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.

paramyxovirus家族的成员仙台病毒（SEV）含有大约15千碱基的单核负RNA RNA基因组。为了阐明SEV的转录和复制机制，我们使用纯化的病毒颗粒开发了一种有效而忠实的体外转录系统。我们先前已经表明，在体外mRNA合成SEV几乎完全取决于添加细胞蛋白（宿主因子）。在本研究中，我们进一步冻结并表征了宿主因子活性，并获得了以下结果。 1）我们从牛脑中纯化了宿主因子活性，该活性在牛脑中刺激了体外SEV mRNA合成，并发现该活性由四种互补蛋白质因子，微管蛋白，磷酸甘油酸激酶，烯醇酶，烯醇酶和未识别的P24组成。 2）我们表明小管蛋白通过从RNP中去除病毒M蛋白并将自己整合到复合物中，参与转录起始复合物的形成。另一方面，发现磷酸甘油酸激酶，烯醇酶和p24在RNA shain伸长步骤上起作用。此外，3）我们证明了重组病毒L蛋白具有帽甲基酶（mRNA鸟嘌呤-7-甲基转移酶）的活性。

项目成果

E.Kajita, M.Wakiyama, K.Miura, K.Mizumoto, T.Oka, I.Komuro, T.Miyata, H.Yatsuki, K.Hori, and K.Shiokawa: "Isolation and characterization of Xenopus laevis aldolase B cDNA and expression patterns of aldolase A, B, and C genes in adult tissues, oocytes and

E.Kajita、M.Wakiyama、K.Miura、K.Mizumoto、T.Oka、I.Komuro、T.Miyata、H.Yatsuki、K.Hori 和 K.Shiokawa：“非洲爪蟾醛缩酶 B 的分离和表征

Furukawa, T., Konishi, F., Shitoh, K., Kojima, M., Nagai, H., Tsukamoto, T: "Evaluation of screening strategy for detecting hereditary nonpolyposis colorectal"Cancer. 94. 911-920 (2002)

Furukawa, T.、Konishi, F.、Shitoh, K.、Kojima, M.、Nagai, H.、Tsukamoto, T：“检测遗传性非息肉病性结直肠癌的筛查策略的评估”。

Honda, A., Mizumoto, K., Ishihama, A: "Minimum molecular architectures for transcription and replication of the influenza"Proc. Natl. Acad. Sci. USA. 99. 13166-13171 (2002)

Honda, A.、Mizumoto, K.、Ishihama, A：“流感转录和复制的最小分子结构”Proc。

T.Yamochi et al.: "p7Oik3-1 is phosphcnylated by cyclin-dependent kinase 3 (cdk 3)"Eur. J. Biochem.. 268. 6076-6082 (2001)

T.Yamochi 等人：“p7Oik3-1 被细胞周期蛋白依赖性激酶 3 (cdk 3) 磷酸化”Eur。

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。