Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection
Genome-Wide Screening Uncovers the Significance of N-Sulfation of Heparan Sulfate as a Host Cell Factor for Chikungunya Virus Infection
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DOI：
10.1128/jvi.00432-17
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发表时间：
2017-07-01
影响因子：
5.4
通讯作者：
Maeda, Yusuke
中科院分区：
文献类型：
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作者：
Tanaka, Atsushi;Tumkosit, Uranan;Maeda, Yusuke
基孔肯雅病毒（CHIKV）感染的分子机制的特点很差。在这项研究中，我们使用全基因组筛选分析了CHIKV感染的宿主因素。将引入外显子捕获载体的人单倍体HAP 1细胞用携带CHIKV E3至E1包膜蛋白的水泡性口炎病毒假型攻击。在对假型病毒感染具有抗性的细胞中富集的基因的分析揭示了硫酸乙酰肝素（HS）的N-硫酸化对于临床分离的CHIKV Thai#16856株对HAP 1细胞的感染性的关键作用。敲除催化HS的N-硫酸化的NDST 1大大降低了CHIKV Thai#16856株的结合和感染性，但不降低日本脑炎病毒（JEV）和黄热病病毒（YFV）的感染性。虽然糖胺聚糖通常是CHIKV、JEV和YFV的有效感染性所需的，如通过使用B3 GAT 3敲除细胞所示，但N-硫酸盐的向性对CHIKV是特异性的。硫酸软骨素（CS）在NOSTI敲除的HAP 1细胞中的表达没有恢复CHIKV Thai#16856菌株的结合及其假型的感染性，但恢复了真实CHIKV Thai#16856的感染性，表明CS在CHIKV结合后的后续步骤中起作用。在该筛选中富集的基因中，我们发现TM 9 SF 2对于HS的N-硫酸化至关重要，因此对于CHIKV感染至关重要，因为它参与NDST 1的适当定位和稳定性。确定的意义和相关蛋白质的N-硫酸化的HS可能有助于了解CHIKV的传播，细胞向性和pathogenicity.IMPORTANCE最近爆发的基孔肯雅热的机制增加了其临床重要性。基孔肯雅病毒（CHIKV）利用宿主糖胺聚糖有效地结合其靶细胞。然而，感染所需的糖胺聚糖的亚结构尚未被表征。在这里，我们揭示了硫酸乙酰肝素中的N-硫酸盐对于临床CHIKV菌株有效感染HAP 1细胞是必不可少的，并且硫酸软骨素并不有助于CHIKV结合，但在HAP 1细胞的后续步骤中发挥作用。通过比较使用中国仓鼠卵巢细胞的先前报道，我们显示，沿着另一个观察结果，即在包膜E2中携带Arg 82的CHIKV的增强的感染性不依赖于HAP 1细胞中的糖胺聚糖，CHIKV的感染方式在宿主细胞中不同。我们还表明，TM 9 SF 2是CHIKV感染HAP 1细胞所必需的，因为它通过确保NDST 1活性参与硫酸乙酰肝素的N-硫酸化。
The molecular mechanisms underlying chikungunya virus (CHIKV) infection are poorly characterized. In this study, we analyzed the host factors involved in CHIKV infection using genome-wide screening. Human haploid HAP1 cells, into which an exon-trapping vector was introduced, were challenged with a vesicular stomatitis virus pseudotype bearing the CHIKV E3 to El envelope proteins. Analysis of genes enriched in the cells resistant to the pseudotyped virus infection unveiled a critical role of N-sulfation of heparan sulfate (HS) for the infectivity of the clinically isolated CHIKV Thai#16856 strain to HAP1 cells. Knockout of NDST1 that catalyzes N-sulfation of HS greatly decreased the binding and infectivity of CHIKV Thai#16856 strain but not infectivity of Japanese encephalitis virus (JEV) and yellow fever virus (YFV). While glycosaminoglycans were commonly required for the efficient infectivity of CHIKV, JEV, and YFV, as shown by using B3GAT3 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in NOSTI-knockout HAP1 cells did not restore the binding of CHIKV Thai#16856 strain and the infectivity of its pseudotype but restored the infectivity of authentic CHIKV Thai#16856, suggesting that CS functions at later steps after CHIKV binding. Among the genes enriched in this screening, we found that TM9SF2 is critical for N-sulfation of HS and therefore for CHIKV infection because it is involved in the proper localization and stability of NDST1. Determination of the significance of and the relevant proteins to N-sulfation of HS may contribute to understanding mechanisms of CHIKV propagation, cell tropism, and pathogenesis.IMPORTANCE Recent outbreaks of chikungunya fever have increased its clinical importance. Chikungunya virus (CHIKV) utilizes host glycosaminoglycans to bind efficiently to its target cells. However, the substructure in glycosaminoglycans required for [HM/infection have not been characterized. Here, we unveil that N-sulfate in heparan sulfate is essential for the efficient infection of a clinical CHIKV strain to HAP1 cells and that chondroitin sulfate does not help the CHIKV binding but does play roles at the later steps in HAP1 cells. We show, by comparing previous reports using Chinese hamster ovary cells, along with another observation that enhanced infectivity of CHIKV bearing Arg82 in envelope E2 does not depend on glycosaminoglycans in HAP1 cells, that the infection manner of CHIKV varies among host cells. We also show that TM9SF2 is required for CHIKV infection to HAP1 cells because it is involved in the N-sulfation of heparan sulfate through ensuring NDST1 activity.