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

流感病毒 RNA 聚合酶的分子解剖及其在病毒复制分析中的应用。

• 批准号: 07670354
• 负责人: TOYODA Tetsuya
• 金额: $ 1.41万
• 依托单位: KURUME UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670354/
• 关键词: INFLUENZA VIRUS; RNA POLYMERASE; SUBUNIT; TRANSCRIPTION; REPLICATION; ANTIBODY; バイナリー複合体

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.

流感病毒RNA聚合酶具有亚基结构PB 1-PB 2-PA，参与RNA基因组的转录和复制。为了研究每个亚基的功能，我们使用携带第8基因组片段两端的短模型RNA模板和含有每个组合的亚基的昆虫细胞核提取物进行体外转录。我们证明了至少含有PB 1亚基的核提取物在有或没有二核苷酸ApG的模型RNA模板上催化RNA合成，这一结果直接表明PB 1亚基是流感病毒RNA聚合酶的催化亚基。测定了所有三种P蛋白上的亚基-亚基接触位点。结果表明，PB 1-PB 2和PB 1-PA形成二元复合物，而PB 2-PA不形成二元复合物，说明PB 1是病毒RNA聚合酶组装的核心亚基。PB 1的C端158个氨基酸与PB 2的N端249个氨基酸结合，而PB 1的N端140个氨基酸与PA的C端2/3区域结合。

项目成果

Makoto Kobayashi, Tetsuya Toyoda, and Akira Ishihama.: "Influenza virus PB1 protein is the minimal and essential subunit of RNA polymerase." Arch.Virol.141. 525-539 (1996)

Makoto Kobayashi、Tetsuya Toyoda 和 Akira Ishihama：“流感病毒 PB1 蛋白是 RNA 聚合酶的最小且必需的亚基。”

Tetsuya Toyoda, Djanybek M.Adyshev, Makoto Kobayashi, Akira Iwata, Susumu Ueda, and Akira Ishihama.: The subunit-subnit contact sites of the influenza virus RNA polymerase.In 'Options for the control of influenza III.(L.E.Brown, A.W.Hampson and R.G.Webste

Tetsuya Toyoda、Djanybek M.Adyshev、Makoto Kobayashi、Akira Iwata、Susumu Ueda 和 Akira Ishihama。：流感病毒 RNA 聚合酶的亚基-亚基接触位点。在“控制 III 型流感的选项”中。（L.E.Brown，A.W.

Masanobu Chinami: "Nucleic acid binding at zinc finger-like motif of human papillomavirus type16 E7 oncoprotein." J. virol. method. (in press). (1996)

Masanobu Chinami：“人乳头瘤病毒 16 型 E7 癌蛋白的锌指样基序上的核酸结合。”

豊田哲也: "ウイルスRNAポリメラーゼの分子解剖" 久留米医学会雑誌. 59. 165-172 (1996)

丰田哲也：“病毒 RNA 聚合酶的分子解剖学”久留米医学会杂志 59. 165-172 (1996)。

L.E.Brown,et al.eds: "Options for the control of influenza III." Elsevier Science B.V.,Amsterdam, 860 (1996)

L.E.Brown 等人编着：“控制 III 型流感的选择”。