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)

Human immunodeficiency virus type 1 (HIV-1) gene expression is regulated by both cellular transcription factors and Tat. The ability of Tat to stimulate transcriptional elongation is dependent on its binding to TAR RNA in conjunction with cyclin T1 and CDK9. A variety of other cellular factors that bind to the HIV-1 long terminal repeat, including NF-kappaB, SP1, LBP, and LEF, are also important in the control of HIV-1 gene expression. Although these factors have been demonstrated to regulate HIV-1 gene expression by both genetic and biochemical analysis, in most cases a direct in vivo demonstration of their role on HIV-1 replication has not been established. Recently, the efficacy of RNA interference in mammalian cells has been shown utilizing small interfering RNAs (siRNAs) to result in the specific degradation of host mRNAs and decreases the levels of their corresponding proteins. In this study, we addressed whether siRNAs directed against either HIV-1 tat or reverse transcriptase or the NF-kappaB p65 subunit could specifically decrease the levels of these proteins and thus alter HIV-1 replication. Our results demonstrate the specificity of siRNAs for decreasing the expression of these viral and cellular proteins and inhibiting HIV-1 replication. These studies suggest that RNA interference is useful in exploring the biological role of cellular and viral regulatory factors involved in the control of HIV-1 gene expression.
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PMID:RNA interference directed against viral and cellular targets inhibits human immunodeficiency Virus Type 1 replication. 1243 22

Human immunodeficiency virus type 1 integrase (HIV-1 IN) is an essential enzyme for effective viral replication. Therefore, IN inhibitors are being sought for chemotherapy against AIDS. We had previously identified a series of salicylhydrazides as potent inhibitors of IN in vitro (Neamati, N.; et al. J. Med. Chem. 1998, 41, 3202-3209.). Herein, we report the design, synthesis, and antiviral activity of three novel mercaptosalicylhydrazide (MSH) derivatives. MSHs were effective against the IN catalytic core domain and inhibited IN binding to HIV LTR DNA. They also inhibited catalytic activities of IN in IN-DNA preassembled complexes. Site-directed mutagenesis and molecular modeling studies suggest that MSHs bind to cysteine 65 and chelate Mg(2+) at the active site of HIV-1 IN. Contrary to salicylhydrazides, the MSHs are 300-fold less cytotoxic and exhibit antiviral activity. They are also active in Mg(2+)-based assays, while IN inhibition by salicylhydrazides is strictly Mn(2+)-dependent. Additionally, in target and cell-based assays, the MSHs have no detectable effect on other retroviral targets, including reverse transcriptase, protease, and virus attachment, and exhibit no detectable activity against human topoisomerases I and II at concentrations that effectively inhibit IN. These data suggest that MSHs are selective inhibitors of HIV-1 IN and may serve as leads for antiviral therapeutics.
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PMID:Metal-dependent inhibition of HIV-1 integrase. 1247 50

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), like all retroviral RTs, is a versatile DNA polymerase that can copy both RNA and DNA templates. In spite of extensive investigations into the structure-function of this enzyme, the structural basis for this dual template specificity is poorly understood. Biochemical studies with two mutations in HIV-1 RT that affect residues contacting the template-primer now provide some insight into this specialized property. The mutations are N255D and N265D, both adjoining the minor groove-binding track, in the thumb region. The N265D substitution led to a loss of processive polymerization on DNA but not on RNA, whereas N255D drastically reduced processive synthesis on both templates. This differential template usage was accompanied by a rapid dissociation of the N265D variant on DNA but not RNA templates, whereas the N255D variant rapidly dissociated from both templates. Molecular dynamics modeling suggested that N265D leads to a loss of template strand-specific hydrogen bonding, indicating that this is a key determinant of the differential template affinity. The N255D substitution caused local changes in conformation and a consequent loss of interaction with the primer, leading to a loss of processive synthesis with both templates. We conclude that N265 is part of a subset of template-enzyme contacts that enable RT to utilize DNA templates in addition to RNA templates and that such residues play an important role in facilitating processive DNA synthesis on both RNA and DNA templates.
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PMID:Mutations proximal to the minor groove-binding track of human immunodeficiency virus type 1 reverse transcriptase differentially affect utilization of RNA versus DNA as template. 1271 77

