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Activity Control of Viral RNA Polymerase by Viral RNA Effecter

病毒RNA效应子对病毒RNA聚合酶活性的控制

基本信息

批准号：
15570150
负责人：
HONDA Ayae
金额：
$ 2.5万
依托单位：
Nippon Institute for Biological Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

The genome of influenza virus consists of eight segments of negative-strand RNA. Both transcription and replication of the viral genome is catalyzed by the influenza virus RNA-dependent RNA polymerase, which is composed of three viral protein subunits, PB1, PB2 and PA. The aim of this research was to understand how the functions of influenza virus RNA-dependent RNA polymerase were regulated. We succeeded the purification of enzymatically active RNA-dependent RNA polymerase from insect cells co-infected with three species of the recombinant baculovirus, each expressing one of three subunits. We analyzed the activity and specificity of the purified RNA polymerase, and got several novel findings as follows :1)The purified RNA polymerase gained the enzyme activity, as measured by endonucleolytic cleavage of capped RNA, only when viral RNA (vRNA) was added. We then proposed a model that vRNA has an "effector" role in controlling the enzyme activity.2)This finding encouraged us to identify t … More he essential RNA element needed for the effector function. Various recombinant plasmids were constructed, which express the chimeric promoters between vRNA and cRNA. After analysis of the effector activity for these artificial promoters, we found that the bulge structure in the promoter region was necessary for activation of the RNA polymerase.3)The RNA polymerase required primers, ApG or capped poly(A), for vRNA-dependent synthesis of RNA as in the case of viral RNP-associated RNA polymerase. This indicates that the RNA polymerase carries the specificity of transcriptase. However, the activity of cRNA-dependent unprimed RNA synthesis was very weak, in agreement with our finding that a host factor(s) is involved in the functional conversion of trascriptase to replicase. Candidates of the putative host factor have been identified by yeast two-hybrid screening.4)Primer Binding site on PB1 was identified using radioactive primer (ApG), which was synthesized using ATP analogue and radioactive GTP. After cross-linking of the radioactive primer, we identified the primer-binding site after digestion of PB1 protein by protease. The primer-binding region was located close to the catalytic site of RNA synthesis on the PB1 subunit Less

流感病毒的基因组由八段负链RNA组成。病毒基因组的转录和复制都由流感病毒RNA依赖性RNA聚合酶催化，该聚合酶由三个病毒蛋白亚基PB1、PB2和PA组成。本研究的目的是了解流感病毒RNA依赖的RNA聚合酶的功能是如何调节的。我们成功地纯化酶活性的RNA依赖的RNA聚合酶从昆虫细胞共感染的三种重组杆状病毒，每个表达三个亚基之一。对纯化的RNA聚合酶进行了活性和特异性分析，得到以下新的发现：（1）纯化的RNA聚合酶只有在加入病毒RNA（vRNA）时才具有酶活性。然后，我们提出了一个模型，即vRNA在控制酶活性方面具有“效应器”作用。 ...更多信息效应子功能所需的基本RNA元件。构建了表达vRNA和cRNA嵌合启动子的各种重组质粒。在分析这些人工启动子的效应子活性后，我们发现启动子区域中的凸起结构对于RNA聚合酶的激活是必需的。3）RNA聚合酶需要引物ApG或加帽的poly（A），以用于与病毒RNP相关的RNA聚合酶一样的vRNA依赖性RNA合成。这表明RNA聚合酶具有转录酶的特异性。然而，依赖于cRNA的未引发RNA合成的活性非常弱，这与我们的发现一致，即宿主因子参与了转录酶向复制酶的功能转化。4）利用ATP类似物和放射性GTP合成的放射性引物（ApG）鉴定了PB1上的引物结合位点。在放射性引物交联后，我们确定了PB1蛋白酶消化后的引物结合位点。引物结合区位于PB1亚基上RNA合成的催化位点附近。