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: UMLS:C0019158 (hepatitis)
30,205 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In studies of both gene therapy and gene function, transgene expression can be modulated at both the transcriptional and post-transcriptional levels. In a previous study, we optimized the transcriptional regulatory elements for adenovirus (Ad) vectors to mediate efficient transgene expression, including promoter, enhancer, intron, and poly(A) sequence. In the present study, we systematically investigated the ability of the Woodchuck hepatitis virus post-transcriptional regulation element (WPRE) to enhance the expression of the luciferase gene, as a model gene, in the context of Ad vectors. We found that the WPRE in the sense orientation cloned between the luciferase gene and the poly(A) sequence stimulated 2- to 7-fold more luciferase expression in vitro and 2- to 50-fold more in the liver, kidney and lung of mouse than occurred without the use of the WPRE. The most efficient Ad vector in this study, which contained the improved CMV promoter (the conventional CMV promoter with the intron A) and the WPRE, showed more than 700-fold luciferase expression in mouse liver than did the Ad vector containing the conventional CMV promoter but no WPRE. These results indicate that inclusion of the WPRE, combined with the optimization of transcriptional regulatory elements in Ad vectors, will permit a given therapeutic goal to be achieved with substantially fewer viral particles. This information would be helpful for the construction of adenovirus vectors for studies regarding both gene therapy and gene function.
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PMID:Woodchuck hepatitis virus post-transcriptional regulation element enhances transgene expression from adenovirus vectors. 1278 24

Correction of diseases may be achieved by delivery of genes to stem cells and developing organ systems. Our previous studies demonstrated life-long expression after in utero injection of adeno-associated virus (AAV) serotype 2 in mice. In the present studies, we compared levels of expression using the elongation factor 1alpha (EF1alpha) or the CMV promoter in AAV2 and AAV5 linked to luciferase via intraperitoneal injection in day 15 fetuses in utero. An additional AAV construct also contained the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). The level and distribution of luciferase expression were assessed by in vivo bioluminescence and luminometric assays. All mice exhibited luciferase expression for >15 months. In vivo, luciferase expression from AAV5 was greater than that produced from AAV2. Vectors containing the CMV promoter produced higher levels of gene expression in all tissues examined compared to EF1alpha-directed vectors. The WPRE increased expression in vitro fourfold and in vivo eightfold. These studies demonstrate that by modifying the promoter and serotype, increases in the efficiency of AAV-directed expression may be achieved. The efficacy of rAAV-mediated gene delivery in utero supports the potential of these vectors for future therapies.
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PMID:Comparison of gene expression after intraperitoneal delivery of AAV2 or AAV5 in utero. 1284 32

Murine coronavirus mouse hepatitis virus (MHV) causes encephalitis and demyelination in the central nervous system of susceptible rodents. Astrocytes are the major target for MHV persistence. However, the mechanisms by which astrocytes survive MHV infection and permit viral persistence are not known. Here we performed DNA microarray analysis on differential gene expression in astrocyte DBT cells by MHV infection and found that the mRNA of the proapoptotic gene BNip3 was significantly decreased following MHV infection. This finding was further confirmed by quantitative reverse transcription-polymerase chain reaction, Western blot analysis, and BNip3-promoter-luciferase reporter system. Interestingly, infection with live and ultraviolet light-inactivated viruses equally repressed BNip3 expression, indicating that the down-regulation of BNip3 expression does not require virus replication and is mediated during cell entry. Furthermore, treatment of cells with chloroquine, which blocks the acidification of endosomes, significantly inhibited the repression of the BNip3 promoter activity induced by the acidic pH-dependent MHV mutant OBLV60, which enters cells via endocytosis, indicating that the down-regulation of BNip3 expression is mediated by fusion between viral envelope and cell membranes during entry. Deletion analysis showed that the sequence between nucleotides 262 and 550 of the 588-base-pair BNip3 promoter is necessary and sufficient for driving the BNip3 expression and that it contains signals that are responsible for MHV-induced down-regulation of BNip3 expression in DBT cells. These results may provide insights into the mechanisms by which MHV evades host antiviral defense and promotes cell survival, thereby allowing its persistence in the host astrocytes.
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PMID:Down-regulation of transcription of the proapoptotic gene BNip3 in cultured astrocytes by murine coronavirus infection. 1459 95

