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NS蛋白的磷酸化

小麦丛矮病毒是在中国发现的一种植物弹状病毒 ,病毒基因组是由一条单链负链RNA组成并编码 5种病毒结构蛋白质 :表面糖蛋白G、膜基质蛋白M、核衣壳蛋白N、大蛋白L和所谓非结构蛋白NS。后来的研究证明 ,在弹状病毒的模式病毒———水泡性口膜炎病毒中 ,NS蛋白也是一种结构蛋白 ,而且在成熟的病毒粒子中以各种磷酸化形式存在 ,并且证明NS的磷酸化和去磷酸化对病毒基因组的转录和复制的调控起重要的作用。用体外磷酸化方法证明 ,结合于小麦丛矮病毒的核衣壳上的NS蛋白可以被磷酸化 ;同时也证明 ,从大肠杆菌中表达的小麦丛矮病毒的NS蛋白 ,只有在病毒核衣壳存在下才可以体外被磷酸化 ;从而证明 ,小麦丛矮病毒或植物弹状病毒的NS蛋白也是一种磷酸化蛋白质 ,在成熟病毒粒子中可能存在磷酸化和非磷酸化两种形式。病毒的L蛋白除以前报道的具有RNA聚合酶活力外 ,也具有蛋白激酶的活力。     

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。     

中国小麦丛矮病毒与日本北方禾谷花叶病毒相关性的研究

从过去各自独立进行的研究工作中了解到,中国小麦丛矮病毒(WRSV)与日本北方禾谷花叶病毒(NCMV)有很大的相似性,但尚无直接证据证明这两种病毒之间的关系。本文从下述三个方面证明这两种病毒是相同的:(1)奥氏双扩散法证明,WRSV的核衣壳抗血清能同时与WRSV和NCMV核衣壳形成清晰的沉淀带,并且异源形成的相邻沉淀带能互相吻合;(2)WRSV和NCMV核衣壳制剂的SDS聚丙烯酰胺凝胶电泳证明两者的N蛋白分子量一致的,均与我们过去报道的N_2蛋白的分子量相等;(3)从WRSV和NCMV的完整病毒及核衣壳的横纹周期间距直接比较,两者也是一致的。至于WRSV和NCMV是完全相同的病毒,还是同一种病毒的不同株系,则尚需对两者的寄主范围等作进一步的研究。     

小麦丛矮病毒（WRSV）的近全长cDNA基因文库的构建

用大肠杆菌Poly（A）聚合酶,在纯化的小麦丛矮病毒的单链基因组RNA3＇末端加多聚腺苷酸,以此RNA作模板,12－18寡聚脱氧胸核着酸作引物,合成cNDA后,分别用限制性内切酶PstI和EcoRI对该cDNA酶切,同时再分别用PstI单酶和SmaI与EcoRI双酶对pUC载体酶切,经连接、转化感受态细胞、筛选和酶切鉴定后,共得到10种不同大小的非定向克隆,其大小之和约14kb,同时还得到定向克隆47个。已知N蛋白基因3＇端含有SacI酶切位点,故在用限制性内切酶SacI对10种插入片段进行分析后发现,仅有约6kb的插入片段含有SacI位点,再用合成的与WRSVNN蛋白mRNA3＇端顺序相同的引物对该克隆进行顺序分析证明,它含有WRSVN蛋白、G蛋白、M蛋白和部分L蛋白基因。对47个定向克隆进行顺序测定后,用DNA顺序分析软件将它与其它几种弹状病毒基因组的3＇前导（leader）和5＇拖尾（trailer）RNA进行比较后发现,有两个定向克隆分别含有它们的3＇前导或5＇拖尾的高保守区顺序,构建了一个近全长的小麦丛矮病毒（WRSV）cDNA文库。     

