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)

We have developed a noninvasive method for SIVsm virion RNA detection in feces of captive sooty mangabeys (SMs) (Cercocebus atys). Employing this method to investigate the natural history of SIVsm in endangered SMs is useful for understanding the diversity and evolution of SIVsm and HIV-2. The fecal samples of 61 wild-living SMs and 14 chimpanzees (Pan troglodytes verus) were studied. Samples were collected in rural Sierra Leone in 1993. One SM sample tested positive by reverse transcriptase-PCR. No viral sequence was detected in the feces of 14 chimpanzees. Phylogenetic analysis of the env sequence obtained from SM#13 showed that it clustered within the SIVsm lineage that includes SIVsmH4, B670, and PBj, confirming a direct connection between SIVsm from West Africa and an American-based colony of SM. The virus, designated as SIVsmSL93g, supports a link between the SIVB670/SIVsmH4/SIVPbj lineage and SMs living in Northern Sierra Leone in 1993. The discovery of this strain in a wild-living SM also indicates that noninvasive methods can be used for SIV detection from monkey feces collected in the field.
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PMID:A link between SIVsm in sooty mangabeys (SM) in wild-living monkeys in Sierra Leone and SIVsm in an American-based SM colony. 1565 Apr 27

CMKLR1 (chemoattractant-like receptor 1) is a G-protein-coupled receptor implicated in cartilage and bone development and is expressed in organs like the parathyroid gland, brain, and lung. The receptor is also expressed in dendritic cells and in macrophages where it acts as a co-receptor for entry of HIV/SIV isolates into human CD4(+) cells. Recently, a protein named "chemerin" (also known as TIG2) was isolated from human inflammatory fluids and hemofiltrate and found to be the endogenous ligand for CMKLR1. We have previously described the genomic organization of the cmklr1 gene and characterized its promoter in mouse neuroblastoma NB4 1A3 cells. In the present study we identify a second transcript, cmklr1b, in mouse microglia BV2 cells. Cmklr1b is transcribed from an alternative promoter with a transcription start site located 6780 bp downstream of the previously identified exon 1 (cmklr1a). The cmklr1b promoter lacks a TATA box but contains two CCAAT boxes in opposite directions. 5' Deletion analysis of the promoter region in BV2 cells using a luciferase reporter gene assay indicates two regions, between 623-755 bp and 56-125 bp upstream of transcription start site, to be important for promoter function. The proximal promoter region includes both CCAAT boxes, and site-directed mutagenesis separately within these elements revealed that only the forward CCAAT element was important for transcription. Although the forward CCAAT element is essential for transcription electrophoretic mobility shift and super-shift assays demonstrated that both CCAAT elements actually bind nuclear proteins from BV2 cells and identified the binding factor as NFY. Real-time reverse transcriptase-PCR experiments of cmklr1b expression in all-trans retinoic acid (ATRA)- stimulated BV2 cells showed strong up-regulation of receptor transcript. Luciferase reporter gene assay of the promoter in ATRA-stimulated BV2 cells confirmed that transcriptional activity of the cmklr1b promoter is increased by ATRA. However, deletion analysis could not identify an ATRA-responsive element within the promoter region suggesting that gene activation is likely to occur through alternative mechanisms. The results emphasise a possible role of cmklr1 in bone modelling.
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PMID:The mouse chemerin receptor gene, mcmklr1, utilizes alternative promoters for transcription and is regulated by all-trans retinoic acid. 1579 32

Simian immunodeficiency virus (SIVcpz) from the chimpanzee subspecies Pan troglodytes troglodytes has been linked phylogenetically to the origin of HIV-1. Related but distinct SIVcpz strains have also been found in P. t. schweinfurthii , suggesting that SIVcpz may have coevolved among the four chimpanzee subspecies. However, SIVcpz strains from P. t. verus and P. t. vellerosus have not yet been identified. To better understand the epidemiology and natural history of SIVcpz among chimpanzees, we tested serum samples from 1415 chimpanzees housed at eight U.S. research centers and six zoos. Records indicated that 264 (18.6%) of the chimpanzees were African-born. Subspecies identities for 161 chimpanzees, based on analysis of mitochondrial DNA sequences, were found to be P. t. troglodytes (n = 14), P. t. schweinfurthii (n = 3), P. t. verus (n = 143), and P. t. vellerosus (n = 1). All samples were screened for HIV/SIV antibodies by using an HIV-1/2 peptide- based enzyme immunoassay (EIA). Reactive samples were tested further by Western blot (WB). Eight sera (0.57%) were EIA reactive, but none was HIV-1/2 WB positive. Two samples were HIV-1 WB indeterminate. Both samples tested negative for SIVcpz and HIV-1 sequences by reverse transcriptase PCR, suggesting an absence of infection. We also tested sera available from 8 male sexual partners, 6 offspring, and 12 cage mates of a known SIVcpz-infected chimpanzee. All samples were negative, suggesting that SIVcpz may not be easily transmitted to close contacts. Our data show that this large population of chimpanzees is not infected with SIVcpz. The absence of SIVcpz infection in P. t. verus suggests that SIVcpz may not be endemic to this subspecies and implies that SIVcpz may have been introduced more recently into the chimpanzee subspecies following divergence.
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PMID:The epidemiology of simian immunodeficiency virus infection in a large number of wild- and captive-born chimpanzees: evidence for a recent introduction following chimpanzee divergence. 1592 95

