小麦丛矮病毒核衣壳的分离及其核酸、蛋白质组成的研究

结合有机溶剂沉淀、聚乙二醇沉淀及差速离心和等电点沉淀等方法可以获得小麦丛矮病毒核衣壳的纯化制剂。应用多种分离方法可以把核酸或蛋白质从核衣壳分离出来。碱水解及S_1核糖核酸酶酶解实验证明小麦丛矮病毒的基因组为单链RNA,双向纸电泳及层析方法测定其碱基组成比例为A=30.3,G=16.3,C=16.7,U=36.6。SDS聚丙烯酰胺凝胶电泳可分离到6～7个蛋白组分,其中相应于一般弹状病毒的核衣壳组分为L,分子量140,000,N分子量46,000,NS分子量40,000。     

小麦丛矮病毒核衣壳的分离及其核酸、蛋白质组成的研究

结合有机溶剂沉淀、聚乙二醇沉淀及差速离心和等电点沉淀等方法可以获得小麦丛矮病毒核衣壳的纯化制剂。应用多种分离方法可以把核酸或蛋白质从核衣壳分离出来。碱水解及S_1核糖核酸酶酶解实验证明小麦丛矮病毒的基因组为单链RNA,双向纸电泳及层析方法测定其碱基组成比例为A=30.3,G=16.3,C=16.7,U=36.6。SDS聚丙烯酰胺凝胶电泳可分离到6～7个蛋白组分,其中相应于一般弹状病毒的核衣壳组分为L,分子量140,000,N分子量46,000,NS分子量40,000。     

小麦丛矮病毒N蛋白的酶加工现象

经SDS-聚丙烯酰胺梯度电泳可以从提纯的小麦丛矮病毒中分离出五种结构蛋白。其中,在N蛋白区域又可分辨出分子量相差2KD的两条蛋白蒂,N_1=46K,N_2=44K。从电泳中分离得到的N_1及N_2蛋白经同位素~(125)I标记后的双向指纹图谱证明没有明显差异,为同一种蛋白质。又通过N末端分析证明N_1的末端为Ser.,N_2为His,初步断定N_1与N_2是前体与酶解产物之间的关系。实验还证明小麦丛矮病毒的核衣壳制剂具有专一酶解N_1至N_2的能力,首次证明了植物弹状病毒的核衣壳具有蛋白水解酶的活力。本文还提出了N蛋白的酶加工现象在弹状病毒的复制和转录的调控过程中可能起重要作用的设想。     

小麦丛矮病毒糖蛋白(G蛋白)的研究

应用SDS-聚丙烯酰胺电泳可以从小麦丛矮病毒中分离出5种结构蛋白。经过碘酸—Schiff′s试剂染色证明,其中分子量为66K的是糖蛋白,是组成病毒外膜突起的G蛋白。应用不同的植物凝集素对完整病毒进行凝集反应试验证实,只有ConA凝集素对病毒有凝集作用,葡萄糖有抑止凝集的作用。G蛋白氨基酸组成分析证明,酸性氨基酸的含量较高。用同位素~(125)I标记的G蛋白和N蛋白的双向图谱表明,植物弹状病毒的结构蛋白之间无共同的肽段。说明植物弹状病毒和动物弹状病毒一样,其蛋白也是由同一病毒基因组的不同片段转录和翻译产生的。     

水稻矮缩病病毒粒子装配模型

水稻矮缩病病毒(RDV)具有多种蛋白质和RNA构成的核衣壳结构, 但是特异性的RNA与蛋白质、蛋白质与蛋白质之间的相互作用和结合, 即有关病毒粒子的装配机理还没有完全阐明. 从细胞内和细胞外两个研究层次详细研究了RNA与蛋白质相互结合情况, 研究发现, P7蛋白能特异并牢固地与RDV基因组的全部12条双链RNA结合; 推定为RNA聚合酶的P1蛋白、鸟苷酰转移酶的P5蛋白和P7蛋白被包裹在病毒核内, 根据体外结合实验, 它们能与病毒转录产物mRNA结合; 从病毒感染的组织中能够提取得到完整的、有感染活性的病毒粒子, 以及缺失P1, P5, P7蛋白和基因组双链RNA, 没有感染活性的空壳病毒粒子; 在病毒粒子中P7蛋白能够与P1蛋白和P3内衣壳蛋白形成蛋白复合物. 这些结果揭示了RDV病毒粒子装配的一种可能的模型, 即核心蛋白和病毒mRNA首先相互结合形成一个整体, 以筛选和富集病毒RNA, 接着包装成为完整的、具有感染活性的病毒粒子.     