Hepatitis C virus (HCV) infection causes hepatitis, hepatocellular carcinoma, and B-cell lymphomas in a significant number of patients. Previously we have shown that HCV infection causes double-stranded DNA breaks and enhances the mutation frequency of cellular genes, including proto-oncogenes and immunoglobulin genes. To determine the mechanisms, we studied in vitro HCV infection of cell culture. Here we report that HCV infection activated the immunologic (type II) isoform of nitric oxide (NO) synthase (NOS), i.e., inducible NOS (iNOS), thereby inducing NO, which in turn induced DNA breaks and enhanced the mutation frequencies of cellular genes. Treatment of HCV-infected cells with NOS inhibitors or small interfering RNA specific for iNOS abolished most of these effects. Expression of the core protein or nonstructural protein 3 (NS3), but not the other viral proteins, in B cells or hepatocytes induced iNOS and DNA breaks, which could be blocked by NOS inhibitors. The core protein also enhanced the mutation frequency of cellular genes in hepatocytes derived from HCV core transgenic mice compared with that in control mice. The iNOS promoter was activated more than fivefold in HCV-infected cells, as revealed by a luciferase reporter assay driven by the iNOS promoter. Similarly, the core and NS3 proteins also induced the same effects. Therefore, we conclude that HCV infection can stimulate the production of NO through activation of the gene for iNOS by the viral core and NS3 proteins. NO causes DNA breaks and enhances DNA mutation. This sequence of events provides a mechanism for HCV pathogenesis and oncogenesis.
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PMID:Hepatitis C virus infection activates the immunologic (type II) isoform of nitric oxide synthase and thereby enhances DNA damage and mutations of cellular genes. 1528 Apr 91

Antisense technology, including ribozyme and small interfering RNA, is being developed to mediate the down-regulation of specific intracellular genes. It was observed in this study that both antiluciferase ribozymes and short hairpin RNAs (shRNAs) could significantly reduce the activity of exogenously expressed luciferase in primary hippocampal neurons in a viral titer-dependent manner. shRNAs were more effective gene-silencing agents than ribozymes, although they exhibited some nonspecific gene-silencing effects at high viral titers. We also attempted to increase ribozyme efficacy by using a woodchuck hepatitis posttranscriptional regulatory element (WPRE) in the ribozyme expression cassette. The results showed that adenoviral vectors encoding specific ribozymes could silence the cellular expression of luciferase and endogenous procaspase-3 significantly. Furthermore, the antiprocaspase-3 ribozyme was shown to inhibit staurosporine-mediated cell death. The addition of a WPRE did not, however, increase or decrease ribozyme activity. As far as we are aware, this is the first example of adenovirally mediated delivery of hammerhead ribozymes being used to manipulate gene expression in primary neurons. The results therefore suggest that hammerhead ribozymes may be useful tools for studying neuronal gene function and have potential as therapeutic agents to treat CNS diseases.
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PMID:Assessing adenoviral hammerhead ribozyme and small hairpin RNA cassettes in neurons: inhibition of endogenous caspase-3 activity and protection from apoptotic cell death. 1565 76

Despite favorable DNA transfer efficiency, gene expression from recombinant adeno-associated virus (rAAV2) vectors in the lung has been variable in the context of cystic fibrosis (CF) gene therapy. This is due, in part, to the large size of the CF transmembrane regulator (CFTR)-coding sequence which necessitates the use of compact endogenous promoter elements versus stronger exogenous promoters. We evaluated the possibility that gene expression from rAAV could be improved by using AAV capsid serotypes with greater tropism for the apical surface of airway cells (i.e. rAAV5 or rAAV1) and/or using strong promoters such as the cytomegalovirus (CMV) enhancer/chicken beta-actin hybrid (Cbeta) promoter. The relative activity of the CMV immediate-early (CMVie) promoter, the Cbeta promoter, and the Cbeta promoter with a downstream woodchuck hepatitis virus post-transcriptional regulatory element (wpre) were assessed in vitro and in vivo in C57\Bl6 mice using human alpha-1 antitrypsin (hAAT) as a secreted reporter. In vivo, the Cbeta-AAT-wpre group achieved maximum serum levels of 1.5 mg/ml of hAAT. AAV capsid serotypes were then compared in vivo utilizing the transcriptionally optimized CB-wpre cassette in rAAV serotype 1, 2 or 5 capsids (rAAV1, rAAV2, and rAAV5), utilizing luciferase as a reporter to compare expression over a wide dynamic range. The pulmonary luciferase levels at 8 weeks were similar in rAAV5 and rAAV1 groups (2.9 x 10(6) relative light units (RLU)/g tissue and 2.7 x 10(6) RLU/g tissue, respectively), both of which were much higher than rAAV2. Although the advantage of rAAV5 over rAAV2 in the lung has already been described, the availability of another serotype (rAAV1) capable of efficient gene transfer in the lung could be useful.
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PMID:Enhancing rAAV vector expression in the lung. 1583 34

