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)

Active recombinant reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) with an amino-terminal extension containing a hexa-histidine sequence has been prepared in milligram quantities in a pure heterodimeric (p66/p51) form by coordinated applications of immobilized metal affinity chromatography (IMAC) and HIV-1 protease treatment. The precursor protein, isolated from extracts of recombinant Escherichia coli by IMAC in a predominantly unprocessed form (p66), migrated on sodium dodecyl sulfate-polyacrylamide gels as a 66-kDa band with minor heterogeneity at lower relative molecular mass. Incubation of this protein with recombinant HIV-1 protease produced a stable heterodimeric RT that was purified in a single step by IMAC. The purified protein retained both RT and RNase H activity, and kinetic parameters (Km and Vmax) were measured with both RNA-dependent DNA polymerization and RNase H activity assays. Carboxyl-terminal sequencing of purified heterodimeric RT indicated that one subunit is intact p66, whereas the other, p51, is a truncated form of p66 that terminates at residue Phe440. Analysis of the HIV-1 protease digest revealed two cleavage sites, at Tyr483-Leu484 and Tyr532-Leu533, in addition to the site at Phe440-Tyr441 that is cleaved to produce p51.
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PMID:Purification and characterization of heterodimeric human immunodeficiency virus type 1 (HIV-1) reverse transcriptase produced by in vitro processing of p66 with recombinant HIV-1 protease. 137 37

Camptothecin (CPT), a topoisomerase I-specific inhibitor, was found in this study to inhibit the replication of equine infectious anemia virus (EIAV) in chronically infected CF2Th cells (designated CF2Th/EIAV). By measuring viral reverse transcriptase activity in the culture medium, we demonstrated that treatment for 1 h with noncytotoxic doses of this drug inhibited production by 32 to 52%, whereas continuous exposure to this drug resulted in an 85 to 92% inhibition. No effect on the viability or growth rate of the cells was detected in any of these treatments. Indirect immunofluorescence analysis of the CPT-treated CF2Th/EIAV cells with anti-p26 capsid protein antibodies showed 60 to 85% reduction in the immunofluorescence-positive cells following drug treatment, and radioimmunoprecipitation analysis of these cells showed a comparable decrease of the pr55gag precursor protein. These data suggest that CPT acts as an anti-EIAV agent to block virus replication in the chronically infected cells.
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PMID:The topoisomerase I inhibitor, camptothecin, inhibits equine infectious anemia virus replication in chronically infected CF2Th cells. 164 21

We constructed a recombinant vaccinia virus carrying the entire gag and pol genes of human immunodeficiency virus type 1 (HIV-1). The main gene product detected in the lysates of infected CV-1 and SW480 cells was the gag precursor protein. However, in the culture fluid of infected SW480 cells, but not of infected CV-1 cells, reverse transcriptase (RT) activity was detected. The highest RT activity was found at a density of 1.15 g/ml and this fraction contained many round particles with diameters of 100-150 nm. In contrast to the infected cell lysates, the particles contained the processed gag and pol proteins, suggesting that particle formation may be a prerequisite for efficient processing of the gag precursor by the HIV protease encoded in the pol gene. Particles were also recovered from the culture fluid of SW480 cells infected with another recombinant vaccinia virus carrying only the gag gene. These particles contained the unprocessed gag precursor, indicating that the gag precursor alone was sufficient for particle production.
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PMID:Production of human immunodeficiency virus (HIV)-like particles from cells infected with recombinant vaccinia viruses carrying the gag gene of HIV. 168 17

Newly developed substrate analogue peptidomimetics are able to inhibit the human immunodeficiency virus, HIV-1 proteinase at nanomolar concentration. In HIV infected cell culture they exhibit antiviral activity. We have analyzed the non-infectious HIV particles produced in chronically HIV infected cell culture in presence of one of these inhibitors. The total production of virus particles was not substantially reduced in drug treated cultures, compared to non-inhibited control cultures, but the infectivity of these virus particles was reduced about 100 fold. The processing of gag and gag-pol protein precursor was inhibited; only borderline activity of reverse transcriptase (RT) could be detected in these particles and they contained nonprocessed gag precursor protein. Thin section electron microscopy of inhibitor-treated, HIV-infected cells revealed reduced viral cytopathogenicity and both inhibition of particle assembly and incomplete maturation of the particles formed. The HIV particles produced in the presence of the proteinase inhibitor were studded with envelope glycoprotein knobs and often comprised multiple budding regions, but were morphologically immature.
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PMID:Analysis of non-infectious HIV particles produced in presence of HIV proteinase inhibitor. 192 79

