Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants
Opposing Roles of Double-Stranded RNA Effector Pathways and Viral Defense Proteins Revealed with CRISPR-Cas9 Knockout Cell Lines and Vaccinia Virus Mutants
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DOI：
10.1128/jvi.00869-16
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发表时间：
2016-09-01
影响因子：
5.4
通讯作者：
Moss, Bernard
中科院分区：
文献类型：
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作者：
Liu, Ruikang;Moss, Bernard
牛痘病毒（VACV）的脱帽酶和细胞核糖核酸外切酶Xrn 1催化mRNA降解的连续步骤，并防止双链RNA（dsRNA）积累，而病毒E3蛋白可以结合dsRNA。我们发现，dsRNA和E3共定位在细胞质内的病毒工厂与脱帽酶突变体以及野生型VACV感染的细胞，他们与抗体共沉淀。E3缺失突变体诱导蛋白激酶R（PKR）和真核翻译起始因子α（eIF 2 α）磷酸化更早，更强的脱帽酶突变体，即使更少的dsRNA，导致更深刻的影响病毒基因表达。通过成簇规则间隔的短回文重复序列-Cas9（CRISPR-Cas9）对人类HAP 1和A549细胞进行遗传修饰，以确定相同的途径是否限制E3和脱帽突变体。E3突变体在PKR敲除（KO）HAP 1细胞中复制，其中RNA酶L本质上是无活性的，但在A549细胞中仅具有PKR和RNA酶L的双敲除（DKO），这表明两种途径同等地降低复制，并且没有额外的dsRNA途径是至关重要的。相反，脱帽酶突变体的复制在DKO A549细胞中显著增加（尽管低于野生型病毒的复制），但在DKO HAP 1细胞中没有，其中病毒蛋白质合成的增加较小。Xrn 1 KO A549细胞存活，但对VACV不允许;然而，野生型和突变型病毒在三KO细胞中复制，其中RNA酶L和PKR也被灭活。由于KO的PKR和RNase L是足以使VACV复制在E3或Xrn 1的情况下，不良的复制脱帽突变体，特别是在HAP 1 DKO，细胞表示额外的translational defects.重要的是，病毒已经发展的方式来防止或抵消级联的抗病毒反应，双链RNA（dsRNA）在宿主细胞中触发。我们发现，在用牛痘病毒（VACV）脱帽酶突变体和野生型病毒感染的细胞中产生过量的dsRNA，并与细胞质病毒工厂中的病毒E3蛋白共定位。通过CRISPR-Cas9基因编辑制备在蛋白激酶R和RNase L dsRNA效应子途径和/或细胞5'外切核酸酶Xrnl中的任一者或两者中有缺陷的新型人细胞系。这两种途径的失活是必要的，足以使E3突变体的完全复制和逆转的缺陷引起的失活的Xrn 1，而脱帽酶突变体仍然表现出基因表达的缺陷。该研究为VACV蛋白的功能提供了新的见解，并且CRISPR-Cas9修饰的人类细胞系的良好表征小组应该具有研究先天dsRNA途径的广泛适用性。
Vaccinia virus (VACV) decapping enzymes and cellular exoribonuclease Xrn1 catalyze successive steps in mRNA degradation and prevent double-stranded RNA (dsRNA) accumulation, whereas the viral E3 protein can bind dsRNA. We showed that dsRNA and E3 colocalized within cytoplasmic viral factories in cells infected with a decapping enzyme mutant as well as with wild-type VACV and that they coprecipitated with antibody. An E3 deletion mutant induced protein kinase R (PKR) and eukaryotic translation initiation factor alpha (eIF2 alpha) phosphorylation earlier and more strongly than a decapping enzyme mutant even though less dsRNA was made, leading to more profound effects on viral gene expression. Human HAP1 and A549 cells were genetically modified by clustered regularly interspaced short palindromic repeat-Cas9 (CRISPR-Cas9) to determine whether the same pathways restrict E3 and decapping mutants. The E3 mutant replicated in PKR knockout (KO) HAP1 cells in which RNase L is intrinsically inactive but only with a double knockout (DKO) of PKR and RNase L in A549 cells, indicating that both pathways decreased replication equivalently and that no additional dsRNA pathway was crucial. In contrast, replication of the decapping enzyme mutant increased significantly (though less than that of wild-type virus) in DKO A549 cells but not in DKO HAP1 cells where a smaller increase in viral protein synthesis occurred. Xrn1 KO A549 cells were viable but nonpermissive for VACV; however, wild-type and mutant viruses replicated in triple-KO cells in which RNase L and PKR were also inactivated. Since KO of PKR and RNase L was sufficient to enable VACV replication in the absence of E3 or Xrn1, the poor replication of the decapping mutant, particularly in HAP1 DKO, cells indicated additional translational defects.IMPORTANCEViruses have evolved ways of preventing or counteracting the cascade of antiviral responses that double-stranded RNA (dsRNA) triggers in host cells. We showed that the dsRNA produced in excess in cells infected with a vaccinia virus (VACV) decapping enzyme mutant and by wild-type virus colocalized with the viral E3 protein in cytoplasmic viral factories. Novel human cell lines defective in either or both protein kinase R and RNase L dsRNA effector pathways and/or the cellular 5' exonuclease Xrn1 were prepared by CRISPR-Cas9 gene editing. Inactivation of both pathways was necessary and sufficient to allow full replication of the E3 mutant and reverse the defect cause by inactivation of Xrn1, whereas the decapping enzyme mutant still exhibited defects in gene expression. The study provided new insights into functions of the VACV proteins, and the well-characterized panel of CRISPR-Cas9-modified human cell lines should have broad applicability for studying innate dsRNA pathways.