【作者】 姚敏；

【导师】 陶小荣；

【作者基本信息】 南京农业大学 ， 植物病理学， 2013， 博士

【摘要】 水稻条纹病毒(Rice stripe virus,RSV)作为纤细病毒属的典型成员,是引起水稻条纹叶枯病的病原物,在灰飞虱体内循回并增殖,以持久方式经卵传播,在我国广大稻区已经造成了极其严重的危害。通过几十年的努力,研究者在RSV的很多方面取得了巨大的成果,如基因组结构及其编码策略和蛋白功能、病毒粒子形态、抗病育种等,但还有很多方面值得更深入地认识和研究。RSV是一个负义链RNA病毒,其RNA5’端并不存在帽子结构,而其mRNA5’末端却有一段非病毒来源的RNA序列,那么这些短的、非病毒的帽子结构的来自什么地方,又有什么规律可寻呢?另一个值得关注的问题是RSV病毒粒子形态,RSV与布尼亚病毒科病毒的亲缘关系十分近,但二者的病毒粒子形态却差别非常大,这是又是什么原因造成的呢?为了解答这两个疑惑,本研究开展了以下两方面的研究：1.水稻条纹病毒mRNA转录起始机制的研究RNA帽子结构存在于所有真核生物mRNA和大多数病毒基因组RNA的5’端,它对于RNA的有效剪切、RNA出核转运和RNA自身稳定具有重要的作用。但是分段负义链RNA病毒的RNA5’端不存在帽子结构,而其mRNA5’末端却有一段非病毒来源的RNA序列,RSV mRNA5’端同样存在这样的序列,RSV是否和其他负义链RNA病毒一样通过“抓帽”机制获得这些帽子结构。为了探明RSV mRNA5’端这些短的、非病毒的帽子结构的来源机制,本研究将RSV和具5,端帽子结构的正义链RNA病毒黄瓜花叶病毒(Cucumber mosaic virus, CMV)共侵染本氏烟,结果发现前者可“窃取”后者RNA5’端的帽子结构并起始自身RNA的转录,而且优先切割在与RSV RNA3’端多碱基互补的位点。源于CMV的RNA帽子长度达到12-20nt(nucleotides)时可以起始RSV长距离转录,但长度为12-16nt的RNA前导序列最适合RSV长距离转录起始,可产生了更多的CMV-RSV嵌合体。虽然从CMV RNA切割而来的初始帽子长度较短(10-13nt),但是这些源于CMV帽子结构、可起始RSV mRNA长距离转录的RNA前导序列包含多达5个额外的AC重复。经序列分析发现这些AC重复通过引物重配机制添加到CMV RNA初始的、较短的帽子上,而且每经历一次引物重配即可增加一个AC到RNA帽子结构上,如此循环,较短的RNA帽子逐步增长至较长的帽子结构(12-20nt)。值得注意的是,源于CMV RNA1/2的初始RNA帽子(10或11nt)不可以直接进行转录延伸,只有当其帽子长度增至12nt以上才可以被用于直接延伸。这些结果表明引物重配机制可以使较短的CMV RNA帽子序列逐步增长至较长且合适长度的RSV RNA帽子结构,从而促使形成较稳定的转录起始复合体,有利于RSV RNA转录起始并长距离延伸。2. RSV NSvc2蛋白在植物细胞中的亚细胞定位研究纤细病毒属病毒与布尼亚病毒科病毒具有极高的亲缘关系,但是纤细病毒的病毒粒子呈细丝状且不具包膜,而布尼亚病毒的病毒粒子是具包膜的球状粒子。在布尼亚病毒的球状病毒粒子形成过程中其糖蛋白起了十分重要的作用,是决定病毒粒子形态的关键因子。RSV基因组RNA2的互补链vcRNA2编码的一个多聚蛋白RSV NSvc2蛋白与布尼亚病毒的糖蛋白具有很高的同源性,且具有许多共同的特征,但病毒粒子形态却截然不同,是否是其糖蛋白的特性发生改变从而导致其病毒粒子形态的变化。为了阐明这个问题,首先通过序列分析发现：RSV NSvc2蛋白具有4个跨膜区域(6-26aa、269-291aa、362-379aa和807-827aa),其中2个区域是信号肽(1-18aa和362-379aa),而且其成熟过程在382aa位点发生切割形成NSvc2-N(41kDa)和NSvc2-C (51kDa)两个成熟蛋白。为了进一步明确RSV病毒粒子形态的成因,本研究在本氏烟中更深入地研究了NSvc2的特征,结果发现：在RSV自然侵染水稻和本氏烟的过程中糖蛋白NSvc2的确发生切割,形成两个成熟蛋白NSvc2-N和NSvc2-C,而且NSvc2-N单独表达时定位于高尔基体,而NSvc2-C单独表达时却积累于内质网,但是一旦二者共同表达时,NSvc2-N即可把Svc2-C从内质网招募到高尔基体上,而且这种从内质网到高尔基体的转运是依赖有功能的细胞早期分泌途径COP I和COP Ⅱ,且定位于高尔基体的NSvc2可与高尔基体一起沿着内质网运动。进一步通过缺失和重组嵌合突变体发现NSvc2的高尔基体定位信号位于NSvc2-N的C端区域(269-315aa),涵盖了NSvc2-N跨膜区和相邻的24aa的胞浆尾部。这些结果表明RSV糖蛋白NSvc2蛋白在植物细胞中定位于高尔基体,与布尼亚病毒科病毒糖蛋白的细胞学特征相同,为RSV的认识提供了新的视角,有望寻找到RSV病毒粒子线状形态的真正成因。更多还原

