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)

As an important enzyme in DNA synthesis, dUTPase is present in a wide variety of organisms and viruses and has been identified as a component of the equine infectious anemia virus (EIAV) pol gene. The role of EIAV dUTPase, designated DU, in virus replication in vitro and in vivo was investigated with a recently described infectious molecular clone of EIAV. A deletion mutant that was deficient in dUTPase activity was constructed, and its replication kinetics was examined in fetal equine kidney (FEK) cells and primary equine bone marrow macrophage (EBMM) cells. In FEK cells, which are permissive for EIAV replication, the mutant virus replicated as well as the parental virus. In primary cultures of EBMM cells, which are primary targets of EIAV infection in vivo, the DU mutant showed delayed replication kinetics and replicated to a lower extent than did the parental virus. As the multiplicity of infection decreased, the difference between the parental and mutant viruses increased, such that at the lowest multiplicity of infection tested, there was over a 100-fold difference in virus production. The mutant virus was also much less cytopathic. The role of DU in replication in vivo was examined using a Shetland pony model of EIAV infection. Shetland ponies that were infected with the parental and mutant viruses showed transient virus RNA levels in plasma approximately 5 to 10 days postinfection. The peak virus levels in plasma (as measured by a quantitative reverse transcriptase PCR assay) were 10- to 100-fold lower in the mutant virus-infected animals than in the animals infected with the parental virus. However, ponies infected with the mutant virus mounted similar antibody responses despite the marked differences in virus replication. These studies demonstrate that EIAV DU is important for the efficient replication of the virus in macrophages in vitro and in vivo and suggests that variations in the DU sequence could markedly affect the biological and pathogenic properties of EIAV.
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PMID:Replication in vitro and in vivo of an equine infectious anemia virus mutant deficient in dUTPase activity. 770 12

Phosphorothioate analogs of oligodeoxynucleotides at a concentration of 2 microM protected Himalayan tahr cells from infection by caprine arthritis encephalitis virus (CAEV) and equine dermis cells from infection by equine infectious anemia virus (EIAV). The characteristics of this inhibition against these lentiviruses are similar to those previously described for the inhibition of HIV-1 in ATH8 cells [17]. Thus, the 28-mer homo-oligomer of cytidine [S-(dC)28] was at least as effective as three anti-sense sequences targeted to the LTR, gag, and env regions of CAEV. The effectiveness of homo-oligomers of equal length was in the order C >> A > T, and a random 28-copolymer with a composition of 2C:1G was as effective as S-(dC)28. Shorter oligonucleotides were less effective (28 > 14 > 5 mers) for all base compositions tested. While replication of a simian type D retrovirus was inhibited by S-(dC)28, this compound did not inhibit the cytopathogenicity of two type C retroviruses, amphotropic murine leukemia virus (MuLV), and baboon endogenous virus, when they were tested in the same cell lines used to support the replication of lentiviruses. Southern blot analysis of the high molecular weight DNA of drug-treated CAEV-infected cells showed that S-(dC)28 was acting at or before the reverse transcription step. Our present data and the earlier finding that S-(dC)28 is a potent in vitro inhibitor of the MuLV reverse transcriptase [15] suggest that S-(dC)28 is acting very early in the replication cycle of these lentiviruses. Since MuLV reverse transcriptase is inhibited in vitro, but its replication is not blocked in permissive cells, our data suggest that the phosphorothioate oligonucleotides are preventing virus attachment.
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PMID:Phosphorothioate oligonucleotides inhibit the replication of lentiviruses and type D retroviruses, but not that of type C retroviruses. 782 17

