請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/95771
標題: 西瓜銀斑病毒之核鞘蛋白與病毒基因體體外組裝之探討
In-vitro encapsidation of genomic RNA segments of watermelon silver mottle virus with its nucleocapsid protein
作者: 陳樂融
Yue- Rong Tan
關鍵字: 核鞘蛋白
病毒基因體體外組裝
RNP
NP
in vitro encapsidation, RNP
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摘要: 逆向遺傳學系統對於了解植物RNA病毒的複製週期和致病機制至關重要。然而,目前尚未有人成功地建構出具有感染力的Orthotospovirus屬的病毒載體。過去研究顯示,將西瓜銀斑病毒(watermelon silver mottle virus, WSMoV)L,M和S全長cDNA接種到表現核鞘蛋白(NP)和RNA聚合酶(L)蛋白的轉基因煙草植物中,研究顯示此方法無法建立該病毒之感染性。因此,我們假設從病毒基因體構築之cDNA構建體轉錄的mRNA形成穩定的病毒核糖核蛋白(ribonucleoprotein, RNP)對於在寄主植物感染過程中保護病毒基因組RNA是不可或缺的。本研究目的是探討WSMoV RNP體外組裝的條件。為了測定體外RNA的組裝能力,將試管內轉錄的帶有GFP基因片段的WSMoV S GFP-RNA與由大腸桿菌表達的重組核鞘蛋白(rNP)進行組裝。我們首先發現超高速離心純化之WSMoV成分之具有感染性的分管(P25 infectious fraction)與未純化之WSMoV病毒原液接種於葵藜後有相似的密集單斑形成。更近一步利用西方墨點法可於純化之WSMoV分管成分中偵測到大量的L、NSs、NP和NSm蛋白。此外,抗體中和實驗 (neutralization assay)顯示針對L、NSs、NP和NSm蛋白的抗體抑制了P25的感染力。由此可見,這四種病毒蛋白具有非常重要的複製核心功能。透過電子顯微鏡觀察此具有感染性的分管成分P25可看到完整的核鞘蛋白體,而此RNP可以保護RNA不受RNase的分解。後續實驗顯示細菌表現的rNP可以跟P25抽取的病毒RNA以及試管內轉錄的S GFP-RNA進行體外組裝,並於電子顯微鏡下觀察到形成長鏈狀的RNP特徵,顯示病毒RNA可以被rNP包裹成核鞘蛋白複合體RNP。此人工合成的RNP可以被利用接種試驗,以分析此人工RNP是否會在具有感染力分管存在時進行複製。
Reverse genetics studies are pivotal for understanding replication cycle and pathogenesis of plant RNA viruses. However, successful infectious clones for negative-sense RNA viruses of the multipartite genus Orthotospovirus have yet to be created. Introduction of watermelon silver mottle virus (WSMoV) L, M and S full-length cDNAs into transgenic Nicotiana benthamiana plants expressing nucleocapsid protein (NP) and RNA polymerase (L) in previous studies showed no infectivity. We hypothesized that formation of stable viral ribonucleoprotein (RNP) with the mRNAs transcribed from infiltrated cDNA constructs is essential to protect the viral genomic RNAs during bioassay for transcription and replication. Thus, the objective of this study was directed to determine the conditions for the in vitro assembly of WSMoV RNP. Our inoculation results of a subcellular fraction of WSMoV designated an infectious fraction P25 portrayed numerous local lesions on Chenopodium quinoa plants similar to those induced by the wild type WSMoV. In this infectious fraction, abundant amounts of L protein, NSs protein, NP, and NSm protein were detected by western blotting. The infectivity of this fraction, however, was greatly reduced when treated with PAb of L, NSs, NSm, and N proteins in neutralization assay. RNP from the P25 fractions was also resistant to RNase digestion. Therefore, bacteria-expressed rNP was used for the in vitro assembly of the viral RNA with the in vitro transcribed S GFP-RNA carrying GFP ORF which replaces the N ORF. Electron microscopy results of the encapsidated products indicated that bacteria-expressed rNP can successfully assemble both viral RNA and in vitro transcribed S GFP-RNA into long and linear RNP-like structures. Taken together, our results suggested that native and transcribed single-stranded orthotospoviral RNAs can be assembled in vitro using bacteria-expressed rNP. The RNP-like structures may be used in generating reverse genetics system of WSMoV.
URI: http://hdl.handle.net/11455/95771
文章公開時間: 10000-01-01
顯示於類別:植物病理學系

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