Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia.
Department of Microbiology, La Trobe University, Melbourne, VIC, Australia.
PLoS Pathog. 2018 Apr 30;14(4):e1007029. doi: 10.1371/journal.ppat.1007029. eCollection 2018 Apr.
Positive-sense RNA virus intracellular replication is intimately associated with membrane platforms that are derived from host organelles and comprised of distinct lipid composition. For flaviviruses, such as West Nile virus strain Kunjin virus (WNVKUN) we have observed that these membrane platforms are derived from the endoplasmic reticulum and are rich in (at least) cholesterol. To extend these studies and identify the cellular lipids critical for WNVKUN replication we utilized a whole cell lipidomics approach and revealed an elevation in phospholipase A2 (PLA2) activity to produce lyso-phosphatidylcholine (lyso-PChol). We observed that the PLA2 enzyme family is activated in WNVKUN-infected cells and the generated lyso-PChol lipid moieties are sequestered to the subcellular sites of viral replication. The requirement for lyso-PChol was confirmed using chemical inhibition of PLA2, where WNVKUN replication and production of infectious virus was duly affected in the presence of the inhibitors. Importantly, we could rescue chemical-induced inhibition with the exogenous addition of lyso-PChol species. Additionally, electron microscopy results indicate that lyso-PChol appears to contribute to the formation of the WNVKUN membranous replication complex (RC); particularly affecting the morphology and membrane curvature of vesicles comprising the RC. These results extend our current understanding of how flaviviruses manipulate lipid homeostasis to favour their own intracellular replication.
正链 RNA 病毒的细胞内复制与源自宿主细胞器的膜平台密切相关，这些膜平台由独特的脂质组成。对于黄病毒，如西尼罗河病毒株 Kunjin 病毒（WNVKUN），我们观察到这些膜平台源自内质网，并且富含（至少）胆固醇。为了扩展这些研究并确定对 WNVKUN 复制至关重要的细胞脂质，我们利用全细胞脂质组学方法，发现磷脂酶 A2（PLA2）活性升高，产生溶血磷脂酰胆碱（lyso-PChol）。我们观察到 PLA2 酶家族在 WNVKUN 感染的细胞中被激活，并且生成的溶血磷脂酰胆碱脂质部分被隔离到病毒复制的亚细胞部位。使用 PLA2 的化学抑制证实了 lyso-PChol 的需求，在抑制剂存在的情况下，WNVKUN 的复制和传染性病毒的产生受到适当影响。重要的是，我们可以通过添加外源性 lyso-PChol 物质来挽救化学诱导的抑制。此外，电子显微镜结果表明，lyso-PChol 似乎有助于形成 WNVKUN 膜复制复合物（RC）；特别是影响 RC 组成的囊泡的形态和膜曲率。这些结果扩展了我们对黄病毒如何操纵脂质稳态以促进自身细胞内复制的理解。
