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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This study demonstrates that cells expressing a dysfunctional analog of a herpes simplex virus (HSV) capsid protein inhibits HSV replication. Vero cell lines expressing HSV-1 capsid protein VP19c/beta-galactosidase fusion proteins were constructed and tested for their kinetics of expression, intracellular location, and ability to interfere with HSV replication. Two chimeric genes were constructed for these studies. The larger chimeric gene encodes the amino terminal 327 amino acids (aa) of VP19c fused to the carboxy terminal 1026 aa of beta-galactosidase, and the shorter chimeric gene encodes VP19c aa 1-30 and 302-327 fused to the carboxy-terminal 1026 aa of beta-galactosidase. Cell lines V32G-1 and V32G-2 containing the larger and the shorter chimeric genes, respectively, were isolated after cotransfection with plasmid pSV2-neo DNA, cell selection, and limiting-dilution cloning. The chimeric VP19c/beta-galactosidase genes resident in V32G-1 and V32G-2 cell lines were induced by early gene products of superinfecting wild-type HSV-1 and HSV-2, but were not constitutively expressed. The hybrid proteins expressed in infected V32G-1 and V32G-2 cells both colocalized with infected cell protein 8 (ICP8) into virus-replicative compartments in the cell nuclei. HSV-1 and HSV-2 growth in V32G-1 cells (which express the larger chimeric gene) was significantly reduced compared to growth in V32G-2 and control Vero cells. The data suggest that the larger VP19c/beta-galactosidase hybrid protein interferes with virus capsid assembly or morphogenesis in a competitive manner. Results also demonstrate that a small portion of VP19c containing the predicted endoplasmic reticulum signal sequence for this capsid protein (aa 1-30) promotes incorporation of the VP19c/beta-galactosidase fusion proteins into nuclear viral replication compartments.
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PMID:Transinhibition of herpes simplex virus replication by an inducible cell-resident gene encoding a dysfunctional VP19c capsid protein. 794 1

We have previously shown that local destruction of neural tissue by wild-type herpes simplex virus type 1 (HSV-1) is attenuated by intracerebral infusion of nerve growth factor (NGF). To investigate the effect of NGF on the extent of neurolysis and efficacy of neuronal gene transfer mediated by an HSV-1 amplicon vector system in vivo, rats were stereotaxically injected in the striatum with an amplicon preparation, pHSVlac. This amplicon contains the Escherichia coli lacZ gene under the transcriptional control of the HSV-1 immediate early 4/5 promoter and is packaged by an HSV-1 helper virus carrying a deletion in the immediate early 3 gene. Vector injection was followed by continuous intracerebral infusion of NGF-beta (total dose 5 micrograms) or vehicle solution over 7 days. Animals were sacrificed at the end of the 7-day infusion period for histological analysis of the brains. A distinct zone of inflammation and necrosis surrounded the injection site in all vector-inoculated animals. The volume of striatal tissue destruction was significantly smaller in NGF-treated animals (1.27 +/- 0.19 mm3; mean +/- SEM) than in the vehicle-treated controls (2.16 +/- 0.37 mm3; P < 0.05 by t-test). Immunohistochemical staining for HSV and beta-galactosidase (beta-Gal) in vehicle-treated animals revealed that many striatal cells harbored HSV antigens (3,678 +/- 636), but only a small number expressed the reporter gene at 7 days post-injection (294 +/- 60). NGF infusion did not significantly affect the number of HSV-immunoreactive cells (4,224 +/- 618), or the number of cells expressing beta-Gal (330 +/- 72) at this time.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Effect of exogenous nerve growth factor on neurotoxicity of and neuronal gene delivery by a herpes simplex amplicon vector in the rat brain. 794 48

Feline herpesvirus type 1 (FHV-1) mutants were constructed, carrying a beta-galactosidase marker gene integrated into the region downstream of the gene encoding the homologue of glycoprotein C (gC) of herpes simplex virus type 1. In cell culture, no differences in replication were observed between mutants and the parent FHV-1 strain. However, in experimentally infected cats, mutants caused fewer clinical signs after oronasal administration although they replicated to the same extent as the parental strain. Sequence analysis in the region of the UL segment surrounding the insertion site revealed an open reading frame (ORF 2) encoding a putative polypeptide of 21K. RNA analysis indicated a corresponding transcript of 0.8 kb that was detected late after infection of cells in culture. This particular UL locus downstream of the gC gene has not been thoroughly investigated in any of the herpesviruses. The putative gene product showed only limited evolutionary conservation since similarity could be found only with the assumed homologue of equine herpesvirus type 1. Further characterization of this newly identified FHV-1 gene involved in virulence may provide insight into the development of disease owing to herpesvirus infection.
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PMID:The gene downstream of the gC homologue in feline herpes virus type 1 is involved in the expression of virulence. 796 20