Human immunodeficiency virus type 1 (HIV-1) variants were selected for resistance against the (+) and (-) enantiomers of a novel nucleoside analogue, 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC), using the infectious molecular clone HXB2D grown in the MT-4 line of human T cells. The variants selected with (+) dOTC were approximately 6-7-fold less sensitive than wild-type virus to this drug. Cloning and sequencing of the complete reverse transcriptase (RT) coding region of these variants identified the M1841 mutation and further selection with virus containing the M1841 substitution led to the appearance of an M184V mutation. In contrast, selection experiments performed with (-) dOTC yielded variants capable of growing in drug concentrations as high as 100 microM, but phenotypic analysis of these viruses revealed near wild-type 50% inhibitory concentration (IC50) values for this compound. Site-directed mutagenesis experiments in which the M1841 and M184V mutations were introduced into HXB2D confirmed the importance of these mutations when viruses were grown in MT4 cells. However, wild-type IC50 values in regard to both (-) and (+) dOTC were obtained when these recombinant viruses were grown in cord blood mononuclear cells (CBMC). Clinical isolates of HIV-1 resistant to lamivudine and containing the M184V substitution also displayed low-level resistance to both (-) and (+) dOTC when grown in CBMC. Finally, cell-free RT assays were performed in the presence of either (-) dOTC triphosphate, (+) dOTC triphosphate, or the triphosphate of a racemic mixture of (+) and (-) dOTC with wild-type and mutated M184V-containing recombinant RT. The data demonstrate chain termination effects of these compounds with regard to both wild-type and mutated enzyme and that the latter was approximately twofold less sensitive than the former to these drugs.
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PMID:Selection and characterization of HIV-1 variants resistant to the (+) and (-) enantiomers of 2'-deoxy-3'-oxa-4'-thiocytidine (dOTC). 1273 57

Human immunodeficiency virus type 2 (HIV-2) infection is a serious problem in West Africa and Asia. However, there have been relatively few studies of HIV-2 reverse transcriptase (RT), a potential target for antiviral therapy. Detailed knowledge of HIV-2 RT activities is critical for development of specific high-throughput screening assays of potential inhibitors. Here, we have conducted a systematic evaluation of HIV-2 RT function, using assays that model specific steps in reverse transcription. Parallel studies were performed with HIV-1 RT. In general, under standard assay conditions, the polymerase and RNase H activities of the two enzymes were comparable. However, when the RT concentration was significantly reduced, HIV-2 RT was less active than the HIV-1 enzyme. HIV-2 RT was also impaired in its ability to catalyze secondary RNase H cleavage in assays that mimic tRNA primer removal during plus-strand transfer and degradation of genomic RNA fragments during minus-strand DNA synthesis. In addition, initiation of plus-strand DNA synthesis was much less efficient with HIV-2 RT than with HIV-1 RT. This may reflect architectural differences in the primer grip regions in the p66 (HIV-1) and p68 (HIV-2) palm subdomains of the two enzymes. The implications of our findings for antiviral therapy are discussed.
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PMID:Human immunodeficiency virus type 2 reverse transcriptase activity in model systems that mimic steps in reverse transcription. 1280 62

Human immunodeficiency virus type 1 (HIV-1) subtype C viruses have been found to almost exclusively use the chemokine receptor CCR5 as a coreceptor for entry, even in patients with advanced AIDS. We have characterized subtype C virus isolates from 28 patients from Harare, Zimbabwe, 20 of whom were receiving antiretroviral treatment. Virus from 10 of the treated patients induced syncytium formation (SI virus) when cultured with MT2 cells. Only non-syncytium-inducing (NSI) virus was cultured from the peripheral blood mononuclear cells of the eight patients who had not received treatment. The majority of these subtype C SI viruses were capable of using both CCR5 and CXCR4 as coreceptors for viral entry, and the consensus V3 loop sequences from the SI viruses displayed a high net charge compared to those of NSI viruses. While those on treatment had reverse transcriptase (RT) and protease mutations, there was no clear association between RT and protease drug resistance mutations and coreceptor tropism. These results suggest that CXCR4-tropic viruses are present within the quasispecies of patients infected with subtype C virus and that antiretroviral treatment may create an environment for the emergence of CXCR4 tropism.
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PMID:High frequency of syncytium-inducing and CXCR4-tropic viruses among human immunodeficiency virus type 1 subtype C-infected patients receiving antiretroviral treatment. 1280 70

