Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane
Gag Induces the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Microdomains at HIV-1 Assembly Sites on the Plasma Membrane
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DOI：
10.1128/jvi.00743-11
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发表时间：
2011-10-01
影响因子：
5.4
通讯作者：
Ono, Akira
中科院分区：
文献类型：
--
作者：
Hogue, Ian B.;Grover, Jonathan R.;Ono, Akira
HIV-1结构蛋白Gag与两种质膜微域--脂筏微域和富含TEM的微域相关联，这两种微域都被认为是HIV-1组装的平台。然而，各种研究表明，在没有HIV-1感染的情况下，脂筏和TEM是不同的微域。为了测量GAG对微域行为的影响，我们利用了两种分析方法：抗体介导的共配实验和Forster共振能量转移(FRET)分析，该方法测量了在没有抗体介导的修补的情况下活细胞中微域标记的聚集。我们在两种方法中都发现，在膜结合的GAG存在的情况下，脂筏和TEM在更大程度上共生和聚集，这表明GAG诱导了脂筏和TEM的结合。GAG膜结合基序的取代表明，虽然GAG膜结合是诱导脂筏与TEM结合的必要条件，但GAG的酰化或磷脂酰肌醇-(4，5)-二磷酸的结合是足够的。最后，在诱导膜弯曲方面有缺陷的GAG衍生物似乎更不能诱导脂筏和透射电子显微镜的结合。相关荧光和扫描电子显微镜对组装位点的高分辨率分析表明，聚集的脂筏和TEM主要发生在完整的细胞表面病毒样颗粒处，而跨膜RAFT标记蛋白似乎与点状GAG荧光有关，即使在没有细胞表面颗粒的情况下也是如此。综上所述，这些结果表明不同的膜微域组件在组装过程中以一种循序渐进的方式招募。
The HIV-1 structural protein Gag associates with two types of plasma membrane microdomains, lipid rafts and tetraspanin-enriched microdomains (TEMs), both of which have been proposed to be platforms for HIV-1 assembly. However, a variety of studies have demonstrated that lipid rafts and TEMs are distinct microdomains in the absence of HIV-1 infection. To measure the impact of Gag on microdomain behaviors, we took advantage of two assays: an antibody-mediated copatching assay and a Forster resonance energy transfer (FRET) assay that measures the clustering of microdomain markers in live cells without antibody-mediated patching. We found that lipid rafts and TEMs copatched and clustered to a greater extent in the presence of membrane-bound Gag in both assays, suggesting that Gag induces the coalescence of lipid rafts and TEMs. Substitutions in membrane binding motifs of Gag revealed that, while Gag membrane binding is necessary to induce coalescence of lipid rafts and TEMs, either acylation of Gag or binding of phosphatidylinositol-(4,5)-bisphosphate is sufficient. Finally, a Gag derivative that is defective in inducing membrane curvature appeared less able to induce lipid raft and TEM coalescence. A higher-resolution analysis of assembly sites by correlative fluorescence and scanning electron microscopy showed that coalescence of clustered lipid rafts and TEMs occurs predominately at completed cell surface virus-like particles, whereas a transmembrane raft marker protein appeared to associate with punctate Gag fluorescence even in the absence of cell surface particles. Together, these results suggest that different membrane microdomain components are recruited in a stepwise manner during assembly.