Dooher Julia E, Schneider Bobbie L, Reed Jonathan C, Lingappa Jaisri R
Department of Pathobiology, University of Washington, 1959 NE Pacific Street, Seattle, WA 98195, USA.
Traffic. 2007 Mar;8(3):195-211. doi: 10.1111/j.1600-0854.2006.00524.x.
In primate cells, assembly of a single HIV-1 capsid involves multimerization of thousands of Gag polypeptides, typically at the plasma membrane. Although studies support a model in which HIV-1 assembly proceeds through complexes containing Gag and the cellular adenosine triphosphatase ABCE1 (also termed HP68 or ribonuclease L inhibitor), whether these complexes constitute true assembly intermediates remains controversial. Here we demonstrate by pulse labeling in primate cells that a population of Gag associates with endogenous ABCE1 within minutes of translation. In the next approximately 2 h, Gag-ABCE1 complexes increase in size to approximately that of immature capsids. Dissociation of ABCE1 from Gag correlates closely with Gag processing during virion maturation and occurs much less efficiently when the HIV-1 protease is inactivated. Finally, quantitative double-label immunogold electron microscopy reveals that ABCE1 is recruited to sites of assembling wild-type Gag at the plasma membrane but not to sites of an assembly-defective Gag mutant at the plasma membrane. Together these findings demonstrate that a population of Gag present at plasma membrane sites of assembly associates with ABCE1 throughout capsid formation until the onset of virus maturation, which is then followed by virus release. Moreover, the data suggest a linkage between Gag-ABCE1 dissociation and subsequent events of virion production.
在灵长类动物细胞中，单个HIV-1衣壳的组装涉及数千个Gag多肽的多聚化，通常发生在质膜处。尽管研究支持一种模型，即HIV-1组装通过包含Gag和细胞腺苷三磷酸酶ABCE1（也称为HP68或核糖核酸酶L抑制剂）的复合物进行，但这些复合物是否构成真正的组装中间体仍存在争议。在这里，我们通过在灵长类动物细胞中进行脉冲标记证明，一群Gag在翻译后几分钟内就与内源性ABCE1结合。在接下来约2小时内，Gag-ABCE1复合物的大小增加到接近未成熟衣壳的大小。ABCE1从Gag上解离与病毒体成熟过程中Gag的加工密切相关，当HIV-1蛋白酶失活时，解离效率要低得多。最后，定量双标记免疫金电子显微镜显示，ABCE1被招募到质膜处组装野生型Gag的位点，但不被招募到质膜处组装缺陷型Gag突变体的位点。这些发现共同表明，在衣壳形成直至病毒成熟开始的整个过程中，存在于组装质膜位点的一群Gag都与ABCE1结合，随后病毒释放。此外，数据表明Gag-ABCE1解离与病毒体产生的后续事件之间存在联系。