A total of 81 cell clones persistently infected with the LAV-1 or HTLV-IIIB strain of human immunodeficiency virus type 1 (HIV-1) was isolated from cells which were obtained by serial passage of some proliferating MT-4 cells after a drastic cytolysis of most cells by HIV-1-infection. These cell clones were classified into 8 types (I to VIII) in terms of the expression of HIV-1 antigens, syncytium formation capacity, and reverse transcriptase activity and infectivity of virus particles in the culture fluid. Type I cell clones were producers of infectious HIV-1 particles, while types II to VIII cell clones did not produce infectious HIV-1 or were producers of uninfectious defective HIV-1 particles. Immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis (PAGE) showed that the gag precursor protein in L-2 cell clone (type IV) was not cleaved to mature gag proteins, while the env precursor protein on L-3 cell clone (type III) was not cleaved to mature env protein. H-7 cell clone (type VIII) did not express any HIV-1 antigen. All these cell clones after the superinfection with infectious HIV-1 synthesized intact gag and env proteins, which were, at least in part, related to the HIV-1 genome persistently present in the cell clones before the superinfection, resulting in production of infectious HIV-1. For example, it was found that L-2 cell clone contained a single copy of the LAV-1 genome per haploid cell and produced doughnut-shaped particles. On the other hand, the cell clone isolated from the L-2 cell clone superinfected with infectious HTLV-IIIB contained the integrated HTLV-IIIB genome in addition to the LAV-1 genome present before the superinfection, and produced intact HIV-1 particles in addition to doughnut-shaped particles from a single cell. These results indicate that complementation and/or genetic recombination events in the superinfected cells may account for the production of infectious intact HIV-1 virions.
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PMID:Production of infectious particles from defective human immunodeficiency virus type 1 (HIV-1)-producing cell clones by superinfection with infectious HIV-1. 200 Nov 75

Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits. The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene. The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase. The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms. The proteins formed by several Moloney MuLV mutants were analyzed. Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.
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PMID:Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli. 242 63

Recent studies suggest that hepatitis B virus (HBV), despite being a DNA virus, replicates via an RNA intermediate (R. H. Miller, P. L. Marion, and S. W. Robinson, Virology 139:64-72, 1984; J. Summers and W. S. Mason, Cell 29:403-415, 1982). The HBV life cycle is therefore a permuted version of the RNA retroviral life cycle. Sequence homology between retroviral reverse transcriptase and the putative HBV polymerase gene product suggests the presence of an HBV reverse transcriptase (H. Toh, H. Hajashida, and T. Miyata, Nature (London) 305:827-829, 1983). As yet, there has been no direct evidence that reverse transcriptase activity is present in the viral particle. We used activity gel analysis to detect the in situ catalytic activities of DNA polymerases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our studies demonstrated that HBV-like particles secreted by a differentiated human hepatoma cell line transfected with genomic HBV DNA contain two major polymerase activities which migrate as approximately 90- and approximately 70-kilodalton (kDa) proteins. This demonstrated, for the first time, that HBV-like particles contain a novel DNA polymerase-reverse transcriptase activity. Furthermore, we propose that the 70-kDa reverse transcriptase may be produced by proteolytic self-cleavage of the 90-kDa precursor protein.
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PMID:Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles. 244 93

Recombinant HIV-1 reverse transcriptase (RT) was stably overproduced as a soluble protein in Escherichia coli using a double-plasmid expression system in which an RT precursor protein was expressed and processed in vivo by HIV-1 protease produced in trans. The RT thus produced consisted of an equimolar mixture of two polypeptides, p66 and p51, which were copurified to greater than 90% homogeneity and were found to share a common NH2 terminus as judged by sequence analysis of the polypeptide mixture. The observed sequence confirmed correct in vivo cleavage by protease at the protease-RT polyprotein junction to yield an NH2 terminus identical to that of genuine viral RT (M. M. Lightfoote et al. (1986) J. Virol. 60, 771-775; F. diMarzo Veronese et al. (1986) Science 231, 1289-1291). The bacterially expressed RT had a specific activity similar to that of viral RT and inhibition studies with phosphonoformate confirmed that it was indistinguishable from the viral enzyme with respect to sensitivity to this inhibitor. Polymerase activated gel analysis of the mixture indicated that p66 was associated with a higher level of RT activity than p51. RNase H activated gel analysis suggested that the purified preparation of recombinant RT was free of endogenous E. coli RNase H, and that the RNase H activity of RT was exclusively associated with the p66 polypeptide, supporting the hypothesis that the RNase H domain is located in the COOH-terminal region of the molecule.
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PMID:Recombinant HIV-1 reverse transcriptase: purification, primary structure, and polymerase/ribonuclease H activities. 247 69

We have identified the major structural core proteins of Ty virus-like particles (Ty-VLPs) and shown that they are generated by proteolytic cleavage of the primary translation product of TYA, p1. This precursor protein is therefore functionally similar to the gag precursor of retroviruses. Cleavage is mediated by a Ty-encoded protease located at the 5' region of TYB and is accompanied by a change in particle morphology. p1 contains sufficient information for the assembly of a pre-Ty-VLP complex, which does not require the presence of either Ty protease or reverse transcriptase. The results indicate that the requirements and pathway of Ty-VLP formation reflect the initial stages of mammalian retroviral assembly and further support the idea of a common origin for Ty elements and retroviruses.
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PMID:The functions and relationships of Ty-VLP proteins in yeast reflect those of mammalian retroviral proteins. 303 May 64

We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli.
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PMID:Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus. 329 93


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