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Expression of tegument protein pp65 of human cytomegalovirus (CMV) and its application to the analysis of viral-specific cellular immunity in CMV-infected individuals

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ARCHIVES OF VIROLOGY 2002年第12期147卷 2405-2417页

作者： Tanaka, N Kimura, H Hoshino, Y Nishikawa, K Kojima, S Nishiyama, Y Morishima, T Nagoya Univ Grad Sch Med Dept Pediat Nagoya Aichi Japan Nagoya Ekisaikai Hosp Dept Pediat Nagoya Aichi Japan Nagoya Univ Sch Med Dis Mechanism & Control Res Inst Virol Lab Nagoya Aichi 466 Japan Nagoya Univ Sch Med Dept Hlth Sci Nagoya Aichi 466 Japan

Investigations into human cytomegalovirus (CMV)-specific cellular immunity are important to better understand and manage CMV infections. CMV phosphoprotein pp65 is thought to be a major antigen for CMV-specific cellular immunity. We newly synthesized protein pp65 with a baculovirus expression system and purified it via metal affinity chromatography in a soluble form. The recombinant protein pp65 was antigenic in an enzyme immuno-linked assay for pp65-specific IgG in sera from 196 children. Traditional lymphoproliferation assays have shown that pp65 protein promotes specific lymphoproliferation in CMV-seropositive donors. Using an intracellular cytokine detection system, we showed that this recombinant protein stimulated CD4-positive T cells to express interferon-gamma. The results of these assays using protein pp65 were comparable with the use of CMV whole antigen. pp65- and CMV-specific cellular immunity, and CMV DNA load were also compared in four recipients of unrelated cord blood transplantation. The delay in re-constitution in CMV-specific cellular immunity was associated with reactivation of CMV. These results indicated that the recombinant protein pp65 can be used to study specific immunity in CMV infections.

关键词：抗原病毒/生物合成抗原病毒/免疫学杆状病毒科/遗传学血液成分输血 CD4阳性T淋巴细胞/免疫学细胞培养的巨细胞病毒/遗传学巨细胞病毒/分离和提纯巨细胞病毒/代谢巨细胞病毒感染/血液巨细胞病毒感染/免疫学巨细胞病毒感染/治疗 DNA 病毒/分析酶联免疫吸附测定遗传载体免疫球蛋白G/血液干扰素Ⅱ型/分析磷蛋白类/生物合成磷蛋白类/免疫学重组蛋白质类/生物合成病毒载量病毒基质蛋白质类/生物合成病毒基质蛋白质类/免疫学青少年儿童儿童学龄前女(雌)性人类婴儿男(雄)性

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Artificial recruitment of Sp1 or TBP can replace the role of IE1 in the synergistic transactivation by IE1 and IE2

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第2期269卷 302-308页

作者： Kim, JM Hong, Y Kim, S Seoul Natl Univ Inst Mol Biol & Genet Kwan Ak Gu Seoul 151742 South Korea

The IE1 and IE2 proteins of human cytomegalovirus transactivate various viral and cellular promoters in a synergistic manner, but the mechanism of their action has not been web elucidated. Here we have examined the IE1-IE2 synergy by artificial recruitment of either Spl or TBP to the promoter. We found that in the presence of Sp1, the synergistic effect of IE1 on IE2-mediated transactivation dramatically decreased. Furthermore, a 117-amino acids glutamine-rich fragment of Spl, which can interact with dTAF(II)110 and hTAF(II)130, was sufficient to replace the role of IE1 in IE1-IE2 synergism. It was also found that TBP recruitment to the promoter markedly decreased the synergistic effect of El on IE2-mediated transactivation. These results suggested that in the context of the synergism between Hi and IE2, the function of IE1 might overlap with that of Spl, for example by recruiting the TFIID complex. (C) 2000 Academic Press.

