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:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human T-cell leukemia (or lymphotropic) virus type II (HTLV-II) was isolated from eight HTLV-seropositive patients, six of whom were also infected with human immunodeficiency virus, by cocultivation of peripheral blood mononuclear cells (PBMCs) with BJAB, a continuous B-cell line. Restriction endonuclease mapping of the proviruses demonstrated consistent differences among isolates, and two distinct physical map patterns were observed. The results suggest the existence of two closely related molecular subtypes of HTLV-II, which are tentatively designated HTLV-IIa and HTLV-IIb. This finding was supported by preliminary nucleotide sequence analysis of the env gene region encoding the transmembrane glycoprotein gp21, which showed consistent differences between the two proposed virus subtypes. Exploitation of differences in restriction endonuclease sites allowed polymerase chain reaction amplification to detect and differentiate the two subtypes in fresh PBMCs of HTLV-seropositive intravenous drug abusers (IVDAs). The results of these studies confirm that HTLV-II infection is the prominent HTLV infection in seropositive IVDAs and also show that infection with both subtypes occurs. The finding of genetic heterogeneity in the HTLV-II group of viruses may have important implications for studies on its role in human disease and will be useful in characterizing the viruses present in newly discovered endemic foci in New World indigenous populations.
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PMID:Multiple isolates and characteristics of human T-cell leukemia virus type II. 134 96

Isolation of a Rochalimaea-like organism from a febrile patient infected with human immunodeficiency virus was confirmed. Analysis of 16S rRNA gene sequences, together with polymerase chain reaction and restriction endonuclease length polymorphism analysis of a portion of the citrate synthase gene, demonstrated that the agent is closely related to members of the genus Rochalimaea and that the isolate is genotypically identical to the presumptive etiologic agent of bacillary angiomatosis. However, the same genotypic analyses readily differentiated the new isolate from isolates of other recognized Rochalimaea species as well as other genera of bacteria previously suggested as putative etiologic agents of bacillary angiomatosis and related syndromes. We propose that the novel species be referred to as Rochalimaea henselae sp. now.
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PMID:Characterization of a novel Rochalimaea species, R. henselae sp. nov., isolated from blood of a febrile, human immunodeficiency virus-positive patient. 137 15

Five strains of human immunodeficiency virus type 1 (HIV-1) were isolated from five Japanese hemophilia patients. Two isolates, HIV-1[GUN-1] and HIV-1[GUN-2], were from brother patients with hemophilia B and the other three isolates, HIV-1[GUN-3], HIV-1[GUN-4], and HIV-1[GUN-5], were from hemophilia A patients. Another HIV-1 strain, HIV-1[GUN-6], was isolated from a Canadian male homosexual with AIDS. The restriction endonuclease cleavage maps of the proviral genomes of these six HIV-1 strains revealed that they were apparently different from each other. The phylogenetic trees constructed using restriction maps and nucleotide sequences were quite similar, indicating that phylogenetic analyses of Japanese HIV-1 isolates can be done using restriction maps of the proviruses. Phylogenetic analyses showed that they were more closely related to HIV-1s which had been reported to be isolated from homosexual patients in the United States than those isolated from African patients. In particular, GUN-1 and GUN-2 isolates were on the branch of a San Francisco isolate, ARV2, while GUN-5 and GUN-6 isolates were on the branch of HTLV-IIIB-related isolates.
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PMID:Six strains of human immunodeficiency virus type 1 isolated in Japan and their molecular phylogeny. 140 18

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.
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PMID:The cytopathic effect of HIV is associated with apoptosis. 168 28

The gag and pol genes of the human immunodeficiency virus type 1 (HIV-1) (ref. 1) are translated as two polyproteins, Pr55gag and Pr160gag-pol (refs 2-6), which are subsequently cleaved by the action of a virus-encoded protease into the four structural gag proteins of the virion core (p17, p24, p7 and p6) and the pol-encoded enzymes essential for retrovirus replication (protease, reverse transcriptase, ribonuclease H, and endonuclease). Mutational inactivation of the proteases of HIV-1 and other retroviruses results in immature, non-infectious virions, indicating that exogenous inhibition of the protease may represent an attractive approach to anti-AIDS therapy. Here we demonstrate that synthetic peptide analogues, which are potent inhibitors of purified HIV-1 protease, inhibit the processing of the viral polyproteins in cultures of HIV-1-infected T lymphocytes and attenuate viral infectivity.
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PMID:Inhibition of HIV-1 protease in infected T-lymphocytes by synthetic peptide analogues. 168 46

Mutations were introduced by recombinant DNA techniques into 9 genes of an infectious molecular clone of human immunodeficiency virus type 1. The 24 mutants generated were characterized biochemically and biologically by transfection and infection experiments. None of the mutants which have mutations in gag (p17, p24, and p15 regions), pol (protease, reverse transcriptase, and endonuclease domains), env (gp120 region), tat, or rev were infectious, whereas vif, vpr, vpu, some of env (gp41) and nef mutants could grow in human CD4+ cells to various degrees. Of the non-infectious mutants, only endonuclease (pol) and gp41 mutants exhibited normal phenotypes with respect to the production of functional reverse transcriptase, the expression of gag, pol, and env proteins, and the generation of progeny virions, when examined in transient assays. All infectious mutants killed the CD4+ cells with the exception of a mutant carrying a defect in the vif gene.
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PMID:Generation and characterization of the human immunodeficiency virus type 1 mutants. 170 90

