Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/35932
標題: 竹嵌紋病毒負股3'端序列於病毒複製其基因體之功能分析
The Functional Studies of the 3'-terminal Nucleotides of BaMV Minus-Strand RNA in the Synthesis of Genomic RNA
作者: 陳怡靜
Chen, Yi-Jing
關鍵字: dsRNA;BaMV;genomic RNA synthesis
出版社: 生物科技學研究所
摘要: 
複製是病毒感染寄主細胞後的主要工作。已知正股RNA病毒利用在負股3'端多加上一個unpaired guanosine,以增加病毒複製聚合酶(RNA-dependent RNA polymerase, RdRp)的複製效率,例如胡瓜嵌紋病毒(Cucumber mosaic virus)及雀麥嵌紋病毒(Brome mosaic virus)。其複製的過渡形式(replicative intermediate)中,負股的3'端皆存在一個unpaired guanosine。竹嵌紋病毒為正股RNA植物病毒,隸屬於Flexiviridae科中之Potexvirus屬。我們想知道,是否竹嵌紋病毒也是利用此策略來增加RdRp的複製其基因體的數量。因此,我們分離出病毒的複製的過渡形式RNA,以3' RACE的方式分析竹嵌紋病毒負股的3'端序列。定序結果顯示竹嵌紋病毒負股RNA的3'端,也具有non-template guanosine。推測竹嵌紋病毒也利用相同的複製策略來增加RdRp的複製效率。
為了了解這個 unpaired guanosine是否的確在竹嵌紋病毒複製過程中扮演重要的功能,我們進行體外(in vitro)複製分析(RdRp assay)。然而結果顯示,具有extra guanosine 的負股RNA相對於不含extra nucleotide的負股RNA,RdRp並沒有較有效率的合成正股RNA in vitro。我們認為,此額外的guanosine的功能,可能在於病毒複製的細微調控,或是具有其他功能。
此外,根據先前實驗顯示,竹嵌紋病毒負股3'端,Ba-77,會形成二級結構做為複製基因體的啟動子。但是針對病毒基因體複製過程中,Ba-77的序列對於病毒核酸複製酶在合成起始的影響仍未知。因此,我們針對負股核酸3'端RNA序列,進行體外複製分析,以了解這些序列在病毒RNA複製過程中的重要性及其可能扮演的角色。而根據結果,負股核酸3'端的第一個核苷酸對於病毒核酸聚合酶是很重要的,將其置換為其他核苷酸將會對病毒RNA的合成造成嚴重的影響。因此我們認為RdRp 以helicase-like domain和Ba-77之large stem-loop結合後,RdRp domain因此辨認第一個核苷酸,開始進行複製正股的工作。
另外,針對竹嵌紋病毒負股3'端,序列U的長度對病毒複製的影響。先前本實驗室已經證實,此U的長度對於病毒在protoplast中會影響病毒正股RNA的累積,此病毒RNA累積量減少的現象,並非因為減少了病毒RdRp產量的緣故,因此,很有可能是因為影響了病毒複製。為了證實這個可能性,我們進行體外複製實驗 (in vitro RdRp replication assay),但是在in vitro中並無法明顯顯示出序列U長度在病毒複製中的重要性。

It has been found in a few positive-sense RNA viruses such as Brome mosaic virus (BMV) and Cucumber mosaic virus (CMV) that there is the presence of an unpaired guanosine residue at the 3'-end of the minus-strand RNA of the replicative intermediate for enhancing the synthesis efficiency of RNA-dependent RNA polymerase (RdRp) in vitro. Bamboo mosaic virus (BaMV) is a positive-strand RNA virus belonging to the genus Potexvirus of the family Flexiviridae. We were interested to know if the same strategy is employed by BaMV. We selectively isolated the replicative intermediate and subjected them to 3' RACE of the minus-strand RNA, cloned and sequenced the RNA. Results showed that BaMV indeed has a non-template G at the 3-end of minus-strand during viral RNA replication. However, no significant enhanced the RdRp polymerization efficiency in vitro with the template containing an extra nucleotide, irrespective of the type of the extra nucleotide.
Furthermore, we were also interested in the function of the 3' sequences of BaMV minus-strand RNA in viral replication. So we did the in vitro RdRp assay in the context of the first nucleotide of the 3' minus RNA template. The result shows that the first nucleotide of minus-strand RNA is very important for RdRp synthesis. It cannot be substituted by any other nucleotides. We suggest after RdRp bind to Ba-77 by its helicase-like domain at the large stem-loop, the first nucleotide of minus-strand RNA will be recognized by polymerase domain and start polymerization. It has been known U-length of 3' minus strand is important for viral RNA accumulation. We also try to analyze the U-length in 3' minus strand by RdRp assay to understand if the role of
U-length is involved in the synthesis of RdRp. But it doesn't show the strong relationship in vitro.
URI: http://hdl.handle.net/11455/35932
Appears in Collections:生物科技學研究所

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