弹状病毒研究的新进展Ⅰ.病毒的基因结构

弹状病毒组是与人类疾病及工农业生产密切相关的一个重要病毒组,例如VSV（水泡性口膜炎病毒）引起的家畜口膜炎,RV（狂犬病毒）引起的狂犬病,IHNV（传染性造血器官坏死病毒）引起的鲑鱼造血器官坏死病,以及对我国农业生产有重大危害的小麦丛矮病毒引起的小麦丛矮病和由水稻黄矮病毒引起的水稻黄矮病等。弹状病毒可以从两个方面的特征区别于其它病毒：首先是形态特征,大多数弹状病毒有子弹状形态,少数为两个子弹状以底部相连的杆状形态;其次,弹状病毒基因组由一条不分节段的负链RNA构成。目前已知的弹状病毒超过100种。弹状病毒…     

SARS冠状病毒中的核衣壳蛋白

严重急性呼吸综合征(SARS)的元凶是一种新冠状病毒,研究病毒结构蛋白的功能有助于了解病毒的感染、复制和包装等生理过程。其中核衣壳蛋白是SARS冠状病毒中含量最丰富和最保守的结构蛋白,自身聚合后包被病毒RNA基因组形成螺旋状核壳体是SARS冠状病毒成熟的关键步骤;核衣壳蛋白能与病毒或宿主细胞中多种蛋白质相互作用,还能影响宿主细胞的多个通路。因此核衣壳蛋白是一个重要的多功能蛋白质,参与了病毒感染、复制和病毒包装等过程。     

水稻齿叶矮缩病毒双链RNA的体外翻译

水稻齿叶矮缩病毒(Rice Ragged Stunt Virus,以下简称RRSV)的基因组双链RNA经羟甲基汞变性处理后,能在兔网状红细胞体外翻译体系中有效地指导蛋白质生物合成。利用该病毒的抗血清免疫沉淀体外翻译产物,结合SDS-聚丙烯酰胺凝胶电泳分析,证明RRSV基因组RNA体外翻译的主要产物有十种左右,其中八种能被该病毒抗血清专一沉淀。用纯化的水稻齿叶矮缩病毒直接作蛋白质电泳分析,证明上述八种体外翻译产物与该病毒结构蛋白的分子量相当。Schiff试剂染色实验未能检测出糖蛋白。     

猪传染性胃肠炎病毒n基因的原核表达及重组蛋白的免疫活性分析

猪传染性胃肠炎(transmissible gastroenteritis,TGE)是由猪传染性胃肠炎病毒(transmissible gas-troenteritis virus,TGEV)引起的一种急性、高度接触性传染病,以呕吐、水样腹泻、脱水和对2周龄以内仔猪高度致死率为特征[1]。猪传染性胃肠炎病毒隶属于冠状病毒科冠状病毒属,是引起仔猪病毒性腹泻的重要病原,其基因组为单股正链的有感染性不分节段的RNA,TGEV结构蛋白主要由S、N、Ms、M蛋白组成[2]。其中n基因指导合成病毒的核衣壳蛋白(N),它是一种磷酸化的蛋白,存在于病毒粒子的内部,其分子质量为47kD[3],与病毒基因组组成核衣壳;N…     

杆状病毒核衣壳组装相关蛋白研究进展

杆状病毒生命周期中会产生包埋型和芽生型两种病毒粒子,这两种病毒粒子的包膜组成存在明显的差异,但拥有相同的核衣壳结构.杆状病毒核衣壳是由衣壳蛋白和杆状病毒基因组两部分组成,核衣壳的正常组装对两种病毒粒子的形成都是不可或缺的,因此核衣壳的正常组装在病毒的整个感染传播过程中发挥着重要作用.尽管越来越多参与核衣壳组装的蛋白被鉴定出来,目前还有许多核衣壳组装细节不明了,例如这些衣壳蛋白之间的互作关系是怎样的,宿主通过何种方式参与到病毒核衣壳组装过程等.本文主要以杆状病毒模式物种苜蓿银纹夜蛾核型多角体病毒(Autographa californica multiple nucleopolyhedrovirus,AcMNPV)为例综述了参与杆状病毒核衣壳组装的相关蛋白,并对一些参与核衣壳运输有关的核衣壳蛋白也做了阐述.     