This paper describes the construction of a recombinant Marek's disease virus serotype 1 (MDV1) in which the Escherichia coli lacZ gene was inserted into the open reading frame homologous to the US10 gene of herpes simplex virus 1 (HSV1). The recombinant virus replicated as well in cell culture as the parental MDV1 K-554 strain. Chickens immunized with the virus were protected against challenge with virulent MDV1, and produced a high level of antibodies against beta-galactosidase as well as against MDV1 antigens. The antibody titres persisted for at least 16 weeks. These results demonstrate that the US10 gene of MDV1 is an effective site for the insertion of foreign genes from which to construct a polyvalent live vaccine for poultry.
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PMID:Construction of recombinant Marek's disease virus type 1 (MDV1) expressing the Escherichia coli lacZ gene as a possible live vaccine vector: the US10 gene of MDV1 as a stable insertion site. 797 37

Two steps of gene targeting were used to replace the p53 gene with the E. coli beta-galactosidase (lacZ) gene in mouse embryonic stem (ES) cells. The first targeting vector consisted of neo and herpes simplex virus thymidine kinase (HSV-tk) genes as a neo-tk cassette in the middle of the targeting vector. At the first targeting, the homologous recombinants became G418 resistant and ganciclovir (GANC) sensitive and were selected by G418 alone. At the second targeting, homologous recombination reciprocally exchanged the neo-tk casette in the ES cell chromosome with the lacZ fragment in the second targeting vector and thus made the ES cells GANC resistant. We obtained two ES cell clones, in which the p53 gene for both had been replaced with a totally non-homologous sequence of the lacZ gene. The germ-line transmission of the manipulated ES cells also demonstrated that the entire procedure had no detrimental effects on ES cells at all.
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PMID:Gene replacement of the p53 gene with the lacZ gene in mouse embryonic stem cells and mice by using two steps of homologous recombination. 804 55

The infectious laryngotracheitis virus (ILTV) gene encoding a homologue to the glycoprotein C gene of herpes simplex virus has been sequenced and identified based on its genomic location, comparative analysis to other gC proteins, and the identification of a glycosylated protein product. Located near the small subunit ribonucleotide reductase gene, the ILTV gC gene is 1242 bp in length and is predicted to encode a membrane glycoprotein containing a characteristic N-terminal hydrophobic signal sequence, five potential N-linked glycosylation sites, and C-terminal transmembrane and cytoplasmic domains. Antibodies raised in rabbits against a Cro-ILTV-beta-galactosidase fusion protein expressed in Escherichia coli recognize a 60-kDa ILTV-specific glycoprotein from infected cell extracts. Transcriptional analysis, using a portion of the open reading frame as a probe, identified a 1.55-kb transcript expressed with late gene kinetics. Comparison to other herpesvirus gC proteins revealed limited amino acid sequence homology and the absence of a charged extracellular region, which would normally interact with cell surface proteoglycans.
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PMID:Identification and characterization of the infectious laryngotracheitis virus glycoprotein C gene. 805 58

The herpes simplex virus type 1 (HSV-1) major capsid protein VP5 gene (UL19) is expressed with beta gamma (gamma 1 [leaky late]) kinetics. We have previously described the construction of recombinant HSV-1 in which the VP5 promoter was engineered to control the expression of the bacterial beta-galactosidase gene as a reporter (C.-J. Huang, S. A. Goodart, M. K. Rice, J. F. Guzowski, and E. K. Wagner, J. Virol. 67:5109-5116, 1993). Here we describe further mutational analysis in recombinant viruses. We have precisely defined the boundaries of the VP5 promoter and identified two regions important for both the level and the kinetics of expression. The 5' boundary was located at -48 relative to the initiation site of transcription by analyzing a series of nested deletions in the upstream sequence, and although a number of cis-acting sites influencing transient expression have been identified upstream of this point, these sites have no role in promoter activity during productive infection. Deletion of an Sp1-binding site located between -48 and the TATA box at -30 greatly reduced VP5 promoter activity late but not early after infection. A cis-acting element whose sequence resembles the human immunodeficiency virus type 1 initiator was located between -2 and +10 in the VP5 sequence by characterizing a series of deletions and site-directed block mutations downstream the TATA box. This element defines the 3' limit of the VP5 promoter, and like the upstream element, disruption of this element also inhibited promoter activity late in the productive cycle.
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PMID:The herpes simplex virus type 1 major capsid protein (VP5-UL19) promoter contains two cis-acting elements influencing late expression. 805 55

