School of Basic Medical Sciences, Tsinghua University, Beijing, China.
State Key Laboratory of Virology, Wuhan Institute of Virology, Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, Hubei, China.
PLoS Pathog. 2024 Aug 22;20(8):e1012444. doi: 10.1371/journal.ppat.1012444. eCollection 2024 Aug.
The Ebola virus (EBOV) has emerged as a significant global health concern, notably during the 2013-2016 outbreak in West Africa. Despite the clinical approval of two EBOV antibody drugs, there is an urgent need for more diverse and effective antiviral drugs, along with comprehensive understanding of viral-host interactions. In this study, we harnessed a biologically contained EBOVΔVP30-EGFP cell culture model which could recapitulate the entire viral life cycle, to conduct a genome-wide CRISPR/Cas9 screen. Through this, we identified PIK3C3 (phosphatidylinositide 3-kinase) and SLC39A9 (zinc transporter) as crucial host factors for EBOV infection. Genetic depletion of SLC39A9 and PIK3C3 lead to reduction of EBOV entry, but not impact viral genome replication, suggesting that SLC39A9 and PIK3C3 act as entry factors, facilitating viral entry into host cells. Moreover, PIK3C3 kinase activity is indispensable for the internalization of EBOV virions, presumably through the regulation of endocytic and autophagic membrane traffic, which has been previously recognized as essential for EBOV internalization. Notably, our study demonstrated that PIK3C3 kinase inhibitor could effectively block EBOV infection, underscoring PIK3C3 as a promising drug target. Furthermore, biochemical analysis showed that recombinant SLC39A9 protein could directly bind viral GP protein, which further promotes the interaction of viral GP protein with cellular receptor NPC1. These findings suggests that SLC39A9 plays dual roles in EBOV entry. Initially, it serves as an attachment factor during the early entry phase by engaging with the viral GP protein. Subsequently, SLC39A9 functions an adaptor protein, facilitating the interaction between virions and the NPC1 receptor during the late entry phase, prior to cathepsin cleavage on the viral GP. In summary, this study offers novel insights into virus-host interactions, contributing valuable information for the development of new therapies against EBOV infection.
埃博拉病毒（EBOV）已成为一个重大的全球健康关注点，特别是在 2013 年至 2016 年西非爆发期间。尽管已经有两种 EBOV 抗体药物获得临床批准，但仍迫切需要更多多样化和有效的抗病毒药物，并全面了解病毒-宿主相互作用。在这项研究中，我们利用了一种生物学上受限制的 EBOVΔVP30-EGFP 细胞培养模型，该模型可以重现整个病毒生命周期，进行全基因组 CRISPR/Cas9 筛选。通过这种方法，我们确定了 PIK3C3（磷脂酰肌醇 3-激酶）和 SLC39A9（锌转运体）是 EBOV 感染的关键宿主因素。SLC39A9 和 PIK3C3 的基因缺失会导致 EBOV 进入减少，但不会影响病毒基因组复制，表明 SLC39A9 和 PIK3C3 作为进入因子，促进病毒进入宿主细胞。此外，PIK3C3 激酶活性对于 EBOV 病毒粒子的内化是不可或缺的，可能是通过调节内吞作用和自噬膜运输，这已被先前认为对于 EBOV 内化是必不可少的。值得注意的是，我们的研究表明 PIK3C3 激酶抑制剂可以有效地阻断 EBOV 感染，凸显了 PIK3C3 作为一个有前途的药物靶点。此外，生化分析表明，重组 SLC39A9 蛋白可以直接结合病毒 GP 蛋白，这进一步促进了病毒 GP 蛋白与细胞受体 NPC1 的相互作用。这些发现表明 SLC39A9 在 EBOV 进入中起双重作用。最初，它通过与病毒 GP 蛋白结合，作为早期进入阶段的附着因子。随后，SLC39A9 作为衔接蛋白，在病毒 GP 蛋白的天冬氨酸蛋白酶切割之前，促进病毒粒子与 NPC1 受体之间的相互作用。总之，这项研究提供了病毒-宿主相互作用的新见解，为开发针对 EBOV 感染的新疗法提供了有价值的信息。