2',3'-didehydro-3'-deoxythymidine (d4T) is a pyrimidine analogue and inhibitor of reverse transcriptase with potent in vitro activity against human immunodeficiency virus (HIV). A phase I trial of d4T was conducted in 41 HIV-infected patients, 12 with AIDS and 29 with AIDS-related complex (ARC). Thirty-six patients were evaluatable. The maximum tolerated dose was 2 mg/kg/day. The dose-limiting toxicity was sensory peripheral neuropathy, which occurred in 20 patients (55%). Four patients (11%) developed hepatotoxicity. Five (14%) developed anemia requiring a transfusion but not discontinuation of drug. The mean +/- SE plasma elimination half-life at all dose levels was 1.2 +/- 0.09 h. Increased or stable absolute CD4 counts were seen in most patients. The majority of patients with detectable serum p24 antigen levels had a persistent decrease by 6 months. d4T is a promising drug for patients with AIDS or ARC. This clinical trial is continuing to determine the minimal effective dose.
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PMID:2',3'-didehydro-3'-deoxythymidine (d4T) in patients with AIDS or AIDS-related complex: a phase I trial. 809 63

The cis- and trans-acting components of the Rev regulatory pathway employed by equine infectious anemia virus (EIAV) to regulate and coordinate viral gene expression were examined in complementation experiments. Viral protein expression and mRNA expression were compared in cells transiently transfected with wild-type or mutant proviruses in combination with Rev expression plasmids. Mutation of the predicted rev gene abolished Gag protein synthesis, and this defect was complemented, in trans, by Rev. Analysis of viral mRNAs from transfected cells confirmed that EIAV expresses five major mRNAs: the full-length and singly spliced mRNAs contain introns and encode viral structural proteins while the three fully spliced mRNAs, encoding nonstructural genes, are generated by alternative splicing. Compared to cells transfected with the wild-type provirus, the intron-containing mRNAs produced from the rev-minus mutant were present at reduced levels in the nuclear RNA fraction and were not detected in the cytoplasm. This pattern of viral mRNA synthesis was restored to the wild-type pattern by providing Rev in trans. In contrast to the intron-containing mRNAs, cytoplasmic accumulation of the multiply spliced class of mRNAs was independent of Rev. Closer examination of the multiply spliced class of viral mRNAs by reverse transcriptase-PCR analysis revealed a Rev-dependent alternative splicing phenomenon. In the absence of Rev, proviruses expressed a four-exon mRNA at high levels; the addition of Rev caused both a decrease in the levels of the four-exon mRNA and the appearance of a related mRNA lacking exon 3. The cis-acting RNA elements that mediate Rev responsiveness were studied with deleted proviruses, which revealed that EIAV contains at least two elements located near the ends of envelope gene. Unlike the Rev-responsive elements in other retroviruses, the cis-acting regions of EIAV do not appear to form complex secondary structures.
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PMID:Equine infectious anemia virus trans-regulatory protein Rev controls viral mRNA stability, accumulation, and alternative splicing. 815 75

The putative dUTPase domain was deleted from the polymerase (pol) gene of equine infectious anemia virus (EIAV) to produce a recombinant delta DUpol Escherichia coli expression cassette and a delta DU proviral clone. Expression of the recombinant delta DUpol polyprotein yielded a properly processed and enzymatically active reverse transcriptase, as determined by immunoblot analysis and DNA polymerase activity gels. Transfection of delta DU provirus into feline (FEA) cells resulted in production of virus that replicated to wild-type levels in both FEA cells and fetal equine kidney cells. In contrast, the delta DU virus replicated poorly (less than 1% of wild-type levels) in primary equine macrophage cultures, as measured by reverse transcriptase assays. Preparations of delta DU virus contained negligible dUTPase activity, which confirms that virion-associated dUTPase is encoded in the pol gene region between the RNase H domain and integrase, as has been demonstrated previously for feline immunodeficiency virus (J. H. Elder, D. L. Lerner, C. S. Hasselkus-Light, D. J. Fontenot, E. Hunter, P. A. Luciw, R. C. Montelaro, and T. R. Phillips, J. Virol. 66:1791-1794, 1992). Our results suggest that virus-encoded dUTPase is dispensable for virus replication in dividing cells in vitro but may be required for efficient replication of EIAV in nondividing equine macrophages, the natural host cells for this virus.
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PMID:Characterization of equine infectious anemia virus dUTPase: growth properties of a dUTPase-deficient mutant. 838 67

