PDF(Pubmed)
Abstract：
The current study analyzed the intersecting biophysical, biochemical, and functional properties of extracellular particles (EPs) with the human immunodeficiency virus type-1 (HIV-1) beyond the currently accepted size range for HIV-1. We isolated five fractions (Frac-A through Frac-E) from HIV-infected cells by sequential differential ultracentrifugation (DUC). All fractions showed a heterogeneous size distribution with median particle sizes greater than 100 nm for Frac-A through Frac-D but not for Frac-E, which contained small EPs with an average size well below 50 nm. Synchronized and released cultures contained large infectious EPs in Frac-A, with markers of amphisomes and viral components. Additionally, Frac-E uniquely contained EPs positive for CD63, HSP70, and HIV-1 proteins. Despite its small average size, Frac-E contained membrane-protected viral integrase, detectable only after SDS treatment, indicating that it is enclosed in vesicles. Single particle analysis with dSTORM further supported these findings as CD63, HIV-1 integrase, and the viral surface envelope (Env) glycoprotein (gp) colocalized on the same Frac-E particles. Surprisingly, Frac-E EPs were infectious, and infectivity was significantly reduced by immunodepleting Frac-E with anti-CD63, indicating the presence of this protein on the surface of infectious small EPs in Frac-E. To our knowledge, this is the first time that extracellular vesicle (EV) isolation methods have identified infectious small HIV-1 particles (smHIV-1) that are under 50 nm. Collectively, our data indicate that the crossroads between EPs and HIV-1 potentially extend beyond the currently accepted biophysical properties of HIV-1, which may have further implications for viral pathogenesis.
摘要：
当前的研究分析了交叉的生物物理，生物化学，和具有人类免疫缺陷病毒1型（HIV-1）的细胞外颗粒（EP）的功能特性超出了HIV-1目前接受的大小范围。我们通过顺序差异超速离心（DUC）从HIV感染的细胞中分离出五个级分（Frac-A至Frac-E）。所有的级分均表现出不均匀的粒度分布，Frac-A至Frac-D的中值粒度大于100nm，但Frac-E的中值粒度不大于100nm。其含有平均尺寸远低于50nm的小EP。同步和释放的培养物在Frac-A中含有大量的传染性EP，具有两性体和病毒成分的标记。此外,Frac-E独特地含有对CD63、HSP70和HIV-1蛋白呈阳性的EPs。尽管它的平均尺寸很小，Frac-E含有膜保护的病毒整合酶，只有在SDS处理后才能检测到，表明它被囊泡包围。使用dSTORM的单颗粒分析进一步支持了这些发现，如CD63，HIV-1整合酶，和病毒表面包膜(Env)糖蛋白(gp)共定位在相同的Frac-E颗粒上。令人惊讶的是,Frac-EEP具有传染性，通过用抗CD63免疫消耗Frac-E，感染性显着降低，表明该蛋白存在于Frac-E中的感染性小EP表面。据我们所知,这是细胞外囊泡（EV）分离方法首次鉴定出50nm以下的感染性小HIV-1颗粒（smHIV-1）。总的来说，我们的数据表明,EP和HIV-1之间的交叉点可能超出了目前公认的HIV-1的生物物理特性,这可能对病毒的发病机制有进一步的影响.
