Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation
Genetic and biochemical studies of poliovirus cis-acting replication element cre in relation to VPg uridylylation
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DOI：
10.1128/jvi.74.22.10371-10380.2000
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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：
Rieder, E;Paul, AV;Wimmer, E
除了位于5 '-和3'-非翻译区的高度保守的茎环结构外，小核糖核酸病毒的基因组复制需要位于编码区的顺式作用RNA元件（称为cre）（K. L. McKnight和S. M.柠檬，J. Virol。70：1941-1952，1996; P. E. Lobert，N. Escriou，J. Ruelle，and T. Natl. Acad. Sci. USA 96：11560-11565，1999; I. Goodfellow，Y. Chaudhry，A.作者：Richard，J. Almond，W. Barclay和D. J. Evans，J. Virol. 74：4590-4600，2000）。cre元件似乎是负链RNA合成所必需的，其机制尚不清楚。我们已经发现，脊髓灰质炎病毒的cre元件（定位于1型脊髓灰质炎病毒的2C编码区; 2C中的核苷酸4444至4505），其与3型脊髓灰质炎病毒的cre元件同源，优先用作在反应中由3D（pol）催化的VPg体外尿苷酰化的模板，该反应由3CD（pro）极大地刺激（A. V. Paul，E.里德尔，D. W. Rim，J.H.货车Boom和E.维默，J.病毒。74：10359-10370，2000）。在这里，我们报告了消除或严重减少体外VPg-尿苷酰化反应和体内产生复制表型的突变之间的直接相关性。没有任何遗传变化显着影响翻译或多蛋白加工。PV-cre（2C）环中保守AAACA序列的第一个A（A4472 C）的取代定位消除了cre RNA作为VPg尿苷酰化模板的能力，并消除了RNA感染性。第二个A（A4473 C; AAACA）的突变严重降低了VPgpUpU的产量，只有在回复到野生型序列后才恢复RNA感染性。第三个A（A4474 G; AAACA）的取代的效果不太严重，但降低了VPg尿苷酰化和病毒产量。PV-cre（2C）上茎区域内碱基配对的破坏也影响VPg的尿苷酰化。从茎中含有突变的转录物衍生的病毒是活的或准感染性的。
In addition to highly conserved stem-loop structures located in the 5'- and 3'-nontranslated regions, genome replication of picornaviruses requires cis-acting RNA elements located in the coding region (termed cre) (K. L. McKnight and S. M. Lemon, J. Virol. 70:1941-1952, 1996; P. E. Lobert, N. Escriou, J. Ruelle, and T. Michiels, Proc. Natl. Acad. Sci. USA 96:11560-11565, 1999; I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590-4600, 2000). cre elements appear to be essential for minus-strand RNA synthesis by an as-yet-unknown mechanism. We have discovered that the cre element of poliovirus (mapping to the 2C coding region of poliovirus type 1; nucleotides 4444 to 4505 in 2C), which is homologous to the cre element of poliovirus type 3, is preferentially used as a template for the in vitro uridylylation of VPg catalyzed by 3D(pol) in a reaction that is greatly stimulated by 3CD(pro) (A. V. Paul, E. Rieder, D. W. Rim, J. H. van Boom, and E. Wimmer, J. Virol. 74:10359-10370, 2000). Here we report a direct correlation between mutations that eliminate, or severely reduce, the in vitro VPg-uridylylation reaction and produce replication phenotypes in vivo. None of the genetic changes significantly influenced translation or polyprotein processing. A substitution mapping to the first A (A4472C) of a conserved AAACA sequence in the loop of PV-cre(2C) eliminated the ability of the cre RNA to serve as template for VPg uridylylation and abolished RNA infectivity. Mutagenesis of the second A (A4473C; AAACA) severely reduced the yield of VPgpUpU and RNA infectivity was restored only after reversion to the wild type sequence. The effect of substitution of the third A (A4474G; AAACA) was less severe but reduced both VPg uridylylation and virus yield. Disruption of base pairing within the upper stem region of PV-cre(2C) also affected uridylylation of VPg. Virus derived from transcripts containing mutations in the stem was either viable or quasi-infectious.