Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0023418 (leukemia)
 93,477 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human T-cell leukemia virus type-2 (HTLV-2) integrase (IN) catalyzes the insertion of the viral genome into the host chromosome. HTLV-2 IN was expressed as an N-terminal hexa-histidine tagged protein in the methylotrophic yeast Pichia pastoris and as a C-terminal hexa-histidine fusion in Escherichia coli. Maximal IN expression was observed at 48h post-induction for the yeast system and 2h post-induction for E. coli. Effective purification strategies were developed using non-ionic and zwitterionic detergents for initial protein extraction, followed by a one-step nickel-chelating chromatography purification. IN from both sources was routinely greater than 90% pure with yields exceeding 1.5mg of purified IN per liter of culture for P. pastoris. The relative pI was defined for both INs, pH 5.0-5.4, by 2D-gel electrophoresis. Specific activities for IN purified from E. coli and P. pastoris were calculated from in vitro 3(') processing assays and were comparable. In vitro IN assays were also performed to optimize reaction buffer pH and metal concentrations for both 3(') processing and strand transfer assays. Strand transfer was optimal from pH 6.2-6.8, more than 1.5 pH units below the optimal 3(') processing pH of 8.3. IN from both sources showed no enhancement in activity with MnCl(2) concentrations greater than 5mM. The specific activity of P. pastoris purified IN was 0.35 product (pmol)/h/microg IN, and E. coli produced IN was 0.48 product (pmol)/h/microg IN.
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PMID:A comparative study of the human T-cell leukemia virus type 2 integrase expressed in and purified from Escherichia coli and Pichia pastoris. 1213 62

Retroviral integrase (IN) is the viral enzyme responsible for the integration of viral DNA into host cellular DNA. In vitro, recombinant IN protein is able to catalyze the 3'-processing, strand transfer and disintegration activities. In order to analyze the importance of specific residues of ALSV (Avian leukemia and sarcoma viruses) IN protein, we introduced 31 amino acid substitutions either in residues previously shown by others to be involved in IN oligomerization or in selected conserved and non-conserved residues through the IN sequence. We tested, in vitro, the three catalytic activities of these mutants as well as their capacity to bind DNA. We found that (i) 88% of the substitutions occurring on well-conserved residues have an effect on IN activities (ii) two mutants (S85T in the central catalytic domain and N197C in the C-terminal domain) present a reduced efficiency of DNA binding compared to the wild type protein. Moreover, all mutations made on the dimer interface of C-terminal domain present reduced activities, suggesting an important role of this part of the protein. Finally, for some mutations, we observed differences between the ALSV and HIV (Human immunodeficiency virus) IN corresponding residues.
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PMID:Analysis of conserved and non-conserved amino acids critical for ALSV (Avian leukemia and sarcoma viruses) integrase functions in vitro. 1220 15

Mutations affecting either the N- or C-terminal regions of the Gag protein p12 block replication of Moloney murine leukemia virus (M-MuLV). Viruses carrying mutations in this portion of gag can mediate the assembly and release of virions but are unable to successfully carry out the early phase of the M-MuLV life cycle. Wild-type and mutant viruses were found to synthesize similar levels of linear viral DNA in both cytoplasmic and nuclear fractions, and there were no significant differences in either the density or sedimentation of the viral protein-nucleic acid complexes. Analysis of the termini of the linear viral DNAs showed that the 3' ends of the mutant viral DNA were processed normally by the integrase. Further, the preintegration complexes extracted from the cytoplasm of cells infected with the mutant viruses were competent for integration into target DNA in vitro. Nevertheless, no circular viral DNAs were detected in cells infected by the mutants, and functional proviruses were not formed. These results suggest that p12 has an unexpected role in the early phase of the life cycle and is needed after viral DNA synthesis to deliver the incoming DNA to the correct location and in the appropriate state to permit either circularization or integration of the viral DNA in vivo.
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PMID:Characterization of Moloney murine leukemia virus p12 mutants blocked during early events of infection. 1236 23