Five cassettes of the pol gene of human immunodeficiency virus 1 were constructed and inserted under the control of the polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus by homologous recombination. The first cassette polF contains the full-length pol open reading frame; the second cassette pol100 starts with the first AUG codon of the pol gene and deletes 103 amino acids from the amino terminus of the pol gene product; the third cassette pol97 deletes the entire protease coding sequence; the fourth cassette pol66 deletes both the protease and endonuclease/integrase coding sequences; and the fifth cassette pol51 contains the reverse transcriptase coding sequences plus 39 3'-terminal nucleotides of the RNase H coding sequences. We have expressed these five forms of the pol gene in Spodoptera frugiperda SF9 cells and have analyzed for both reverse transcriptase and RNase H activities. The polF construct expressed several processed forms, 66 kDa, 51 kDa, and 34 kDa proteins, that were detected only by Western blot. In contrast, pol100, pol97, pol66, and pol51 products were expressed at high levels and were readily detectable in gels by staining. The levels of expression of these four products were estimated to be greater than 150 mg/liter of culture (5 x 10(8) cells). Activity gel analyses showed that the pol100, pol97, pol66, and pol51 products possess reverse transcriptase activity; however, only pol97 and pol66 have RNase H activity. Our results demonstrate that many forms, including partially cleaved forms of human immunodeficiency virus 1 pol gene products, possess reverse transcriptase activity but only certain forms have RNase H activity.
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PMID:Enzyme activities in four different forms of human immunodeficiency virus 1 pol gene products. 171 Dec 3

In order to characterize novel human immunodeficiency virus type 1 (HIV-1) continuous epitopes, we designed a simple method, based on recombinant DNA, providing a complete set of peptides derived from HIV-1. A library (4 x 10(4) clones) was first constructed in a new expression/secretion vector, using as inserts small fragments of HIV-1 DNA (50-150 bp) generated by random DNAse I cleavage. This peptide library, expressed in the yeast Saccharomyces cerevisiae, was screened with sera of HIV-1 infected individuals and human and murine anti-HIV-1 monoclonal antibodies. Plasmids from immunoreactive colonies were recovered and the sequences of the HIV-1 derived inserts were determined. By using human sera, we have detected classical HIV-1 epitopes and identified two novel major epitopes, which may be used to improve diagnostic tests, localized in the p24 core protein and in the endonuclease. In addition, four minor epitopes were also defined by screening the library with monoclonal antibodies: in the protease, in the p17 core protein, in gp120 and near the C-terminal of gp41. This method is general and can be used for any protein from which a cloned cDNA is available.
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PMID:A peptide library expressed in yeast reveals new major epitopes from human immunodeficiency virus type 1. 171 77

The human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT) is a protein of 66 kDa, p66, which contains two domains, an amino-terminal DNA polymerase and an RNase H at the carboxy terminus of the molecule. In order to characterize the mode of action of the RNase H, two previously described mutant enzymes were used, with substitutions in the highly conserved histidine 539, which was mutated to the neutral amino acid asparagine and to the negatively charged aspartate. The purified wild-type (wt) and mutant (mt) enzyme activities are analyzed here using RNA-DNA hybrids consisting of in vitro transcribed RNA that harbors the polypurine tract (PPT) from HIV-1 and DNA oligonucleotides complementary to the PPT or to other regions of the RNA. Analysis of the radioactively labeled RNA of these model hybrids after RNase H treatment indicates that both, wt and mt enzymes, are capable of cleaving the RNA in an endonucleolytic manner. The mt enzymes exhibit a severely reduced exonuclease activity. They are more sensitive towards salt and competition with excess of unlabeled hybrid, suggesting a reduced substrate binding affinity. DNA elongation by the RT is coupled with RNA hydrolysis by the 3'-5' exonuclease of the wt RNase H. The RNase Hmt of the mt enzymes, however, does not exhibit such processive 3'-5' exonuclease activity during DNA synthesis but gives rise to sporadic endonucleolytic cuts, whereas the RT is not affected. The endonuclease activities of the RNase H mt enzymes exhibit cleavage preferences in the absence or presence of DNA synthesis different from those of the wt enzyme. They cannot recognize specific sequences required to generate a PPT-primer and therefore cannot initiate plus-strand DNA synthesis in vitro at the 3' end of the PPT, which is essential for viral replication.
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PMID:Mutations of a conserved residue within HIV-1 ribonuclease H affect its exo- and endonuclease activities. 171 5

We have examined the ribonuclease H (RNase H) activity of human immunodeficiency virus reverse transcriptase (HIV-RT) using a natural sequence 83-nucleotide-long RNA template to which was hybridized a DNA oligomer. This substrate configuration allowed for the simultaneous electrophoretic resolution of 5'-, 3'-, and internally derived RNase H cleavage products. Assays performed in the presence of excess challenger RNA to sequester the RT permitted the analysis of products resulting from a single round of binding of RT to substrate. Substrate cleavage was highly sensitive to ionic strength, showing greatest activity at low KCl concentrations. The increase in cleavage correlated with an increase in the half-life of the enzyme on the RNA-DNA hybrid from approximately 31 s to 6.2 min at 80 and 5 mM KCl, respectively. Internally derived cleavage products generated in challenged reactions were primarily 2-9 nucleotides in length. These lengths indicate that the products were generated by an endo- rather than an exonuclease activity. The directionality and processivity of the endonuclease were also determined by examination of cleavage products from challenged reactions. Although the lengths of 5'-derived products markedly decreased with time, no change in the size distribution of 3'-derived products was observed, indicating that cleavage proceeded processively in the 3' to 5' direction. The 5'-derived products were shortened more in reactions performed under conditions allowing multiple versus single enzyme-binding events, suggesting that the endonuclease action of a single enzyme is not processive enough to generate the maximum possible amount of cleavage on each substrate. Therefore, HIV-RT displays a partially processive 3' to 5' endonuclease activity.
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PMID:Human immunodeficiency virus reverse transcriptase displays a partially processive 3' to 5' endonuclease activity. 172 2


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