Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1L-C7L- Mutant.
Identification of Restriction Factors by Human Genome-Wide RNA Interference Screening of Viral Host Range Mutants Exemplified by Discovery of SAMD9 and WDR6 as Inhibitors of the Vaccinia Virus K1L-C7L- Mutant.
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DOI：
10.1128/mbio.01122-15
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发表时间：
2015-08-04
期刊：
影响因子：
6.4
通讯作者：
Moss B
中科院分区：
文献类型：
--
作者：
Sivan G;Ormanoglu P;Buehler EC;Martin SE;Moss B
如果病毒已经抵消了宿主的防御机制，则旨在识别限制病毒复制的宿主因素的RNA干扰（RNAi）筛选可能会失败。为了克服这一限制，我们正在研究使用病毒宿主范围突变体，表现出受损的复制在nonpermissive细胞。该策略选择了一种缺失C7 L和K1 L基因（K1 L-C7 L-）的痘苗病毒（VACV）突变体，该突变体在晚期基因表达之前的一个步骤中消除了人类细胞中的复制。我们在感染VACV K1 L − C7 L −突变体的HeLa细胞中进行了人类全基因组小干扰RNA（siRNA）筛选，该突变体表达由晚期启动子调控的绿色荧光蛋白。该阳性选择筛选具有显著低的背景水平，并导致从约20，000个测试的细胞中鉴定出显著增强绿色荧光蛋白表达的一些细胞基因，特别是SAMD 9和WDR 6。通过针对SAMD 9或WDR 6的多个siRNA使突变病毒的复制成为可能。此外，SAMD 9和WDR 6成簇规则间隔短回文重复序列（CRISPR）/Cas9敲除HeLa细胞系允许K1 L-C7 L-突变体的复制，与siRNA数据一致。外源性SAMD 9或干扰素调节因子1的表达限制了K1 L-C7 L-突变体在SAMD 9-/-细胞中的复制。免疫沉淀表明SAMD 9与K1和C7蛋白存在独立的相互作用。敲除WDR 6并没有降低SAMD 9的水平，也没有检测到WDR 6与SAMD 9、C7和K1蛋白的相互作用，这表明这些限制因子独立地起作用，但可能在相同的先天防御途径中起作用。微生物病原体与细胞的共同进化导致了一场军备竞赛，入侵者和宿主不断地争夺优势。因此，如果病原体已经产生了有效的反应，传统的siRNA筛选可能无法揭示重要的免疫机制。然而，宿主限制性病毒突变体失去了一个或多个防御基因，这些基因是它们在非允许细胞中复制所需的。通过筛选人类基因组文库的短RNA，抑制在nonpermissive细胞中的个别宿主基因的表达，我们确定SAMD 9和WDR 6作为主要的限制因素，防止复制的牛痘病毒突变体，并建议，主机范围的筛选通常可以用于调查的主机-病原体相互作用。
RNA interference (RNAi) screens intended to identify host factors that restrict virus replication may fail if the virus already counteracts host defense mechanisms. To overcome this limitation, we are investigating the use of viral host range mutants that exhibit impaired replication in nonpermissive cells. A vaccinia virus (VACV) mutant with a deletion of both the C7L and K1L genes, K1L−C7L−, which abrogates replication in human cells at a step prior to late gene expression, was chosen for this strategy. We carried out a human genome-wide small interfering RNA (siRNA) screen in HeLa cells infected with a VACV K1L−C7L− mutant that expresses the green fluorescent protein regulated by a late promoter. This positive-selection screen had remarkably low background levels and resulted in the identification of a few cellular genes, notably SAMD9 and WDR6, from approximately 20,000 tested that dramatically enhanced green fluorescent protein expression. Replication of the mutant virus was enabled by multiple siRNAs to SAMD9 or WDR6. Moreover, SAMD9 and WDR6 clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 knockout HeLa cell lines were permissive for replication of the K1L−C7L− mutant, in agreement with the siRNA data. Expression of exogenous SAMD9 or interferon regulatory factor 1 restricted replication of the K1L−C7L− mutant in the SAMD9−/− cells. Independent interactions of SAMD9 with the K1 and C7 proteins were suggested by immunoprecipitation. Knockout of WDR6 did not reduce the levels of SAMD9 and interactions of WDR6 with SAMD9, C7, and K1 proteins were not detected, suggesting that these restriction factors act independently but possibly in the same innate defense pathway. The coevolution of microbial pathogens with cells has led to an arms race in which the invader and host continuously struggle to gain the advantage. For this reason, traditional siRNA screens may fail to uncover important immune mechanisms if the pathogens have already developed effective responses. However, host-restricted viral mutants have lost one or more defense genes needed for their replication in nonpermissive cells. By screening human genome libraries of short RNAs that inhibit the expression of individual host genes in nonpermissive cells, we identified SAMD9 and WDR6 as major restriction factors that prevented replication of a vaccinia virus mutant and suggest that host range screening can be generally useful for the investigation of host-pathogen interactions.