus type 1 (HIV-1) rapidly develops resistance to lamivudine during monotherapy, typically resulting in the appearance at position 184 in reverse transcriptase (RT) of isoleucine instead of the wild-type methionine (M184I) early in therapy, which is later replaced by valine (M184V). M184V reduces viral susceptibility to drug in vitro by approximately 100-fold, but also results in a lower processivity of RT. We show that a drop in absolute viral fitness associated with the outgrowth of M184V results in a drop in viral load only in individuals with high CD4(+) counts, from whom we estimate the relative fitness of M184V in the presence of drug to be approximately 10% of that of the wild type prior to therapy. The timing of emergence of the M184V mutant varies widely between infected individuals. From analysis of the frequency of M184I and M184V mutants determined at multiple time points in seven individuals during lamivudine therapy, we estimated the fitness advantage of M184V over M184I during therapy to be approximately 23% on average. We have also estimated the average ratio of the frequencies of the two mutants prior to therapy to be 0. 2:1, with a range from 0.12:1 to 0.33:1. We have found that the differences between individuals in the rate of evolution of lamivudine resistance arise due to genetic drift affecting the relative frequency of M184I and M184V prior to therapy. These results show that stochastic effects can be significant in HIV evolution, even when there is large fitness difference between mutant and wild-type variants.
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PMID:Evolution of lamivudine resistance in human immunodeficiency virus type 1-infected individuals: the relative roles of drift and selection. 1086 35

The antiretroviral nucleoside analog 2',3'-dideoxy-3'-thiacytidine (3TC) is a potent inhibitor of wild-type human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). A methionine-to-valine or methionine-to-isoleucine substitution at residue 184 in the HIV-1 YMDD motif, which is located at the RT active site, leads to a high level of resistance to 3TC. We sought to determine whether 3TC can inhibit the replication of wild-type murine leukemia virus (MLV), which contains V223 at the YVDD active site motif of the MLV RT, and of the V223M, V223I, V223A, and V223S mutant RTs. Surprisingly, the wild type and all four of the V223 mutants of MLV RT were highly resistant to 3TC. These results indicate that determinants outside the YVDD motif of MLV RT confer a high level of resistance to 3TC. Therefore, structural differences among similar RTs might result in widely divergent sensitivities to antiretroviral nucleoside analogs.
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PMID:Wild-type and YMDD mutant murine leukemia virus reverse transcriptases are resistant to 2',3'-dideoxy-3'-thiacytidine. 1086 83

