In vitro synthesis of minus-strand RNA by an isolated Cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3′ end of the virus RNA
In vitro synthesis of minus-strand RNA by an isolated Cereal yellow dwarf virus RNA-dependent RNA polymerase requires VPg and a stem-loop structure at the 3′ end of the virus RNA
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DOI：
10.1128/jvi.01050-06
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发表时间：
2006-11-01
影响因子：
5.4
通讯作者：
Buck, Kenneth W.
中科院分区：
文献类型：
--
作者：
Osman, Toba A. M.;Coutts, Robert H. A.;Buck, Kenneth W.
小麦黄矮病病毒(CyDV)RNA含有一个5‘端基因组连锁蛋白(VPG)。我们在细菌中表达了CyDV基因组的VPG区，并用纯化的蛋白(BVPg)制备了一种抗血清，该抗血清能够检测感染CyDV的燕麦提取液中的游离VPG。用BAL-31核酸酶处理环状病毒膜结合的RNA聚合酶，得到模板依赖的RNA依赖的RNA聚合酶(RdRp)。RdRp是模板特异性的，能够利用来自CyDV正链和负链RNA的模板，但不能利用三种无关病毒：红三叶坏死性花叶病毒、黄瓜花叶病毒和烟草花叶病毒的模板。RdRp催化的RNA合成需要3‘端的Gu序列和bVPg的存在。此外，在正链RNA模板上合成负链RNA需要在CyDV RNA的3‘末端附近存在一个假定的茎环结构。碱基配对的茎、茎中的单核苷酸(A)凸起和四环序列都是模板活性所必需的。有证据表明，体外负链合成是由模板3‘端的bVPg引发的。这些数据与RdRp与茎环结构结合的模型一致，该结构定位活性位点以识别3‘-末端GU序列，通过在VPG中添加A残基来启动RNA合成。
Cereal yellow dwarf virus (CYDV) RNA has a 5 '-terminal genome-linked protein (VPg). We have expressed the VPg region of the CYDV genome in bacteria and used the purified protein (bVPg) to raise an antiserum which was able to detect free VPg in extracts of CYDV-infected oat plants. A template-dependent RNA-dependent RNA polymerase (RdRp) has been produced from a CYDV membrane-bound RNA polymerase by treatment with BAL 31 nuclease. The RdRp was template specific, being able to utilize templates from CYDV plus- and minus-strand RNAs but not those of three unrelated viruses, Red clover necrotic mosaic virus, Cucumber mosaic virus, and Tobacco mosaic virus. RNA synthesis catalyzed by the RdRp required a 3 '-terminal GU sequence and the presence of bVPg. Additionally, synthesis of minus-strand RNA on a plus-strand RNA template required the presence of a putative stem-loop structure near the 3 ' terminus of CYDV RNA. The base-paired stem, a single-nucleotide (A) bulge in the stem, and the sequence of a tetraloop were all required for the template activity. Evidence was produced showing that minus-strand synthesis in vitro was initiated by priming by bVPg at the 3 ' end of the template. The data are consistent with a model in which the RdRp binds to the stem-loop structure which positions the active site to recognize the 3 '-terminal GU sequence for initiation of RNA synthesis by the addition of an A residue to VPg.