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 analyzed transmission of (LTR, v-src, LTR) cryptic structure integrated in the H-19 mammalian tumor cell line. From this cell line different isolates of transforming virus were rescued in heterokaryons produced by fusion with chicken fibroblasts infected by replication-competent avian leukosis virus RAV-1. One of them (F6) was used for the transformation of avian cells in the absence of the helper virus. In four transformed cell lines studied, the (LTR, v-src, LTR) structure was again integrated at a unique position in the cell DNA of each line. This indicated that the (LTR, v-src, LTR) structure is transmitted by the helper virus without recombination. This point has been further supported by the finding that a src-containing species corresponding in size to the nonpolyadenylated src mRNA is present in the RNA isolated from the rescued F6 transforming virus which might serve as template for the synthesis of (LTR, v-src, LTR) structure by the reverse transcriptase provided by RAV-1.
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PMID:Transmission of (LTR, v-src, LTR) without recombination with a helper virus. 301 94

The analysis of retroviral mutants has played a critical role in the development of our understanding of the complex viral life cycle. The most fundamental result of that analysis has been the definition of the replication functions encoded by the viruses. From a biochemical examination of a particular step in the life cycle it is difficult to determine, for example, whether that step is catalyzed by a viral or a host enzyme; but the isolation of a viral mutant defective in that step can firmly establish that a viral function is involved. In this way many facts about the viruses have been established. We know that reverse transcriptase is encoded by the virus; that RNAase H and DNA polymerase activities reside on the same gene product; that processing of many precursor proteins is mediated by a viral proteinase; and that establishment of the integrated provirus requires a viral protein. The list of functions mediated by viral enzymes has largely been defined by the mutants isolated and studied in various laboratories. The second significant result of the studies of viral mutants has been the assignation of the replication functions to particular viral genes, and then more specifically to particular domains of these genes. Mutants and viral variants have been essential in the determination, for example, that the gag protein is the critical gene product for the assembly of a virion particle; that the env protein is the determinant of species specificity of infection; or that the LTR is a major determinant of tissue tropism and leukemogenicity. The subdivisions of functions within a given gene have similarly hinged on mutants. Genetic mapping was needed to establish that P30 is the most important region for assembly; that the proteinase and integrase functions reside, respectively, in the 5' and 3' portions of the pol gene; and that the glycosylated gag protein is dispensable for replication. A third important area of knowledge has depended heavily on viral mutants: the determination of host functions and proteins that interact with viral proteins. Variant viruses with altered or restricted host ranges serve to define differences between pairs of different host cells, and the mapping of the viral mutations serves to define the viral protein important in that interaction with the host. These studies are only in their infancy, but it is clear that substantial efforts will be made to further analyze these host functions.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Mutants of murine leukemia viruses and retroviral replication. 303 30

The basis of the specific binding of tRNATrp by avian myeloblastosis virus reverse transcriptase was studied by chemical and enzymatic modification of the RNA. Binding does not depend on recognition of the tryptophan anticodon since molecules cleaved in the anticodon are stably bound by the enzyme. Modification of pseudouridine residues in the tRNA destroys binding to reverse transcriptase. These results are consistent with a model in which reverse transcriptase-tRNATrp interaction occurs not at the anticodon, but at regions in the tRNA which contain or are stabilized by pseudouridine residues.
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PMID:Structural features required for the binding of tRNATrp to avian myeloblastosis virus reverse transcriptase. 619 93

A porcine prorelaxin gene has been constructed partly by synthetic means and partly from its natural messenger RNA. A gene coding for the 32 N-terminal amino acids including a chain initiator methionine codon (B gene) was synthesised and inserted in a plasmid at a site downstream from a tryptophan promoter in such a way that its expression is under the control of the trp promoter. DNA corresponding to the rest of the prorelaxin was prepared using reverse transcriptase extension of a primer complementary to relaxin mRNA and joined at a suitable restriction site to the B gene. Transformation of E. coli with this plasmid followed by suitable induction resulted in the synthesis of a new protein identified as prorelaxin by its size and its antigenic similarity to relaxin.
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PMID:Cloning and expression of a porcine prorelaxin gene in E. coli. 619 93

