检索结果-内蒙古大学图书馆

Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321

JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第39期274卷 28011-28018页

作者： Reed, KE Rice, CM Washington Univ Sch Med Dept Mol Microbiol St Louis MO 63110 USA

The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser(2321), a result verified by two additional, independent methods: (i) substitution of Ala for Ser(2321) and the concomitant disappearance of the major in vivo phosphorylated peptides and corresponding in vitro phosphorylated peptides;and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser(2321) with the major in vivo phosphorylated NS5A peptides. Site directed mutagenesis of Ser(2321) suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser(2321) (PLPPPRS2321 PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.

关键词：氨基酸序列氨基酸取代细胞系共有序列仓鼠亚科肝炎病毒属/代谢丝裂原激活蛋白激酶类/代谢分子序列数据诱变定点肽碎片/化学肽碎片/分离和提纯肽类/化学合成肽类/化学肽类/代谢磷肽类/化学磷肽类/分离和提纯磷酰化蛋白质丝氨酸苏氨酸激酶/代谢 RNA复制酶/代谢重组融合蛋白质类/化学重组融合蛋白质类/代谢序列比对序列同源性氨基酸丝氨酸转染病毒非结构蛋白质类/化学病毒非结构蛋白质类/代谢动物

来源：评论

学校读者我要写书评

暂无评论

Purified NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B dependence for cleavage of substrates with dibasic amino acids in vitro

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第14期275卷 9963-9969页

作者： Yusof, R Clum, S Wetzel, M Murthy, HMK Padmanabhan, R Univ Kansas Med Ctr Dept Biochem & Mol Biol Kansas City KS 66160 USA Univ Alabama Birmingham Ctr Macromol Crystallog Birmingham AL 35294 USA

Dengue virus type 2 NS3, a multifunctional protein, has a serine protease domain (NS3pro) that requires the conserved hydrophilic domain of NS2B for protease activity in cleavage of the polyprotein precursor at sites following two basic amino acids. In this study, we report the expression of the NS2B-NS3pro precursor in Escherichia cole as a fusion protein with a histidine tag at the N terminus. The precursor was purified from insoluble inclusion bodies by Ni2+ affinity and gel filtration chromatography under denaturing conditions. The denatured precursor was refolded to yield a purified active protease complex. Biochemical analysis of the protease revealed that its activity toward either a natural substrate, NS4B-NS5 precursor, or the fluorogenic peptide substrates containing two basic residues at P1 and P2, was dependent on the presence of the NS2B domain. The peptide with a highly conserved Gly residue at P3 position was 3-fold more active as a substrate than a Gin residue at this position. The cleavage of a chromogenic substrate with a single Arg residue at P1 was NS2B-independent. These results suggest that heterodimerization of the NS3pro domain with NS2B generates additional specific interactions with the P2 and P3 residues of the substrates.

关键词：氨基酸序列氨基酸类二氨基/代谢色谱法亲和色谱法凝胶克隆分子辅酶类/代谢保守序列登革热病毒/酶学内肽酶类/代谢酶激活大肠杆菌动力学蛋白质前体/分离和提纯蛋白质前体/代谢 RNA解螺旋酶类重组融合蛋白质类/分离和提纯重组融合蛋白质类/代谢丝氨酸内肽酶类/代谢底物特异性病毒非结构蛋白质类/化学病毒非结构蛋白质类/分离和提纯病毒非结构蛋白质类/代谢

来源：评论

学校读者我要写书评

暂无评论

Solution structure of substrate-based ligands when bound to hepatitis C virus NS3 protease domain

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 1999年第26期274卷 18618-18624页

作者： LaPlante, SR Cameron, DR Aubry, N Lefebvre, S Kukolj, G Maurice, R Thibeault, D Lamarre, D Llinàs-Brunet, M Boehringer Ingelheim Canada Ltd Dept Chem Bio Mega Res Div Laval PQ H7S 2G5 Canada Boehringer Ingelheim Canada Ltd Dept Biol Sci Bio Mega Res Div Laval PQ H7S 2G5 Canada

The interactions of the NS3 protease domain with inhibitors that are based on N-terminal cleavage products of peptide substrates were studied by NMR methods. Transferred nuclear Overhauser effect experiments showed that these inhibitors bind the protease in a well defined, extended conformation. Protease-induced line-broadening studies helped identify the segments of inhibitors which come into contact with the protease. A comparison of the NMR data of the free and protease-bound states suggests that these ligands undergo rigidification upon complexation. This work provides the first structure of an inhibitor when bound to NS3 protease and should be valuable for designing more potent inhibitors.

