Effects of Influenza A Virus NS1 Protein on Protein Expression: the NS1 Protein Enhances Translation and Is Not Required for Shutoff of Host Protein Synthesis

The influenza A virus NS1 protein, a virus-encoded alpha/beta interferon (IFN-α/β) antagonist, appears to be a key regulator of protein expression in infected cells. We now show that NS1 protein expression results in enhancement of reporter gene activity from transfected plasmids. This effect appears to be mediated at the translational level, and it is reminiscent of the activity of the adenoviral virus-associated I (VAI) RNA, a known inhibitor of the antiviral, IFN-induced, PKR protein. To study the effects of the NS1 protein on viral and cellular protein synthesis during influenza A virus infection, we used recombinant influenza viruses lacking the NS1 gene (delNS1) or expressing truncated NS1 proteins. Our results demonstrate that the NS1 protein is required for efficient viral protein synthesis in COS-7 cells. This activity maps to the amino-terminal domain of the NS1 protein, since cells infected with wild-type virus or with a mutant virus expressing a truncated NS1 protein—lacking approximately half of its carboxy-terminal end—showed similar kinetics of viral and cellular protein expression. Interestingly, no major differences in host cell protein synthesis shutoff or in viral protein expression were found among NS1 mutant viruses in Vero cells. Thus, another viral component(s) different from the NS1 protein is responsible for the inhibition of host protein synthesis during viral infection. In contrast to the earlier proposal suggesting that the NS1 protein regulates the levels of spliced M2 mRNA, no effects on M2 protein accumulation were seen in Vero cells infected with delNS1 virus.甲型流感病毒NS1蛋白是病毒编码的α/β干扰素（IFN-α/β）拮抗剂，被认为是感染宿主细胞内蛋白质表达的关键调控因子。我们的研究证实，NS1蛋白的表达可增强转染质粒的报告基因活性。该效应似乎通过翻译水平的调控介导，其活性特征与腺病毒病毒相关I（VAI）RNA的功能相似——后者是已知的抗病毒、干扰素诱导型PKR蛋白（PKR protein）的抑制剂。为探究甲型流感病毒感染过程中NS1蛋白对病毒及宿主细胞蛋白质合成的影响，我们使用了两类重组甲型流感病毒：一类为缺失NS1基因的delNS1病毒，另一类为表达截短型NS1蛋白的重组病毒。实验结果表明，NS1蛋白是COS-7细胞中高效合成病毒蛋白所必需的。该功能活性定位于NS1蛋白的氨基末端结构域：感染野生型病毒，或感染表达截短型NS1蛋白（缺失其近半数羧基末端区域）的突变病毒的细胞，其病毒与宿主蛋白质的表达动力学特征均无显著差异。值得注意的是，在Vero细胞中，不同NS1突变病毒之间的宿主蛋白质合成关闭效应以及病毒蛋白表达水平均无显著差异。由此可见，病毒感染过程中抑制宿主蛋白质合成的作用，并非由NS1蛋白介导，而是由其他一种或多种病毒成分所调控。与此前“NS1蛋白可调控剪接型M2 mRNA水平”的假说不同，在感染delNS1病毒的Vero细胞中，并未观察到M2蛋白积累量存在明显变化。