两篇Nature：RNA干扰分析流感病毒

　　

生物通报道：去年的甲流让人记忆深刻，2010年2－3月国内新增甲型H1N1流感重症和死亡病例数降至较低水平，不过世卫组织向各国发出警告：甲流高峰期还未结束，防治工作不能松懈。

尽管甲流疫情随着气候和相应的防治措施已渐渐不再像之前那样受到关注，但是相关的科学研究和临床研究并没有停下脚步，在2月的Nature杂志上，两篇文章分别利用全基因组RNA干扰方法，筛选发现了甲流病毒复制所需的人类宿主因子。

来自伯纳姆医学研究所（Burnham institute for medical research）的研究人员发现了甲流流感病毒在肺上皮细胞系中复制所需295个宿主基因。其中219个是野生型病毒高效生长所需的，23个是病毒进入所需的。

甲流病毒是一种上呼吸道病毒，它钻进人类细胞后利用人类细胞器进行自我复制，其子代病毒破壳而出，再感染其他的健康细胞。从结构上来说，甲型流感病毒就像是个球，球上布满触角，它们是血凝素蛋白质(H1)神经氨酸酶(N1)，而这些H1和N1蛋白质是会引起人体免疫系统攻击的抗原。

甲流RNA只能编码11种蛋白，因此需要利用宿主复制来完成其生命周期，了解这些宿主复制基因对于掌握甲流病毒的特性，以及防治和治疗甲流具有重要的意义。

研究人员利用了一种系统分析方法，以全基因组RNA干扰筛选为基础，识别出了295个宿主基因。其中219个是野生型病毒高效生长所需的，23个是病毒进入所需的。

另外一篇文章由马普研究院传染病研究所的研究人员完成，他们发现了影响病毒复制的287个宿主基因。研究分析证实，其中168个基因（占59%）要么抑制H1N1流感病毒（119个基因），要么抑制当前流行的起源于猪的甲流病毒（121个基因），重叠率为60%。 这些研究应能为用于治疗流感病毒感染的、由宿主因子引导的抗病毒药物提供几个潜在目标。

除此之外，近期也获得许多甲流病毒研究新成果，比如研究人员发现120种参与抵抗甲流的蛋白因子分别与内涵体酸化、囊泡、线粒体代谢功能、RNA剪接有关联。其中的一个重要发现是，干扰素诱导的跨膜蛋白（interferon-inducible transmembrane proteins）IFITM1, IFITM 2和IFITM 3对甲流H1N1病毒早期的复制过程有重要的作用。

当IFITM3的产生被中断后，H1N1的复制出现惊人的增加。因此研究人员开始猜测，这些蛋白是一些天然的抗病毒蛋白。当IFITM3不存在时，这些病毒的复制增加5-10倍。病毒蛋白水平越高复制的越快。IFITM3的作用在这方面确实很突出。 研究人员在其它细胞类型以及不同的H1N1毒株上发现IFITM3有相同的作用，包括人类和小鼠的肺细胞。在增加IFITM3时发现，H1N1的复制完全被抑制了。 这项研究证明了通过广泛筛查去发现与病毒复制有关的细胞蛋白的优势。

IFITM3基因位于11号染色体上，其次排列的是IFITM1 和IFITM2基因。尽管试验中IFITM3的作用最强，但如果所有这三种蛋白都大量产生，就会抑制H1N1病毒以及其它流感病毒。

让研究人员感到吃惊的是，这些蛋白还能抑制完全不同病毒的复制，包括西尼罗河病毒和登革热病毒。然而，IFITM并不能有效对抗所有的病毒，而且研究人员也不能准确知道这些蛋白存在的真正意义。它们的活性程度不同，所以它们可能对不同病毒有特异性。

（生物通：万纹）

原文摘要：

Human host factors required for influenza virus replicationnear-final version

Influenza A virus is an RNA virus that encodes up to 11 proteins and this small coding capacity demands that the virus use the host cellular machinery for many aspects of its life cycle1. Knowledge of these host cell requirements not only informs us of the molecular pathways exploited by the virus but also provides further targets that could be pursued for antiviral drug development. Here we use an integrative systems approach, based on genome-wide RNA interference screening, to identify 295 cellular cofactors required for early-stage influenza virus replication. Within this group, those involved in kinase-regulated signalling, ubiquitination and phosphatase activity are the most highly enriched, and 181 factors assemble into a highly significant host–pathogen interaction network. Moreover, 219 of the 295 factors were confirmed to be required for efficient wild-type influenza virus growth, and further analysis of a subset of genes showed 23 factors necessary for viral entry, including members of the vacuolar ATPase (vATPase) and COPI-protein families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase kinase 3 (GSK3)-β. Furthermore, 10 proteins were confirmed to be involved in post-entry steps of influenza virus replication. These include nuclear import components, proteases, and the calcium/calmodulin-dependent protein kinase (CaM kinase) IIβ (CAMK2B). Notably, growth of swine-origin H1N1 influenza virus is also dependent on the identified host factors, and we show that small molecule inhibitors of several factors, including vATPase and CAMK2B, antagonize influenza virus replication.

Genome-wide RNAi screen identifies human host factors crucial for influenza virus replication

Influenza A virus, being responsible for seasonal epidemics and reoccurring pandemics, represents a worldwide threat to public health1. High mutation rates facilitate the generation of viral escape mutants, rendering vaccines and drugs directed against virus-encoded targets potentially ineffective2. In contrast, targeting host cell determinants temporarily dispensable for the host but crucial for virus replication could prevent viral escape. Here we report the discovery of 287 human host cell genes influencing influenza A virus replication in a genome-wide RNA interference (RNAi) screen. Using an independent assay we confirmed 168 hits (59%) inhibiting either the endemic H1N1 (119 hits) or the current pandemic swine-origin (121 hits) influenza A virus strains, with an overlap of 60%. Notably, a subset of these common hits was also essential for replication of a highly pathogenic avian H5N1 strain. In-depth analyses of several factors provided insights into their infection stage relevance. Notably, SON DNA binding protein (SON)3 was found to be important for normal trafficking of influenza virions to late endosomes early in infection. We also show that a small molecule inhibitor of CDC-like kinase 1 (CLK1)4 reduces influenza virus replication by more than two orders of magnitude, an effect connected with impaired splicing of the viral M2 messenger RNA. Furthermore, influenza-virus-infected p27 -/- (cyclin-dependent kinase inhibitor 1B; Cdkn1b) mice accumulated significantly lower viral titres in the lung, providing in vivo evidence for the importance of this gene. Thus, our results highlight the potency of genome-wide RNAi screening for the dissection of virus–host interactions and the identification of drug targets for a broad range of influenza viruses.