Dynamics of HIV-1 assembly and release.
Dynamics of HIV-1 assembly and release.
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DOI：
10.1371/journal.ppat.1000652
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发表时间：
2009-11
期刊：
影响因子：
6.7
通讯作者：
Lamb DC
中科院分区：
文献类型：
--
作者：
Ivanchenko S;Godinez WJ;Lampe M;Kräusslich HG;Eils R;Rohr K;Bräuchle C;Müller B;Lamb DC
人类免疫缺陷病毒（HIV）的组装和释放发生在感染细胞的质膜上，并由Gag多蛋白驱动。以前的研究使用生化方法和静态图像分析病毒形态发生，而直到最近才缺乏动态和动力学信息。利用宽视场和全内反射荧光显微镜，我们以高时间分辨率研究了荧光标记的HIV-1在活细胞质膜上的组装和释放。Gag组装成与单个病毒粒子相对应的离散簇。从同一位置形成多个粒子的现象很少被观察到。使用与Gag融合的光转换荧光蛋白，我们确定组装优先由最近附着在质膜上或直接从细胞质溶胶到达的Gag分子成核。膜结合和细胞质衍生的Gag多蛋白对芽的生长都有贡献。在它们最初出现后，组装位点在单个细胞的质膜上积累了1-2小时。组装动力学非常迅速：出芽部位的Gag分子数量增加，速率常数为~ 5×10−3 s−1，达到饱和指数，对应于单个病毒粒子组装完成90%需要8-9分钟。在组装开始后约1,500±700 s观察到细胞外颗粒的释放。病毒招募细胞ESCRT机制成分的能力，或经历蛋白水解成熟的能力，或Vpu的缺失，都没有显著改变组装动力学。人类免疫缺陷病毒（HIV）颗粒在被感染细胞的质膜上形成并释放。在这里，我们利用荧光标记的HIV结构蛋白分析了HIV组装和释放的动力学。我们确定病毒蛋白外壳的组装发生在组装位点成核后的8-9分钟内，病毒颗粒是单独形成的，而不是由大的斑块形成的。在组装位点成核后约25分钟观察到病毒粒子释放。因此Gag壳的组装似乎只占粒子形成所需时间的一小部分，这表明穿越膜和裂变是病毒粒子形成的限速阶段。利用病毒Gag蛋白的光转换标记，我们确定了驱动新组装位点成核和芽生长的Gag分子优先从Gag分子的细胞质池和最近附着在膜上的Gag中招募。没有观察到Gag的细胞内组装或囊状运输。所描述的结果为我们对病毒释放的了解增加了必要的动态信息，并为干扰病毒复制的这一阶段提供了实验基础。
Assembly and release of human immunodeficiency virus (HIV) occur at the plasma membrane of infected cells and are driven by the Gag polyprotein. Previous studies analyzed viral morphogenesis using biochemical methods and static images, while dynamic and kinetic information has been lacking until very recently. Using a combination of wide-field and total internal reflection fluorescence microscopy, we have investigated the assembly and release of fluorescently labeled HIV-1 at the plasma membrane of living cells with high time resolution. Gag assembled into discrete clusters corresponding to single virions. Formation of multiple particles from the same site was rarely observed. Using a photoconvertible fluorescent protein fused to Gag, we determined that assembly was nucleated preferentially by Gag molecules that had recently attached to the plasma membrane or arrived directly from the cytosol. Both membrane-bound and cytosol derived Gag polyproteins contributed to the growing bud. After their initial appearance, assembly sites accumulated at the plasma membrane of individual cells over 1–2 hours. Assembly kinetics were rapid: the number of Gag molecules at a budding site increased, following a saturating exponential with a rate constant of ∼5×10−3 s−1, corresponding to 8–9 min for 90% completion of assembly for a single virion. Release of extracellular particles was observed at ∼1,500±700 s after the onset of assembly. The ability of the virus to recruit components of the cellular ESCRT machinery or to undergo proteolytic maturation, or the absence of Vpu did not significantly alter the assembly kinetics. Human immunodeficiency virus (HIV) particles are formed and released at the plasma membrane of the infected cell. Here, we analyzed the dynamics of HIV assembly and release making use of fluorescently labeled HIV structural proteins. We determined that assembly of the viral protein shell occurs within ∼8–9 min after nucleation of an assembly site and virus particles are formed individually and not from large patches. Virion release was observed ∼25 min after nucleation of the assembly site. Assembly of the Gag shell thus appears to constitute only a minor part of the period required for particle formation indicating that traversing the membrane and fission are the rate-limiting stages in virion formation. Using a photoconvertible label in the viral Gag protein, we established that the Gag molecules driving nucleation of a new assembly site and in bud growth are recruited preferentially from the cytosolic pool of Gag molecules and from recently membrane-attached Gag. No intracellular assembly or vesicular trafficking of Gag was observed. The described results add essential dynamic information to our picture of virus release and provide an experimental basis for interfering with this stage of virus replication.
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DOI：
10.1083/jcb.200303200
发表时间：
2003-09-29
期刊：
The Journal of cell biology
影响因子：
--
作者：
Larson DR;Ma YM;Vogt VM;Webb WW
通讯作者：
Webb WW
影响因子：
3.4
作者：
Gladnikoff, Micha;Rousso, Itay
通讯作者：
Rousso, Itay
影响因子：
5.4
作者：
KONVALINKA, J;LITTERST, MA;KRAUSSLICH, HG
通讯作者：
KRAUSSLICH, HG
DOI：
10.1073/pnas.0511136103
发表时间：
2006-02-21
影响因子：
11.1
作者：
Brügger, B;Glass, B;Kräusslich, HG
通讯作者：
Kräusslich, HG
影响因子：
48
作者：
Manley, Suliana;Gillette, Jennifer M.;Lippincott-Schwartz, Jennifer
通讯作者：
Lippincott-Schwartz, Jennifer