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)

Bovine leukemia virus (BLV) encodes at least two regulatory proteins, Rex and Tax. Tax, the transactivating protein, stimulates the long terminal repeat to promote viral transcription and may be involved in tumorigenesis. Rex is involved in the transition from early expression of regulatory proteins to later expression of viral structural proteins. We have targeted ribozymes against the mRNA encoding Rex and Tax. The ribozymes consist of the hammer-head catalytic motif flanked by antisense sequences that hybridize with the complementary rex/tax mRNA. To evaluate cleavage in a cell-free system, we transcribed portions of rex/tax mRNA and incubated them with synthetic RNA ribozymes. A ribozyme was identified that cleaves > 80% of the target RNA. Synthetic DNA encoding this ribozyme was cloned into the expression vector pRc/RSV and transfected into BLV-infected bat lung cells. Intracellular cleavage of rex/tax mRNA was confirmed by reverse transcriptase PCR. In cells expressing the ribozyme, viral expression was markedly inhibited. Expression of the BLV core protein p24 was inhibited by 61%, and reverse transcriptase activity in supernatant was inhibited by 92%. Ribozyme inhibition of BLV expression suggests that cattle expressing these sequences may be able to control BLV replication.
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PMID:Ribozyme cleaves rex/tax mRNA and inhibits bovine leukemia virus expression. 824 89

The level of genetic variation of human immunodeficiency virus type 1 (HIV-1), a member of the lentivirus genus of the Retroviridae family, is high relative to that of retroviruses in some other genera. The high error rates of purified HIV-1 reverse transcriptase in cell-free systems suggest an explanation for this high genetic variation. To test whether the in vivo rate of mutation during reverse transcription of HIV-1 is as high as predicted by cell-free studies, and therefore higher than that rates of mutation of retroviruses in other genera, we developed an in vivo assay for detecting forward mutations in HIV-1, using the lacZ alpha peptide gene as a reporter for mutations. This system allows the rates and types of mutations that occur during a single cycle of replication to be studied. We found that the forward mutation rate for HIV-1 was 3.4 x 10(-5) mutations per bp per cycle. Base substitution mutations predominated; G-to-A transition mutations were the most common base substitution. The in vivo mutation rates for HIV-1 are three and seven times higher than those previously reported for two other retroviruses, spleen necrosis virus and bovine leukemia virus, respectively. In contrast, our calculated in vivo mutation rate for HIV-1 is about 20-fold lower than the error rate of purified HIV-1 reverse transcriptase, with the same target sequence. This finding indicates that HIV-1 reverse transcription in vivo is not as error prone as predicted from the fidelity of purified reverse transcriptase in cell-free studies. Our data suggest that the fidelity of purified HIV-1 reverse transcriptase may not accurately reflect the level of genetic variation in a natural infection.
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PMID:Lower in vivo mutation rate of human immunodeficiency virus type 1 than that predicted from the fidelity of purified reverse transcriptase. 754 46

Human T-cell leukemia virus types I (HTLV-I) and II (HTLV-II) and bovine leukemia virus contain a region of approximately 600 nucleotides located 3' to the env gene and 5' to the last exon of the tax and rex regulatory genes. This region was originally termed nontranslated or untranslated (UT) since it did not appear to be expressed. Several studies have identified novel mRNAs in HTLV-I-, HTLV-II-, a bovine leukemia virus-infected cells that splice into open reading frames (ORFs) contained in the UT region and, thus, have the potential to produce proteins that might contribute to the biological properties of these viruses. The HTLV-II infectious molecular clone pH6neo has several ORFs in the UT region (nucleotides 6641 to 7213) and a large ORF which overlaps the third exon of tax/rex. To investigate the importance of these ORF-containing sequences on viral replication and transformation in cell culture, proviral clones containing deletions in UT (pH6neo delta UT) or a stop codon insertion mutation (pH6neoST) were constructed. Lymphoid cells were transfected with mutant proviral constructs, and stable cell clones, designated 729pH6neo delta UT and 729pH6neoST, were characterized. Viral protein production, reverse transcriptase activity, and the capacity to induce syncytia were indistinguishable from cells transfected with the wild-type clone. Finally, 729pH6neo delta UT- and 729pH6neoST-producer cells cocultured with primary blood T lymphocytes resulted in cellular transformation characteristic of HTLV. These results indicate that putative protein-coding sequences between env and the last exon of tax/rex are not required for viral replication or transformation in cell culture.
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PMID:Human T-cell leukemia virus type II nucleotide sequences between env and the last exon of tax/rex are not required for viral replication or cellular transformation. 798 33