We earlier reported that immunization of macaques with a reverse transcriptase-deleted SHIV(KU2) (DeltartSHIV(KU2)) plasmid that contained HIV-1(HXB2) env and SIV gag-nef induced protection against AIDS caused by challenge virus SHIV89.6P with a heterologous env. We further deleted vif and integrase from DeltartSHIV(KU2) and substituted the 3'LTR with SV40 poly A sequences, creating Delta4SHIV(KU2) (M) and a parallel construct containing gag-nef of HIV-1(SF2), Delta4SHIV(KU2) (H). Six macaques received two intramuscular injections of the (M) DNA, and another six received three injections of the (H) DNA. Three of the latter group received two post-challenge boosts with (M) DNA vaccine. Seven virus control macaques were inoculated with SHIV89.6P. All twelve immunized macaques were challenged with SHIV89.6P virus, and CMI responses were measured by ELISPOT assays. Virus control animals all developed progressive infection, whereas vaccinated macaques from both groups controlled virus replication, with plasma viral loads dropping to undetectable levels between weeks 6 and 126 p.i. This DNA vaccine was efficacious even though it encoded Env, Gag, and Nef that were genetically distinct from the proteins in the challenge virus. The DNA vaccine induced broad-based protection without using viral proteins to boost the immunity.
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PMID:Immunoprophylaxis against AIDS in macaques with a lentiviral DNA vaccine. 1665 Apr 48

Monitoring of viral load in macaques has usually been carried out using in-house PCR-based methods. A novel viral load (VL) kit (ExaVir Load) based on the measurement of lentivirus reverse transcriptase (RT) activity provides a potential alternative to methods that measure plasma viral RNA. RT is a fundamental and conserved activity of all retroviruses and the method should theoretically detect RT from all lentiviruses. To test this we compared VL measured by a commercially available RT kit with an in-house QC RT-PCR in macaques infected with SIV and SHIV. Both RT and RNA levels were measured over time in both sets of macaques. Results indicated that the relationship between both tests was strong for SIV and SHIV (r = 0.95 and r = 0.92, p < 0.0001, respectively). The VL trends also followed each other, indicating that both techniques measured the same process of viral replication. Furthermore, the RT load obtained using standardized control plasma samples supplied by NIBSC gave values close to the designated VL. However, when comparing RT load with QC RT-PCR a consistently three to five time higher level was obtained with the RT assay, highlighting potential differences in assay calibration. Even so, the data suggest that the RT assay is both sensitive and robust for use in the SIV/SHIV macaque model, particularly where molecular-based assays for SIV VL determinations are not easily available. The assay is also a commercially available kit and hence has the potential to reduce the variability seen between laboratories using in-house PCR.
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PMID:Reverse transcriptase viral load correlates with RNA in SIV/SHIV-infected macaques. 1698 19

Following infection by HIV or SIV, reverse transcriptase (RT) directs the conversion of the single-stranded RNA genome into a double-stranded DNA molecule that integrates into the host cell genome. RT encodes for several immunogenic epitopes that are desirable for inclusion in a human vaccine for HIV infection, however, issues of safety have dampened enthusiasm for inclusion of an enzymatically-active RT molecule into an AIDS vaccine. In this study, virally-regulated, replication-incompetent lentiviral particles were expressed from DNA plasmids. The sequences for integrase, Vpr, Vif, Nef, and the long terminal repeats (LTRs) were deleted and mutations were engineered into capsid to decreases RNA packaging. Virus-like particles incorporated no RT (HIV-VLP DeltaRT or SHIV-VLP DeltaRT) or contained a full-length enzymatically-inactivated RT molecule (HIV-VLP or SHIV-VLP). Each secreted VLP was enveloped with a lipid bilayer derived from primate cells with embedded, native viral envelopes in similar concentrations as infectious virions. BALB/c mice were vaccinated (weeks 0, 3, and 6) with purified VLPs via intranasal inoculation in the presence of cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODNs). All VLPs, with or without RT, elicited both robust humoral and cellular immune responses to Gag, Pol, and Env antigens. Therefore, the lack of RT enhances the safety of these VLPs for use in future human clinical trials without a significant reduction in the overall immunogenicity of these VLP immunogens.
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PMID:Lentivirus-like particles without reverse transcriptase elicit efficient immune responses. 1707 23