我国禾谷类病毒病的病原问题——Ⅹ.应用酶联免疫吸附测定法检测小麦丛矮病传毒昆虫带毒率

使用酶联免疫吸附测定法(ELISA)检测小麦丛矮病传毒昆虫带毒率。酶用碱性磷酸酯酶,抗原用分离净化的小麦丛矮病毒的核衣壳及其降解物,抗体取抗血清丙种球蛋白部分,交联剂用戊二醛。检测191头灰飞虱的结果与生物接种测定的符合率约为86%。讨论了提高灵敏度的潜力及大田检测的前景。     

我国禾谷类病毒病的病原问题 Ⅹ.应用酶联免疫吸附测定法检测小麦丛矮病传毒昆虫带毒率

使用酶联免疫吸附测定法(ELl8A)检测小麦丛矮病传毒昆虫带毒率。酶用碱性磷酸酯酶,抗原用分离净化的小麦丛矮病毒的核衣壳及其降解物,抗体取抗血清丙种球蛋白部分,交联剂用戊二醛。检测191头灰飞虱的结果与生物接种测定的符合率约为86%。讨论了提高灵敏度的潜力及大田检测的前景。     

小麦丛矮病毒NS蛋白基因的克隆及序列分析

利用与Ｎ蛋白ｍＲＮＡ３＇末端顺序相同的２０寡聚核苷酸引物,通过点杂交、限制性内切酶分析从小麦丛矮病毒（ＷＲＳＶ）ｃＤＮＡ文库中筛选到编码Ｎ蛋白基因下游顺序的ｃＤＮＡ克隆。序列分析表明,该ｃＤＮＡ片段含有一编码的４０ｋＤ蛋白的开放读框。将该读框的全长ｃＤＮＡ经ＰＣＲ扩增后,克隆到ｐＧＥＸ－３Ｘ上,在大肠杆菌ＤＥ３中用ＩＰＴＧ诱导表达,经蛋白质印迹鉴定,该基因为小麦丛矮病毒ＮＳ蛋白基因。     

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…     

P-protein of Chandipura virus is an N-protein-specific chaperone that acts at the nucleation stage

The nucleocapsid protein N of Chandipura virus is prone to aggregation in vitro. We have shown that this aggregation occurs in two phases in a nucleation-dependent manner. Electron microscopy suggests that the aggregated state may have a ring-like structure. Using a GFP fusion, we have shown that the N-protein also aggregates in vivo. The P-protein suppresses the N-protein aggregation efficiently, both in vitro and in vivo. Increased lag phase in the presence of the P-protein suggests that chaperone-like action of the P-protein occurs before the nucleation event. The P-protein, however, does not exert any chaperone-like action against other proteins, suggesting that it binds to the N-protein specifically. Surface plasmon resonance and fluorescence enhancement indeed suggest that the P-protein binds tightly to the native N-protein. The P-protein is thus an N-protein-specific chaperone which inhibits the nucleation phase of N-protein aggregation, thus keeping a pool of encapsidation-competent N-protein for viral maturation.     

Expression of a recombinant DNA gene coding for the vesicular stomatitis virus nucleocapsid protein.

A cDNA clone containing the entire vesicular stomatitis virus nucleocapsid gene was assembled by fusing portions of two partial clones. When the cDNA clone was inserted into a new general-purpose eucaryotic expression vector and introduced into appropriate host cells, abundant N-protein synthesis ensued. The expressed protein was indistinguishable from authentic N protein produced during vesicular stomatitis virus infections. The recombinant N protein was recognized by a polyclonal antibody and two different monoclonal antibodies and could not be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis from authentic N. Our results suggest that the recombinant N protein produced in transfected cells rapidly aggregates into high-molecular-weight complexes in the absence of vesicular stomatitis virus genomic RNA.     

Kinetic, quantitative, and functional analysis of multiple forms of the vesicular stomatitis virus nucleocapsid protein in infected cells.

