Activity Control of Viral RNA Polymerase by Viral RNA Effecter

流感病毒基因组由8段负链RNA组成。病毒基因组的转录和复制都是由流感病毒RNA依赖的RNA聚合酶催化的，该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依赖性RNA聚合酶的功能是如何被调控的。我们成功地从三种重组杆状病毒共感染的昆虫细胞中纯化了酶活性RNA依赖RNA聚合酶，每种杆状病毒表达三个亚基中的一个。我们分析了纯化的RNA聚合酶的活性和特异性，得到了以下几个新发现：1)纯化的RNA聚合酶只有在加入病毒RNA （vRNA）时才能获得酶活性，这是通过对带帽RNA的核内溶裂解来测定的。然后我们提出了一个模型，vRNA在控制酶活性方面具有“效应”作用。2)这一发现鼓励我们进一步确定效应功能所需的基本RNA元件。构建了多种表达vRNA与cRNA嵌合启动子的重组质粒。在分析了这些人工启动子的效应活性后，我们发现启动子区域的凸起结构是RNA聚合酶激活所必需的。3) RNA聚合酶需要引物，如ApG或capped poly(A)，用于RNA依赖的RNA合成，如病毒RNA相关RNA聚合酶。这说明RNA聚合酶具有转录酶的特异性。然而，RNA依赖的非引物RNA合成活性非常弱，这与我们的发现一致，即一个宿主因子参与了转录酶到复制酶的功能转化。通过酵母双杂交筛选已确定了候选宿主因子。4)利用ATP类似物和放射性GTP合成的放射性引物（ApG）确定PB1上的结合位点。将放射性引物交联后，用蛋白酶消化PB1蛋白，确定引物结合位点。引物结合区位于PB1亚基Less上的RNA合成催化位点附近

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