Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg
Identification of an RNA hairpin in poliovirus RNA that serves as the primary template in the in vitro uridylylation of VPg
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DOI：
10.1128/jvi.74.22.10359-10370.2000
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发表时间：
2000-11-01
影响因子：
5.4
通讯作者：
Wimmer, E
中科院分区：
文献类型：
--
作者：
Paul, AV;Rieder, E;Wimmer, E
正链脊髓灰质炎病毒RNA复制的第一步是合成互补的负链。该过程由 UMP 与末端蛋白 VPg 共价连接启动，产生 VPgpU 和 VPgpUpU。我们之前已经证明，这些产品可以通过仅需要合成 VPg、UTP、poly(A)、纯化的脊髓灰质炎病毒 RNA 聚合酶 3D(pol) 和 Mg2+ 的反应在体外制备（A. V. Paul、J. H. van Boom、D. Filippov 和 E. Wimmer，Nature 393：280-284，1998）。由于这种多聚腺苷酸依赖性过程无法赋予脊髓灰质炎病毒RNA复制足够的特异性，因此我们开发了一种新的测定方法来寻找与可以提供此功能的病毒或细胞因子结合的病毒RNA模板。我们现在在蛋白质 2C 的编码区发现了一个小 RNA 发夹，作为 PV1(M) RNA 中的位点，该位点被用作 VPg 体外尿苷酰化的主要模板。最近在脊髓灰质炎病毒 RNA 中描述了这种发夹是起始负链 RNA 合成的必需结构 (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000)。添加纯化的病毒蛋白 3CD(pro) 会强烈刺激以 cre(2C) RNA 转录物或全长 PV1(M) RNA 为模板的尿苷酰化反应。从小基因组中删除 cre(2C) RNA 序列会消除它们在反应中充当模板的能力。 HRV14 RNA (K. L. McKnight 和 S. M. Lemon, RNA 4:1569-1584, 1998) 和脊髓灰质炎病毒 cre(2C) 中 VP1 编码区中的类似信号可以在测定中进行功能性交换。在 cre(2C) 模板上专门制作的 VPgpUpU 前体可能被转移到充当脊髓灰质炎病毒 RNA 合成引物的位点的机制仍有待确定。
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 Mg2+ (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,vith 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.