Human immunodeficiency virus type 1 (HIV-1) based gene transfer systems are gaining in popularity due to their ability to transduce terminally differentiated and non-dividing cells. Oncoretroviral vectors based on Moloney murine leukemia virus (MoMLV), on the other hand, can only transduce dividing cells. The reasons for increased ability of lentivirus vectors to transduce such cells has been attributed to several of the viral proteins (integrase, matrix and Vpr) that are purported to be involved in the nuclear import of the pre-integration complex (PIC). Nuclear import is also augmented by a unique triple stranded DNA region created during reverse transcription of the incoming viral RNA in the target cell (discussed in chapter 3). This chapter deals with the rationale behind the design of human immunodeficiency virus type 1 (HIV-1) based packaging systems with an emphasis on some recent advances in the field for the creation of safe and efficient HIV-1 based vectors. The review covers trans-acting proteins and cis-sequences required for the deployment of HIV-1 vectors for gene transfer. This is a rapidly advancing field that with further refinements may soon allow the utilization of HIV-1 based and/or other lentivirus vectors in a clinical setting.
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PMID:HIV-1 vector systems. 1246 62

Human T-cell leukemia virus type 1 (HTLV-1) is a type C human retrovirus and is the causative agent of adult T-cell leukemia and other diseases. The enzymatic and structural proteins of HTLV-I are synthesized as part of a Gag-Pro-Pol precursor polyprotein, and the mature proteins are released by proteolytic processing catalyzed by HTLV-I protease. The locations of most of the proteolytic cleavage sites are known, however, the site that creates the N-terminus of HTLV-1 integrase has not been previously identified. A 15 residue peptide corresponding to junction of the C-terminus of RNaseH and N-terminus of integrase (DALLITPVLQLSPAF-OH) was incubated with HTLV-1 protease. Analysis of the cleavage products by LC-MS revealed fragments Ac-DALLITPVLQL-OH and H(2)N-SPAF-OH were produced, indicating cleavage between the leucine and serine. This is the first physical identification of the N-terminal amino acid sequence of the integrase of HTLV-1.
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PMID:Identification of the RT-RH/IN cleavage site of HTLV-I. 1250 78

The retroviral integrase (IN) carries out the integration of the viral DNA into the host genome. Both IN and the DNA sequences at the viral long-terminal repeat (LTR) are required for the integration function. In this report, a series of minor groove binding hairpin polyamides targeting sequences within terminal inverted repeats of the Moloney murine leukemia virus (M-MuLV) LTR were synthesized, and their effects on integration were analyzed. Using cell-free in vitro integration assays, polyamides targeting the conserved CA dinucleotide with cognate sites closest to the terminal base pairs were effective at blocking 3' processing but not strand transfer. Polyamides which efficiently inhibited 3' processing and strand transfer targeted the LTR sequences through position 9. Polyamides that inhibited integration were effective at nanomolar concentrations and showed subnanomolar affinity for their cognate LTR sites. These studies highlight the role of minor groove interactions within the LTR termini for retroviral integration.
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PMID:Inhibition of Moloney murine leukemia virus integration using polyamides targeting the long-terminal repeat sequences. 1275 29

Classically, the 5' and 3' long terminal repeats (LTRs) are considered necessary but not sufficient for retroviral integration. Recently, we reported that inclusion of these and additional elements from Moloney murine leukemia virus (MoMLV) facilitated transgene integration, without retroviral integrase, when placed in an adenoviral context (AdLTR-luc vector) (Nat. Biotech. 18 (2000), 176; Biochem. Biophys. Res. Commun. 300 (2003), 115). To help understand this nonhomologous DNA recombination event, we constructed another vector, AdELP-luc, with 2.7 kb of MoMLV elements identically placed into an E1-deleted adenovirus type 5 backbone upstream of a luciferase cDNA reporter gene. Unlike AdLTR-luc, no MoMLV elements were placed downstream of the expression cassette. AdELP-luc readily infected epithelial cells in vitro. Southern hybridizations with DNA from cloned cells showed that disruption of the MoMLV sequences occurred. One cell clone, grown in vitro without any special selection medium for 9 months, exhibited stable vector integration and luciferase activity. Importantly, both Southern hybridization and FISH analyses showed that in addition to the MoMLV elements and expression cassette, substantial adenoviral sequence downstream of the luciferase cDNA was genomically integrated. These results suggest that the 2.7 kb of MoMLV sequence included in AdELP-luc have cis-acting functions and mediates an unusual integration event.
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PMID:Inclusion of Moloney murine leukemia virus elements upstream of the transgene cassette in an E1-deleted adenovirus leads to an unusual genomic integration in epithelial cells. 1295 13

