Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292.
Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7292.
Proc Natl Acad Sci U S A. 2017 Jun 20;114(25):6587-6592. doi: 10.1073/pnas.1619519114. Epub 2017 May 10.
The are a diverse family of RNA viruses including many pathogens of medical and veterinary importance. Classically considered "nonenveloped," recent studies show that some picornaviruses, notably hepatitis A virus (HAV; genus Hepatovirus) and some members of the Enterovirus genus, are released from cells nonlytically in membranous vesicles. To better understand the biogenesis of quasi-enveloped HAV (eHAV) virions, we conducted a quantitative proteomics analysis of eHAV purified from cell-culture supernatant fluids by isopycnic ultracentrifugation. Amino acid-coded mass tagging (AACT) with stable isotopes followed by tandem mass spectrometry sequencing and AACT quantitation of peptides provided unambiguous identification of proteins associated with eHAV versus unrelated extracellular vesicles with similar buoyant density. Multiple peptides were identified from HAV capsid proteins (53.7% coverage), but none from nonstructural proteins, indicating capsids are packaged as cargo into eHAV vesicles via a highly specific sorting process. Other eHAV-associated proteins ( = 105) were significantly enriched for components of the endolysosomal system (>60%, < 0.001) and included many common exosome-associated proteins such as the tetraspanin CD9 and dipeptidyl peptidase 4 (DPP4) along with multiple endosomal sorting complex required for transport III (ESCRT-III)-associated proteins. Immunoprecipitation confirmed that DPP4 is displayed on the surface of eHAV produced in cell culture or present in sera from humans with acute hepatitis A. No LC3-related peptides were identified by mass spectrometry. RNAi depletion studies confirmed that ESCRT-III proteins, particularly CHMP2A, function in eHAV biogenesis. In addition to identifying surface markers of eHAV vesicles, the results support an exosome-like mechanism of eHAV egress involving endosomal budding of HAV capsids into multivesicular bodies.
甲型肝炎病毒（HAV；属 Hepatovirus）和某些肠病毒属成员等一些小核糖核酸病毒，经最近研究显示，它们是以膜囊泡的形式非裂解性地从细胞中释放出来的。为了更好地了解准包膜甲型肝炎病毒（eHAV）病毒粒子的生物发生过程，我们通过等密度超离心法，对从细胞培养上清液中纯化的 eHAV 进行了定量蛋白质组学分析。采用稳定同位素编码的氨基酸质量标记（AACT），结合串联质谱测序和 AACT 对肽段的定量分析，明确鉴定了与 eHAV 相关的蛋白，而与具有相似浮力密度的无关细胞外囊泡进行了区分。从 HAV 衣壳蛋白中鉴定出了多个肽段（覆盖度为 53.7%），但非结构蛋白中没有鉴定到，表明衣壳蛋白通过高度特异的分选过程被包装成 eHAV 囊泡的货物。其他与 eHAV 相关的蛋白（= 105）富含内体溶酶体系统的成分（>60%，<0.001），包括许多常见的外泌体相关蛋白，如四跨膜蛋白 CD9 和二肽基肽酶 4（DPP4），以及多个内体分选复合物必需的运输蛋白 III（ESCRT-III）相关蛋白。免疫沉淀证实 DPP4 可在细胞培养中产生的 eHAV 表面或急性甲型肝炎患者血清中呈现。通过质谱法未鉴定到 LC3 相关肽段。RNAi 消耗研究证实，ESCRT-III 蛋白，特别是 CHMP2A，在 eHAV 的生物发生中起作用。除了鉴定出 eHAV 囊泡的表面标志物外，该结果还支持了 eHAV 出芽的外泌体样机制，即 HAV 衣壳通过内体出芽进入多泡体。
