Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein.
Selective Degradation of Host RNA Polymerase II Transcripts by Influenza A Virus PA-X Host Shutoff Protein.
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DOI：
10.1371/journal.ppat.1005427
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发表时间：
2016-02
期刊：
影响因子：
6.7
通讯作者：
Gaglia MM
中科院分区：
文献类型：
--
作者：
Khaperskyy DA;Schmaling S;Larkins-Ford J;McCormick C;Gaglia MM
甲型流感病毒（IAVs）通过宿主关闭过程抑制宿主基因表达。宿主关闭限制了宿主的先天免疫反应，也可能使翻译装置转向病毒蛋白的生产。多种IAV蛋白调控宿主关闭，包括PA-X，一种尚未完全鉴定的核糖核酸酶。我们报道PA-X选择性靶向宿主RNA聚合酶II （Pol II）转录的mrna，同时保留Pol I和Pol III的产物。有趣的是，我们发现PA-X也可以靶向细胞核中Pol ii转录的RNA，包括注定不被翻译的非编码RNA，以及具有阻断核糖体装载的RNA发夹结构的报告转录物。转录物降解可能发生在细胞核中，因为PA-X在细胞核中富集，其核定位与靶RNA水平的降低相关。宿主mrna在pa - x介导的核内裂解后的完全降解依赖于宿主5 ' -> - 3 ' -切外切酶Xrn1。IAV mrna在结构上与宿主mrna相似，但在3 '端通过病毒RNA依赖的RNA聚合酶复合物的作用进行合成和修饰。用野生型IAV或重组pa - x缺陷病毒感染细胞表明，IAV mrna在感染过程中抵抗pa - x介导的降解。同时，PA-X的缺失导致特定病毒mrna合成的改变和病毒蛋白积累的减少。总的来说，这些结果显著提高了我们对IAV宿主关闭的理解，并表明PA-X通过在细胞核中辨别Pol ii依赖性RNA生物发生的某些方面而导致宿主mrna的选择性降解。所有病毒都依赖宿主成分将病毒mrna转化为蛋白质。一些病毒，包括甲型流感病毒，编码触发RNA破坏的因子。具有这种功能的甲型流感病毒因子被称为PA-X。PA-X限制宿主mrna和蛋白质在感染细胞中的积累，抑制宿主对感染的反应，但迄今为止其确切的作用机制尚不清楚。在这里，我们报道PA-X选择性地靶向细胞mrna，同时保留病毒mrna，从而损害宿主基因表达并确保病毒mrna优先进入蛋白质合成机制。我们证明，PA-X切割的mrna的完全降解依赖于宿主因子Xrn1， PA-X可能在细胞核中起作用。有趣的是，PA-X靶向似乎对宿主RNA聚合酶II的产物具有选择性，而典型的mRNA加工是切割所必需的。尽管病毒mrna不受pa - x介导的降解，但pa - x缺陷病毒在某些病毒mrna的合成上表现出缺陷，并减少了病毒蛋白的积累。因此，pa - x介导的宿主关闭影响病毒基因表达的效率。这些研究极大地促进了我们对这种重要的病毒宿主关闭蛋白的理解，并可能为限制甲型流感病毒感染的发病机制提供未来的机会。
Influenza A viruses (IAVs) inhibit host gene expression by a process known as host shutoff. Host shutoff limits host innate immune responses and may also redirect the translation apparatus to the production of viral proteins. Multiple IAV proteins regulate host shutoff, including PA-X, a ribonuclease that remains incompletely characterized. We report that PA-X selectively targets host RNA polymerase II (Pol II) transcribed mRNAs, while sparing products of Pol I and Pol III. Interestingly, we show that PA-X can also target Pol II-transcribed RNAs in the nucleus, including non-coding RNAs that are not destined to be translated, and reporter transcripts with RNA hairpin structures that block ribosome loading. Transcript degradation likely occurs in the nucleus, as PA-X is enriched in the nucleus and its nuclear localization correlates with reduction in target RNA levels. Complete degradation of host mRNAs following PA-X-mediated endonucleolytic cleavage is dependent on the host 5’->3’-exonuclease Xrn1. IAV mRNAs are structurally similar to host mRNAs, but are synthesized and modified at the 3’ end by the action of the viral RNA-dependent RNA polymerase complex. Infection of cells with wild-type IAV or a recombinant PA-X-deficient virus revealed that IAV mRNAs resist PA-X-mediated degradation during infection. At the same time, loss of PA-X resulted in changes in the synthesis of select viral mRNAs and a decrease in viral protein accumulation. Collectively, these results significantly advance our understanding of IAV host shutoff, and suggest that the PA-X causes selective degradation of host mRNAs by discriminating some aspect of Pol II-dependent RNA biogenesis in the nucleus. All viruses depend on host components to convert viral mRNAs into proteins. Several viruses, including influenza A virus, encode factors that trigger RNA destruction. The influenza A virus factor that serves in this capacity is known as PA-X. PA-X limits accumulation of host mRNAs and proteins in infected cells and suppresses host responses to infection, but to date its precise mechanism of action remains obscure. Here we report that PA-X selectively targets cellular mRNAs, while sparing viral mRNAs, thereby compromising host gene expression and ensuring priority access of viral mRNAs to the protein synthesis machinery. We demonstrate that complete degradation of mRNAs cut by PA-X is dependent on the host factor Xrn1 and that PA-X likely works in the cell’s nuclei. Interestingly, PA-X targeting appears to be selective for products of host RNA polymerase II, and canonical mRNA processing is required for cleavage. Even though viral mRNAs are spared from PA-X-mediated degradation, PA-X-deficient viruses displayed defects in the synthesis of certain viral mRNAs and decreased viral protein accumulation. Thus, PA-X-mediated host shutoff influences the efficiency of viral gene expression. These studies significantly advance our understanding of this important viral host shutoff protein and may provide future opportunities to limit the pathogenesis of influenza A virus infection.