STRUCTURE-FUNCTION RELATIONSHIP OF VIRAL RNA DEPENDENT RNA POLYMERASE.

甲型流感病毒依赖RNA的NA聚合酶由PB1、PB2和PA三个亚基组成。PB1是NA合成的催化亚基。PB2赋予PB1转录酶活性。PA提供复制酶活性，并执行最近发现的独特的蛋白酶活性。为了定位其功能域，我们产生了位点特异性抗体。RNA合成活性定位于PB2的1-159位氨基酸和PA的501-617位氨基酸。依赖CaP-I的核糖核酸酶活性定位于PB2的1-159和305-559氨基酸，PB1的1-222氨基酸和PA的601-716氨基酸。帽子结合位点定位于PB2的402-559位氨基酸，我们先前确定PB1的C端158个氨基酸与PB2的N端1 249个氨基酸结合，而PB1的N端140个氨基酸与PA的C端2/3区域结合。这一次，我们缩小了PB2的28个残基和PB1的180个残基的PB1-PB2结合位点，缩小了PB1的25个残基和PA200个残基的PB1-PA结合部位。为了将我们的知识扩展到其他病毒依赖RNA的RNA聚合酶，我们建立了丙型肝炎病毒RNA聚合酶的体外转录系统。

The RNA-dependent NA polymerase of influenza A virus consists of three subunits, pB1, PB2 and PA. PB1 is a catalytic subunit of NA synthesis. PB2 confers a transcriptase activity to PB1. PA provides replicase activity and performs a recently identified unique protease activity. In order to map its functional domains, we generated site-specific antibodies. RNA synthesis activity was mapped in amino acids 1-159 of PB2 and 501-617 of PA. Cap-I dependent RNase activity was mapped in amino acids 1-159 and 305-559 of PB2, 1-222 of PB1, and 601-716 of PA. Cap-binding site was mapped in amino acids 402-559 of PB2.We previously determined that the C-terminal 158 amino acids of PB1 bound to the N-termina1 249 amino acid stretch of PB2, while the N-terminal 140 amino acids ofPB1 bound to the C-terminal two third region of PA. This time, we have narrowed the PB1-PB2 binding site in 28 residues of PB2 and 180 residues of PB1, and the PB1-PA binding site in 25 residues of PB1 and about 200 residues of PA.In order to expand our knowledge into other viral RNA-dependent RNA polymerases, we established in vitro transcription system of hepatitis C RNA polymerase.