PDF(Pubmed)
Abstract：
Rift Valley fever phlebovirus (RVFV) is a zoonotic pathogen that causes Rift Valley fever (RVF) in livestock and humans. Currently, there is no licensed human vaccine or antiviral drug to control RVF. Although multiple species of animals and humans are vulnerable to RVFV infection, host factors affecting susceptibility are not well understood. To identify the host factors or genes essential for RVFV replication, we conducted CRISPR-Cas9 knockout screening in human A549 cells. We then validated the putative genes using siRNA-mediated knock-downs and CRISPR-Cas9-mediated knock-out studies. The role of a candidate gene in the virus replication cycle was assessed by measuring intracellular viral RNA accumulation, and the virus titers were analyzed using plaque assay or TCID50 assay. We identified approximately 900 genes with potential involvement in RVFV infection and replication. Further evaluation of the effect of six genes on viral replication using siRNA-mediated knock-downs revealed that silencing two genes (WDR7 and LRP1) significantly impaired RVFV replication. For further analysis, we focused on the WDR7 gene since the role of the LRP1 gene in RVFV replication was previously described in detail. WDR7 knockout A549 cell lines were generated and used to dissect the effect of WRD7 on a bunyavirus, RVFV, and an orthobunyavirus, La Crosse encephalitis virus (LACV). We observed significant effects of WDR7 knockout cells on both intracellular RVFV RNA levels and viral titers. At the intracellular RNA level, WRD7 affected RVFV replication at a later phase of its replication cycle (24 h) when compared with the LACV replication, which was affected in an earlier replication phase (12 h). In summary, we identified WDR7 as an essential host factor for the replication of two different viruses, RVFV and LACV, both of which belong to the Bunyavirales order. Future studies will investigate the mechanistic role through which WDR7 facilitates phlebovirus replication.
摘要：
裂谷热静脉病毒（RVFV）是一种人畜共患病原体，可引起牲畜和人类的裂谷热（RVF）。目前,没有许可的人类疫苗或抗病毒药物来控制RVF。尽管多种动物和人类都容易感染RVFV，影响易感性的宿主因素尚不清楚。为了确定RVFV复制所必需的宿主因子或基因，我们在人A549细胞中进行了CRISPR-Cas9基因敲除筛选。然后，我们使用siRNA介导的敲除和CRISPR-Cas9介导的敲除研究验证了推定的基因。通过测量细胞内病毒RNA积累来评估候选基因在病毒复制周期中的作用。并使用噬斑测定或TCID50测定分析病毒滴度。我们鉴定了大约900个可能参与RVFV感染和复制的基因。使用siRNA介导的敲减对六个基因对病毒复制的影响的进一步评估表明，沉默两个基因（WDR7和LRP1）显着损害了RVFV复制。为了进一步分析，我们专注于WDR7基因,因为LRP1基因在RVFV复制中的作用之前已经有详细描述.产生WDR7敲除A549细胞系，并用于剖析WRD7对布尼亚病毒的影响，RVFV,和一种直鼻病毒，LaCrosse脑炎病毒（LACV）。我们观察到WDR7敲除细胞对胞内RVFVRNA水平和病毒滴度的显著影响。在细胞内RNA水平，与LACV复制相比，WRD7在其复制周期的后期（24小时）影响了RVFV复制，在较早的复制阶段（12小时）受到影响。总之,我们确定WDR7是两种不同病毒复制的必需宿主因子，RVFV和LACV，两者都属于Bunyavirales命令。未来的研究将研究WDR7促进静脉病毒复制的机制作用。
