【作者】 崔晓艳；

【导师】 孙广宇；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2010， 博士

【摘要】 马铃薯Y病毒属(Potyvirus)是植物病毒中最大的的属,有200多种确定和暂定种,约占已知植物病毒的30%。许多种在世界范围内对农业生产造成重大经济损失。该属病毒基因组为正义单链RNA,长约10kb,5?-末端共价结合基因组连接蛋白(viral protein, genome-linked,VPg),3?-末端为多聚腺苷酸,只包含一个开放阅读框架(ORF),翻译成一个约360 kDa的多聚蛋白,最终裂解成11个成熟的病毒蛋白,病毒编码蛋白的功能极其复杂。Potyviruses的复制机制尚不明确,但已知正链RNA病毒的复制与植物细胞内膜系统相关,因此研究病毒编码的膜相关蛋白成为研究病毒复制机理的突破口。本文通过研究该属病毒编码的3个膜相关蛋白(6K2蛋白、P3蛋白、P3-PiPo蛋白)的功能,理解其在病毒复制过程中的作用,为研究potyviruses的复制机制提供理论依据。主要内容如下:1、已知6K2蛋白含有一个疏水区,是该属病毒编码的复制相关的膜蛋白。利用酵母双杂交系统(YTHS),从本氏烟(Nicotiana benthamiana) cDNA文库中筛选到与大豆花叶病毒伦敦分离物(SMV-L) 6K2特异性结合的22个寄主蛋白。通过共聚焦显微镜观察对其中8个寄主蛋白进行了亚细胞定位,并发现其中叶绿体蛋白ATO与6K2共定位到叶绿体外膜上。双分子荧光互补试验(BiFC)证实了ATO与SMV-L 6K2在叶绿体外膜上有互作关系,推测ATO可能参与了SMV-L 6K2诱导ER增生形成的膜状小泡转运到叶绿体外膜的过程,勾勒出ATO与SMV-L 6K2互作在病毒复制中作用的假设模型。2、P3蛋白是该属病毒编码的除6K2蛋白外的另一个膜蛋白,为了探明P3蛋白的功能,我们利用共聚焦显微镜,进行了烟草蚀纹病毒(TEV) P3在本氏烟(Nicotiana benthamiana)中的亚细胞定位及与寄主细胞器的共定位研究,得到以下主要结论:TEV-P3分布在内质网(ER)上,形成与高尔基体(Golgi)相关的小颗粒;P3从ER到Golgi的转运依赖于COPI和COPII转运机制;这些小颗粒起源于ERES。突变缺失试验进一步分析得出TEV P3蛋白中C-端跨膜区及邻近的氨基酸序列是负责与Golgi结合的区域。细胞显微录像发现TEV P3在胞质内利用肌动球蛋白动力系统(Actomyosin)运动。根据试验证据提出,TEV P3可能在病毒的复制及运动过程中起了一个桥梁的作用,锚定与膜相关的病毒复制复合物(VRC)到肌动蛋白丝。3、P3-PiPo蛋白是在P3蛋白阅读框内新发现的编码蛋白,功能未知。利用酵母双杂交方法研究大豆花叶病毒伦敦分离物(SMV-L) P3-PiPo蛋白与该病毒自身编码的其他蛋白之间的相互作用,发现只有NIb可与P3-PiPo发生相互作用,经双分子荧光互补试验(BiFC)显示P3-PiPo/NIb在细胞膜上发生互作。利用酵母双杂交体系分析证明P3-PiPo与NIb的互作位点主要集中在PiPo区域。在本氏烟(Nicotiana benthamiana)中的亚细胞定位试验发现,P3-PiPo蛋白延细胞膜形成极小的点状物,可与NIb共定位于细胞核膜,且与NIb、6K2-NIa共定位于叶绿体边缘小泡。该小泡是病毒侵染过程中形成的VRC的位点,据此推测P3-PiPo参与病毒的复制。综上所述,通过对该属病毒编码的膜相关蛋白6K2、P3、P3-PiPo的功能研究,推测了6K2蛋白与寄主蛋白的互作在病毒复制过程中的功能及作用机制;提出了P3蛋白在病毒复制和运动中的作用机理;得出了P3-PiPo蛋白与病毒复制相关。极大地推动了potyviruses复制机理的研究。更多还原

