Langelier Charles, von Schwedler Uta K, Fisher Robert D, De Domenico Ivana, White Paul L, Hill Christopher P, Kaplan Jerry, Ward Diane, Sundquist Wesley I
Department of Biochemistry, 15 N. Medical Drive East, Room 4100, University of Utah School of Medicine, Salt Lake City, UT 84112-5650, USA.
J Virol. 2006 Oct;80(19):9465-80. doi: 10.1128/JVI.01049-06.
The budding of many enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), requires some of the same cellular machinery as vesicle formation at the multivesicular body (MVB). In Saccharomyces cerevisiae, the ESCRT-II complex performs a central role in MVB protein sorting and vesicle formation, as it is recruited by the upstream ESCRT-I complex and nucleates assembly of the downstream ESCRT-III complex. Here, we report that the three subunits of human ESCRT-II, EAP20, EAP30, and EAP45, have a number of properties in common with their yeast orthologs. Specifically, EAP45 bound ubiquitin via its N-terminal GRAM-like ubiquitin-binding in EAP45 (GLUE) domain, both EAP45 and EAP30 bound the C-terminal domain of TSG101/ESCRT-I, and EAP20 bound the N-terminal half of CHMP6/ESCRT-III. Consistent with its expected role in MVB vesicle formation, (i) human ESCRT-II localized to endosomal membranes in a VPS4-dependent fashion and (ii) depletion of EAP20/ESCRT-II and CHMP6/ESCRT-III inhibited lysosomal targeting and downregulation of the epidermal growth factor receptor, albeit to a lesser extent than depletion of TSG101/ESCRT-I. Nevertheless, HIV-1 release and infectivity were not reduced by efficient small interfering RNA depletion of EAP20/ESCRT-II or CHMP6/ESCRT-III. These observations indicate that there are probably multiple pathways for protein sorting/MVB vesicle formation in human cells and that HIV-1 does not utilize an ESCRT-II-dependent pathway to leave the cell.
许多包膜RNA病毒的出芽，包括1型人类免疫缺陷病毒（HIV-1），需要一些与多泡体（MVB）处囊泡形成相同的细胞机制。在酿酒酵母中，ESCRT-II复合物在MVB蛋白分选和囊泡形成中发挥核心作用，因为它被上游的ESCRT-I复合物招募并促使下游ESCRT-III复合物组装成核。在此，我们报告人类ESCRT-II的三个亚基EAP20、EAP30和EAP45具有许多与其酵母直系同源物相同的特性。具体而言，EAP45通过其N端的EAP45中类GRAM泛素结合（GLUE）结构域结合泛素，EAP45和EAP30都结合TSG101/ESCRT-I的C端结构域，而EAP20结合CHMP6/ESCRT-III的N端一半。与其在MVB囊泡形成中的预期作用一致，（i）人类ESCRT-II以VPS4依赖的方式定位于内体膜，（ii）EAP20/ESCRT-II和CHMP6/ESCRT-III的缺失抑制了表皮生长因子受体的溶酶体靶向和下调，尽管程度小于TSG101/ESCRT-I的缺失。然而，EAP20/ESCRT-II或CHMP6/ESCRT-III的有效小干扰RNA缺失并未降低HIV-1的释放和感染性。这些观察结果表明，人类细胞中可能存在多种蛋白质分选/MVB囊泡形成途径，并且HIV-1不利用依赖ESCRT-II的途径离开细胞。
