MOLECULAR DISSECTION OF THE INFLUENZA VIRUS RNA POLYMERASE AND ITS APPLICATION TO ANALYSIS OF VIRAL REPLICATION.

具有亚基结构PB1-PB2-PA的流感病毒RNA聚合酶参与RNA基因组的转录和复制。为了研究每个亚基的功能，我们在体外使用携带第8基因组片段末端和每个组合含有亚基的昆虫细胞核提取物的短模型RNA模板进行转录。我们证明了含有至少PB1亚基的核提取物在含有或不含二核苷酸ApG的模型RNA模板上催化RNA合成。这一结果直接表明PB1亚基是流感病毒RNA聚合酶的催化亚基。通过转染不同组合的编码野生型和序列缺失突变体的P蛋白亚基，并与亚基特异性抗体共免疫沉淀，确定了所有3种P蛋白上的亚基-亚基接触位点。结果表明，PB1-PB2和PB1-PA之间形成二元配合物，而PB2-PA之间不形成二元配合物。因此，我们得出结论，PB1是病毒RNA聚合酶组装的核心亚基。PB1的c端158个氨基酸与PB2的n端249个氨基酸相结合，而PB1的n端140个氨基酸与PA的c端2 / 3区相结合。在没有PB1亚基的情况下表达时，未检测到PB2-PA结合。

Infuluenza virus RNA polymerase with the subunit structure PB1-PB2-PA is involved in both transcription and replication of the RNA genome. In order to study the function of each subunit we performed transcription in vitro using short model RNA templates carrying both termini of 8th genome segment and insect cell nuclear extracts containing subunits at each combination. And we demonstrated that the nuclear extracts containing at least PB1 subunit catalyzed RNA synthesis on model RNA templates with or without dinucleotide ApG.This result directly indicate that PB1 subunit is a catalytic subunit of influenza virus RNA polymerase.By transfection of various combinations of cDNA encoding wild-type and serial deletion mutants of each P protein subunit and co-immunoprecipitation with subunit-specific antibodies, the subunit-subunit contact sites on all the three P proteins were determined. Results indicate that the binary complexes are formed between PB1-PB2 and PB1-PA but not PB2-PA.Therefore, we concluded that PB1 is a core subunit for assembly of the viral RNA polymerase. The C-terminal 158 amino acids of PB1 bound to the N-terminal 249 amino acid stretch of PB2, while the N-terminal 140 amino acids of PB1 bound to the C-terminal two third region of PA.PB2-PA binding was not detected when they were expressed in the absence of PB1 subunit.