【Abstract】 Rice stripe virus (RSV), the type member of the genus Tenuivirus, caused significant losses in rice fields in China and is known to be transmitted by Laodelphax striatellus in a persistent, circulative-propagative manner. Currently, RSV raised serious concerns and was focused on the genome structure and its coding strategy, the function of proteins, resistance breeding and other aspects, but many aspects are worthy of in-depth knowledge and research. RSV, a negative-sense strand RNA virus, has no cap structure at its RNAs5’ terminal, whereas there is a non-viral RNA sequence at its mRNAs5’terminal. It was asked where do these short, non-viral cap structures come and what rules can be found it? Another issue of concern is the morphology of RSV particles. RSV and the viruses of Bunyaviridae have a close phylogenetic relationship, but how to explain the phenomena that the morphologies of their particles are largely different. To answer these doubts, two aspects were carried out in this study as follows:1.RSV mRNA transcription initiation mechanismRNA cap structure found at the5’end of eukaryotic mRNA and the majority of the viral genomic RNA. The cap has several important biological roles, such as protecting mRNA from degradation by5’exoribonucleases and directing pre-mRNA splicing and mRNA export from the nucleus. But all segemet, negative-sense strand RNA virus have no cap structure at its RNAs5’terminal, whereas there is a non-viral RNA sequence at its mRNAs5’terminal, as well as RSV. But whether RSV acquiring cap structures from cellular mRNAs by’cap snatching’has not been determined.To ascertain the origin of these short and non-viral cap structure, in this study RSV and Cucumber mosaic virus (CMV), a sense strand RNA virus having the cap structure at its RNAs5’end, were co-infected Nicotiana benthamiana. CMV RNAs were found to serve as cap donors for rice stripe virus (RSV) transcription initiation during their co-infection of N. benthamiana. The5’end of CMV RNAs was cleaved preferentially at residues that had multiple-base complementarity to the3’end of the RSV template. The length requirement for CMV capped primers to be suitable for elongation varied between12and20nt (nucleotides), and those of12-16nt were optimal for elongation and generated more CMV-RSV chimeric mRNA transcripts. The original cap donors that were cleaved from CMV RNAs were predominantly short (10-13nt). However, the CMV capped RNA leaders that underwent long-distance elongation were found to contain up to five repetitions of additional AC dinucleotides. Sequence analysis revealed that these AC dinucleotides were used to increase the size of short cap donors in multiple prime-and-realign cycles. Each prime-andrealign cycle added an AC dinucleotide onto the capped RNA leaders; thus, the original cap donors were gradually converted to longer capped RNA leaders (of12-20nt). Interestingly, the original10nt (or11nt) cap donor cleaved from CMV RNA1/2did not undergo direct extension; only capped RNA leaders that had been increased to>12nt were used for direct elongation. These findings suggest that this repetitive priming and realignment may serve to convert short capped CMV RNA leaders into longer, more suitable sizes to render a more stabilized transcription complex for elongation during RSV transcription initiation.2-Subcellular localization of RSV NSvc2proteins in plant cellsViruses of Tenuivirus have a close phylogenetic relationship with the viruses in the family of Bunyaviridae, but Tenuiviruses adopt a long filamentous morphology while Bunyaviruses are membrane-enveloped sphere particles. For the viruses in Bunyaviridae, targeting of glycoproteins to the Golgi apparatus plays a pivotal role in the maturation of membrane-enveloped sphere particle. NSvc2glycoprotein encoded by RSV vcRNA2, exhibiting high homology with glycoproteins of Bunyaviruses, has many characteristics in common with glycoprotein of the viruses in the family Bunyaviridae. However, their viron morphologies are different. We will ask whether the characteristics of RSV glycoprotein NSvc2resulted in the difference the virion morphology between RSV and viruses in the family Bunyaviridae.To support this speculation, the amino acids sequences of RSV NSvc2were analyzed firstly. The results showed that RSV NSvc2has four transmembrane (TM) domains (6-26aa,269-291aa,362-379aa and807-827aa). And two of them (1-18aa and362-379aa) were also predicted to be a signal peptide. RSV NSvc2was cut at382aa into two mature proteins amino-terminal NSvc2(NSvc2-N,41kDa) and carboxyl-terminal NSvc2(NSvc2-C,51kDa). To further clarify the causes of RSV particle morphology, we described the characteristics of RSV NSvc2in N. benthamiana. The results supported that RSV NSvc2could be processed really into two mature proteins NSvc2-N and NSvc2-C. Meanwhile, we demonstrated that NSvc2-N glycoprotein targeted to the Golgi apparatus whereas NSvc2-C accumulated in the ER membrane in N. benthamiana cells. Upon co-expression, NSvc2-N redirected NSvc2-C from ER to Golgi apparatus. The targeted NSvc2glycoproteins moved together with Golgi stacks along the ER track. Targeting of NSvc2glycoproteins to Golgi apparatus was strictly dependent on functional anterograde traffic out of the ER to Golgi or retrograde transport route. Further analysis of expressed truncated and chimeric NSvc2proteins demonstrated that the Golgi targeting signal mapped to a region of NSvc2-N (amino-acid269-315) encompassing the transmembrane domain and the adjacent24amino-acids of the cytosolic tail. All these data determined that RSV NSvc2targeted into Golgi stack. These cytological features of RSV glycoproteins are similar with the viruses of Bunyaviridae. Our findings provide new insights into the intracellular targeting of RSV glycoproteins in plant cells, and the real causes of RSV filamentous particles will be expected to find.更多还原