Paul A V, Rieder E, Kim D W, van Boom J H, Wimmer E
Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, Stony Brook, New York 11794, USA.
J Virol. 2000 Nov;74(22):10359-70. doi: 10.1128/jvi.74.22.10359-10370.2000.
The first step in the replication of the plus-stranded poliovirus RNA is the synthesis of a complementary minus strand. This process is initiated by the covalent attachment of UMP to the terminal protein VPg, yielding VPgpU and VPgpUpU. We have previously shown that these products can be made in vitro in a reaction that requires only synthetic VPg, UTP, poly(A), purified poliovirus RNA polymerase 3D(pol), and Mg(2+) (A. V. Paul, J. H. van Boom, D. Filippov, and E. Wimmer, Nature 393:280-284, 1998). Since such a poly(A)-dependent process cannot confer sufficient specificity to poliovirus RNA replication, we have developed a new assay to search for a viral RNA template in conjunction with viral or cellular factors that could provide this function. We have now discovered a small RNA hairpin in the coding region of protein 2C as the site in PV1(M) RNA that is used as the primary template for the in vitro uridylylation of VPg. This hairpin has recently been described in poliovirus RNA as being an essential structure for the initiation of minus strand RNA synthesis (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). The uridylylation reaction either with transcripts of cre(2C) RNA or with full-length PV1(M) RNA as the template is strongly stimulated by the addition of purified viral protein 3CD(pro). Deletion of the cre(2C) RNA sequences from minigenomes eliminates their ability to serve as template in the reaction. A similar signal in the coding region of VP1 in HRV14 RNA (K. L. McKnight and S. M. Lemon, RNA 4:1569-1584, 1998) and the poliovirus cre(2C) can be functionally exchanged in the assay. The mechanism by which the VPgpUpU precursor, made specifically on the cre(2C) template, might be transferred to the site where it serves as primer for poliovirus RNA synthesis, remains to be determined.
正链脊髓灰质炎病毒RNA复制的第一步是合成互补负链。这个过程由UMP共价连接到末端蛋白VPg起始，产生VPgpU和VPgpUpU。我们之前已经表明，这些产物可以在体外反应中合成，该反应仅需要合成的VPg、UTP、聚（A）、纯化的脊髓灰质炎病毒RNA聚合酶3D（pol）和Mg(2+)（A. V. Paul、J. H. van Boom、D. Filippov和E. Wimmer，《自然》393:280 - 284，1998）。由于这样一个依赖聚（A）的过程不能赋予脊髓灰质炎病毒RNA复制足够的特异性，我们开发了一种新的检测方法，以寻找与可能提供此功能的病毒或细胞因子结合的病毒RNA模板。我们现在发现，蛋白2C编码区的一个小RNA发夹结构是PV1（M）RNA中用作VPg体外尿苷酸化主要模板的位点。最近在脊髓灰质炎病毒RNA中已描述这个发夹结构是负链RNA合成起始的必需结构（I. Goodfellow、Y. Chaudhry、A. Richardson、J. Meredith、J. W. Almond、W. Barclay和D. J. Evans，《病毒学杂志》74:4590 - 4600，2000）。以cre(2C) RNA转录本或全长PV1（M）RNA作为模板的尿苷酸化反应，通过添加纯化的病毒蛋白3CD(pro)而受到强烈刺激。从小基因组中删除cre(2C) RNA序列消除了它们在反应中作为模板的能力。HRV14 RNA的VP1编码区中的类似信号（K. L. McKnight和S. M. Lemon，《RNA》4:1569 - 1584，1998）和脊髓灰质炎病毒cre(2C)在该检测中可以进行功能交换。在cre(2C)模板上特异性合成的VPgpUpU前体可能转移到其作为脊髓灰质炎病毒RNA合成引物的位点的机制，仍有待确定。