The reverse transcriptase-RNA dependent DNA polymerase of Bovine Leukemia Virus (BLV) was isolated and characterized. The enzyme has a molecular weight of about 80kd and the isoelectric point is 7.6. The enzyme prefers magnesium, as a divalent cation using synthetic homopolymeric template primer poly (C) oligo (dG). Monoclonal antibodies directed against reverse transcriptase of human immunodeficiency virus type I (HIV-I) did not crossreact with the isolated polymerase.
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PMID:Characterization of the RNA dependent DNA polymerase of bovine leukemia virus. 891 42

Genetically simplified derivatives of complex retroviruses that replicate in animal models are useful tools to study the role of the complex regulatory genes in virus infection and pathogenesis and were proposed as a novel approach toward the development of vaccines against complex retroviruses. Previously we developed genetically simple derivatives of bovine leukemia virus (BLV) that can replicate in tissue culture independently of the BLV regulatory proteins, Tax and Rex, and the RIII and GIV open reading frames (K. Boris-Lawrie and H. M. Temin, J. Virol. 69:1920-1924, 1995). These derivatives are encoded on novel, hybrid retrovirus genomes that contain transcriptional control sequences of a simple retrovirus and gag-pol or env genes of the complex BLV. The first-generation simple BLV derivatives replicate as complementary viruses (coviruses) by using separate gag-pol or env genomes, and therefore virus spread is limited to cells that are infected with both covirus genomes. Here we describe a second-generation simple BLV derivative that is encoded on a single hybrid genome. We show the virus to be replication competent by successive passage on D17 target cells and by analysis of viral RNA and proteins in the infected cells. Furthermore, we evaluate the immunogenicity and infectivity of the simple BLV derivatives in a BLV animal model. Small groups of rats were injected either with virus-producing cells or with proviral DNA. Western immunoblot analysis revealed that antibodies against the major viral antigenic determinants are induced in response to either method of introduction and that seroconversion is sustained in most of the rats for at least 6 months (the duration of the study). The magnitudes of the antiviral responses were similar in rats infected with the first-generation simple BLV coviruses, the second-generation replication-competent derivative, or wild-type BLV. Wild-type BLV typically infects peripheral blood mononuclear cells (PBMC), and the simple BLV derivatives were also found to infect PBMC as demonstrated by PCR amplification of proviral sequences and reverse transcriptase PCR amplification of viral RNA in treated rats. These results establish that simple BLV derivatives lacking tax and rex are infectious and immunogenic in rats. These viruses will be useful tools in comparative studies with BLV to evaluate the role of tax and rex in maintenance of virus load and in disease outcome.
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PMID:In vivo study of genetically simplified bovine leukemia virus derivatives that lack tax and rex. 899 77

The hormone regulation of viruses has been of great interest since the discovery of glucocorticoid stimulation of mouse mammary tumor virus via a hormone response element in the viral long terminal repeat (LTR) promoter region. This report describes the investigation of the hormone responsiveness of bovine leukemia virus (BLV), an oncogenic retrovirus that infects dairy and beef cattle worldwide. It is a member of the human T cell leukemia (HTLV)/BLV group of retroviruses, which encode a protein, Tax, that is essential for regulating transcription of their own proviruses and for transforming host cells. We investigated the responsiveness of BLV to the hormones 17 beta-estradiol, progesterone, prolactin, insulin, and dexamethasone, a potent glucocorticoid. Only dexamethasone, in combination with insulin or insulin/prolactin, consistently stimulated BLV expression, as measured by reverse transcriptase activity, RNA blot hybridization (Northern blots), and CAT (chloramphenicol acetyltransferase) reporter assays of cell lines transiently or stably transfected with the BLV LTR. This effect required the presence of glucocorticoid receptors and Tax. This is the first report of hormone responsiveness in a virus of the HTLV/BLV group.
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PMID:Hormone regulation of bovine leukemia virus via the long terminal repeat. 943 16

