A stable HeLa cell line that inducibly expresses poliovirus 2Apro:: Effects on cellular and viral gene expression
A stable HeLa cell line that inducibly expresses poliovirus 2Apro:: Effects on cellular and viral gene expression
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DOI：
10.1128/jvi.74.5.2383-2392.2000
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发表时间：
2000-03-01
影响因子：
5.4
通讯作者：
Carrasco, L
中科院分区：
文献类型：
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作者：
Barco, A;Feduchi, E;Carrasco, L
获得了在四环素调控下可诱导表达脊髓灰质炎病毒蛋白水解酶Za基因的HeLa细胞克隆(2A7d)。2A(Pro)的合成可诱导2A7d细胞发生严重的形态变化。去除四环素一天后，细胞聚集在一起，几个小时后死亡。脊髓灰质炎病毒2A型(PRO)裂解两种形式的启动因子eIF4G，在蛋白酶诱导几个小时后，导致对CAPPED-mRNA翻译的广泛抑制。Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone，是一种选择性的2A(PRO)抑制剂，既可以阻止eIF4G的切割，也可以阻止翻译的抑制，但不能阻止细胞死亡。2A(Pro)的表达仍然允许脊髓灰质炎病毒的复制和含有微小核糖体病毒前导序列的mRNAs的翻译，而痘苗病毒的复制被显著抑制。：在2A7d-ON细胞中，转染的封顶mRNA的翻译被阻止，而带有微小RNA病毒内部核糖体进入位点(IRES)序列的mRNA的荧光素酶合成被2A(Pro)的存在增强。此外，在2A7d细胞中合成2A(Pro)补充了脊髓灰质炎病毒2A(Pro)缺陷变体的翻译缺陷。这些结果表明，脊髓灰质炎病毒2A(PRO)的表达模仿了脊髓灰质炎病毒感染细胞的一些表型特征，如细胞圆形、抑制蛋白质合成和增强IRES驱动的翻译。该细胞系构成了进一步分析2A型(PRO)功能、补充脊髓灰质炎病毒2A型(PRO)突变体和测试抗病毒化合物的有用工具。
A HeLa cell clone (2A7d) that inducibly expresses the gene for poliovirus protease ZA (2A(pro)) under the control of tetracycline has been obtained. Synthesis of 2A(pro) induces severe morphological changes in 2A7d cells. One day after tetracycline removal, cells round up and a few hours later die. Poliovirus 2A(pro) cleaves both forms of initiation factor eIF4G, causing extensive inhibition of capped-mRNA translation a few hours after protease induction. Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, a selective inhibitor of 2A(pro), prevents both eIF4G cleavage and inhibition of translation but not cellular death. Expression of 2A(pro) still allows both the replication of poliovirus and the translation of mRNAs containing a picornavirus leader sequence, while vaccinia virus replication is drastically inhibited.:Translation of transfected capped mRNA is blocked in 2A7d-On cells, while luciferase synthesis from a mRNA bearing a picornavirus internal ribosome entry site (IRES) sequence is enhanced by the presence of 2A(pro). Moreover, synthesis of 2A(pro) in 2A7d cells complements the translational defect of a poliovirus 2A(pro)-defective variant. These results show that poliovirus 2A(pro) expression mimics some phenotypical characteristics of poliovirus-infected cells, such as cell rounding, inhibition of protein synthesis and enhancement of IRES-driven translation. This cell line constitutes a useful tool to further analyze 2A(pro) functions, to complement poliovirus 2A(pro) mutants, and to test antiviral compounds.