The genes IE175, IE63, IE68, IE12, UL29 and UL19 of herpes simplex virus types 1 and 2 were cloned. Fragments of these genes were cloned into open reading frame expression vector pEX1-3. Recombinant proteins containing amino acid sequences of ICP4, ICP27, ICP22, ICP47, major DNA-binding protein and major capsid protein were expressed in E. coli cells by fusion with cro-beta-galactosidase proteins.
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PMID:[The production of recombinant polypeptides from the herpes simplex viruses types 1 and 2]. 809 48

To achieve gene delivery to sensory neurons of the trigeminal ganglion, thymidine kinase-negative (TK-) herpes simplex viruses (HSV) containing the reporter gene lacZ (the gene for E. coli beta-galactosidase) downstream of viral (in vectors RH116 and tkLTRZ1) or mammalian (in vector NSE-lacZ-tk) promoters were inoculated onto mouse cornea and snout. Trigeminal ganglia were removed 4, 14, 30, and 60 days after inoculation with vectors and histochemically processed with 5-bromo-4-chloro-3 indolyl-beta-galactoside (X-Gal). With vector tkLTRZ1, large numbers of labeled neurons were observed in rostromedial and central trigeminal ganglion at 4 days after inoculation. A gradual decline in the number of labeled neurons was observed with this vector at subsequent time points. With vectors RH116 and NSE-lacZ-tk, smaller numbers of labeled neurons were seen at 4 days following inoculation than were observed with vector tkLTRZ1. No labeled neurons could be observed at 14 days after inoculation with vectors RH116 and NSE-lacZ-tk. Immunocytochemistry for E. coli beta-galactosidase and in situ hybridization to HSV latency-associated transcripts revealed labeled neurons in regions of the trigeminal ganglion similar to that observed with X-Gal staining. A comparable distribution of labeled neurons in trigeminal ganglion was also observed after application of the retrograde tracer Fluoro-Gold to mouse cornea and snout. These data provide evidence that retrogradely transported tk- herpes virus vectors can be used to deliver a functional gene to sensory neurons in vivo in an anatomically predictable fashion.
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PMID:Comparative efficacy of expression of genes delivered to mouse sensory neurons with herpes virus vectors. 810 60

Competitive quantitative PCRs were used to examine the consequences of stereotactically injecting a highly attenuated herpes simplex virus type 1 mutant into rat brains. This mutant virus, designated RR1CAT/RR2lacZ, was engineered so that coding sequences of the genes UL39 and UL40 specifying the subunits of the viral ribonucleotide reductase were replaced by the chloramphenicol acetyltransferase (CAT) and the lacZ gene coding sequences, respectively. Stereotactic injection of this virus into the hippocampal region of the rat brain resulted in a localized infection. Viral gene products were visualized by immunochemical, cytochemical, or in situ hybridization techniques in the injected hippocampal region at 2 days postinjection. Viral genomes, represented by glycoprotein B (gB), latency-associated transcript (LAT), and lacZ sequences could be amplified by PCR from templates obtained by scraping hippocampal tissue off single 10-microns frozen sections. Both gB message and LAT could be detected by reverse transcriptase (RT)-PCR. At day 7 postinjection, neither CAT message, gB message, nor beta-galactosidase activity could be visualized by the same techniques, although viral DNA was detected by PCR and LAT could be detected by RT-PCR. A similar pattern was seen at 8 weeks, suggesting that latency was established by the mutant virus in cells of the injected hippocampus. By competitive quantitative PCR, hippocampal sections were determined to contain 2.6 x 10(5) genome equivalents (represented by the gB gene) on day 2, 6.2 x 10(4) on day 7, and 8.3 x 10(4) at 8 weeks. By competitive quantitative RT-PCR, the numbers of LAT molecules at the same time points were 3.2 x 10(6), 1.3 x 10(6), and 1.2 x 10(6), respectively. The numbers of LAT molecules per genome equivalent were 12.5, 20.3, and 14.5, respectively, being approximately the same for each of the three time points. The data permit the conclusion that the RR mutant virus establishes latency in the rat brain with the persistence of the viral genome and the production of LAT molecules. Once latency is established, the numbers of viral genomes and LAT RNA molecules remain constant. Thus the competitive quantitative PCR and RT-PCR techniques provide very sensitive and reliable methods to quantitate viral DNA and RNA present in infected tissue.
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PMID:Competitive quantitative PCR analysis of herpes simplex virus type 1 DNA and latency-associated transcript RNA in latently infected cells of the rat brain. 810 47


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