A large variety of natural products have been described as anti-HIV agents, and for a portion thereof the target of interaction has been identified. Cyanovirin-N, a 11-kDa protein from Cyanobacterium (blue-green alga) irreversibly inactivates HIV and also aborts cell-to-cell fusion and transmission of HIV, due to its high-affinity interaction with gp120. Various sulfated polysaccharides extracted from seaweeds (i.e., Nothogenia fastigiata, Aghardhiella tenera) inhibit the virus adsorption process. Ingenol derivatives may inhibit virus adsorption at least in part through down-regulation of CD4 molecules on the host cells. Inhibition of virus adsorption by flavanoids such as (-)epicatechin and its 3-O-gallate has been attributed to an irreversible interaction with gp120 (although these compounds are also known as reverse transcriptase inhibitors). For the triterpene glycyrrhizin (extracted from the licorice root Glycyrrhiza radix) the mode of anti-HIV action may at least in part be attributed to interference with virus-cell binding. The mannose-specific plant lectins from Galanthus, Hippeastrum, Narcissus, Epipac tis helleborine, and Listera ovata, and the N-acetylgl ucosamine-specific lectin from Urtica dioica would primarily be targeted at the virus-cell fusion process. Various other natural products seem to qualify as HIV-cell fusion inhibitors: the siamycins [siamycin I (BMY-29304), siamycin II (RP 71955, BMY 29303), and NP-06 (FR901724)] which are tricyclic 21-amino-acid peptides isolated from Streptomyces spp that differ from one another only at position 4 or 17 (valine or isoleucine in each case); the betulinic acid derivative RPR 103611, and the peptides tachyplesin and polyphemusin which are highly abundant in hemocyte debris of the horseshoe crabs Tachypleus tridentatus and Limulus polyphemus, i.e., the 18-amino-acid peptide T22 from which T134 has been derived. Both T22 and T134 have been shown to block T-tropic X4 HIV-1 strains through a specific antagonism with the HIV corecept or CXCR4. A number of natural products have been reported to interact with the reverse transcriptase, i.e., baicalin, avarol, avarone, psychotrine, phloroglucinol derivatives, and, in particular, calanolides (from the tropical rainforest tree, Calophyllum lanigerum) and inophyllums (from the Malaysian tree, Calophyllum inophyllum). The natural marine substance illimaquinone would be targeted at the RNase H function of the reverse transcriptase. Curcumin (diferuloylmethane, from turmeric, the roots/rhizomes of Curcuma spp), dicaffeoylquinic and dicaffeoylt artaric acids, L-chicoric acid, and a number of fungal metabolites (equisetin, phomasetin, oteromycin, and integric acid) have all been proposed as HIV-1 integrase inhibitors. Yet, we have recently shown that L-c hicoric acid owes its anti-HIV activity to a specific interaction with the viral envelope gp120 rather than integrase. A number of compounds would be able to inhibit HIV-1 gene expression at the transcription level: the flavonoid chrysin (through inhibition of casein kinase II, the antibacter ial peptides melittin (from bee venom) and cecropin, and EM2487, a novel substance produced by Streptomyces. (ABSTRACT TRUNCATED)
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PMID:Current lead natural products for the chemotherapy of human immunodeficiency virus (HIV) infection. 1093 47

Several studies have indicated that the genetic diversity of human T-cell leukemia virus type 1 (HTLV-1), a virus associated with adult T-cell leukemia, is significantly lower than that of other retroviruses, including that of human immunodeficiency virus type 1 (HIV-1). To test whether HTLV-1 variation is lower than other retroviruses, a tractable vector system has been developed to measure reverse transcription accuracy in one round of HTLV-1 replication. This system consists of a HTLV-1 vector that contains a cassette with the neomycin phosphotransferase (neo) gene, a bacterial origin of DNA replication, and the lacZalpha peptide gene region (the mutational target). The vector was replicated by trans-complementation with helper plasmids. The in vivo mutation rate for HTLV-1 was determined to be 7 x 10(-6) mutations per target base pair per replication cycle. The majority of the mutations identified were base substitution mutations, namely, G-to-A and C-to-T transitions, frameshift mutations, and deletion mutations. Mutation of the methionine residue in the conserved YMDD motif of the HTLV-1 reverse transcriptase to either alanine or valine (i.e., M188A or M188V) led to a factor of two increase in the rate of mutation, indicating the role of this motif in enzyme accuracy. The HTLV-1 in vivo mutation rate is comparable to that of bovine leukemia virus (BLV), another member of the HTLV/BLV genus of retroviruses, and is about fourfold lower than that of HIV-1. These observations indicate that while the mutation rate of HTLV-1 is significantly lower than HIV-1, this lower rate alone would not explain the low diversity in HTLV-1 isolates, supporting the hypothesis that HTLV-1 replicates primarily as a provirus during cellular DNA replication rather than as a virus via reverse transcription.
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PMID:In vivo analysis of human T-cell leukemia virus type 1 reverse transcription accuracy. 1100 Feb 22