Zidovudine is a dideoxynucleoside analogue of thymidine. It acts by interfering with viral reverse transcriptase, thereby inhibiting human immunodeficiency virus (HIV) replication. Zidovudine has been shown in clinical trials to prolong survival of patients with acquired immune deficiency syndrome (AIDS) and advanced AIDS-related complex (ARC), and to delay progression to ARC or AIDS in patients with earlier disease. At the present time it is suggested that zidovudine be initiated when the CD4 lymphocyte count is less than 500 cells/mm3. Recent studies have suggested a delay in the development of AIDS in patients with CD4 counts over 500 cells/mm3, but ongoing studies will require confirmation. The adverse reactions associated with zidovudine have been well described. It appears that haematological toxicity is associated with both the dose and stage of disease. Anaemia may present more often within the first 3 months of therapy, whereas neutropenia can occur early or late. Mild headache and gastrointestinal intolerance may occur early and in some cases limit tolerance to the drug. A number of neurological adverse reactions have been reported rarely including seizures and dose-reduction encephalopathy. The most significant late adverse reaction is that of myopathy, which occurs in patients receiving zidovudine for more than 6 months. With careful monitoring, the adverse reactions of zidovudine are manageable and patient tolerance of the medication is acceptable.
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PMID:Zidovudine toxicity. Clinical features and management. 848 Dec 17

In order to investigate the modes of DNA synthesis supported by the 66 and 51 kDa subunits of equine infectious anemia virus reverse transcriptase (EIAV RT), recombinant p66 polypeptides containing a modified ribonuclease H (RNase H) domain were purified and evaluated. Defined heteropolymeric template-primer combinations and high-resolution gel electrophoresis provided a qualitative evaluation of DNA polymerase and RNase H activities, while DNase I footprinting revealed features of replication complexes containing the truncated enzymes. Removal of alpha-helix E' and the conserved beta 5'-alphaE' "His-loop" in p66delta20 RT uncouples the RNase H activities, alters affinity for template-primer and dictates how the replicating enzyme responds to secondary structure on both DNA and RNA templates. Despite these alterations, DNase I footprinting shows no major difference in the overall structure of DNA-directed DNA synthesis complexes. In contrast, removing 47 C-terminal residues, which includes alpha-helix D', beta-strand 5' and alpha-Helix E', yields an enzyme with distributive DNA polymerase properties closely resembling the purified p51 subunit.
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PMID:Involvement of C-terminal structural elements of equine infectious anemia virus reverse transcriptase in DNA polymerase and ribonuclease H activities. 864 20

Control of equine infectious anemia (EIA) is currently based on detection of anti-EIA virus (EIAV) antibodies. However, serologic diagnostic methods may give false-negative results in infected horses that fail to respond adequately or are in the early stages of infection. We developed a reverse transcriptase nested PCR (RT-nPCR) assay for the detection of viral gag gene sequences in plasma from EIAV-infected horses. The ability of RT-nPCR to detect field strains of EIAV was investigated by assaying plasma samples from 71 horses stabled on EIA quarantine ranches. Positive PCR signals were detected in 63 of 63 horses with EIAV antibody test-positive histories on approved serologic tests, demonstrating that RT-nPCR was probably directed against highly conserved sequences in the viral genome. The RT-nPCR assay, agar gel immunodiffusion test, and conventional virus isolation were compared for detection of early infection in 12 experimentally infected ponies. Viral gag sequences were detected in all 12 animals by 3 days postinfection (p.i.) by RT-nPCR, whereas virus could not be routinely isolated on cell culture until 9 to 13 days p.i. and EIAV antibodies could not be detected by agar gel immunodiffusion until 20 to 23 days p.i. Finally, specificity of the RT-nPCR assay was examined by testing plasma from 43 horses with serologic test-negative histories and no known contact with EIAV-infected animals. Viral gag sequences were not detectable in this control group. These data suggest that the EIAV RT-nPCR assay effectively detects EIAV and is more sensitive than current standard methods for detection of early stages of infection.
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PMID:Detection of equine infectious anemia viral RNA in plasma samples from recently infected and long-term inapparent carrier animals by PCR. 873 2