Dephosphorylation of West Nile virus capsid protein enhances the processes of nucleocapsid assembly

West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins and it encapsidates the viral RNA to form the nucleocapsid. It is known to be a multifunctional protein involved in assembly and apoptosis. WNV C protein was previously found to be phosphorylated in infected cells and bioinformatic analysis revealed 5 putative phosphorylation sites at serine 26, 36, 83, 99 and threonine 100. Phosphorylation was abolished through mutagenesis of these putative phosphorylation sites to investigate how phosphorylation could affect the processes of nucleocapsid assembly like RNA binding, oligomerization and cellular localization. It was found that phosphorylation attenuated its RNA binding activity. Although oligomerization was not inhibited by mutagenesis of the putative phosphorylation sites, the rate of dimerization and oligomerization was affected. Hypophosphorylation of C protein reduced its nuclear localization efficiency and hence enhanced cytoplasmic localization. This study also revealed that although WNV C is phosphorylated in infected cells, the relative level of phosphorylation is reduced over the course of an infection to promote RNA binding and nucleocapsid formation in the cytoplasm. This is the first report to describe how dynamic phosphorylation of WNV C protein modulates the processes involved in nucleocapsid assembly.     

Constitutively phosphorylated residues in the NS protein of vesicular stomatitis virus

The NS protein of vesicular stomatitis virus is an auxiliary protein in the virus core (nucleocapsid) that plays a role in virus-specific RNA synthesis. NS exhibits a variety of phosphorylated forms, and the degree of phosphorylation correlates with the rate of RNA synthesis. However, chymotryptic peptide mapping has indicated that all forms of NS share a common cluster of phosphorylated residues. To locate these residues in the primary structure of the molecule, we performed a series of residue-specific chemical and enzymatic cleavages and separated radiophosphate-labeled peptides by gel electrophoresis. The data indicate that the constitutively phosphorylated sites in NS molecules reside in the amino-terminal region of the molecule, between residues 35 and 78. The previously reported resistance of the phosphoamino acids in this region to dephosphorylation by exogenous phosphatase suggests that this domain is embedded within the tertiary structure of the molecule or involved in quaternary interactions. In contrast, the amino acid residues that are phosphorylated secondarily, making NS more active in RNA synthesis, reside in more exposed regions of the molecule.     

Phosphorylation of bluetongue virus nonstructural protein 2 is essential for formation of viral inclusion bodies

In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.     

Phosphorylation of rubella virus capsid regulates its RNA binding activity and virus replication

Rubella virus is an enveloped positive-strand RNA virus of the family TOGAVIRIDAE: Virions are composed of three structural proteins: a capsid and two membrane-spanning glycoproteins, E2 and E1. During virus assembly, the capsid interacts with genomic RNA to form nucleocapsids. In the present study, we have investigated the role of capsid phosphorylation in virus replication. We have identified a single serine residue within the RNA binding region that is required for normal phosphorylation of this protein. The importance of capsid phosphorylation in virus replication was demonstrated by the fact that recombinant viruses encoding hypophosphorylated capsids replicated at much lower titers and were less cytopathic than wild-type virus. Nonphosphorylated mutant capsid proteins exhibited higher affinities for viral RNA than wild-type phosphorylated capsids. Capsid protein isolated from wild-type strain virions bound viral RNA more efficiently than cell-associated capsid. However, the RNA-binding activity of cell-associated capsids increased dramatically after treatment with phosphatase, suggesting that the capsid is dephosphorylated during virus assembly. In vitro assays indicate that the capsid may be a substrate for protein phosphatase 1A. As capsid is heavily phosphorylated under conditions where virus assembly does not occur, we propose that phosphorylation serves to negatively regulate binding of viral genomic RNA. This may delay the initiation of nucleocapsid assembly until sufficient amounts of virus glycoproteins accumulate at the budding site and/or prevent nonspecific binding to cellular RNA when levels of genomic RNA are low. It follows that at a late stage in replication, the capsid may undergo dephosphorylation before nucleocapsid assembly occurs.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Hepatitis C virus NS5A protein is phosphorylated by casein kinase II

Hepatitis C virus (HCV) has a positive-strand RNA genome that encodes a polyprotein, which is post-translationally processed by cellular and viral proteinases into three structural and six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists as two phosphorylated forms of 56 kDa and 58 kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from rat-1 cells by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein. This protein kinase was associated through the N-terminus of NS5A and was not regulated by cell cycle. The GST-NS5A was also phosphorylated in vitro by the purified casein kinase II (CKII), a member of the CMCG kinase family. Since CKII and the NS5A-associated protein kinase have the same molecular size and property by In-gel kinase assay and an inhibitor treatment test, we conclude that HCV NS5A protein is phosphorylated by CKII.     

Vesicular stomatitis virus N and NS proteins form multiple complexes.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.