Paul Aniko V, Yin Jiang, Mugavero JoAnn, Rieder Elizabeth, Liu Ying, Wimmer Eckard
Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, Stony Brook, New York 11790, USA.
J Biol Chem. 2003 Nov 7;278(45):43951-60. doi: 10.1074/jbc.M307441200. Epub 2003 Aug 22.
Poliovirus RNA replication is initiated when a molecule of UMP is covalently linked to the hydroxyl group of a tyrosine in the terminal protein VPg. This reaction can be reproduced in vitro with an assay that utilizes two purified viral proteins, RNA polymerase 3Dpol and viral protein 3CDpro, synthetic VPg, UTP, and Mg2+. The template for the reaction is either poliovirus RNA or transcripts of a small RNA hairpin, termed cre(2C), located in the coding sequence of protein 2CATPase. The products of the reaction are VPgpU and VPgpUpU, the primers used by 3Dpol for RNA synthesis. With mutant template RNAs in this assay we determined the precise initiation site. Our results indicate that 1) 3Dpol does not possess strict specificity toward the nucleotide it links to VPg, 2) A-5 of the conserved 1GXXXAAAXXXXXXA14 sequence in the loop is the template nucleotide for the linkage of both the first and second UMPs to VPg, 3) VPgpUpU is synthesized by a "slide-back" mechanism, and 4) A-6 provides specificity to the reaction during the slide-back step and also modulates the uridylylation reaction. In additional experiments we determined the effect of mutations in the 5AAA7 sequence of cre(2C) on viral growth, RNA replication, and on the activity of the 2CATPase protein. Furthermore, we observed that the spacing between G-1 and A-5 and the size of the loop affect the yield but not the nature of the VPg-linked products.
当一分子尿苷一磷酸（UMP）与末端蛋白VPg中酪氨酸的羟基共价连接时，脊髓灰质炎病毒RNA复制启动。该反应可在体外通过一种检测方法重现，该方法利用两种纯化的病毒蛋白、RNA聚合酶3Dpol和病毒蛋白3CDpro、合成的VPg、尿苷三磷酸（UTP）和镁离子（Mg2+）。该反应的模板可以是脊髓灰质炎病毒RNA或位于2CATP酶编码序列中的一个小RNA发夹结构（称为cre(2C)）的转录本。反应产物是VPgpU和VPgpUpU，它们是3Dpol用于RNA合成的引物。通过该检测方法中使用的突变模板RNA，我们确定了精确的起始位点。我们的结果表明：1）3Dpol对其连接到VPg的核苷酸不具有严格的特异性；2）环中保守的1GXXXAAAXXXXXXA14序列中的A-5是第一个和第二个UMP与VPg连接的模板核苷酸；3）VPgpUpU是通过“回滑”机制合成的；4）A-6在回滑步骤中为反应提供特异性，并且还调节尿苷酰化反应。在其他实验中，我们确定了cre(2C)的5AAA7序列中的突变对病毒生长、RNA复制以及2CATP酶蛋白活性的影响。此外，我们观察到G-1和A-5之间的间距以及环的大小会影响VPg连接产物的产量，但不影响其性质。
