Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration
Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration
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DOI：
10.1128/jvi.00388-14
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发表时间：
2014-08-01
影响因子：
5.4
通讯作者：
Lozach, Pierre-Yves
中科院分区：
文献类型：
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作者：
Meier, Roger;Franceschini, Andrea;Lozach, Pierre-Yves
布尼亚病毒科是一个大家族的有包膜动物病毒，其中许多是重要的新兴病原体。布尼亚病毒如何进入和感染哺乳动物细胞在很大程度上仍不清楚。我们使用了两个全基因组沉默屏幕与不同的小干扰RNA（siRNA）库，以调查在布尼亚病毒Uukuniemi病毒（UUKV），晚穿透病毒感染人类细胞过程中所需的宿主蛋白。对文库的序列分析显示，筛选中的许多siRNA不仅通过沉默预期的靶标，而且通过以微小RNA（miRNA）样方式沉默其他基因来抑制感染。通过使用合成的miRNAs（miRs）证实了siRNA中的7个核苷酸的种子区可以引起感染的扰动。所测试的miR之一miR-142- 3 p显示出通过调节v-SNARE VAMP 3来干扰传入病毒的细胞内运输，这是两种siRNA筛选共享的强烈命中。破伤风毒素灭活VAMP 3导致感染阻断。在固定和活细胞中使用基于荧光的技术，我们发现病毒在内化后5分钟进入VAMP 3（+）内体囊泡，并且在此后15分钟共定位最大。此时，LAMP 1与含有VAMP 3（+）病毒的内体结合。在耗尽VAMP 3的细胞中，病毒主要被捕获在LAMP 1阴性隔室中。总之，我们的研究结果表明，UUKV依赖于VAMP 3的渗透，通过内吞网络的病毒运输增加了复杂性的指示。重要信息布尼亚病毒代表了全球人类和牲畜日益增长的威胁。不幸的是，人们对这些新出现的病原体知之甚少。我们在这里报告的第一个人类全基因组siRNA屏幕布尼亚病毒。筛选结果鉴定出562种在细胞进入和病毒复制中具有潜在作用的宿主细胞因子。为了证明我们的方法的鲁棒性，我们确认并分析了v-SNARE VAMP 3在Uukuniemi病毒进入和感染中的作用。所获得的信息为未来研究布尼亚病毒感染的细胞生物学和新的抗病毒策略奠定了基础。此外，通过阐明大规模siRNA筛选中的严重警告，我们的实验和生物信息学程序将在过去和未来的高内容筛选数据的综合分析中具有价值。
The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3(+) endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3(+) virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network.IMPORTANCE Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we confirmed and analyzed the role of the v-SNARE VAMP3 in Uukuniemi virus entry and infection. The information gained lays the basis for future research into the cell biology of bunyavirus infection and new antiviral strategies. In addition, by shedding light on serious caveats in large-scale siRNA screening, our experimental and bioinformatics procedures will be valuable in the comprehensive analysis of past and future high-content screening data.