Human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) are the causative agents of AIDS. HIV-2 is prevalent at moderate to high rates in West African countries, such as Senegal, Guinea, Gambia, and Cape Verde. Diagnosis of HIV-2 is made with a positive HIV-1/HIV-2 ELISA or simple/rapid assay, followed by one or two confirmatory tests specific for HIV-2. Following CD(4)(+)T cell counts, HIV-2 viral burden and clinical signs and symptoms of immunodeficiency are beneficial in monitoring HIV-2 disease progression. Although non-nucleoside reverse transcriptase inhibitors are ineffective in treating HIV-2, nucleoside reverse transcriptase inhibitors and protease inhibitors can be effective in dual and triple antiretroviral regimens. Their use can decrease HIV-2 viral load, increase CD(4)(+)T cell counts and improve AIDS-related symptoms. HIV-2 resistance to various nucleoside reverse transcriptase inhibitors and protease inhibitors, including zidovudine, lamivudine, ritonivir and indinavir, has been identified in some HIV-2 infected patients on antiretroviral therapy. The knowledge of HIV-2 peculiarities, when compared to HIV-1, is crucial to helping diagnose and guide the clinician in the choice of the initial antiretroviral regimen and for monitoring therapy success.
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PMID:Diagnosis, antiretroviral therapy, and emergence of resistance to antiretroviral agents in HIV-2 infection: a review. 1280 87

Human immunodeficiency virus type 1 isolates having dipeptide insertions in the fingers subdomain of the reverse transcriptase (RT) show high level resistance to 3 '-azido-3 '-deoxythymidine (AZT) and other nucleoside analogues. Insertions are usually associated with thymidine analogue resistance mutations, such as T215Y. The resistance phenotype correlates with increased ATP-dependent phosphorolytic activity, which facilitates removal of thymidine analogues from inhibitor-terminated primers. In this report, we show that substituting Thr, Ser, or Asn for Tyr-215 in a multidrug-resistant RT, bearing a Ser-Ser insertion between codons 69 and 70, leads to AZT and stavudine resensitization through the loss of the ATP-mediated removal activity. The mutation D67N, which is rarely found in insertion-containing strains, had no effect on excision and a minor influence on resistance. Substituting Tyr-215 had a larger effect than deleting the dipeptide insertion. The presence of both the insertion and mutation T215Y in the wild-type BH10 RT conferred significant ATP-mediated removal activity and moderate resistance to AZT. However, resistance levels and unblocking activities were lower than those observed with the multidrug-resistant enzyme. Removal reactions can be inhibited by the next complementary dNTP. Both Tyr-215 and the dipeptide insertion affect RT-DNA.DNA-dNTP ternary complex formation, an effect that was not detected in the presence of foscarnet. Based on crystal structures of binary and ternary complexes of HIV-1 RT, we propose that Tyr-215 exerts its action by facilitating a proper orientation of the pyrophosphate donor molecule, whereas the effects on dNTP binding are indirect and could be related to significant conformational changes occurring during polymerization.
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PMID:Molecular determinants of multi-nucleoside analogue resistance in HIV-1 reverse transcriptases containing a dipeptide insertion in the fingers subdomain: effect of mutations D67N and T215Y on removal of thymidine nucleotide analogues from blocked DNA primers. 1504 90

Human immunodeficiency virus type 1 (HIV-1) isolates derived from HIV-infected, treatment-naive Ugandan infants were propagated and tested for sensitivity to antiretroviral (ARV) drugs. Although most subtype A and D isolates displayed inhibition profiles similar to those of subtype B strains, a subtype D isolate identified as D14-UG displayed high-level resistance to nevirapine in peripheral blood mononuclear cell cultures (>2,000-fold) and in MT4 cell cultures ( approximately 800-fold) but weaker resistance to delavirdine ( approximately 13-fold) and efavirenz ( approximately 8-fold) in MT4 cell cultures. To investigate the possible mechanism for this resistance to nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs), the RT coding region in pol was sequenced and compared to the consensus RT sequence of NNRTI-resistant and NNRTI-sensitive subtype A, B, and D HIV-1 isolates. D14-UG did not contain the classic amino acid substitutions conferring NNRTI resistance (e.g., Y181C, K103N, and G190A) but did have some putative sites associated with drug resistance, I135L, T139V, and V245T. Wild-type and mutated protease-RT genes from D14-UG and an NNRTI-sensitive subtype D isolate from Uganda (D13-UG) were cloned into pNL4-3 to produce recombinant viruses and to determine the effects of the mutations on susceptibility to ARV drugs, specifically, NNRTIs. The results showed that I135L and/or V245T mutations can confer high-level resistance to nevirapine and delavirdine as well as low level cross-resistance to efavirenz. Finally, ex vivo fitness analyses suggested that NNRTI-resistant sites 135L and 245T in wild-type isolate D14-UG may reduce RT fitness but do not have an impact on the fitness of the primary HIV-1 isolate.
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PMID:Characterization of a subtype D human immunodeficiency virus type 1 isolate that was obtained from an untreated individual and that is highly resistant to nonnucleoside reverse transcriptase inhibitors. 1511 18

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.
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PMID:Structural and biochemical effects of human immunodeficiency virus mutants resistant to non-nucleoside reverse transcriptase inhibitors. 1518 41


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