Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have studied the process of Moloney murine leukemia virus (M-MuLV) assembly by characterization of core (gag) protein mutants and analysis of wild-type (wt) gag proteins produced by cells in the presence of the ionophore monensin. Our genetic studies involved examination of linker insertion mutants of a Gag-beta-galactosidase (Gag-beta-gal) fusion protein, GBG2051, which is incorporated into virus particles when expressed in the presence of wt viral proteins. Analysis indicated that the amino-terminal two-thirds of the gag matrix domain is essential for targeting of proteins to the plasma membrane; mutant proteins localized to the cytoplasm or were trapped on intracellular membranes. Mutations through most of the coding region of the gag capsid domain generated proteins which were released from cells in membrane vesicles but not in virions. In contrast, linker insertions into p12gag or carboxy-terminal portions of the matrix or capsid coding regions did not affect assembly of fusion proteins into virus particles. Monensin, which blocks vesicular transport, inhibited gag protein intracellular transport and release from cells. Our results suggest that a significant proportion of M-MuLV myristylated gag proteins travel via vesicles to the cell surface. Specific matrix protein polypeptide regions and myristic acid modification are both necessary for appropriate gag protein transport, while capsid protein interactions appear to mediate the final phase of virion formation.
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PMID:Transport and assembly of gag proteins into Moloney murine leukemia virus. 169 96

A novel method for discovery of HIV-1 protease inhibitors in complex biological samples has been developed. The assay is based on two specific reagents: a recombinant protein constituted by a portion of the HIV-1 Gag polyprotein comprising the p17-p24 cleavage site, fused to E. coli beta-galactosidase, and a monoclonal antibody which binds the fusion protein in the Gag region. Binding occurs only if the fusion protein has not been cleaved by the HIV-1 protease. The assay has been adapted for the screening of large numbers of samples in standard 96-well microtiter plates. Using this method about 12000 microbial fermentation broths have been tested and several HIV-1 protease inhibitory activities have been detected. One of these has been studied in detail.
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PMID:A high throughput assay for inhibitors of HIV-1 protease. Screening of microbial metabolites. 200 37

The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.
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PMID:Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization. 210 12

It has been shown recently that BCG can be used as a live recombinant vaccine to stimulate immune responses. Proliferative or cytotoxic T-cell responses against several viral proteins such as HIV Gag, Env or Nef were obtained after parenteral immunization with BCG expressing these proteins. Antibody responses were also obtained after immunization of mice with recombinant BCG strain which expressed lac Z under the control of a promoter sequence isolated from Mycobacterium paratuberculosis. We have used this recombinant vaccine in guinea-pigs to investigate the influence of various routes of immunization on the immunogenicity of a foreign antigen expressed by recombinant BCG. Guinea-pigs were immunized by oral, respiratory or intradermal routes and proliferative responses, delayed-type hypersensitivity and antibody responses specific for beta-galactosidase were followed for 16 weeks. Results demonstrated that humoral and cellular immune responses specific for beta-galactosidase can be produced in all groups of guinea-pigs. However, the respiratory and especially the oral route of administration induced higher local and systemic immune responses than the intradermal route of immunization. Moreover, the oral immunization of mice with this recombinant BCG induced IgA responses which could be detected in both sera and intestinal secretions. Therefore, this study demonstrates for the first time that oral immunization with recombinant BCG can induce strong cellular and humoral immune responses.
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PMID:Oral immunization with recombinant BCG induces cellular and humoral immune responses against the foreign antigen. 829 80

Assembly of type C retroviruses such as Moloney murine leukemia virus (M-MuLV) ordinarily occurs at the plasma membranes of infected cells and absolutely requires the particle core precursor protein, Pr65gag. Previously we have shown that Pr65gag is membrane associated and that at least a portion of intracellular Pr65gag protein appears to be routed to the plasma membrane by a vesicular transport pathway. Here we show that intracellular particle formation can occur in M-MuLV-infected cells. M-MuLV immature particles were observed by electron microscopy budding into and within rough endoplasmic reticulum, Golgi, and vacuolar compartments. Biochemical fractionation studies indicated that intracellular Pr65gag was present in nonionic detergent-resistant complexes of greater than 150S. Additionally, viral RNA and polymerase functions appeared to be associated with intracellular particles, as were Gag-beta-galactosidase fusion proteins which have the capacity to be incorporated into virions. Immature intracellular particles in postnuclear lysates could be proteolytically processed in vitro to mature forms, while extracellular immature M-MuLV particles remained immature as long as 10 h during incubations. The occurrence of M-MuLV-derived intracellular particles demonstrates that Pr65gag can associate with intracellular membranes and indicates that if a plasma membrane Pr65gag receptor exists, it also can be found in other membrane compartments. These results support the hypothesis that intracellular particles may serve as a virus reservoir during in vivo infections.
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PMID:Assembly and composition of intracellular particles formed by Moloney murine leukemia virus. 835 Mar 94

The activity in vivo of HIV-1 proteinase (PR) was analysed in the baculovirus expression system, using eight different constructs of the prt gene under the control of polyhedrin (PH) promoters of various strengths. None of the active PRs was expressed in substantial quantities, and only PH-fused and/or non-functional PR mutants accumulated in high amounts in insect cells. However, enough PR activity was generated from a lengthened PR construct in insect cells to process Gag polyprotein substrate co-expressed in the same cells in trans. Fusion of the first 58 residues from the PH sequence to the PR N terminus did not significantly change its activity and specificity of cleavage of the Gag substrate. When analysed under mild denaturing conditions, PH-fused or unfused full-length PR point mutants, as well as PH-fused or unfused C-terminal deletion mutants, showed a propensity to multimerize, with a predominant occurrence of dimers. The incorporation of PR into Gag particles was studied using eight Gag-PR fusion constructs, all containing a non-functional PR mutant. The PR domain was fused to the C-terminal p6 domain of Gag (p6gag), or translated in frame with NCp7 (as in frameshifted Gag-Pol polyprotein) and followed by downstream sequences of increasing lengths from the Pol domain or the bacterial beta-galactosidase. The results suggested that the presence of the p6gag domain was detrimental to the encapsidation of polyprotein-embedded PR.
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PMID:Proteolytic activity in vivo and encapsidation of recombinant human immunodeficiency virus type 1 proteinase expressed in baculovirus-infected cells. 901 Feb 96