We have investigated four members of a three-generation Dutch family for a suspected hemoglobinopathy. Chronic hemolysis and a moderate macrocytic normochromic anemia with slight morphological abnormalities of the red cells was observed in all four. Hemoglobin chain synthesis in vitro and separation of the globin chains by reversed phase high performance liquid chromatography revealed an abnormal beta-globin species in addition to the normal alpha and beta chains. The decreased amount of normal beta-globin and the low amount of unidentified protein suggested an unstable beta-globin variant. An abnormal band was detected by isoelectrofocusing. In one family member tested, the hemoglobin in an erythrocyte lysate had decreased heat stability. All carriers were positive in the isopropanol hemoglobin instability test. Treatment of erythrocytes with methylviolet gave rise to microgranular inclusions. Nucleotide sequencing of the polymerase chain reaction-amplified beta-globin gene revealed a heterozygous single base pair T-->C mutation at codon 75, which changes the normal CTG codon for leucine to a CCG codon for proline. This variant has previously been identified as Hb Atlanta or beta 75(E19)Leu-->Pro. The mutation creates a new Msp I restriction site, which was used to confirm the diagnosis in all four family members. A quantitative reverse transcriptase polymerase chain reaction procedure for determining the relative amounts of mRNA transcripts for the normal and abnormal globin chain showed a comparable stability for both transcripts.
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PMID:A Dutch family with Hb Atlanta [beta 75(E19)Leu-->Pro]. 893 61

Structural studies of authentic HIV reverse transcriptase (RT) suggest a role for the p51 carboxyl terminus in forming an active RNase H conformation [Rodgers, D. W., Gamblin, S. J., Harris, B. A., Ray, S., Culp, J. S., Hellmig, B., Woolf, D. J., Debouck, C. & Harrison, S. C. (1995) Proc. Natl. Acad. Sci. USA 92, 1222-1226]. We have purified mutant RT heterodimers containing deletion of 5, 9, or 13 amino acids from the p51 carboxyl terminus. These "selectively deleted" heterodimers have been analyzed for changes in RNA-dependent DNA polymerase activity, RNase H activity, and the ability to catalyze DNA strand transfer. As deletions extended into the p51 subunit, a decrease in the stability of the RT-DNA complex was apparent. The largest effect was observed for p66/p51Delta13 RT, which showed a 3-fold decrease relative to wild-type RT. RNase H activity was measured by digestion of the RNA in a 5' 32P-labeled RNA/DNA hybrid. Deletion of 5 or 9 amino acids from p51 had little effect on synthesis-dependent and synthesis-independent RNase H activities. In contrast, deletion of 13 amino acids from p51 increased the length of the hydrolysis products of both RNase H activities by 8-10 bp, thus changing the spatial relationship between the polymerase and RNase H active sites from a distance of 17-18 bp to 26-27 bp. The Delta13 derivative was also incapable of efficient DNA strand transfer. This defect in strand transfer could be suppressed by the 71-amino acid form of HIV nucleocapsid protein (NC) but not by the 55-amino acid form (NC55) or by equine infectious anemia virus NC. These results provide evidence for the existence of a specific complex between RT and NC and are discussed in terms of the role of this complex in proviral DNA synthesis.
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PMID:Mutations in HIV reverse transcriptase which alter RNase H activity and decrease strand transfer efficiency are suppressed by HIV nucleocapsid protein. 919 28


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