Replication of simple retroviruses depends on the recruitment of a single large primary transcript toward splicing, transport/packaging and translation regulations. In this respect, we studied the novel SD' 4.4 kb RNA of murine leukemia retroviruses (MLV) which results from alternative splicing of the primary transcript. We showed that SD' RNA was required for optimal replication since expression of a pre-spliced SD' RNA trans-complemented the impaired infectivity of a SD'-defective mutant. We monitored the fate of this novel transcript throughout early and late events of the viral life cycle. SD' RNA was specifically incorporated into virions demonstrating that the unspliced RNA was not the unique viral RNA present in virions. Furthermore, SD' RNA was reverse transcribed and its DNA copy integrated into the host genome, thus constituting a new splice donor-associated retroelement (SDARE) in infected cells. Finally, we showed that SD' mRNA encoded a 50 kDa polyprotein, and to a lower extent an additional 60 kDa polyprotein, which harbored Gag and integrase domains.
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PMID:A new retroelement constituted by a natural alternatively spliced RNA of murine replication-competent retroviruses. 1297 Jan 98

During replicative cycle of retroviruses, the reverse-transcribed viral DNA is integrated into the cell DNA by the viral integrase (IN) enzyme. The central core domain of IN contains the catalytic site of the enzyme and is involved in binding viral ends and cell DNA as well as dimerization. We previously performed single amino acid substitutions in the core domain of an Avian Leukemia and Sarcoma Virus (ALSV) IN [Arch. Virol. 147 (2002) 1761]. Here, we modeled the resulting IN mutants and analyzed the ability of these mutants to mediate concerted DNA integration in an in vitro assay, and to form dimers by protein-protein cross-linking and size exclusion chromatography. The N197C mutation resulted in the inability of the mutant to perform concerted integration that was concomitant with a loss of IN dimerization. Surprisingly, mutations Q102G and A106V at the dimer interface resulted in mutants with higher efficiencies than the wild-type IN in performing two-ended concerted integration of viral DNA ends. The G139D and A195V mutants had a trend to perform one-ended DNA integration of viral ends instead of two-ended integration. More drastically, the I88L and L135G mutants preferentially mediated nonconcerted DNA integration although the proteins form dimers. Therefore, these mutations may alter the formation of IN complexes of higher molecular size than a dimer that would be required for concerted integration. This study points to the important role of core domain residues in the concerted integration of viral DNA ends as well as in the oligomerization of the enzyme.
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PMID:Mutational analyses of the core domain of Avian Leukemia and Sarcoma Viruses integrase: critical residues for concerted integration and multimerization. 1497 25

Human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and Moloney murine leukemia virus (MoMLV) integrases were stably expressed to determine their intracellular trafficking. Each lentiviral integrase localized to cell nuclei in close association with chromatin while the murine oncoretroviral integrase was cytoplasmic. Fusions of pyruvate kinase to the lentiviral integrases did not reveal transferable nuclear localization signals. The intracellular trafficking of each was determined instead by the transcriptional coactivator LEDGF/p75, which was required for nuclear localization. Stable small interfering RNA expression eliminated detectable LEDGF/p75 expression and caused dramatic, stable redistribution of each lentiviral integrase from nucleus to cytoplasm while the distribution of MoMLV integrase was unaffected. In addition, endogenous LEDGF/p75 coimmunoprecipitated specifically with each lentiviral integrase. In vitro integration assays with preintegration complexes (PICs) showed that endogenous LEDGF/p75 is a component of functional HIV-1 and FIV PICs. However, HIV-1 and FIV infection and replication in LEDGF/p75-deficient cells was equivalent to that in control cells, whether cells were dividing or growth arrested. Two-long terminal repeat circle accumulation in nondividing cell nuclei was also equivalent to that of LEDGF/p75 wild-type cells. Virions produced in LEDGF/p75-deficient cells had normal infectivity. We conclude that LEDGF/p75 fully accounts for cellular trafficking of diverse lentiviral, but not oncoretroviral, integrases and is the main lentiviral integrase-to-chromatin tethering factor. While lentiviral PIC nuclear import is unaffected by LEDGF/p75 knockdown, this protein is a component of functional lentiviral PICs. A role in HIV-1 integration site distribution merits investigation.
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PMID:LEDGF/p75 determines cellular trafficking of diverse lentiviral but not murine oncoretroviral integrase proteins and is a component of functional lentiviral preintegration complexes. 1530 44


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