Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.
Glucosylceramide is essential for Heartland and Dabie bandavirus glycoprotein-induced membrane fusion.
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DOI：
10.1371/journal.ppat.1011232
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发表时间：
2023-03
期刊：
影响因子：
6.7
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中科院分区：
文献类型：
--
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由于气候变化，新出现的蜱传人畜共患病毒大量繁殖，包括中心带病毒（HRTV）和大别带病毒（DBV）。作为高致死率的出血热病原菌，HRTV 和 DBV 已被认为是危险的病毒病原体，可能导致未来广泛流行。尽管存在严重的健康问题，但病毒感染的机制在很大程度上仍不清楚。 HRTV 和 DBV Gn 和 Gc 是感染期间早期进入事件所需的病毒表面糖蛋白。鞘糖脂，包括半乳糖神经酰胺 (GalCer)、葡萄糖神经酰胺 (GlcCer) 和乳糖神经酰胺 (LacCer)，是一类膜脂，在膜结构和病毒生命周期中发挥重要作用。在这里，我们的全基因组 CRISPR/Cas9 敲除筛选确定了鞘糖脂生物合成途径对于 HRTV 和 DBV 感染至关重要。产生 GlcCer 的 UDP-葡萄糖神经酰胺葡萄糖基转移酶 (UGCG) 的缺陷导致用 HRTV 或 DBV Gn/Gc 糖蛋白假型化的重组病毒失去感染性。相反，外源性补充 GlcCer（而非 GalCer 或 LacCer）以剂量依赖性方式恢复病毒进入 UGCG 缺陷细胞。生物物理分析表明，GlcCer 靶向 Gc 的脂质头基结合袋，形成稳定的蛋白质-脂质复合物，使 Gc 蛋白插入宿主溶酶体膜脂质双层中进行病毒融合。诱变表明，Gc 脂质结合袋上的 D841 残基对于 GlcCer 相互作用以及病毒进入至关重要。这些发现揭示了 GlcCer 鞘糖脂在 HRTV 和 DBV Gc 介导的膜融合中的详细机制，并为蜱传病毒感染提供了潜在的治疗靶点。中心带病毒（HRTV）和大别带病毒（DBV）最近分别在美国和亚洲被确定为新出现的蜱传人畜共患病毒。作为高致死率的出血热病原菌，HRTV 和 DBV 已被认为是危险的病毒病原体，可能导致未来广泛流行。尽管存在严重的健康问题，但病毒感染的机制在很大程度上仍不清楚。在这里，我们使用全基因组 CRISPR/Cas9 敲除筛选来确定 HRTV 进入哺乳动物细胞的要求。我们发现鞘糖脂生物合成途径对于 HRTV 和 DBV 感染至关重要。 HRTV 和 DBV 在鞘糖脂生物合成缺陷细胞中的感染性显着降低。我们还发现葡萄糖神经酰胺 (GlcCer) 在 HRTV 糖蛋白介导的膜融合中发挥着至关重要的作用。 GlcCer 靶向宿主溶酶体膜中 HRTV 糖蛋白的脂质头基结合袋，形成稳定的脂质-蛋白质复合物，从而促进病毒融合和进入。我们的研究揭示了 GlcCer 鞘糖脂在 HRTV 和 DBV Gc 介导的膜融合中的详细分子机制，并为蜱传病毒感染提供了潜在的治疗靶点。
Due to climate changes, there has been a large expansion of emerging tick-borne zoonotic viruses, including Heartland bandavirus (HRTV) and Dabie bandavirus (DBV). As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. HRTV and DBV Gn and Gc are viral surface glycoproteins required for early entry events during infection. Glycosphingolipids, including galactosylceramide (GalCer), glucosylceramide (GlcCer) and lactosylceramide (LacCer), are a class of membrane lipids that play essential roles in membrane structure and viral lifecycle. Here, our genome-wide CRISPR/Cas9 knockout screen identifies that glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The deficiency of UDP-glucose ceramide glucosyltransferase (UGCG) that produces GlcCer resulted in the loss of infectivity of recombinant viruses pseudotyped with HRTV or DBV Gn/Gc glycoproteins. Conversely, exogenous supplement of GlcCer, but not GalCer or LacCer, recovered viral entry of UGCG-deficient cells in a dose-dependent manner. Biophysical analyses showed that GlcCer targeted the lipid-head-group binding pocket of Gc to form a stable protein-lipid complex, which allowed the insertion of Gc protein into host lysosomal membrane lipid bilayers for viral fusion. Mutagenesis showed that D841 residue at the Gc lipid binding pocket was critical for GlcCer interaction and thereby, viral entry. These findings reveal detailed mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provide a potential therapeutic target for tickborne virus infection. Heartland bandavirus (HRTV) and Dabie bandavirus (DBV) were recently identified as emerging tick-borne zoonotic viruses in the United States and Asia, respectively. As etiologic agents of hemorrhagic fever with high fatality, HRTV and DBV have been recognized as dangerous viral pathogens that likely cause future wide epidemics. Despite serious health concerns, the mechanisms underlying viral infection are largely unknown. Here, we use genome-wide CRISPR/Cas9 knockout screens to determine the requirements of HRTV entry in mammalian cells. We found that the glycosphingolipid biosynthesis pathway is essential for HRTV and DBV infection. The infectivity of HRTV and DBV in glycosphingolipid biosynthesis-deficient cells was drastically reduced. We also found that glucosylceramide (GlcCer) plays a vital role in HRTV glycoproteins-mediated membrane fusion. The GlcCer targets the lipid-head-group binding pocket of HRTV glycoprotein in the host lysosomal membrane to form a stable lipid-protein complex, thereby facilitating viral fusion and entry. Our study reveals the detailed molecular mechanism of GlcCer glycosphingolipid in HRTV and DBV Gc-mediated membrane fusion and provides a potential therapeutic target for tick-borne virus infection.
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