Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro
Characterization of active reverse transcriptase and nucleoprotein complexes of the yeast retrotransposon Ty3 in vitro
复制标题
DOI：
10.1074/jbc.274.51.36643
复制
发表时间：
1999-12-17
影响因子：
4.8
通讯作者：
Darlix, JL
中科院分区：
文献类型：
--
作者：
Cristofari, G;Gabus, C;Darlix, JL
人类免疫缺陷病毒（HIV）和远亲酵母Ty 3逆转录转座子编码逆转录酶（RT）和一种核酸结合蛋白，称为核衣壳蛋白（NCp），具有一个或两个锌指，分别为HIV-1复制和Ty 3转座所需。在体外，HIV-1 NCp 7与病毒5' RNA和引物tRNA（3）（Lys）的结合可催化类似于病毒粒子核衣壳的核蛋白复合物的形成。核衣壳复合物形成在病毒RNA二聚化和tRNA退火至引物结合位点（PBS）中起作用。RT在这些核蛋白复合物中被募集，并在PBS中合成起始的负链cDNA。最近对酵母Ty 3的研究结果表明，同源NCp 9促进引物tRNA（i）（Met）与5 '-3'二分PBS的退火，使RNA：为了比较逆转录转座子和HIV-1中特异性的cDNA合成，我们建立了一个Ty 3模型系统，该系统包括Ty 3 RNA和5 '-3' PBS，引物tRNA（i）（Met），NCp 9，以及第一次高度纯化的Ty 3 RT。与逆转录病毒RT相反，逆转录转座子Ty 3 RT不能通过自引发指导cDNA合成。我们还表明，Ty 3核蛋白复合物在体外形成，而不是锌指的N末端的NCp 9，是需要复合物的形成，tRNA退火到PBS，RNA二聚化，和引物tRNA指导的cDNA合成由Ty 3 RT。这些结果表明，NCp 9伴侣真正的cDNA合成由RT在酵母Ty 3逆转录转座子，如说明了NCp 7在HIV-1，加强了Ty 3 NCp 9是HIV-1 NCp 7的祖先的概念。
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.