Min Ji-Young, Li Shoudong, Sen Ganes C, Krug Robert M
Institute for Cellular and Molecular Biology, Section of Molecular Genetics and Microbiology, room 2.122, 2500 Speedway, University of Texas at Austin, Austin, TX 78712, USA.
Virology. 2007 Jun 20;363(1):236-43. doi: 10.1016/j.virol.2007.01.038. Epub 2007 Feb 22.
It is not known how influenza A viruses, important human pathogens, counter PKR activation, a crucial host antiviral response. Here we elucidate this mechanism. We show that the direct binding of PKR to the NS1 protein in vitro that results in inhibition of PKR activation requires the NS1 123-127 amino acid sequence. To establish whether such direct binding of PKR to the NS1 protein is responsible for inhibiting PKR activation in infected cells, we generated recombinant influenza A/Udorn/72 viruses expressing NS1 proteins in which amino acids 123/124 or 126/127 are changed to alanines. In cells infected with these mutant viruses, PKR is activated, eIF-2alpha is phosphorylated and viral protein synthesis is inhibited, indicating that direct binding of PKR to the 123-127 sequence of the NS1 protein is necessary and sufficient to block PKR activation in influenza A virus-infected cells. Unexpectedly, the 123/124 mutant virus is not attenuated because reduced viral protein synthesis is offset by enhanced viral RNA synthesis at very early times of infection. These early viral RNAs include those synthesized predominantly at later times during wild-type virus infection, demonstrating that wild-type temporal regulation of viral RNA synthesis is absent in 123/124 virus-infected cells. Enhanced early viral RNA synthesis after 123/124 virus infection also occurs in mouse PKR-/- cells, demonstrating that PKR activation and deregulation of the time course of viral RNA synthesis are not coupled. These results indicate that the 123/124 site of the NS1A protein most likely functionally interacts with the viral polymerase to mediate temporal regulation of viral RNA synthesis. This interaction would occur in the nucleus, whereas PKR would bind to NS1A proteins in the cytoplasm prior to their import into the nucleus.
甲型流感病毒作为重要的人类病原体，如何对抗PKR激活这一关键的宿主抗病毒反应尚不清楚。在此我们阐明了这一机制。我们发现，PKR在体外与NS1蛋白的直接结合导致PKR激活受到抑制，这一过程需要NS1蛋白的123 - 127氨基酸序列。为了确定PKR与NS1蛋白的这种直接结合是否负责抑制感染细胞中的PKR激活，我们构建了表达NS1蛋白的重组甲型流感病毒A/Udorn/72，其中123/124或126/127位氨基酸被替换为丙氨酸。在感染这些突变病毒的细胞中，PKR被激活，eIF - 2α被磷酸化，病毒蛋白合成受到抑制，这表明PKR与NS1蛋白的123 - 127序列直接结合对于阻断甲型流感病毒感染细胞中的PKR激活是必要且充分的。出乎意料的是，123/124突变病毒并未减毒，因为在感染的极早期，病毒蛋白合成的减少被病毒RNA合成的增强所抵消。这些早期病毒RNA包括那些在野生型病毒感染后期主要合成的RNA，这表明在123/124病毒感染的细胞中不存在野生型病毒RNA合成的时间调控。123/124病毒感染后早期病毒RNA合成的增强在小鼠PKR - / - 细胞中也会发生，这表明PKR激活与病毒RNA合成时间进程的失调并不相关。这些结果表明，NS1A蛋白的123/124位点很可能在功能上与病毒聚合酶相互作用，以介导病毒RNA合成的时间调控。这种相互作用将发生在细胞核中，而PKR将在NS1A蛋白导入细胞核之前在细胞质中与其结合。
