CAPSID PROTEIN OF MOLONEY MURINE LEUKEMIA VIRUS: HIV

我们已经开发了一个系统，用于分析组氨酸标记的 （His标记的）逆转录病毒核心（Gag）蛋白，在体外组装在 脂质单层由卵磷脂（PC）加上 新型脂质DHGN。 DHGN显示出通过原子螯合镍 吸收光谱法，和含有DHGN的单分子膜， His标记的蛋白质的结合金缀合物。 使用PC + DHGN 单层，我们检查了N-末端的膜结合阵列 His标记的莫洛尼鼠白血病病毒（M-MuLV）衣壳（CA）蛋白， 体内研究表明，体外衍生的His-MoCA 阵列反映了一些Gag蛋白的相互作用， 组装病毒颗粒 His-MoCA蛋白形成了广泛的 二维（2D）蛋白质晶体，反射高达9.5 A 分辨率 His-MoCA阵列的图像分析二维投影 揭示了一个独特的笼状网络。 的不对称性 网络的各个组成部分导致了两个 六聚体环的类型，围绕无蛋白质的笼孔。 这些 结果预测Gag六聚体构成逆转录病毒核心 子结构和保持架孔尺寸限定了排除限制， 逆转录病毒包膜蛋白或其他质膜的进入 蛋白质转化成病毒颗粒 我们相信2D 结晶方法将允许详细分析逆转录病毒 Gag蛋白和其他His标记的蛋白。

We have developed a system for analysis of histidine-tagged (His-tagged) retrovirus core (Gag) proteins, assembled in vitro on lipid monolayers consisting of egg phosphatidylcholine (PC) plus the novel lipid DHGN. DHGN was shown to chelate nickel by atomic absorption spectrometry, and DHGN-containing monolayers specifically bound gold conjugates of His-tagged proteins. