The retroviral capsid domain dictates virion size, morphology, and coassembly of Gag into virus-like particles
The retroviral capsid domain dictates virion size, morphology, and coassembly of Gag into virus-like particles
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DOI：
10.1128/jvi.79.21.13463-13472.2005
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发表时间：
2005-11-01
影响因子：
5.4
通讯作者：
Vogt, VM
中科院分区：
文献类型：
--
作者：
Ako-Adjei, D;Johnson, MC;Vogt, VM
逆转录病毒结构蛋白Gag能够在活细胞和体外独立组装成病毒样颗粒（VLP）。当通过透射电子显微镜（TEM）观察时，人类免疫缺陷病毒I型（HIV-1）和劳斯肉瘤病毒（RSV）的未成熟VLP在形态上不同。为了更好地理解导致这些区别的Gag-Gag相互作用的性质，我们构建了编码几种RSV/HIV-1嵌合Gag蛋白的载体，用于在昆虫细胞或脊椎动物细胞中表达。我们使用TEM，共聚焦荧光显微镜，和一种新的相关扫描电镜（SEM）共聚焦显微镜技术，研究这些蛋白质的组装特性。大多数嵌合蛋白组装成常规VLP，其中衣壳（CA）结构域是总体颗粒直径和形态的主要决定因素。基质和CA之间的域的存在也影响颗粒形态，通过增加内部电子致密环和VLP膜之间的间距。野生型RSV、HIV-1或鼠白血病病毒Gag的双标记版本在细胞中不共定位。然而，野生型Gag蛋白与具有相同CA结构域的嵌合Gag蛋白广泛共定位，这意味着Gag相互作用由CA介导。这种现象的一个引人注目的例子是由携带HIV-1 CA结构域的RSV Gag的核输出缺陷嵌合体提供的，其本身定位于细胞核，但在野生型HIV-1 Gag存在下重新定位于细胞质。野生型和嵌合Gag蛋白能够共组装成一个单一的VLP相关荧光SEM观察，如果，只有当，CA域是来自同一病毒。这些结果意味着Gag-Gag相互作用的主要选择性由CA结构域决定。
The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type I (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.