Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2.
Inhibition of translation in cells infected with a poliovirus 2Apro mutant correlates with phosphorylation of the alpha subunit of eucaryotic initiation factor 2.
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感染脊髓灰质炎病毒 2Apro 突变体的细胞中翻译的抑制与真核起始因子 2 α 亚基的磷酸化相关。
DOI：
10.1128/jvi.63.12.5069-5075.1989
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发表时间：
1989
影响因子：
5.4
通讯作者：
Racaniello,VR
中科院分区：
文献类型：
--
作者：
O'Neill,RE;Racaniello,VR
在感染性克隆的2APro区插入6个碱基对，在多肽序列中增加亮氨酸和苏氨酸，构建了脊髓灰质炎病毒2型兰辛突变体。由此产生的小斑块突变体2A-2在HeLa细胞中的病毒产量降低，并且合成病毒蛋白的效率低下。在感染早期，感染突变体并没有导致宿主细胞蛋白质合成的特异性抑制，这一缺陷被归因于未能诱导帽结合复杂蛋白p220的切割。在感染变异病毒后的后期，细胞和病毒的蛋白质合成都受到严重抑制。为了解释这种蛋白质合成的全局抑制，我们检测了真核细胞起始因子2的α亚基(eIF-2α)的磷酸化状态。在R2-2A-2和野生型病毒感染的细胞中，eIF-2α都被磷酸化，这表明脊髓灰质炎病毒不编码阻止eIF-2α磷酸化的功能。EIF-2α磷酸化的动力学和程度与感染细胞中双链RNA的产生相关，表明EIF-2α被P1/EIF-2α激酶磷酸化。当R2-2A-2在蛋白激酶抑制剂2-氨基嘌呤存在的情况下感染HeLa细胞时，病毒滴度显著升高，p220发生裂解，宿主细胞蛋白质合成受到特异性抑制。由于2-氨基嘌呤不能抑制eIF-2α的磷酸化，我们认为2-氨基嘌呤通过抑制第二个未知的激酶而挽救了R2-2A-2诱导p220裂解的能力。
A poliovirus type 2 Lansing mutant was constructed by inserting 6 base pairs into the 2Apro region of an infectious cDNA clone, resulting in the addition of a leucine and threonine into the polypeptide sequence. The resulting small-plaque mutant, 2A-2, had a reduced viral yield in HeLa cells and synthesized viral proteins inefficiently. Infection with the mutant did not lead to specific inhibition of host cell protein synthesis early in infection, and this defect was attributed to a failure to induce cleavage of the cap-binding complex protein p220. At late times after infection with the mutant virus, both cellular and viral protein syntheses were severely inhibited. To explain this global inhibition of protein synthesis, the phosphorylation state of the alpha subunit of eucaryotic initiation factor 2 (eIF-2 alpha) was examined. eIF-2 alpha was phosphorylated in both R2-2A-2- and wild-type-virus-infected cells, indicating that poliovirus does not encode a function that blocks phosphorylation of eIF-2 alpha. The kinetics and extent of eIF-2 alpha phosphorylation correlated with the production of double-stranded RNA in infected cells, suggesting that eIF-2 alpha is phosphorylated by P1/eIF-2 alpha kinase. When HeLa cells were infected with R2-2A-2 in the presence of 2-aminopurine, a protein kinase inhibitor, much higher virus titers were produced, cleavage of p220 occurred, and host cell protein synthesis was specifically inhibited. Since phosphorylation of eIF-2 alpha was not inhibited by 2-aminopurine, we propose that 2-aminopurine rescues the ability of R2-2A-2 to induce cleavage of p220 by inhibition of a second as yet unidentified kinase.