Productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane
Productive human immunodeficiency virus type 1 assembly takes place at the plasma membrane
复制标题
DOI：
10.1128/jvi.00308-07
复制
发表时间：
2007-07-01
影响因子：
5.4
通讯作者：
Cohen, Eric A.
中科院分区：
文献类型：
--
作者：
Finzi, Andres;Orthwein, Alexandre;Cohen, Eric A.
GAG蛋白是指导人类免疫缺陷病毒I型(HIV-1)颗粒组装和萌发所必需的，也是足够的。最近的证据表明，针对晚期内体/多囊泡小体(LE/MVB)的GAG发生在病毒颗粒在质膜(PM)发芽之前。然而，在到达稳定状态的目的地之前，GAG遵循的路线仍然是一个有争议的话题。采用亚细胞分离方法，结合脉冲追逐标记，分析了产生HIV-1的HEK 293T细胞的Gag转运。我们的结果表明，大多数新合成的GAG主要针对PM虽然PM靶向的GAG被有效释放，但发现剩余的细胞表面相关的GAG中有很大一部分随后内化到LE/MVB，并在那里积累，因此占LE/MVB相关的GAG的大部分。重要的是，这种GAG在LE/MVB中的积累被发现是胆固醇依赖的，因为它对类固醇结合药物非利平和甲基-对环糊精敏感。这些结果表明，PM是HIV-1在细胞中高效组装的主要部位，也支持细胞内GAG的积累。
Gag proteins are necessary and sufficient to direct human immunodeficiency virus type I (HIV-1) particle assembly and budding. Recent evidence suggests that Gag targeting to late endosomal/multivesicular body (LE/MVB) compartments occurs prior to viral particle budding at the plasma membrane (PM). However, the route that Gag follows before reaching its steady-state destinations still remains a subject of debate. Using a subcellular fractionation method that separates PM from LE/MVB combined with pulse-chase labeling, we analyzed Gag trafficking in HIV-1-producing HEK 293T cells. Our results reveal that the majority of newly synthesized Gag is primarily targeted to the PM. While PM-targeted Gag was efficiently released, a significant fraction of the remaining cell surface-associated Gag was found to be subsequently internalized to LE/MVB, where it accumulated, thus accounting for the majority of LE/MVB-associated Gag. Importantly, this accumulation of Gag in LE/MVB was found to be cholesterol dependent since it was sensitive to the sterol-binding drugs filipin and methyl-p-cyclodextrin. These results point towards the PM as being the primary site of productive HIV-1 assembly in cells that also support Gag accumulation in intracellular compartments.