Cadherins, calcium-dependent cell adhesion molecules, play crucial roles, not only in the maintenance of tissue integrity, but also in the regulation of many aspects of cell behavior. We investigated the expression of "classic" E-, N- and P-cadherins in bone marrow-derived cultured mast cells (BMMC) and peritoneal mast cells (PMC) from mice. Flow cytometric analysis and immunocytochemical staining indicated that E-cadherin was expressed on the cell surface of BMMC and also at lower levels on PMC. N-cadherin was also expressed on the surface of BMMC, but not of PMC, whereas P-cadherin expression was seen in neither cell type. Significant expression of E- and N-cadherin mRNA was observed in BMMC by reverse transcriptase-polymerase chain reaction (RT-PCR), but PMC expressed only E-cadherin mRNA. Western blotting analysis indicated expression of alpha- and beta-catenins and p120-catenin (or p120 cas) in BMMC, whereas PMC showed less intense expression of alpha- and beta-catenins with high levels of p120 expression. Analyses of beta-catenin or E-cadherin immunoprecipitates from BMMC lysate revealed that alpha-catenin, beta-catenin, and E-cadherin were co-precipitated, suggesting that E-cadherin and catenins form a complex in mast cells. Addition of a blocking antibody of homophilic E-cadherin interactions, or a synthetic E-cadherin-binding decapeptide containing the histidine-alanine-valine (HAV) sequence in methylcellulose cultures of gut intraepithelial mononuclear cells or BMMC, significantly suppressed the clonal growth of mast cells. Furthermore, the blocking antibody or synthetic decapeptide significantly suppressed BMMC adhesion to E-cadherin-expressing F9 cell monolayers. These results indicated that E-cadherin and associated cytoplasmic proteins in mast cells might be involved in the regulation of certain stages of mast cell differentiation and cell-cell interactions.
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PMID:E-cadherin and cadherin-associated cytoplasmic proteins are expressed in murine mast cells. 1104 74

The amino acid change V75T in human immunodeficiency virus type 1 reverse transcriptase confers a low level of 2',3'-didehydro-2',3'-dideoxythymidine (stavudine, d4T) resistance in vivo and in vitro. Valine 75 is located at the basis of the fingers subdomain of reverse transcriptase between the template contact point and the nucleotide-binding pocket. V75T reverse transcriptase discriminates 3.6-fold d4T 5'-triphosphate relative to dTTP, as judged by pre-steady state kinetics of incorporation of a single nucleotide into DNA. In addition, V75T increases the DNA polymerization rate up to 5-fold by facilitating translocation along nucleic acid single-stranded templates. V75T also increases the reverse transcriptase-mediated repair of the d4TMP-terminated DNA by pyrophosphate but not by ATP. The V75T/Y146F double substitution partially suppressed both increases in rate of polymerization and pyrophosphorolysis, indicating that the hydroxyl group of Thr-75 interacts with that of Tyr-146. V75T recombinant virus was 3-4-fold d4T-resistant and 3-fold resistant to phosphonoformic acid relative to wild type, confirming that the pyrophosphate traffic is affected in V75T reverse transcriptase. Thus, in addition to nucleotide selectivity V75T defines a type of amino acid change conferring resistance to nucleoside analogues that links translocation rate to the traffic of pyrophosphate at the reverse transcriptase active site.
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PMID:The valine-to-threonine 75 substitution in human immunodeficiency virus type 1 reverse transcriptase and its relation with stavudine resistance. 1113 27

The patterns of resistance-conferring mutations that are selected in HIV-1 reverse transcriptase (RT) by the racemates of 2'-dideoxy-3'-oxa-4'-thiocytidine (+/-)dOTC and its fluorinated derivative (+/-)dOTFC were characterized. Genotypic and phenotypic analyses of HIV-1 clinical isolates and HXB2D variants selected with (+/-)dOTC and (+/-)dOTFC were performed in primary cells and in the MT-2 T cell line. HIV-1 variants selected with (+/-)dOTC or (+/-)dOTFC displayed fivefold decreased susceptibility to the respective compounds. A substitution of methionine to valine was identified at position 184 (M184V) in variants selected with (+/-)dOTC. In contrast, a mutation of lysine to arginine at position 65 (K65R) was found in variants selected with (+/-)dOTFC. These patterns of selected mutations differ from those seen with the individual enantiomers. Studies with mutated recombinant HXB2D-M184V and -K65R confirmed that these mutations are important for phenotypic resistance in MT-2 cells. Clinical isolates that display resistance to (-)2'-deoxy-3'-thiacytidine (3TC) also showed cross-resistance to (+/-)dOTC and (+/-)dOTFC. These studies demonstrate that similar genotypes may be selected by the dOTC and dOTFC compounds to those with the structurally related drug 3TC.
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PMID:Selection of resistance-conferring mutations in HIV-1 by the nucleoside reverse transcriptase inhibitors (+/-)dOTC and (+/-)dOTFC. 1122 93

