Labeau Athena, Simon-Loriere Etienne, Hafirassou Mohamed-Lamine, Bonnet-Madin Lucie, Tessier Sarah, Zamborlini Alessia, Dupré Thierry, Seta Nathalie, Schwartz Olivier, Chaix Marie-Laure, Delaugerre Constance, Amara Ali, Meertens Laurent
INSERM U944, CNRS UMR 7212, Genomes & Cell Biology of Disease Unit, Institut de Recherche Saint-Louis, Université de Paris, Hôpital Saint-Louis, Paris, France.
G5 Evolutionary Genomics of RNA Viruses, Institut Pasteur, Paris, France.
J Virol. 2020 Mar 17;94(7). doi: 10.1128/JVI.01751-19.
Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection. Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.
登革病毒（DENV）是一种由蚊子传播的黄病毒，可引发登革热疾病，这是一个重大的人类健康问题，目前尚无特效疗法。与其他病毒一样，DENV在有效感染过程中严重依赖宿主细胞机制。在本研究中，我们利用单倍体HAP1细胞进行了全基因组CRISPR-Cas9筛选，以确定对DENV感染至关重要的宿主基因。我们鉴定出内质网（ER）驻留的磷酸多萜醇甘露糖合酶（DPMS）复合物的两个亚基DPM1和DPM3，它们是DENV及其他相关黄病毒（如寨卡病毒，ZIKV）的宿主依赖性因子。DPMS复合物催化磷酸多萜醇甘露糖（DPM）的合成，DPM在导致N-糖基化、糖基磷脂酰肌醇（GPI）锚定生物合成以及内质网腔中蛋白质的C-或O-甘露糖基化的途径中作为甘露糖基供体。DPM1催化活性所必需的DXD基序发生突变，会消除DPMS介导的DENV感染。同样，甘露糖基转移酶ALG3（将甘露糖转移至脂连接寡糖，LLO）的基因敲除使细胞对DENV的敏感性降低。我们还证实，在DPMS活性缺陷的细胞中，病毒RNA扩增受到阻碍，并且截短的寡糖被转移至病毒前膜（prM）和包膜（E）糖蛋白上，影响其正确折叠。总体而言，我们的研究为DENV感染的宿主依赖性机制提供了新见解，并支持目前使用糖基化抑制剂治疗DENV感染的治疗方法。由登革病毒（DENV）引起的登革热疾病已成为人类最重要的蚊媒病毒疾病，是全球主要的健康问题。DENV仅编码少数几种蛋白质，依赖宿主细胞机制来完成其生命周期。需要鉴定出对DENV感染至关重要的宿主因子，以提出抗病毒干预的新靶点。通过全基因组CRISPR-Cas9筛选，我们鉴定出DPMS复合物的两个亚基DPM1和DPM3，它们是DENV以及其他相关病毒（如寨卡病毒）复制的重要宿主因子。我们证实DPMS复合物在病毒感染过程中发挥双重作用，既调节病毒RNA复制，又促进病毒结构糖蛋白的折叠/稳定性。这些结果为DENV及其他黄病毒利用宿主分子促进其生命周期提供了见解。
