Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative
Construction and characterization of a fluorescently labeled infectious human immunodeficiency virus type 1 derivative
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DOI：
10.1128/jvi.78.19.10803-10813.2004
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发表时间：
2004-10-01
影响因子：
5.4
通讯作者：
Kräusslich, HG
中科院分区：
文献类型：
--
作者：
Müller, B;Daecke, J;Kräusslich, HG
可以在活细胞中检测到的标签的引入，为直接分析病毒复制的动态过程（如结构蛋白的运输和组装）开辟了新的可能性。我们的目标是生成一种工具，用于分析人类免疫缺陷病毒1型（HIV-1）主要结构蛋白Gag的贩运，以及在真实环境中分析病毒与宿主细胞的相互作用。我们在这里描述了在Gag多蛋白内携带标签的感染性HIV衍生物的构建和表征。基于我们最初的发现，一个短表位标签可以插入Gag的基质结构域的C端附近而不影响病毒的复制，我们构建了在类似位置携带egfp基因的HIV衍生物，从而在真实的病毒环境中表达Gag- egfp融合蛋白。显示正常病毒蛋白组成的颗粒从转染的细胞中释放出来，Gag-EGFP被病毒蛋白酶有效地处理，产生预期的产物。电镜观察到颗粒形貌成熟。改良后的病毒甚至被发现具有传染性，尽管相对传染性降低了。通过制备含有等摩尔量Gag- egfp和Gag的混合颗粒，我们能够获得高度荧光标记的病毒粒子制剂，这些病毒粒子具有正常的形态和完全的野生型传染性，这表明HIV颗粒组装过程具有显着的灵活性。荧光病毒衍生物是研究HIV与活细胞相互作用的有用工具。
The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on our initial finding that a short epitope tag could be inserted near the C terminus of the matrix domain of Gag without affecting viral replication, we constructed HIV derivatives carrying the egfp gene at the analogous position, resulting in the expression of a Gag-EGFP fusion protein in the authentic viral context. Particles displaying normal viral protein compositions were released from transfected cells, and Gag-EGFP was efficiently processed by the viral protease, yielding the expected products. Furthermore, particles with mature morphology were observed by thin-section electron microscopy. The modified virus was even found to be infectious, albeit with reduced relative infectivity. By preparing mixed particles containing equimolar amounts of Gag-EGFP and Gag, we were able to obtain highly fluorescently labeled virion preparations which displayed normal morphology and full wild-type infectivity, demonstrating that the process of HIV particle assembly displays a remarkable flexibility. The fluorescent virus derivative is a useful tool for investigating the interaction of HIV with live cells.