We have expressed the recombinant reverse transcriptase (RT) of bovine leukemia virus (BLV) in bacteria. The gene encoding the RT was designed to start at its 5' end next to the last codon of the mature viral protease, namely the amino terminus of the RT matches the last 26 codons of the pro gene and is coded for by the pro reading frame. The RT sequence extends into the pol gene, utilizing the pol reading frame after overcoming the stop codon by adding an extra nucleotide (thus imitating the naturally occurring frameshift event). Hence we have generated a transframe polypeptide that is a 584-residues-long protein (see Rice, Stephens, Burny, and Gilden (1985) Virology 142, 357-377). This protein was partially purified after adding a six-histidine tag and studied biochemically testing a variety of parameters. The enzyme exhibits all activities typical of RTs, i.e., both RNA- and DNA-dependent DNA polymerase as well as a ribonuclease H (RNase H) activity. Unlike most RTs, the BLV RT is enzymatically active as a monomer even after binding a DNA substrate. The enzyme shows a preference for Mg2+ over Mn2+ in both its DNA polymerase and RNase H activities. BLV RT is relatively resistant to nucleoside triphosphate analogues, which are known to be potent inhibitors of other RTs such as that of HIV.
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PMID:Catalytic features of the recombinant reverse transcriptase of bovine leukemia virus expressed in bacteria. 1036 2

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

We have recently expressed in bacteria the enzymatically active reverse transcriptase (RT) of bovine leukemia virus (BLV) [Perach, M. & Hizi, A. (1999) Virology 259, 176-189]. In the present study, we have studied in vitro two features of the DNA polymerase activity of BLV RT, the processivity of DNA synthesis and the fidelity of DNA synthesis. These properties were compared with those of the well-studied RTs of human immunodeficiency virus type 1 (HIV-1) and murine leukaemia virus (MLV). Both the elongation of the DNA template and the processivity of DNA synthesis exhibited by BLV RT are impaired relative to the other two RTs studied. Two parameters of fidelity were studied, the capacity to incorporate incorrect nucleotides at the 3' end of the nascent DNA strand and the ability to extend these 3' end mispairs. BLV RT shows a fidelity of misinsertion higher than that of HIV-1 RT and lower than that of MLV RT. The pattern of mispair elongation by BLV RT suggests that the in vitro error proneness of BLV RT is closer to that of HIV-1 RT. These fidelity properties are discussed in the context of the various retroviral RTs studied so far.
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PMID:The processivity and fidelity of DNA synthesis exhibited by the reverse transcriptase of bovine leukemia virus. 1184 87

Bovine leukemia virus (BLV) is a widespread specific pathogen of cattle. Analysis of the pol viral gene polymorphism has been used to characterize the polymorphism of BLV isolates at stock-breeding farms in Russia and Ukraine. The fragments of the pol gene corresponding to the reverse transcriptase and integrase 494 and 233 bp in size, respectively, have been used for analysis. Phylogenetic analysis has revealed several variants of BLV clustered with a high bootstrap support in Russia and Ukraine. A new classification of BLV variants is suggested. Comparison of phylograms based on the polymorphism of the nucleotide sequences of the integrase and reverse transcriptase domains did not show topological conflicts. Therefore, recombination between BLV variants has not been found.
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PMID:[Identification and classification of bovine leukemia virus isolates in Russia and Ukraine based on the pol viral gene polymorphism]. 2298 71


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