Multiple forms of the vesicular stomatitis virus nucleocapsid protein N have been detected in infected cells. One form is complexed with the viral NS protein in a 1:1 molar ratio, and the other forms are distinguished by their more rapid sedimentation rates on glycerol gradients. I performed a series of experiments designed to analyze the relationships between these forms of the N protein. Pulse-chase experiments demonstrate that the N protein is made first as the form which binds to the NS protein, forming a 1-to-1 molar complex, and that with increasing times of chase it is either assembled into nucleocapsids or converted to the two higher sedimenting forms. Using a newly developed quantitative immunoblotting procedure, I have quantitated the three differentially sedimenting species of the N protein and have shown that at later times postinfection (6 to 7 h), the faster-sedimenting forms of the N protein account for as much as 50% of the soluble N protein in the cell. The activity of these forms has been assessed, with only the 1-to-1 molar N-NS complex demonstrating the ability to support the replication and encapsidation of viral genomic RNA. A model for the conversion of the N protein from the active N-NS complex into the other forms of the protein is presented, and the possible function of the N-protein self-complexes is discussed.     

Expression of the vesicular stomatitis virus nucleocapsid protein controlled by the lambda PR and PL promoters in Escherichia coli

Expression vectors were constructed in which a cDNA specifying the vesicular stomatitis virus nucleocapsid (VSV N) protein was inserted near the translational initiation region downstream from the thermoinducible P_R or P_L promoter of bacteriophage λ. Expression of the VSV N-protein was determined by a radioimmunoassay with monoclonal antibody prepared against the VSV N-protein. The expression of the VSV N-protein in Escherichia coli was low with either system. However, the deletion of a part of leader sequence from the translational initiation signal to the VSV N-gene resulted in at least 30-fold increase in production of the VSV N-protein. The VSV N-protein in E. coli was also analyzed by radioimmune blot after separation of proteins by gel electrophoresis. Degraded proteins reacted with the antibody were also observed in the cell extracts.     

Delineation and modelling of a nucleolar retention signal in the coronavirus nucleocapsid protein

Unlike nuclear localization signals, there is no obvious consensus sequence for the targeting of proteins to the nucleolus. The nucleolus is a dynamic subnuclear structure which is crucial to the normal operation of the eukaryotic cell. Studying nucleolar trafficking signals is problematic as many nucleolar retention signals (NoRSs) are part of classical nuclear localization signals (NLSs). In addition, there is no known consensus signal with which to inform a study. The avian infectious bronchitis virus (IBV), coronavirus nucleocapsid (N) protein, localizes to the cytoplasm and the nucleolus. Mutagenesis was used to delineate a novel eight amino acid motif that was necessary and sufficient for nucleolar retention of N protein and colocalize with nucleolin and fibrillarin. Additionally, a classical nuclear export signal (NES) functioned to direct N protein to the cytoplasm. Comparison of the coronavirus NoRSs with known cellular and other viral NoRSs revealed that these motifs have conserved arginine residues. Molecular modelling, using the solution structure of severe acute respiratory (SARS) coronavirus N-protein, revealed that this motif is available for interaction with cellular factors which may mediate nucleolar localization. We hypothesise that the N-protein uses these signals to traffic to and from the nucleolus and the cytoplasm.     

Trimeric hantavirus nucleocapsid protein binds specifically to the viral RNA panhandle

Hantaviruses are tripartite negative-sense RNA viruses and members of the Bunyaviridae family. The nucleocapsid (N) protein is encoded by the smallest of the three genome segments (S). N protein is the principal structural component of the viral capsid and is central to the hantavirus replication cycle. We examined intermolecular N-protein interaction and RNA binding by using bacterially expressed Sin Nombre virus N protein. N assembles into di- and trimeric forms. The mono- and dimeric forms exist transiently and assemble into a trimeric form. In contrast, the trimer is highly stable and does not efficiently disassemble into the mono- and dimeric forms. The purified N-protein trimer is able to discriminate between viral and nonviral RNA molecules and, interestingly, recognizes and binds with high affinity the panhandle structure composed of the 3' and 5' ends of the genomic RNA. In contrast, the mono- and dimeric forms of N bind RNA to form a complex that is semispecific and salt sensitive. We suggest that trimerization of N protein is a molecular switch to generate a protein complex that can discriminate between viral and nonviral RNA molecules during the early steps of the encapsidation process.