Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories
Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories
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DOI：
10.1128/jvi.01080-20
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发表时间：
2020-12-01
影响因子：
5.4
通讯作者：
Aizaki, Hideki
中科院分区：
文献类型：
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作者：
Gewaid, Hossam;Aoyagi, Haruyo;Aizaki, Hideki
一些正链RNA病毒产生双膜小泡(DMV)作为复制位点，双膜小泡是膜复制工厂的一种。对这些囊泡生物发生过程中涉及的脂类成分知之甚少。鞘磷脂(SM)是丙型肝炎病毒(HCV)复制所必需的，但SM参与的机制尚不清楚。SM的生物合成始于内质网(ER)，产生神经酰胺，神经酰胺在神经酰胺转移蛋白(CERT)的作用下从内质网转运到高尔基体，在高尔基体中转化为SM。在这项研究中，通过使用小分子抑制剂或通过CERT的敲除(KO)来抑制SM的生物合成，以一种与基因型无关的方式抑制了丙型肝炎病毒的复制。外源性SM或CERT蛋白的异位表达可以挽救丙型肝炎病毒复制的减少，但不能通过无功能的CERT突变体的异位表达来挽救。在用SM生物合成抑制剂处理的稳定的复制子细胞中观察到少量的DMV，或者在CERT-KO细胞中观察到，在一个复制不依赖的系统中，SM对DMV具有重要的意义。体外分离的DMV的SM的降解影响了它们的形态，并分别增加了丙型肝炎病毒RNA和蛋白质对核糖核酸酶和蛋白酶处理的脆弱性。已知可诱发DMV的脊髓灰质炎病毒在CERT-KO细胞中的复制减少，而已知可诱导内陷小泡的登革病毒则没有。综上所述，这些发现表明SM是一些正链RNA病毒产生的DMV的重要组成部分。以往的报道认为鞘磷脂(SM)是丙型肝炎病毒复制所必需的，但其机制尚不清楚。在这项研究中，我们首次证明了SM和SM生物合成途径中的神经酰胺转移蛋白(CERT)对于双膜囊泡(DMV)的生物合成是必不可少的，双膜囊泡是病毒复制的场所。在CERT-KO细胞中，观察到低数量的DMV，这些细胞被导入复制子RNA或在复制不依赖的系统中驱动丙型肝炎病毒蛋白生产的结构。CERT蛋白的异位表达挽救了丙型肝炎病毒的复制，但CERT突变体取消了CERT与囊泡相关膜蛋白相关蛋白(VAP)或磷脂酰肌醇4-磷酸(PI4P)的结合，表明VAP和PI4P在丙型肝炎病毒复制中发挥了新的作用。DMV的生物合成对多种正链RNA病毒的复制具有重要意义。对这一过程的了解有望促进诊断和防病毒的发展。
Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.