Interaction of retrovirus vectors and endogenous retroviruses present in packaging cell lines and target cells may result in unwanted events, such as the formation of recombinant viruses and the mobilization of therapeutic vectors. Using sensitive reverse transcriptase PCR assays, we investigated human and murine gene therapy packaging cell lines for incorporation of endogenous retrovirus transcripts into murine leukemia virus (MLV) vector particles and, conversely, whether vector genomes are incorporated into human endogenous retrovirus (HERV) particles. VL30 endogenous retrovirus sequences were efficiently packaged in particles produced by the murine AM12 packaging system. For every seven MLV-derived beta-galactosidase (beta-Gal) vector genomes present in the particles, one copy of VL30 was also packaged. Although human FLY packaging cells expressed several classes of HERV transcripts (HERV-K, HuRT, type C, and RTVL-H), none was detectable in the MLV vector particles released from the cells. Nonspecific packaging of the MLV Gag-Pol expression vector transcripts was detected in the FLY virions at a low level (1 in 17,000 sequences). These findings indicate that human packaging cells produce retrovirus particles far less contaminated by endogenous viral sequences than murine packaging cells. Human teratocarcinoma cells (GH cells), which produce HERV-K particles, were transduced with an MLV-derived beta-Gal vector. Although both HERV-K and RTVL-H sequences were found in association with the particles, beta-Gal transcripts were not detected, indicating that HERV Gag proteins do not efficiently package MLV-based vectors.
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PMID:Packaging of endogenous retroviral sequences in retroviral vectors produced by murine and human packaging cells. 952 84

Newly developed antiviral compounds consisting of an adamantane derivative chemically linked to a water-soluble polyanionic matrix were shown to inhibit HIV-1 infection in lymphoblastoid cells, HeLa CD4+ beta-galactosidase (MAGI) cells and macrophages. The effect of the compounds was recorded by measuring viral reverse transcriptase activity and p24 by ELISA in culture supernatant and by immunoblotting of cell lysates. In this paper we describe the data obtained with one of the most promising compounds, Amant. Amant was not toxic for the host cells at concentrations as high as 1 mg/ml. The inhibition of HIV-1 replication in MT-4 and MAGI cells was observed when Amant was added either before infection or with the virus (0 h of infection), and was expressed even when the compound added at 0 h was removed 1.5 h after infection. Its inhibitory concentration (IC50) against HIV-1 and HIV-2 replication was 2-6 and 93 microg/ml, respectively. The anti-HIV-1 effect of the compound was gradually decreased when it was added 1 and 2 h post infection, and no inhibition was observed when the compound was added 4 h after infection, suggesting that the compound as a membranotropic drug blocks an early step of replication. It completely prevented the transport of Gag proteins into the nuclei. Pretreatment of the virus with Amant did not reduce its infectious activity. The classical adamantane derivatives amantadine and rimantadine hydrochloride did not inhibit HIV replication.
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PMID:Inhibition of HIV-1 replication by newly developed adamantane-containing polyanionic agents. 1032 46

The incorporation of human immunodeficiency virus type 1 (HIV-1) Gag-beta-galactosidase (Gag-beta-gal; GBG) fusion proteins into HIV virus-like particles in the presence of HIV Gag proteins was studied. HIV Gag-beta-gal fusion constructs were cotransfected individually into COS7 cells with or without an HIV Gag protein expression plasmid. Release of HIV GBG fusion proteins from the cells were measured by assay of the medium versus intracellular beta-gal activities. Analysis indicates that fusion proteins (constructs HIVGBG, GBG 1919 and 1877) retaining the C-terminal portion of the CA and the adjacent NC domains were efficiently assembled into virus-like particles. Fusion proteins with deleted sequences covering the N-terminal portions of the gag sequences (GBG 831, 1147, 1419, 1447, 1511, 1552, 1600, 1630, 1684, 1715, and 1752) were impaired in entry into virus-like particles. The presence of CA major homology region (MHR) in the fusion proteins had no significant effects on inducing fusion protein incorporation when the C-terminal CA sequences in the fusion proteins were truncated (GBG 1841 and 1801). Subcellular fractionation studies indicated that most fusion proteins including the nonmyristylated one were enriched in the crude membrane fraction. Exceptions to this rule were fusion proteins with intact MHR but truncated C-terminal CA sequences, which possessed low levels of membrane association. However, assembly of fusion proteins into HIV Gag particles did not correlate with their subcellular fractionation or immunofluorescence localization patterns. Overall, the studies suggest that the very C-terminal CA and adjacent NC sequences are the primary determinants for incorporation of HIV Gag-beta-gal fusion proteins into virus particles.
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PMID:Sequence requirements for incorporation of human immunodeficiency virus gag-beta-galactosidase fusion proteins into virus-like particles. 1045 53

We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MPsi) made using PCR amplification after cotransformation with GagDeltaPR protein into yeast cells. Colonies with low beta-galactosidase activity were analyzed to locate mutations in MPsi sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.
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PMID:Yeast three-hybrid screening of rous sarcoma virus mutants with randomly mutagenized minimal packaging signals reveals regions important for gag interactions. 1098 63


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