A problem in utilizing herpes simplex virus (HSV) as a vector for expression of foreign genes in CNS neurons has been the inability to facilitate long-term expression of the engineered genes. Previously, we showed that the murine moloney leukemia virus LTR would drive beta-galactosidase (beta-gal) transcription for extended periods from the latent viral genome in sensory, but not motor neurons. In this communication we further evaluate the utility of the LTR promoter for use in long-term expression vectors. Following stereotactic injection of 8117/43 (an ICP4 minus, non-replicating virus with the LTR driving the beta-gal gene, or KD6 (an ICP4 minus non-replicating virus not expressing beta-gal) into the hippocampus of rats, polymerase chain reaction (PCR) analysis of viral DNA after 2 months indicated that latent infections were established. Assaying by both x-gal staining and reverse transcriptase PCR we demonstrate that (1) beta-gal can be detected for at least 6 months in hippocampal neurons, and (2) although the number of beta-gal transcripts in these cells drops considerably by 2 weeks, they can be detected during the period studied. These studies indicate that the LTR promoter is active and affords long-term expression in the CNS, albeit at comparatively low levels compared to those observed at acute times.
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PMID:Long-term expression of a reporter gene from latent herpes simplex virus in the rat hippocampus. 747 33

Three species of E6/E7 cDNAs of human papillomavirus type 16 (HPV16) for the full-length E6/E7 and spliced E6*I/E7 and E6*II/E7 mRNAs were synthesized by reverse transcriptase-(RT-)PCR from RNA of the cervical carcinoma cell line SiHa. Two cDNA mutants carrying point mutations in either a splice donor site or acceptor site within the E6 open reading frame were also constructed. These HPV16 E6/E7 cDNAs were cloned under the SV40 enhancer/promoter and the MMTV LTR to examine the activities of ras-collaborative transformation and induction of cellular DNA synthesis, both of which depend on the E7 gene product. The E6*II/E7 cDNA and two mutated cDNAs deficient in the spliced mRNA transcription showed lower levels of both activities than the full-length E6/E7 and the E6*I/E7 cDNA. The rat cell lines carrying each of the E6/E7 cDNAs contained the E6/E7 mRNA species expected. A small amount of E6*I/E7-sized mRNA was transcribed from a splice-donor site mutant of the E6/E7 cDNA, which turned out to be a transcript derived from a cryptic splice donor site six bases upstream from the conventional site. Among NIH3T3 cells carrying one of the above-mentioned E6/E7 cDNAs, the cells expressing E6*I/E7 mRNA [cells carrying cF(wt) and c*I] produced an amount of E7 protein comparable with those carrying the E7 or E6E7 region. These results suggest that the E6*I/E7 is the mRNA that is important for the efficient expression of E7 product from the HPV16 E6/E7 region.
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PMID:Biologic activity of human papillomavirus type 16 E6/E7 cDNA clones isolated from SiHa cervical carcinoma cell line. 748 85

Reverse transcriptases from both human immunodeficiency viruses type 1 and 2 are obligatory dimers. A tryptophan-rich repeat motif that is highly conserved between these proteins, as well as in the reverse transcriptase from simian immunodeficiency virus, has been postulated to be involved in hydrophobic subunit interactions. A synthetic 19-mer peptide covering part of this tryptophan repeat motif was recently shown to inhibit human immunodeficiency viruses type 1 reverse transcriptase subunit dimerization (Divita, G., Restle, T., Goody, R. S., Chermann, J.-C., and Baillon, J. G. (1994) J. Biol. Chem. 269, 13080-13083). In the present study, we show that the same peptide can also inhibit human immunodeficiency virus type 2 reverse transcriptase subunit dimerization, suggesting that the same inhibitors might be used as agents against both viruses as well as against variants of human immunodeficiency virus type 1 that differ from the variant against which they were developed. Under appropriate experimental conditions, e.g. at acidic pH, this peptide is also able to induce the dissociation of the enzyme from human immunodeficiency virus type 1.
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PMID:Interface peptides as structure-based human immunodeficiency virus reverse transcriptase inhibitors. 749 82

Macrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetyltransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-kappa B binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.
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PMID:Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells. 751 19

Based on presently available information on the structure of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, peptides have been synthesized which correspond to the sequence of a particular region of the protein involved in formation of the active heterodimeric form of the enzyme. Several peptides that are 15-19 amino acids long and that are derived from the so-called connection domain of the reverse transcriptase are able to inhibit dimerization of the enzyme and thus inhibit development of its enzymatic activities. In particular, a tryptophan-rich 19-mer corresponding to residues 389-407 was relatively efficient, showing an apparent dissociation constant in the micromolar range for one or both of the subunits. The sequence of this region is identical for both subunits, since one (molecular mass of 51 kDa) is the proteolytic product of the other (molecular mass of 66 kDa). Dissociation of the preformed heterodimer could not be induced by the peptides, but increasing concentrations reduced the rate of dimerization in a concentration-dependent manner until it became immeasurable at high concentrations. The results suggest that inhibition of dimerization of reverse transcriptase is an attractive approach to chemotherapeutic intervention in HIV infection and that further development of peptide-based inhibition strategies is worth pursuing.
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PMID:Inhibition of human immunodeficiency virus type 1 reverse transcriptase dimerization using synthetic peptides derived from the connection domain. 751 98

We set up a PCR laboratory for the diagnosis of HIV-1. Probably due to the variability of the HIV-genome, classical primers that performed well in some laboratories in the past, did not suffice for detection of HIV-1 strains in Belgian hospitals. Two new primer sets amplifying a fragment in the LTR-gag gene and in the env gene, which perform better on strains seen in Belgium, have been developed and evaluated. One primer set, conceived and evaluated on Belgian strains by the "Instituut voor Tropische Geneeskunde" in Antwerp, was also included. These three primer sets performed superior (92% sensitivity and 100% specificity on 24 samples) than the classical primers (83.5% sensitivity and 56% specificity on 21 samples). Together with a well-studied testing algorithm, they allow the reliable identification of the presence of the HIV-1 genome. To detect resistance of HIV-1 to reverse transcriptase (RT) inhibitors, we developed a set of two overlapping nested PCR primer sets and additional sequencing primers to amplify and sequence the total RNA or DNA RT gene using a direct cycle sequencing approach of the amplified fragment. Some clinical isolates were amplified and sequenced. In HIV-1 isolates from TIBO R82913-treated patients we identified two amino acid mutations (V108I and Y188L) involved in resistance (more than 100-fold reduced sensitivity). In an untreated patient we identified an amino acid variant (I/V 179D) involved in a 7-fold reduced sensitivity to TIBO. Several other amino acid variants, not involved in resistance, were detected in treated and untreated patients. Using this sequencing technique on cultured virus isolates we also observed in one TIBO-treated patient a differential selection among the strains of the original HIV-1 pool. From this patient we isolated and sequenced a completely TIBO sensitive HIV-1 strain after extensive cultivation in cord blood lymphocytes of the original TIBO resistant HIV-1 virus pool. We could however identify the resistant genotype after cultivation of this resistant HIV-1 virus pool on CEM cells. Our study revealed that sequencing investigations on emerging resistance should preferentially be done with uncultured patient samples since viral sequences and virus-drug sensitivities obtained from isolates cultured in vitro may not necessarily correspond to the sequences and sensitivities of the dominant strain in vivo.
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PMID:Polymerase chain reaction (PCR) as a diagnostic tool in HIV infection. 751 11


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