关键词：结合部位细胞系仓鼠亚科巨细胞病毒/代谢即早蛋白质类/代谢即早蛋白质类/生理学 Sp1转录因子/代谢反式激活(遗传学)/生理学转录因子TFIID 转录因子 TFⅡ类/代谢病毒蛋白质类动物

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Surface expression of HLA-E, an inhibitor of natural killer cells, enhanced by human cytomegalovirus gpUL40

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SCIENCE 2000年第5455期287卷 1031-1033页

作者： Tomasec, P Braud, VM Rickards, C Powell, MB McSharry, BP Gadola, S Cerundolo, V Borysiewicz, LK McMichael, AJ Wilkinson, GWG John Radcliffe Hosp Inst Mol Med MRC Human Immunol Unit Oxford OX3 9DS England Univ Wales Coll Med Dept Med Cardiff CF14 4XN S Glam Wales

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/ NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell Lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

关键词：氨基酸序列氨基酸取代抗原 CD 细胞系细胞膜/免疫学细胞培养的保守序列巨细胞病毒/遗传学巨细胞病毒/免疫学巨细胞病毒/代谢细胞毒性免疫减量调节 HLA抗原/免疫学 HLA抗原/代谢组织相容性抗原Ⅰ类/免疫学组织相容性抗原Ⅰ类/代谢杀伤细胞天然/免疫学分子序列数据开放读码框架蛋白分选信号/化学蛋白分选信号/代谢受体免疫/代谢重组融合蛋白质类/化学重组融合蛋白质类/代谢转染增量调节病毒蛋白质类/化学病毒蛋白质类/遗传学病毒蛋白质类/代谢人类

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A protein-based therapeutic for human cytomegalovirus infection

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第6期97卷 2864-2869页

作者： Jean, F Thomas, L Molloy, SS Liu, GP Jarvis, MA Nelson, JA Thomas, G Oregon Hlth & Sci Univ Vollum Inst Portland OR 97201 USA Oregon Hlth & Sci Univ Dept Mol Microbiol & Immunol Portland OR 97201 USA

Current antiviral strategies target viral gene products. Although initially successful, their severe toxicity and susceptibility to circumvention by the generation of drug-resistant variants limit their usefulness. By contrast, the central role of the host cell serine endoprotease furin in the proteolytic activation of numerous pathogens points to the endoprotease as a strategic target for therapeutics. Herein, we show that the production of infectious human cytomegalovirus is dramatically reduced by exogenous addition of a bioengineered serpin, alpha(1)-PDX. This protein is a potent and selective furin inhibitor (K-i = 0.6 nM) and is 10-fold more effective than currently used antiherpetic agents in cell-culture models. The requirement of furin for the processing of envelope glycoproteins from many pathogenic viruses and for the activation of several bacterial toxins suggests that selective inhibitors of furin have potential as broad-based anti-pathogens.

关键词：印迹法蛋白质巨细胞病毒/代谢巨细胞病毒感染/药物疗法剂量效应关系药物成纤维细胞/代谢费林蛋白酶动力学显微镜检查荧光沉淀素试验时间因素肿瘤细胞培养的 α1抗胰蛋白酶/代谢人类

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Human cytomegalovirus UL69 protein is required for efficient accumulation of infected cells in the G₁ phase of the cell cycle

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 2000年第6期97卷 2692-2696页

作者： Hayashi, ML Blankenship, C Shenk, T Princeton Univ Dept Mol Biol Princeton NJ 08544 USA

Human cytomegalovirus blocks cell-cycle progression in the G(1) compartment upon infection of primary human fibroblasts, The virus-coded UL69 protein can institute a G(1) block when expressed in cells in the absence of virus infection. We have constructed a cytomegalovirus mutant, TNsubUL69, that lacks the UL69 coding region. This virus grows slowly in fibroblasts, but produces a wild-type yield after an extended delay. It grows with normal kinetics in cells coinfected with a recombinant retrovirus, retroUL69, which expresses UL69 protein, demonstrating that its growth defect results from the mutation in the UL69 gene. UL69 protein is packaged within virus particles, and it was possible for us to produce two types of virus stocks. TNsubUL69(+pUL69) lacks the UL69 gene but contains UL69 protein in virus particles. It is produced by growth in fibroblasts that are coinfected with retroUL69, TNsubUL69(-pUL69) lacks the UL69 gene and protein. It is produced by growth in fibroblasts that do not contain UL69 protein. The mutant virions lacking both the UL69 gene and protein fail to induce a cell-cycle block with normal efficiency, whereas the mutant particles lacking the gene but containing the protein can institute the block. These results are consistent with the view that the UL69 protein contributes to the cytomegalovirus-induced cell-cycle block, and they suggest that UL69 protein delivered to cells within virions can induce the block without the synthesis of additional UL69 protein encoded by the infecting viral genome.

