The VP2 protein of grass carp reovirus (GCRV) expressed in a baculovirus exhibits RNA polymerase activity
The VP2 protein of grass carp reovirus (GCRV) expressed in a baculovirus exhibits RNA polymerase activity
复制标题
杆状病毒中表达的草鱼呼肠孤病毒（GCRV）的VP2蛋白表现出RNA聚合酶活性
DOI：
10.1007/s12250-014-3366-5
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发表时间：
2014-04-01
影响因子：
5.5
通讯作者：
Fang, Qin
中科院分区：
文献类型：
--
作者：
Fan, Liming;Liu, Huan;Fang, Qin
双壳草鱼呼肠孤病毒（GCRV）能够进行内源RNA转录和加工。基因组序列分析表明，由基因片段 2 (S2) 编码的蛋白质 VP2 是假定的 RNA 依赖性 RNA 聚合酶 (RdRp)。在前期工作中，我们在大肠杆菌中表达了与RNA聚合酶活性相关的VP2功能区（记为rVP2（390-900）），并制备了针对VP2的多克隆抗体。为了表征 GCRV RNA 聚合酶，首先构建了重组全长 VP2 (rVP2)，并在杆状病毒系统中表达为带有 His 标签的融合蛋白。免疫荧光 (IF) 测定以及表达细胞提取物和纯化的 His 标记 rVP2 的免疫印迹 (IB) 分析表明，rVP2 在 Sf9 细胞中成功表达。复制酶活性的进一步表征表明，纯化的 rVP2 和 GCRV 颗粒表现出聚 (C) 依赖性聚 (G) 聚合酶活性。 RNA酶活性需要二价阳离子Mg2+，并且在28℃下达到最佳。结果为进一步研究水生病毒转录和复制过程中的RNA聚合酶奠定了基础。
The double-shelled grass carp reovirus (GCRV) is capable of endogenous RNA transcription and processing. Genome sequence analysis has revealed that the protein VP2, encoded by gene segment 2 (S2), is the putative RNA-dependent RNA polymerase (RdRp). In previous work, we have ex-pressed the functional region of VP2 that is associated with RNA polymerase activity (denoted as rVP2(390-900)) in E. coli and have prepared a polyclonal antibody against VP2. To characterize the GCRV RNA polymerase, a recombinant full-length VP2 (rVP2) was first constructed and expressed in a baculovirus system, as a fusion protein with an attached His-tag. Immunofluorescence (IF) assays, together with immunoblot (IB) analyses from both expressed cell extracts and purified His-tagged rVP2, showed that rVP2 was successfully expressed in Sf9 cells. Further characterization of, the replicase activity showed that purified rVP2 and GCRV particles exhibited poly(C)-dependent poly(G) polymerase activity. The RNA enzymatic activity required the divalent cation Mg2+, and was optimal at 28 degrees C. The results provide a foundation for further studies on the RNA polymerases of aquareoviruses during viral transcription and replication.