Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses
Activation of RNase L is dependent on OAS3 expression during infection with diverse human viruses
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DOI：
10.1073/pnas.1519657113
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发表时间：
2016-02-23
影响因子：
11.1
通讯作者：
Weiss, Susan R.
中科院分区：
文献类型：
--
作者：
Li, Yize;Banerjee, Shuvojit;Weiss, Susan R.
2', 5'-寡聚腺苷酸（2- 5a）合成酶(OAS)-RNase L系统是ifn诱导的抗病毒途径。RNase L的活性取决于由OAS合成的2-5A。尽管人类三种具有酶活性的OAS蛋白- oas1、OAS2和oas3 -在结合dsRNA后合成2-5A，但在病毒感染过程中，究竟是哪一种酶激活了RNase L尚不清楚。我们使用集群规则间隔短回传重复序列(CRISPR)-CRISPR相关蛋白-9核酸酶（Cas9）技术来设计人类a549衍生的细胞系，其中每个OAS基因或RNase L都被敲除。转染poly(rI): poly(rC)（一种合成的病毒dsRNA替代物），或感染来自不同组的四种病毒（西尼罗病毒、辛德比病毒、流感病毒或牛痘病毒），OAS1-KO和OAS2-KO细胞合成的2-5A数量与亲本野生型细胞合成的2-5A数量相似，通过rRNA降解评估RNase L激活。相比之下，OAS3-KO细胞合成了最少的2-5A，并且rRNA保持完整，与感染RNase的L-KO细胞相似。所有四种病毒在OAS3- ko或RNase L-KO A549细胞中复制的滴度都高于亲本、OAS1-KO或OAS2-KO细胞，证明了OAS3的抗病毒作用。在完整细胞中，OAS3对dsRNA的亲和力高于OAS1或OAS2，这与OAS3在RNase L活化中的主导作用一致。最后，对OAS3作为负责RNase L激活的主要OAS异构体的需求并不局限于A549细胞，因为来自另外两种人类细胞系的OAS3- ko细胞也缺乏RNase L激活。
The 2', 5'-oligoadenylate (2-5A) synthetase (OAS)-RNase L system is an IFN-induced antiviral pathway. RNase L activity depends on 2-5A, synthesized by OAS. Although all three enzymatically active OAS proteins in humans-OAS1, OAS2, and OAS3-synthesize 2-5A upon binding dsRNA, it is unclear which are responsible for RNase L activation during viral infection. We used clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein-9 nuclease (Cas9) technology to engineer human A549-derived cell lines in which each of the OAS genes or RNase L is knocked out. Upon transfection with poly(rI): poly(rC), a synthetic surrogate for viral dsRNA, or infection with each of four viruses from different groups (West Nile virus, Sindbis virus, influenza virus, or vaccinia virus), OAS1-KO and OAS2-KO cells synthesized amounts of 2-5A similar to those synthesized in parental wild-type cells, causing RNase L activation as assessed by rRNA degradation. In contrast, OAS3-KO cells synthesized minimal 2-5A, and rRNA remained intact, similar to infected RNase L-KO cells. All four viruses replicated to higher titers in OAS3-KO or RNase L-KO A549 cells than in parental, OAS1-KO, or OAS2-KO cells, demonstrating the antiviral effects of OAS3. OAS3 displayed a higher affinity for dsRNA in intact cells than either OAS1 or OAS2, consistent with its dominant role in RNase L activation. Finally, the requirement for OAS3 as the major OAS isoform responsible for RNase L activation was not restricted to A549 cells, because OAS3-KO cells derived from two other human cell lines also were deficient in RNase L activation.