Gene delivery of IFN-alpha to the liver may represent an interesting strategy to maximize its antiviral efficacy and reduce side effects. We used a recombinant adeno-associated virus (AAV) encoding woodchuck IFN-alpha (AAV-IFN) to treat animals with chronic woodchuck hepatitis virus infection. The vector was given by intraportal or intramuscular route. Long-term transgene expression was detected after intraportal administration of an AAV encoding luciferase. In contrast, in the majority of the animals that received AAV-IFN through the portal vein, the expression of IFN-alpha was transient (30-40 days) and was associated with a significant but transient decrease in viral load. One animal, in which hepatic production of IFN-alpha persisted at high levels, died because of bone marrow toxicity. The disappearance of IFN-alpha expression correlated with the disappearance of AAV genomes from the liver. Intramuscular administration of AAV-IFN resulted in prolonged but fluctuating expression of the cytokine with no significant antiviral effect. In summary, this report shows that long-term expression of IFN-alpha in muscle is feasible but higher interferon levels might be needed to control viral replication. On the other hand, IFN-alpha gene delivery to the liver using an AAV vector induces a significant but transient antiviral effect in the woodchuck model.
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PMID:IFN-alpha gene therapy for woodchuck hepatitis with adeno-associated virus: differences in duration of gene expression and antiviral activity using intraportal or intramuscular routes. 1596 22

Coronaviruses are enveloped, positive-stranded RNA viruses considered to be promising vectors for vaccine development, as (i) genes can be deleted, resulting in attenuated viruses; (ii) their tropism can be modified by manipulation of their spike protein; and (iii) heterologous genes can be expressed by simply inserting them with appropriate coronaviral transcription signals into the genome. For any live vector, genetic stability is an essential requirement. However, little is known about the genetic stability of recombinant coronaviruses expressing foreign genes. In this study, the Renilla and the firefly luciferase genes were systematically analyzed for their stability after insertion at various genomic positions in the group 1 coronavirus feline infectious peritonitis virus and in the group 2 coronavirus mouse hepatitis virus. It appeared that the two genes exhibit intrinsic differences, the Renilla gene consistently being maintained more stably than the firefly gene. This difference was not caused by genome size restrictions, by different effects of the encoded proteins, or by different consequences of the synthesis of the additional subgenomic mRNAs. The loss of expression of the firefly luciferase was found to result from various, often large deletions of the gene, probably due to RNA recombination. The extent of this process appeared to depend strongly on the coronaviral genomic background, the luciferase gene being much more stable in the feline than in the mouse coronavirus genome. It also depended significantly on the particular genomic location at which the gene was inserted. The data indicate that foreign sequences are more stably maintained when replacing nonessential coronaviral genes.
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PMID:Coronaviruses as vectors: stability of foreign gene expression. 1618 77

The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. S(VSV-Cyt), an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and S(MHV-TMDCyt), an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. S(VSV-TMDCyt), a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that S(VSV-TMDCyt) trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.
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PMID:Important role for the transmembrane domain of severe acute respiratory syndrome coronavirus spike protein during entry. 1641 7

Triaryl pyrazoline {[5-(4-chloro-phenyl)-3-thiophen-2-yl-4,5-dihydro-pyrazol-1-yl]-phenyl-methanone} inhibits flavivirus infection in cell culture. The inhibitor was identified through high-throughput screening of a compound library using a luciferase-expressing West Nile (WN) virus infection assay. The compound inhibited an epidemic strain of WN virus without detectable cytotoxicity (a 50% effective concentration of 28 microM and a compound concentration of >or=300 microM required to reduce 50% cell viability). Besides WN virus, the compound also inhibited other flaviviruses (dengue, yellow fever, and St. Louis encephalitis viruses), an alphavirus (Western equine encephalitis virus), a coronavirus (mouse hepatitis virus), and a rhabdovirus (vesicular stomatitis virus). However, the compound did not suppress an orthomyxovirus (influenza virus) or a retrovirus (human immunodeficiency virus type 1). Mode-of-action analyses in WN virus showed that the compound did not inhibit viral entry or virion assembly but specifically suppressed viral RNA synthesis. To examine the mechanism of inhibition of dengue virus, we developed two replicon systems for dengue type 1 virus: (i) a stable cell line that harbored replicons containing a luciferase reporter and a neomycin phosphotransferase selection marker and (ii) a luciferase-expressing replicon that could differentiate between viral translation and RNA replication. Analyses of the compound in the dengue type 1 virus replicon systems showed that it weakly suppressed viral translation but significantly inhibited viral RNA synthesis. Overall, the results demonstrate that triaryl pyrazoline exerts a broad spectrum of antiflavivirus activity through potent inhibition of viral RNA replication. This novel inhibitor could be developed for potential treatment of flavivirus infection.
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PMID:Triaryl pyrazoline compound inhibits flavivirus RNA replication. 1656 47


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