Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.
Localization and functions of native and eGFP-tagged capsid proteins in HIV-1 particles.
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DOI：
10.1371/journal.ppat.1010754
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发表时间：
2022-08
期刊：
影响因子：
6.7
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中科院分区：
文献类型：
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在具有感染性的HIV-1颗粒中，衣壳蛋白(CA)形成一个称为衣壳的锥形外壳，包裹着病毒核糖核蛋白复合体(VRNP)。进入细胞后，衣壳通过一个鲜为人知的过程被分解，称为去涂层，这是释放反向转录的HIV-1基因组并整合到宿主染色质中所必需的。使用间接CA标记技术的单一病毒成像显示，脱壳发生在细胞质或核孔中，而最近一项使用EGFP标记的CA的研究报告称，脱壳发生在细胞核中。为了描述HIV-1的脱壳部位，我们研究了EGFP标记的CA掺入衣壳的机制以及这种荧光标记用于可视化HIV-1脱壳的应用。我们发现，病毒粒子掺入的EGFP标记的CA有效地从衣壳中排除，并且标记的CA的子集是vRNP相关的。这些结果表明，绿色荧光蛋白标记的CA不是衣壳脱壳的直接标志。我们进一步证明了天然CA与vRNP组分的免疫共沉淀，为亚病毒复合体在细胞核内保留EGFP标记和未标记CA提供了基础。此外，我们发现功能性病毒复制复合体可以在核孔上与整合酶相互作用的宿主因子结合，从而抑制感染并在核进口之前展示衣壳的通透性。最后，我们发现含有野生型和突变型CA的HIV-1核心与CA结合宿主辅因子CPSF6的细胞质池和核池的相互作用不同。我们的结果表明，衣壳重塑(包括衣壳完整性的丧失)是HIV-1核进入的主要途径，并为通过与vRNP组分相互作用在细胞核内滞留CA的机制提供了新的见解。HIV-1衣壳解体的时间、位置和机制仍不清楚。通过将几个绿色荧光蛋白(GFP)标记的衣壳蛋白(CA)加入到病毒粒子中，直接标记HIV-1衣壳蛋白可以成像HIV-1衣壳蛋白在病毒感染过程中的时空损失。然而，由于50%的病毒包装的CA蛋白参与形成保护HIV-1基因组的锥形衣壳，少数病毒粒子掺入的EGFP标记的CA蛋白的定位和功能仍不清楚。在这里，我们开发了几种方法来测试EGFP标记的CA蛋白在病毒粒子中的定位和功能。我们发现，绿色荧光蛋白标记的CA蛋白被排除在锥形衣壳之外，并且这些蛋白的一部分通过CA和vRNP组件之间的直接相互作用与病毒核糖核蛋白复合体(VRNPs)相关联。EGFP标记的CA通过vRNP结合保留在细胞核中，不太可能报告HIV-1衣壳的分解。我们还发现，HIV-1衣壳在运输到细胞核的过程中变得通透性和重塑。我们的研究为CA与vRNPs相互作用的能力提供了新的见解，使其保留在细胞核中，并强调衣壳重塑是HIV-1进入细胞核的首选途径。
In infectious HIV-1 particles, the capsid protein (CA) forms a cone-shaped shell called the capsid, which encases the viral ribonucleoprotein complex (vRNP). Following cellular entry, the capsid is disassembled through a poorly understood process referred to as uncoating, which is required to release the reverse transcribed HIV-1 genome for integration into host chromatin. Whereas single virus imaging using indirect CA labeling techniques suggested uncoating to occur in the cytoplasm or at the nuclear pore, a recent study using eGFP-tagged CA reported uncoating in the nucleus. To delineate the HIV-1 uncoating site, we investigated the mechanism of eGFP-tagged CA incorporation into capsids and the utility of this fluorescent marker for visualizing HIV-1 uncoating. We find that virion incorporated eGFP-tagged CA is effectively excluded from the capsid shell, and that a subset of the tagged CA is vRNP associated. These results thus imply that eGFP-tagged CA is not a direct marker for capsid uncoating. We further show that native CA co-immunoprecipitates with vRNP components, providing a basis for retention of eGFP-tagged and untagged CA by sub-viral complexes in the nucleus. Moreover, we find that functional viral replication complexes become accessible to integrase-interacting host factors at the nuclear pore, leading to inhibition of infection and demonstrating capsid permeabilization prior to nuclear import. Finally, we find that HIV-1 cores containing a mixture of wild-type and mutant CA interact differently with cytoplasmic versus nuclear pools of the CA-binding host cofactor CPSF6. Our results suggest that capsid remodeling (including a loss of capsid integrity) is the predominant pathway for HIV-1 nuclear entry and provide new insights into the mechanism of CA retention in the nucleus via interaction with vRNP components. The timing, location and mechanisms of HIV-1 capsid disassembly which is referred to as uncoating remains unclear. Direct labeling of HIV-1 capsids, by incorporating a few green fluorescent proteins (GFP) tagged capsid protein (CA) into virions allows to image the spatio-temporal loss of HIV-1 CA during virus infection. However, the localization and functions of a few virion incorporated eGFP-tagged CA proteins remain unclear, since <50% of virus packaged CA proteins participate to form the conical capsid shell that protects the HIV-1 genome. Here we developed several approaches to test the localization and function of eGFP-tagged CA proteins in virions. We found that eGFP-tagged CA proteins are excluded from the conical capsid shell and that a subset of these proteins is associated with the viral ribonucleoprotein complex (vRNPs), through direct interactions between CA and vRNP components. eGFP-tagged CA is retained in the nucleus by virtue of vRNP association and is unlikely to report on HIV-1 capsid disassembly. We also found that HIV-1 capsids become permeabilized and are remodeled during their transport into the nucleus. Our study provides new insights into the ability of CA to interact with vRNPs for its retention in the nucleus and highlights capsid remodeling as a preferred pathway for HIV-1 entry into the nucleus.
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DOI：
10.1038/nrmicro3503
发表时间：
2015-08
期刊：
Nature reviews. Microbiology
影响因子：
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作者：
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通讯作者：
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影响因子：
5.8
作者：
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DOI：
10.1073/pnas.1419945112
发表时间：
2014-12-30
影响因子：
11.1
作者：
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通讯作者：
Yeager, Mark
DOI：
10.1126/science.aah7002
发表时间：
2017-01-06
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
Ballandras-Colas A;Maskell DP;Serrao E;Locke J;Swuec P;Jónsson SR;Kotecha A;Cook NJ;Pye VE;Taylor IA;Andrésdóttir V;Engelman AN;Costa A;Cherepanov P
通讯作者：
Cherepanov P
影响因子：
48
作者：
de Chaumont, Fabrice;Dallongeville, Stephane;Olivo-Marin, Jean-Christophe
通讯作者：
Olivo-Marin, Jean-Christophe