RNase L Targets Distinct Sites in Influenza A Virus RNAs
RNase L Targets Distinct Sites in Influenza A Virus RNAs
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DOI：
10.1128/jvi.02953-14
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发表时间：
2015-03-01
影响因子：
5.4
通讯作者：
Barton, David J.
中科院分区：
文献类型：
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作者：
Cooper, Daphne A.;Banerjee, Shuvojit;Barton, David J.
甲型流感病毒(IAV)感染受到1型干扰素介导的抗病毒防御以及针对这些防御的病毒对策的影响。当IAVNS1蛋白被禁用时，核糖核酸酶L限制病毒的复制；然而，在这种条件下核糖核酸酶L靶向切割的RNA尚未确定。在这项研究中，我们使用深度测序的方法确定了宿主和感染IAV PR8 Delta NS1的A549细胞中的病毒RNA中的RNA酶L裂解位点。核糖核酸酶L的短发夹状RNA敲除使我们能够区分核糖核酸酶L依赖和L非依赖的切割位点。在IAVRNA片段(正链和负链)中，存在明显的依赖于核糖核酸酶L的切割位点。PB2、PB1和PA基因组RNA中的切割表明病毒RNPs容易被RNase L切割。显著数量的切割被映射到IAV RNAs中的特定区域，包括一些同义位点保守增加的区域。在细胞RNA中，依赖于核糖核酸酶L的切割在RNA的精确位置发生得最频繁。我们的数据显示，当NS1蛋白失活时，核糖核酸酶L针对宿主和病毒RNA中的特定位点来限制流感病毒的复制。重要核糖核酸酶L是干扰素调节和双链RNA激活的抗病毒宿主反应的关键组件。我们试图确定核糖核酸酶L在流感病毒感染过程中是如何发挥其抗病毒活性的。我们通过禁用一种抑制核糖核酸酶L活性的病毒蛋白ns1来增强核糖核酸酶L的抗病毒活性。然后，使用深度测序的方法，我们鉴定了核糖核酸酶L靶向的宿主和病毒RNA。我们发现核糖核酸酶L在非常精确的位置切割病毒RNA和核糖核酸。RNA酶L对IAV RNA的直接切割突显了病毒RNA和抗病毒核酸内切酶之间的亲密斗争。
Influenza A virus (IAV) infections are influenced by type 1 interferon-mediated antiviral defenses and by viral countermeasures to these defenses. When IAV NS1 protein is disabled, RNase L restricts virus replication; however, the RNAs targeted for cleavage by RNase L under these conditions have not been defined. In this study, we used deep-sequencing methods to identify RNase L cleavage sites within host and viral RNAs from IAV PR8 Delta NS1-infected A549 cells. Short hairpin RNA knockdown of RNase L allowed us to distinguish between RNase L-dependent and RNase L-independent cleavage sites. RNase L-dependent cleavage sites were evident at discrete locations in IAV RNA segments (both positive and negative strands). Cleavage in PB2, PB1, and PA genomic RNAs suggests that viral RNPs are susceptible to cleavage by RNase L. Prominent amounts of cleavage mapped to specific regions within IAV RNAs, including some areas of increased synonymous-site conservation. Among cellular RNAs, RNase L-dependent cleavage was most frequent at precise locations in rRNAs. Our data show that RNase L targets specific sites in both host and viral RNAs to restrict influenza virus replication when NS1 protein is disabled.IMPORTANCERNase L is a critical component of interferon-regulated and double-stranded-RNA-activated antiviral host responses. We sought to determine how RNase L exerts its antiviral activity during influenza virus infection. We enhanced the antiviral activity of RNase L by disabling a viral protein, NS1, that inhibits the activation of RNase L. Then, using deep-sequencing methods, we identified the host and viral RNAs targeted by RNase L. We found that RNase L cleaved viral RNAs and rRNAs at very precise locations. The direct cleavage of IAV RNAs by RNase L highlights an intimate battle between viral RNAs and an antiviral endonuclease.