Synthesis of the hepatitis B virus (HBV) DNA genome occurs within the viral nucleocapsid in a mechanistically ordered fashion. The nucleocapsid contains small pores that permit influx of nucleotide triphosphates and metabolites of nucleoside analogues such as lamivudine for DNA synthesis. Lamivudine is a potent inhibitor of HBV and human immunodeficiency virus (HIV) reverse transcriptases, but substitutions of isoleucine or valine for methionine within the tyrosine-methionine-aspartate-aspartate (YMDD) motif are associated with virologic and clinical resistance to lamivudine therapy. Under lamivudine selection pressure, the high viral production rate and the low fidelity viral polymerase contribute to frequent development of the YMDD mutants. However, the pattern and dynamics of emergence of the mutant viruses over the wild-type virus are determined by multiple factors including replication efficiency, host immune response, and availability of replication space. Structural modeling of HIV reverse transcriptase has permitted key insights into the molecular basis of lamivudine resistance of HBV based on evolutionary relatedness of HIV and HBV. The side groups of isoleucine and valine of the YMDD mutants sterically prevent lamivudine from appropriately configuring into the nucleotide binding site of the reverse transcriptase. Aminotransferase flares are associated with lamivudine therapy and may signify clinical resistance with emergence of YMDD mutants. They may also herald the recovery phase with seroconversion and viral clearance. Reconstitution of the endogenous anti-HBV immune response may be equally important in the control of viral replication by lamivudine and other nucleoside analogues.
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PMID:Molecular anatomy and pathophysiologic implications of drug resistance in hepatitis B virus infection. 1166 95

In order to compare the resistance pattern to zidovudine plus lamivudine in zidovudine-experienced patients, we studied three HIV-1-infected patients enrolled in NUCB3004, an open-label trial. Over a 24-week follow-up, the patients were studied for drug sensitivity, reverse transcriptase genotype, viral load (HIV-1 RNA level) and viral phenotype (syncytium inducing (SI) or non-syncytium inducing). Virus isolates derived from peripheral blood mononuclear cells (PBMCs) were tested for changes in drug susceptibility. Proviral DNA in the patients' PBMCs and RNA from plasma and culture supernatant were subjected to amplification and sequencing. All three HIV-1 strains showed a decreased susceptibility to either zidovudine or lamivudine after 24 weeks of therapy. The pattern of DNA genotypic resistance to lamivudine in patient A showed a mutation at codon 184 of the reverse transcriptase-encoding gene (methionine to valine). No HIV-1 strains with lamivudine-related mutations in proviral DNA were found among the isolates obtained from patients B and C. In these two patients, the mutation at codon 184 of the reverse transcriptase-encoding gene appeared in RNA, both in plasma and in culture supernatant. Viral phenotyping revealed the maintenance of the SI phenotype at week 24. Two out of the three patients experienced a reduction in HIV-1 RNA levels after 24 weeks of therapy, and in two out of three there was a rebound in viral load at week 28 together with the onset of the codon 184 mutation in RNA. The degree of phenotypic resistance to both zidovudine and lamivudine correlated with the amino acid changes in RNA and the rapid increase in viral load.
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PMID:Loss of antiviral effect owing to zidovudine and lamivudine double resistance in HIV-1-infected patients in an ongoing open-label trial. 1132 65


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