关键词：腺苷三磷酸酶类/代谢氨基酸序列肝炎病毒属/代谢配体磁共振波谱学模型化学模型分子分子序列数据蛋白质构象 RNA解螺旋酶类/代谢丝氨酸内肽酶类/代谢溶液病毒非结构蛋白质类/代谢

来源：评论

学校读者我要写书评

暂无评论

Probing the active site of the hepatitis C virus serine protease by fluorescence resonance energy transfer

引用

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第20期275卷 15106-15113页

作者： Fattori, D Urbani, A Brunetti, M Ingenito, R Pessi, A Prendergast, K Narjes, F Matassa, VG De Francesco, R Steinkühler, C Ist Ric Biol Mol P Angeletti I-00040 Rome Italy Merck Res Labs West Point PA 19486 USA

A serine protease domain contained within the viral NS3 protein is a key player in the maturational processing of the hepatitis C virus polyprotein and a prime target for the development of antiviral drugs. In the present work, we describe a dansylated hexapeptide inhibitor of this enzyme. Active site occupancy by this compound could be monitored following fluorescence resonance energy transfer between the dansyl fluorophore and protein tryptophan residues and could be used to 1) unambiguously assess active site binding of NS3 protease inhibitors, 2) directly determine equilibrium and pre-steady-state parameters of enzyme-inhibitor complex formation, and 3) dissect, using site-directed mutagenesis, the contribution of single residues of NS3 to inhibitor binding in direct binding assays. The assay was also used to characterize the inhibition of the NS3 protease by its cleavage products. We show that enzyme-product inhibitor complex formation depends on the presence of an NS4A cofactor peptide, Equilibrium and pre-steady-state data support an ordered mechanism of ternary (enzyme-inhibitor-cofactor) complex formation, requiring cofactor complexation prior to inhibitor binding.

关键词：氨基酸序列结合部位丹磺酰化合物能量传递肝炎病毒属/酶学动力学分子序列数据诱变定点寡肽类/化学寡肽类/代谢寡肽类/药理学重组蛋白质类/化学重组蛋白质类/代谢丝氨酸内肽酶类/化学丝氨酸内肽酶类/代谢丝氨酸蛋白酶抑制剂/化学丝氨酸蛋白酶抑制剂/代谢丝氨酸蛋白酶抑制剂/药理学光谱法荧光底物特异性色氨酸病毒非结构蛋白质类/化学病毒非结构蛋白质类/代谢

来源：评论

学校读者我要写书评

暂无评论

In vivo and in vitro phosphorylation of rice dwarf phytoreovirus Pns12 cytoplasmic nonstructural protein

引用

ARCHIVES OF VIROLOGY 1999年第7期144卷 1371-1380页

作者： Suzuki, N Hosokawa, D Matsuura, Y Kikuchi, A Omura, T Akita Prefectural Coll Agr Inst Biotechnol Lab Plant Genet Engn Akita Japan Tokyo Univ Agr & Engn Fac Agr Plant Pathol Lab Tokyo Japan Natl Inst Infect Dis Dept Virol 2 Tokyo Japan Natl Agr Res Ctr Ibaraki Osaka Japan

In vivo and in vitro phosphorylation and intracellular location of rice dwarf phytoreovirus Pns12, which is encoded by one of the twelve dsRNA genome segments, S12, and comprises 312 amino acids, was investigated. When [P-32]phosphoric acid was incorporated into RDV-infected leafhopper cultured cells, labelled Pns12 was immunoprecipitated from the cells by a monospecific anti-Pns12 polyclonal antibody. Recombinant Pns12 was purified from Spodoptera frugiperda cells infected with AcRS12, a baculovirus recombinant carrying a full-length cDNA of RDV S12. Purified Pns12 was also demonstrated to be phosphorylated in vitro by a kinase activity present in host (rice, barley, wheat, leafhopper) cells and non-host (tobacco, spinach, white clover, S. frugiperda, mosquito, mammals) cells as well. Immunocytochemical studies showed Pns12 accumulated in the cytoplasm of infected cells, and frequently localized in a slightly electron-dense patch. These results demonstrated that RDV Pns12 was a cytoplasmic nonstructural phosphoprotein.

