Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins
Hepatitis C virus NS5A colocalizes with the core protein on lipid droplets and interacts with apolipoproteins
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DOI：
10.1006/viro.2001.1225
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发表时间：
2002-01-20
期刊：
影响因子：
3.7
通讯作者：
Lai, MMC
中科院分区：
文献类型：
--
作者：
Shi, ST;Polyak, SJ;Lai, MMC
丙型肝炎病毒 (HCV) 的非结构蛋白 5A (NS5A) 已被证明与多种细胞蛋白相互作用，并参与细胞生长的调节、干扰素抵抗和其他细胞信号传导途径，但 NS5A 在 HCV 发病机制中的作用尚未确定。为了进一步表征这种多功能蛋白，我们发起了肝癌细胞系中 NS5A 的亚细胞定位研究。通过免疫荧光染色和共聚焦显微镜观察，NS5A 定位于核周膜结构，包括内质网 (ER) 和高尔基体。此外，当单独表达或作为 NS3-5B 多蛋白的一部分表达时，它还与细胞质球状结构的表面相关。油红O染色显示这些球状结构是脂滴，HCV核心蛋白也位于其中。通过膜浮选分析进一步证实了 NS5A 与细胞内膜的关联。为了确定 NS5A 是否与任何细胞脂质结合蛋白相互作用，我们在 HepG2 和人肝脏 cDNA 文库中进行了酵母双杂交筛选。载脂蛋白 A1 (apoA1) 是高密度脂蛋白 (HDL) 颗粒的蛋白质成分之一，通过两个独立的筛选过程进行鉴定。 NS5A 和 apoA1 之间的相互作用通过体外 Pull-down 和体内共免疫沉淀实验得到证实。免疫荧光染色显示 NS5A 和 apoA1 在高尔基体中显着共定位。我们的结果建立了 NS5A 与脂滴和 apoA1 的关联，表明 NS5A 与核心蛋白一起可能在脂质代谢紊乱的发病机制中发挥作用，导致丙型肝炎中常见的肝脏脂肪变性。 (C) 2002 Elsevier Science。
The nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) has been shown to interact with a variety of cellular proteins and implicated in the regulation of cell growth, interferon resistance, and other cellular signaling pathways, but the role of NS5A in HCV pathogenesis has not been firmly established. To further characterize this multifunctional protein, we instigated the studies of the subcellular localization of NS5A in a hepatoma cell line. NS5A was localized to the perinuclear membrane structures, including the endoplasmic reticulum (ER) and the Golgi apparatus, by immunofluorescence staining and confocal microscopy. In addition, it was also associated with the surface of cytoplasmic globular structures when expressed alone or as a part of the NS3-5B polyprotein. Oil red O staining revealed that these globular structures were lipid droplets, where the HCV core protein was also localized. The association of NS5A with intracellular membrane was further confirmed by membrane flotation analysis. To determine whether NS5A interacts with any cellular lipid-binding protein, we performed yeast two-hybrid screening in both HepG2 and human liver cDNA libraries. Apolipoprotein A1 (apoA1), one of the protein components of high-density lipoprotein (HDL) particles, was identified by two independent screening processes. The interaction between NS5A and apoA1 was confirmed by both in vitro pull-down and in vivo coimmunoprecipitation experiments. Immunofluorescence staining revealed a significant colocalization of NS5A and apoA1 in the Golgi apparatus. Our results established an association of NS5A with lipid droplets and apoA1, suggesting that NS5A, together with the core protein, may play a role in the pathogenesis of the derangement of lipid metabolism, contributing to liver steatosis commonly observed in hepatitis C. (C) 2002 Elsevier Science.