关键词：细胞周期细胞培养的巨细胞病毒/遗传学巨细胞病毒/代谢 DNA复制/遗传学成纤维细胞/病毒学 G1期即早蛋白质类/遗传学即早蛋白质类/生理学动力学突变 RNA 信使/代谢时间因素病毒蛋白质类/遗传学病毒蛋白质类/生理学人类

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Cytomegalovirus-encoded β chemokine promotes monocyte-associated viremia in the host

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PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 1999年第19期96卷 10881-10886页

作者： Saederup, N Lin, YC Dairaghi, DJ Schall, TJ Mocarski, ES Stanford Univ Sch Med Dept Microbiol & Immunol Stanford CA 94305 USA ChemoCentryx San Carlos CA 94070 USA

Chemokine homologs are encoded by many large DNA viruses, suggesting that they contribute to control of host leukocyte transmigration and trafficking during viral infection. Murine cytomegalovirus carries a CC (beta) chemokine homolog gene giving rise to two related proteins, murine cytomegalovirus chemokine 1 and 2 (MCK-1 and MCK-2). MCK-1 peptide was found to induce calcium signaling and adherence in murine peritoneal macrophages. Cells bearing human chemokine receptor CCR3 and the human macrophage THP1 cell line were responsive to MCK-1. This pattern suggested that MCK-1 might act as an agonist, promoting leukocyte trafficking during viral infection. Consistent with this prediction, MCK-1/MCK-2 mutant viruses exhibit dramatically reduced peak levels of monocyte-associated viremia in experimentally infected mice. Thus, MCK-1/MCK-2 appears to promote host leukocyte migration to initial sites of infection and may be responsible for attracting monocytes or macrophages that efficiently disseminate virus in the host.

关键词：氨基酸序列钙/代谢炎症趋化因子类/遗传学炎症趋化因子类/代谢趋化因子 CC/遗传学趋化因子 CC/生理学巨细胞病毒/遗传学巨细胞病毒/代谢小鼠近交BALBC 模型生物学模型遗传学分子序列数据单核细胞/代谢单核细胞/病毒学腹膜/代谢腹膜/病毒学重组遗传序列同源性氨基酸时间因素病毒蛋白质类病毒血症/遗传学病毒血症/代谢动物人类小鼠

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RECOGNITION OF COMPARTMENTALIZED INTRACELLULAR ANALOGS OF GLYCOPROTEIN-H OF HUMAN CYTOMEGALOVIRUS

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ARCHIVES OF VIROLOGY 1992年第1-4期126卷 67-80页

作者： BOGNER, E RESCHKE, M REIS, B REIS, E BRITT, W RADSAK, K UNIV MARBURG INST VIROLROBERT KOCH STR 1W-3550 MARBURGGERMANY SCH MED DEPT PEDIAT & MICROBIOLBIRMINGHAMAL

Infected cell proteins immunoprecipitated from human cytomegalovirus (HCMV)-infected fibroblasts with glycoprotein H (gH)-specific conformation-dependent monoclonal antibody (mab 14-4 b) were found to consist of three components of 86 kDa, 89 kDa, and 125 kDa (gp 86, 89, and 125). Affinity purified antibodies from human convalescent serum reactive with an NH2-terminal epitope of gH recognized three polypeptides of comparable size in immunoblots, suggesting antigenic relatedness of these three components of the gH-complex. Using subcellular fractions for immunoblotting, gp 86 was identified as an endoglycosidase H (endo H)-sensitive gH-form present in the nuclear fraction whereas gp 89 and gp 125 were endo H-resistant and present in the membrane fraction or in virions. Incomplete endo H-digestion suggested that four of six predicted N-glycosylation sites of the gH molecule were occupied by carbohydrate side chains. Analysis under nonreducing conditions revealed that the compartmentalized as well as virion-associated gH analogs form high molecular weight complexes. The relation of the recognized gH analogs to the processing pathway of gH is discussed.

关键词：细胞培养的克隆分子巨细胞病毒/遗传学巨细胞病毒/代谢巨细胞病毒/超微结构免疫印迹法显微镜检查电子质粒/遗传学重组融合蛋白质类/生物合成重组融合蛋白质类/遗传学重组融合蛋白质类/代谢限制性内切酶图谱法病毒包膜蛋白质类/生物合成病毒包膜蛋白质类/遗传学病毒包膜蛋白质类/代谢人类

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