Mechanism of transcription of Paramyxovirus genome : Purification and characterization of host factors

副粘病毒基因组的转录机制：宿主因子的纯化和表征

• 批准号: 11470080
• 负责人: MIZUMOTO Kiyohisa
• 金额: $ 7.87万
• 依托单位: Kitasato University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11470080/
• 关键词: Paramyxovirus; Sendai virus; negative-strand RNA genome; RNA-dependent RNA polymerase; host factor; transcription; mRNA cap; tubulin; ウイルスmRNA合成; mRNAキャップ構造; キャップメチル化酵素; ウイルスLタンパク質; マイナス鎮RNAゲノム; ウイルスmRNA; mRNAキャッピング; パラミフソウイルス; パラミクソウイルス科

Sendai virus (SeV), a member of Paramyxovirus family, contains a monopartite negative strand RNA genome of approximately 15 kilobases. To elucidate the mechanism of transcription and replication of SeV, we developed an efficient and faithful in vitro transcription system using purified virus particles. We have previously shown that in vitro mRNA synthesis of SeV is almost entirely dependent on the addition of cellular proteins (host factors). In the present study, we further furified and characterized the host factor activity, and obtained the following results. 1) We purified the host factor activity, which stimulates in vitro SeV mRNA synthesis, from bovine brain, and found that the activity consists of four complementary protein factors, tubulin, phosphoglycerate kinase, enolase, and unidentified p24. 2) We showed that tubulin is involved in a transcription initiation complex formation by removing the viral M protein from the RNP, and by being integrated itself into the complex. On the other hand, phosphoglycerate kinase, enolase, and p24 are found to act at RNA shain elongation step. Furthermore, 3) we demonstrated that a recombinant viral L protein exhibits the cap methylase (mRNA guanine-7-methyltransferase) activity.

仙台病毒（SeV）是副粘病毒科的一员，含有约15千碱基的单极负链RNA基因组。为了阐明SeV的转录和复制机制，我们利用纯化的病毒颗粒构建了高效、可靠的体外转录系统。我们之前已经表明，SeV的体外mRNA合成几乎完全依赖于细胞蛋白（宿主因子）的添加。在本研究中，我们进一步对宿主因子活性进行了提纯和表征，得到了以下结果：1)我们从牛脑中纯化了刺激体外SeV mRNA合成的宿主因子活性，发现其活性由四种互补的蛋白因子组成，即微管蛋白、磷酸甘油酸激酶、烯醇化酶和未识别的p24。2)我们发现微管蛋白通过从RNP中去除病毒M蛋白并将自身整合到复合物中参与转录起始复合物的形成。另一方面，磷酸甘油酸激酶、烯醇化酶和p24被发现在RNA链的延伸阶段起作用。此外，3)我们证明了重组病毒L蛋白具有帽甲基化酶（mRNA鸟嘌呤-7-甲基转移酶）活性。

项目成果

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S.Muraoka 等：“木材腐烂真菌 Galerina fasciculata 和 Galerina helvoliceps 中鹅膏菌素的检测和鉴定”Appl。

K.Tsuji et al.: "Differential ellect of ik3-1/Cables on p53-and p73-induced cell death"J. Biol. Chem.. 277. 2951-2957 (2002)

K.Tsuji 等人：“ik3-1/Cables 对 p53 和 p73 诱导的细胞死亡的差异选择”J。

E.Kajita et al.: "Isolation and characterization of Xenopus laevis aldolase B cDNA and expression patterns of aldolase A, B, and C genes in adult tissues, oocytes and embryos of Xenopus laevis."Biochemica et Biophysica Acta. 1493. 101-118 (2000)

E. Kajita 等人：“非洲爪蟾醛缩酶 B cDNA 的分离和表征，以及非洲爪蟾成体组织、卵母细胞和胚胎中醛缩酶 A、B 和 C 基因的表达模式。”生物化学与生物物理学学报。