【Abstract】 Potyvirus is the largest plant virus family, includes more than 200 species,≈30% of known plant viruses, can be responsible for severe economic losses to agriculture world-wide. Potyvirus contains a single-stranded, positivesense [ss(+)] RNA genome of about 10 kb nucleotides. There is a VPg (viral protein genome-linked) covalently linked to its 5’end and a poly(A) tail at the 3’end. The viral genome contains a single open reading frame (ORF), encoding a large polyprotein of about 360 kDa that is ultimately cleaved into 11 viral protein products. The function of viral proteins are complex. Potyvirus replication mechanism is still not clear. As (+)RNA viruses replicate in association with cellular membranes, it is the foundation for replication to investigate the function of potyvirus-encoded membrane-associated proteins. In this paper, to better understand the replication mechanisms of potyviruses, the functional characterization of potyvirus-encoded membrane-associated proteins (6K2 protein, P3 protein, P3-PiPo protein) were investigated. The main contents were as follows:1、6K2 protein contains a central hydrophobic domain, which has been demonstrated to associate with potyviruses replication mechanisms. We used yeast two-hybrid systerm to screen the SMV host proteins from the model plant Nicotiana benthamiana cDNA library that interact with soybean mosaic virus london strain (SMV-L) encoded proteins, a 6-kDa potyvirus membrane protein (6K2). Yeast two-hybrid screening has identified a total of 22 host candidates that interacted with 6K2. Localization and co-localization with 6K2 of 8 selected host proteins have been observed by confocal microscope. ATO protein was co-localized with 6K2 on the chloroplasts, confirmed by the bimolecular fluorescence complementation assay (BiFC) in planta. So we suggest hypothetical model for that ER-derived membranous vesicles induced by 6K2 protein was trafficed to the chloroplast through interaction with ATO, then the VRC was induced into chloroplast.2、Besides of 6K protein, P3 protein of potyviruses also contained two hydrophobic domains, to be suggested viral membrance protein. To explore the possible function of P3, we investigated the subcellular localization of the Tobacco etch virus (TEV) P3 protein in Nicotiana benthamiana leaf epidermal cells. The TEV P3 protein localized on the endoplasmic reticulum (ER) membrane and formed punctate inclusions in association with the Golgi apparatus. The trafficking of P3 to the Golgi was mediated by the early secretory pathway(COPI and COPII). The Golgi-associated punctate structures originated from the ER exit site (ERES). Deletion analyses identified P3 domains required for the retention of P3 at the Golgi. Moreover, the P3 punctate structure was found to traffic along the actin filaments. Taken together, the possible dual roles of P3 in viral replication and movement suggested that this protein may act as bridge to anchor the membrane-bound VRC close to the actin microfilament.3、Recently, P3-PiPo was discovered, which is an overlap protein produced via ribosomal frameshifting or transcriptional slippage at a highly conserved within the P3 cistron. In order to investigate the possible role of P3-PiPo, we investigated the soybean mosaic virus london strain (SMV-L) viral proteins interactions with P3-PiPo by yeast two-hybrid system. The interaction between SMV-L P3-PiPo and its NIb was detected and supported with Bimolecular fluorescence complementation (BiFC). We further find out the PIPO domain responsible for this interaction by YTHS. The subcellular localization of P3-PiPo protein in Nicotiana benthamiana leaf epidermal cells shown a little punctate inclusions along the plasma membrance. Its co-localization with NIb were detected in karyotheca. When co-expressing P3-PiPo with NIb、6K2-NIa, we can observed P3-PiPo together with 6K2-NIa, accumulating in the viral-derived vesicles beside the choropalst, where is the site of VCR. So we can suggest that P3-PiPo was involved in the virus replication.Taken together, the functional characterization of potyvirus-encoded membrane-associated proteins promoted the research of potyviruses replication mechanisms. These data contain that the hypothetical model for that ER-derived membranous vesicles induced by 6K2 protein playing a key role in potyviruses replication through interaction with ATO, that the possible dual roles of P3 in viral replication and movement, that the P3-PiPo involved in the VCR.更多还原