关键词：细胞系细胞质/化学水稻/病毒学磷酰化呼肠病毒科/化学斜纹夜蛾属病毒非结构蛋白质类/分析病毒非结构蛋白质类/代谢动物

来源：评论

学校读者我要写书评

暂无评论

Eukaryotic translation initiation factor 4GI is a cellular target for NS1 protein, a translational activator of influenza virus

引用

MOLECULAR AND CELLULAR BIOLOGY 2000年第17期20卷 6259-6268页

作者： Aragón, T de la Luna, S Novoa, I Carrasco, L Ortín, J Nieto, A Univ Autonoma Madrid CSIC Ctr Nacl Biotecnol E-28049 Madrid Spain Univ Autonoma Madrid CSIC Ctr Mol Biol E-28049 Madrid Spain

Influenza virus NS1 protein is an RNA-binding protein whose expression alters several posttranscriptional regulatory processes, like polyadenylation, splicing, and nucleocytoplasmic transport of cellular mRNAs. In addition, NS1 protein enhances the translational rate of viral, but not cellular, mRNAs. To characterize this effect, we looked for targets of NS1 influenza virus protein among cellular translation factors. We found that NS1 coimmunoprecipitates with eukaryotic initiation factor 4GI (eIF4GI), the large subunit of the cap-binding complex eIF4F, either in influenza virus-infected cells or in cells transfected with NS1 cDNA. Affinity chromatography studies using a purified His-NS1 protein-containing matrix shelved that the fusion protein pulls down endogenous eIF4GI from COS-1 cells and labeled eIF4GI translated in vitro, but not the eIF4E subunit of the eIF4F factor. Similar in vitro binding experiments with eIF4GI deletion mutants indicated that the NS1-binding domain of eIF4GI is located between residues 157 and 550, in a region where no other component of the translational machinery is known to interact. Moreover, using overlay assays and pull-down experiments, we shelved that NS1 and eIF4GI proteins interact directly, in an RNA-independent manner. Mapping of the eIF4GI-binding domain in the NS1 protein indicated that the first 113 N-terminal amino acids of the protein, but not the first 81, are sufficient to bind eIF4GI. The first of these mutants has been previously shown to act as a translational enhancer, while the second is defective in this activity. Collectively, these and previously published data suggest a model where NS1 recruits eIF4GI specifically to the 5' untranslated region (5' UTR) of the viral mRNA, allowing for the preferential translation of the influenza virus messengers.

关键词： 5'非翻译区印迹法蛋白质 COS细胞细胞系色谱法亲和细胞质/代谢 DNA 互补/代谢真核细胞起始因子4G 基因缺失模型遗传学诱变定点肽起始因子类/化学肽起始因子类/遗传学肽起始因子类/生理学质粒沉淀素试验蛋白质结合蛋白质生物合成蛋白质结构三级 RNA/代谢重组融合蛋白质类/代谢转录遗传转录激活转染病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢动物狗

来源：评论

学校读者我要写书评

暂无评论

Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein

引用

VIROLOGY 1999年第1期263卷 30-41页

作者： Tu, H Gao, L Shi, ST Taylor, DR Yang, T Mircheff, AK Wen, YM Gorbalenya, AE Hwang, SB Lai, MMC Univ So Calif Sch Med Dept Mol Microbiol & Immunol Howard Hughes Med Inst Los Angeles CA 90033 USA Univ So Calif Sch Med Dept Physiol Los Angeles CA 90033 USA Univ So Calif Sch Med Dept Biophys Los Angeles CA 90033 USA Univ So Calif Sch Med Dept Ophthalmol Los Angeles CA 90033 USA Shanghai Med Univ Inst Mol Virol Shanghai 200032 Peoples R China NCI Adv Biomed Comp Ctr Frederick MD 21702 USA Hallym Univ Inst Environm & Life Sci Chunchon 200702 South Korea

