Tanaka Atsushi, Tumkosit Uranan, Nakamura Shota, Motooka Daisuke, Kishishita Natsuko, Priengprom Thongkoon, Sa-Ngasang Areerat, Kinoshita Taroh, Takeda Naokazu, Maeda Yusuke
Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.
Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections, Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand.
J Virol. 2017 Jun 9;91(13). doi: 10.1128/JVI.00432-17. Print 2017 Jul 1.
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 E1 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 knockout cells, the tropism for N-sulfate was specific to CHIKV. Expression of chondroitin sulfate (CS) in -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. 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 CHIKV 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.
基孔肯雅病毒（CHIKV）感染的分子机制尚未得到充分阐明。在本研究中，我们通过全基因组筛选分析了参与CHIKV感染的宿主因子。将携带外显子捕获载体的人单倍体HAP1细胞用携带CHIKV E3至E1包膜蛋白的水泡性口炎病毒假型进行攻击。对在抗假型病毒感染的细胞中富集的基因进行分析，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HAP1细胞的感染性具有关键作用。催化HS的N-硫酸化的NDST1基因敲除极大地降低了CHIKV Thai#16856株的结合和感染性，但不影响日本脑炎病毒（JEV）和黄热病病毒（YFV）的感染性。虽然糖胺聚糖是CHIKV、JEV和YFV有效感染所共同需要的，如使用基因敲除细胞所显示的，但对N-硫酸盐的嗜性是CHIKV特有的。硫酸软骨素（CS）在NDST1基因敲除的HAP1细胞中的表达并未恢复CHIKV Thai#16856株的结合及其假型的感染性，但恢复了真实的CHIKV Thai#16856的感染性，这表明CS在CHIKV结合后的后期步骤中起作用。在该筛选中富集的基因中，我们发现TM9SF2对于HS的N-硫酸化至关重要，因此对于CHIKV感染也至关重要，因为它参与了NDST1的正确定位和稳定性。确定HS的N-硫酸化的重要性及相关蛋白可能有助于理解CHIKV传播、细胞嗜性和发病机制。最近基孔肯雅热的爆发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，CHIKV感染所需的糖胺聚糖中的亚结构尚未得到表征。在此，我们揭示硫酸乙酰肝素中的N-硫酸盐对于临床CHIKV株对HAP1细胞的有效感染至关重要，并且硫酸软骨素无助于CHIKV的结合，但在HAP1细胞的后期步骤中起作用。通过比较先前使用中国仓鼠卵巢细胞的报道以及另一项观察结果，即包膜E2中携带Arg82的CHIKV的增强感染性在HAP1细胞中不依赖于糖胺聚糖，我们表明CHIKV的感染方式在不同宿主细胞中有所不同。我们还表明TM9SF2是CHIKV感染HAP1细胞所必需的，因为它通过确保NDST1活性参与硫酸乙酰肝素的N-硫酸化。