Natural and selected resistance of HIV-1 to current anti-HIV drugs continues to pose serious problems to the development of HIV-1 antivirals. The viral reverse transcriptase (RT) is a proven therapeutic target. Single-stranded RNA and DNA (ssRNA and ssDNA) aptamers have been selected that specifically and potently inhibit RT function. In particular, the ssDNA aptamer RT1t49 was previously selected to recognize the RT from a subtype B strain of HIV-1 and binds with a reported K(d) of 4 nM. In the present work, we show that RT1t49 inhibits recombinant RT cloned from diverse branches of the primate lentiviral family. Aptamer concentrations required for half-maximal inhibition of all HIV-1, HIV-2, and SIV(CPZ) RTs assayed were in the low-to mid-nanomolar range for both polymerase and RNase H activities. Using pre-steady-state and order-of-addition kinetic analyses, we also established that this ssDNA aptamer competes with primer-template for access to RT, and that addition of a nucleoside analog RT inhibitor (NRTI) to the in vitro reaction enhanced the overall effectiveness of both drugs, while nonnucleoside analog RT inhibitors (NNRTIs) exhibited simple additivity. This is the first demonstration of universal inhibition of HIV and SIV(cpz) RTs by a nucleic acid aptamer and supports previous reports suggesting that resistance to RT1t49 may be exceptionally infrequent.
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PMID:Single-stranded DNA aptamer RT1t49 inhibits RT polymerase and RNase H functions of HIV type 1, HIV type 2, and SIVCPZ RTs. 1753 Sep 96

Reverse transcriptase (RT) assay is commonly used to detect enzyme activity associated with retroviral-like particles. Previously, detection of RT activity in virus-infected cultures was done using a radioisotope-based assay system. However, assay systems, which detect the antigen directly(as opposed to antibody ELISA assays), have been developed. For diagnostic purposes, RT activity and p24 antigen capture assays are the two most commonly used methods for detection of retroviral infection. More recently, new non-radioactive assay systems have been developed. In this study, four non-radioactive reverse transcriptase kits were evaluated using samples obtained from a chimeric virus, simian/human immunodeficiency virus (SHIV) and SIV-infected cell cultures. The results showed that the magnesium kit was the most appropriate for detection of SIV and SHIV infection in cell culture supernatants.
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PMID:Evaluation of Non-radioactive ELISA assay Kits for Detection of Retroviral Reverse Transcriptase (RT) Activity Associated with Retroviral SIV and HIV. 1765 46

We have identified 1H-benzylindole analogues as a novel series of human immunodeficiency virus (HIV) integrase inhibitors with antiretroviral activities against different strains of HIV type 1 (HIV-1), HIV-2, and simian immunodeficiency virus strain MAC(251) [SIV(MAC(251))]. Molecular modeling and structure-activity relationship-based optimization resulted in the identification of CHI/1043 as the most potent congener. CHI/1043 inhibited the replication of HIV-1(III(B)) in MT-4 cells at a 50% effective concentration (EC(50)) of 0.60 microM, 70-fold below its cytotoxic concentration. Equal activities against HIV-1(NL4.3), HIV-2(ROD), HIV-2(EHO), and SIV(MAC(251)) were observed. CHI/1043 was equally active against virus strains resistant against inhibitors of reverse transcriptase or protease. Replication of both X4 and R5 strains in peripheral blood mononuclear cells was sensitive to the inhibitory effect of CHI/1043 (EC(50), 0.30 to 0.38 microM). CHI/1043 inhibited integrase strand transfer activity in oligonucleotide-based enzymatic assays at low micromolar concentrations. Time-of-addition experiments confirmed CHI/1043 to interfere with the viral replication cycle at the time of retroviral integration. Quantitative Alu PCR corroborated that the anti-HIV activity is based upon the inhibition of proviral DNA integration. An HIV-1 strain selected for 70 passages in the presence of CHI/1043 was evaluated genotypically and phenotypically. The mutations T66I and Q146K were present in integrase. Cross-resistance to other integrase strand transfer inhibitors, such as L-708,906, the naphthyridine analogue L-870,810, and the clinical drugs GS/9137 and MK-0518, was observed. In adsorption, distribution, metabolism, excretion, and toxicity studies, antiviral activity was strongly reduced by protein binding, and metabolization in human liver microsomes was observed. Transport studies with Caco cells suggest a low oral bioavailability.
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PMID:Preclinical evaluation of 1H-benzylindole derivatives as novel human immunodeficiency virus integrase strand transfer inhibitors. 1854 26

The biochemical effects of 2-(ethoxymethylthio)-9-phenyl-cyclohepta[d]pyrimidone (EPCP), a novel non-nucleoside reverse transcriptase inhibitor, have been investigated. Treatment with EPCP (EC(50) of 0.88 nM in CEM x174 cells) significantly inhibited the activity of SIV reverse transcriptase and elevated the percentage of viable cells in an SIV-infected sample in a dose-dependent manner. The percentage of cells accumulated in G1 phase increased significantly from 34.5 to 62.4%, with a concomitant reduction in S-phase from 50.7% in the control to 22.6% in the infected group. This cell cycle profile was restored by treatment with EPCP. SIV upregulated the levels of the caspase-3, p53 and bax proteins, and downregulated the level of bcl-2 in infected cells. The apoptotic effect of SIV was also blocked by treatment with EPCP. The pharmacological effects of EPCP paralleled those of AZT, suggesting the possibility that EPCP might be a novel antiviral agent for SIV.
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PMID:Pyrimidone derivative inhibits simian immunodeficiency virus-induced apoptosis of CEM x174 cells. 1906 83


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