Hepatitis C virus (HCV) NS5A is a phosphoprotein that possesses a cryptic trans-activation activity. To investigate its potential role in viral replication, we searched for the cellular proteins interacting with NS5A protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a newly discovered soluble N-erhylmaleimide-sensitive factor attachment protein receptor-like protein termed human vesicle-associated membrane protein-associated protein of 33 kDa (hVAP-33). In vitro binding assay and in vivo coimmunoprecipitation studies confirmed the interaction between hVAP-33 and NS5A. interestingly hVAP-33 was also shown to interact with NS5B, the Viral RNA-dependent RNA polymerase. NS5A and NS5B bind to different domains of hVAP-53: NS5A binds to the C-terminus, whereas NS5B binds to the N-terminus of hVAP-33. Immunofluorescent staining showed a significant colocalization of hVAP-33 with both NS5A and NS5B proteins. hVAP-33 contains a coiled-coil domain followed by a membrane-spanning domain at its C-terminus. Cell fractionation analysis revealed that hVAP-33 is predominantly associated with the ER, the Golgi complex, and the prelysosomal membrane, consistent with its potential role in intracellular membrane trafficking. These interactions provide a mechanism for membrane association of the HCV RNA replication complex and further suggest that NS5A is a part of the viral RNA replication complex. (C) 1999 Academic Press.

关键词： COS细胞载体蛋白质类/遗传学载体蛋白质类/代谢荧光抗体技术基因文库肝炎病毒属/酶学肝炎病毒属/遗传学肝炎病毒属/代谢肝炎病毒属/生理学膜蛋白质类/遗传学膜蛋白质类/代谢沉淀素试验 RNA复制酶/遗传学 RNA复制酶/代谢亚细胞部分双杂交系统技术膜泡运输蛋白质类病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢病毒复制动物女(雌)性人类兔大鼠

来源：评论

学校读者我要写书评

暂无评论

Epitope-tagging approach to determine the stoichiometry of the structural and nonstructural proteins in the virus particles: Amount of Vpr in relation to Gag in HIV-1

引用

VIROLOGY 2000年第2期268卷 364-371页

作者： Singh, SP Lai, D Cartas, M Serio, D Murali, R Kalyanaraman, VS Srinivasan, A Thomas Jefferson Univ Kimmel Canc Ctr Dept Microbiol & Immunol Philadelphia PA 19107 USA Univ Penn Dept Pathol & Lab Med Philadelphia PA 19104 USA Adv Biosci Labs Inc Kensington MD 20895 USA

We used an epitope-tagging approach to determine the ratio of Gag (structural) to Vpr (nonstructural) in the virus particles directed by human immunodeficiency virus type 1. For this purpose, chimeric Gag and Vpr expression plasmids were constructed with the Flag epitope (DYKDDDDK), and the sequences corresponding to the chimeric protein were Introduced into human immunodeficiency virus type 1 proviral DNA (NL4-3) to determine the ratio in the virus particles when these proteins are expressed in cis. In addition, NL4-3 DNA was modified to disrupt Vpr synthesis to determine the extent of incorporation of Vpr-FL when it is expressed in trans through a heterologous promoter, The analysis of virus particles generated by transfection of proviral DNA into RD cells indicated that (1) the ratio of Gag to Vpr in virus particles, when Vpr-FL is expressed in cis (in the context of proviral DNA), is in the range of 150-200:1 (14-18 molecules of Vpr per virion) and (2) the expression of Vpr-FL in trans showed efficient incorporation with a Gag to Vpr ratio of 5-7:1 (392-550 molecules of Vpr). These results suggest that the presence of the same epitope on different viral proteins may provide an accurate comparison of these proteins in the virus particles. (C) 2000 Academic Press.

