Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity
Critical Role of the PA-X C-Terminal Domain of Influenza A Virus in Its Subcellular Localization and Shutoff Activity
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DOI：
10.1128/jvi.00954-16
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发表时间：
2016-08-01
影响因子：
5.4
通讯作者：
Takimoto, Toru
中科院分区：
文献类型：
--
作者：
Hayashi, Tsuyoshi;Chaimayo, Chutikarn;Takimoto, Toru
PA- x是最近发现的一种流感病毒蛋白，由PA n端191个氨基酸和独特的c端41或61个残基组成。我们和其他人显示PA-X有很强的抑制宿主蛋白质合成能力通过主机mRNA衰减,这是由核酸内切酶活性的n端结构域(b·w·贾格尔h . m .明智的,j·c·卡什k·a·沃尔特斯n . m .遗嘱y l·肖r . l . Dunfee l·m·舒瓦a . Ozinsky g·l·贝尔·r·m·道尔顿a, s . Efstathiou j·f·阿特金斯,a·e·弗斯j . k .陶本伯杰和p .迪加尔,2012年,科学337:199 - 204,http://dx.doi.org/10.1126/science.1222213和e·a·Desmet李建军，李建军，刘建军，等。中国生物医学工程学报，2013,37 (1):388 - 388,http://dx.doi.org/10.1128/JVI.02826-12)。然而，宿主mRNA降解的机制，特别是PA-X靶向mRNA的位置和方式，尚未得到分析。在这项研究中，我们确定了PA-X的定位和c端独特区域在关闭活性中的作用。定量亚细胞定位分析显示，PA-X在细胞质和细胞核中分布均匀。通过对一系列PA-X c端缺失突变体的表征，我们发现前9个氨基酸足以实现核定位，但还需要另外6个残基才能诱导完整PA-X的最大关闭活性。重要的是，PA-X c端缺失突变体的强制核定位增强了关闭活性，突出了核PA-X更有效地降解宿主mrna的能力。然而，PA-X还能抑制体外合成的转染mrna的荧光素酶表达，表明PA-X也能降解细胞质中的mrna。在PA-X c -末端的碱基氨基酸中，195K、198K和199R被鉴定为诱导宿主关闭和核定位的关键残基。总的来说，我们的数据表明，PA-X c端结构域的15个残基在细胞质和细胞核中降解mrna的过程中起着关键作用。甲型流感病毒表达PA-X蛋白抑制宿主基因表达，包括宿主抗病毒基因，从而使病毒在感染细胞中有效复制。然而，对于PA-X如何诱导宿主关闭知之甚少。在本研究中，我们发现PA-X在细胞质和细胞核中都有相同的定位，但PA-X的c端区介导的核定位对关闭活性有显著影响。c端区域的三个基本残基在核定位中起关键作用，但需要额外的基本残基才能达到最大的关闭活性。我们的研究结果表明，PA-X靶向并降解细胞核和细胞质中的mrna，并且PA-X独特的c端区域的前15个残基在关闭活性中起关键作用。
PA-X is a recently identified influenza virus protein that is composed of the PA N-terminal 191 amino acids and unique C-terminal 41 or 61 residues. We and others showed that PA-X has a strong ability to suppress host protein synthesis via host mRNA decay, which is mediated by endonuclease activity in its N-terminal domain (B. W. Jagger, H. M. Wise, J. C. Kash, K. A. Walters, N. M. Wills, Y. L. Xiao, R. L. Dunfee, L. M. Schwartzman, A. Ozinsky, G. L. Bell, R. M. Dalton, A. Lo, S. Efstathiou, J. F. Atkins, A. E. Firth, J. K. Taubenberger, and P. Digard, 2012, Science 337:199-204, http://dx.doi.org/10.1126/science.1222213, and E. A. Desmet, K. A. Bussey, R. Stone, and T. Takimoto, 2013, J Virol 87:3108-3118, http://dx.doi.org/10.1128/JVI.02826-12). However, the mechanism of host mRNA degradation, especially where and how PA-X targets mRNAs, has not been analyzed. In this study, we determined the localization of PA-X and the role of the C-terminal unique region in shutoff activity. Quantitative subcellular localization analysis revealed that PA-X was located equally in both cytoplasm and nucleus. By characterizing a series of PA-X C-terminal deletion mutants, we found that the first 9 amino acids were sufficient for nuclear localization, but an additional 6 residues were required to induce the maximum shutoff activity observed with intact PA-X. Importantly, forced nuclear localization of the PA-X C-terminal deletion mutant enhanced shutoff activity, highlighting the ability of nuclear PA-X to degrade host mRNAs more efficiently. However, PA-X also inhibited luciferase expression from transfected mRNAs synthesized in vitro, suggesting that PA-X also degrades mRNAs in the cytoplasm. Among the basic amino acids in the PA-X C-terminal region, 3 residues, 195K, 198K, and 199R, were identified as key residues for inducing host shutoff and nuclear localization. Overall, our data indicate a critical role for the 15 residues in the PA-X C-terminal domain in degrading mRNAs in both the cytoplasm and nucleus.IMPORTANCEInfluenza A viruses express PA-X proteins to suppress global host gene expression, including host antiviral genes, to allow efficient viral replication in infected cells. However, little is known about how PA-X induces host shutoff. In this study, we showed that PA-X localized equally in both the cytoplasm and nucleus of the cells, but the nuclear localization of PA-X mediated by its C-terminal region has a significant impact on shutoff activity. Three basic residues at the C-terminal region play a critical role in nuclear localization, but additional basic residues were required for maximum shutoff activity. Our findings indicate that PA-X targets and degrades mRNAs in both the nucleus and cytoplasm, and that the first 15 residues of the PA-X unique C-terminal region play a critical role in shutoff activity.