Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 <i>in vitro</i>

作     者：Cristofari, G Gabus, C Ficheux, D Bona, M Le Grice, SFJ Darlix, JL 

作者机构：Ecole Normale Super Lyon INSERM LaboRetro Unite Virol HumaineU412 F-69364 Lyon 07 France Inst Biol & Chim Prot F-69367 Lyon France NCI Frederick Canc Res & Dev Ctr HIV Drug Resistance Program Div Basic SciNIH Frederick MD 21702 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第51期

页      面：36643-36648页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 081704[工学-应用化学] 07[理学] 08[工学] 0817[工学-化学工程与技术] 

基　　金：NIAID NIH HHS [AI31147] Funding Source: Medline 

主　　题：氨基酸序列 真菌蛋白质类/遗传学 HIV逆转录酶/遗传学 分子序列数据 核蛋白类/遗传学 反转录因子/遗传学 酿酒酵母菌/酶学 酿酒酵母菌/遗传学 人类 

摘      要：Human immunodeficiency virus (HIV) and the distantly related yeast Ty3 retrotransposon encode reverse transcriptase (RT) and a nucleic acid-binding protein designated nucleocapsid protein (NCp) with either one or two zinc fingers, required for HIV-1 replication and Ty3 transposition, respectively. In vitro binding of HIV-1 NCp7 to viral 5 RNA and primer tRNA(3)(Lys) catalyzes formation of nucleoprotein complexes resembling the virion nucleocapsid. Nucleocapsid complex formation functions in viral RNA dimerization and tRNA annealing to the primer binding site (PBS). RT is recruited in these nucleoprotein complexes and synthesizes minus-strand cDNA initiated at the PBS. Recent results on yeast Ty3 have shown that the homologous NCp9 promotes annealing of primer tRNA(i)(Met) to a 5 -3 bipartite PBS, allowing RNA:tRNA dimer formation and initiation of cDNA synthesis at the 5 PBS (1), To compare specific cDNA synthesis in a retrotransposon and HIV-1, we have established a Ty3 model system comprising Ty3 RNA with the 5 -3 PBS, primer tRNA(i)(Met), NCp9, and for the first time, highly purified Ty3 RT. Here we report that Ty3 RT is as active as retroviral HIV-1 or murine leukemia virus RT using a synthetic template-primer system, Moreover, and in contrast to what was found with retroviral RTs, retrotransposon Ty3 RT was unable to direct cDNA synthesis by self-priming. We also show that Ty3 nucleoprotein complexes were formed in vitro and that the N terminus of NCp9, but not the zinc finger, is required for complex formation, tRNA annealing to the PBS, RNA dimerization, and primer tRNA-directed cDNA synthesis by Ty3 RT. These results indicate that NCp9 chaperones bona fide cDNA synthesis by RT in the yeast Ty3 retrotransposon, as illustrated for NCp7 in HIV-1, reinforcing the notion that Ty3 NCp9 is an ancestor of HIV-1 NCp7.