关键词：表位作图/方法表位/代谢基因产物 vpr/生物合成基因产物 vpr/遗传学基因产物 vpr/免疫学基因产物 vpr/代谢 HIV核心蛋白质p24/生物合成 HIV核心蛋白质p24/遗传学 HIV核心蛋白质p24/免疫学 HIV核心蛋白质p24/代谢肽类/遗传学肽类/免疫学肽类/代谢质粒/化学合成质粒/免疫学肿瘤细胞培养的病毒非结构蛋白质类/生物合成病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/免疫学病毒非结构蛋白质类/代谢病毒结构蛋白质类/生物合成病毒结构蛋白质类/遗传学病毒结构蛋白质类/免疫学病毒结构蛋白质类/代谢病毒粒子/遗传学病毒粒子/代谢人类

来源：评论

学校读者我要写书评

暂无评论

Loss of dimerisation of the nonstructural protein NS1 of Kunjin virus delays viral replication and reduces virulence in mice, but still allows secretion of NS1

引用

VIROLOGY 1999年第1期264卷 66-75页

作者： Hall, RA Khromykh, AA Mackenzie, JM Scherret, JH Khromykh, TI Mackenzie, JS Univ Queensland Dept Microbiol Brisbane Qld 4072 Australia Royal Childrens Hosp Sir Albert Sakzewski Virus Res Ctr Brisbane Qld 4029 Australia

The flavivirus nonstructural protein NS1 has been implicated in viral RNA replication, although its precise role has not been identified. In its native slate NS1 exists as a heat labile homodimer that is thought to be required for NS1 function and secretion. However, we have recently identified a cDNA clone of KUN virus (FLSD) that replicates efficiently in cell culture but produces and secretes NS1 in monomeric form. Sequence analysis of the NS1 gene in FLSD revealed a single amino acid substitution (proline(250) to leucine) when compared with the parental KUN virus. When site-directed mutagenesis was used to substitute leucine(250) with proline in FLSD to produce the clone 250pro, dimerisation was fully restored. Furthermore, time course experiments revealed that 250pro replicated in Vero cells significantly faster than FLSD and produced 100-fold more infectious virus early (12-24 h) in infection. This correlated with our observations that FLSD required approximately IO-fold more infectious virus than 250pro to produce disease in weanling mice after intraperitoneal inoculation. Taken together our results indicate that mutation from proline to leucine at residue 250 in KUN NS1 ablates dimer formation, slows virus replication, and reduces virulence in mice. (C) 1999 Academic Press.

关键词：氨基酸序列氨基酸取代长尾猴黑保守序列二聚作用脑炎病毒亚组日本/致病力脑炎病毒亚组日本/生理学脑炎虫媒病毒/病理生理学动力学小鼠近交BALBC 分子序列数据诱变定点点突变重组蛋白质类/化学重组蛋白质类/代谢序列比对序列同源性氨基酸 Vero细胞病毒非结构蛋白质类/化学病毒非结构蛋白质类/遗传学病毒非结构蛋白质类/代谢毒力动物小鼠

来源：评论

学校读者我要写书评

暂无评论

De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase

引用

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 2000年第3期268卷 798-803页

作者： Sun, XL Johnson, RB Hockman, MA Wang, QM Eli Lilly & Co Lilly Res Labs Infect Dis Res Indianapolis IN 46285 USA

The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template;the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate. (C) 2000 Academic Press.

关键词： 3'非翻译区基因组病毒肝炎病毒属/遗传学肝炎病毒属/代谢聚胞苷酸类/代谢聚尿苷酸类/代谢 RNA复制酶/代谢 RNA 病毒/生物合成 RNA 病毒/遗传学重组蛋白质类/代谢底物特异性病毒非结构蛋白质类/代谢

来源：评论

学校读者我要写书评

暂无评论

建议与咨询留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

时间限定

文献类型

馆藏选择

核心期刊

语言

文献类型

帮助

文字说明：

检索规则说明：

检索范例：

分类表

所选分类

限定检索结果

文献类型

馆藏范围

日期分布

学科分类号

主题

机构

作者

语言

请选择保存的检索档案：

请选择收藏分类：

通借通还

建议与咨询 留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

时间限定

文献类型

馆藏选择

核心期刊

语言

文献类型

帮助

文字说明：

检索规则说明：

检索范例：

分类表

所选分类

限定检索结果

文献类型

馆藏范围

日期分布

学科分类号

主题

机构

作者

语言

请选择保存的检索档案： 新增检索档案 确定 取消

请选择收藏分类： 新增自定义分类 确定 取消

通借通还

建议与咨询留下您的常用邮箱和电话号码，以便我们向您反馈解决方案和替代方法

请选择保存的检索档案：

请选择收藏分类：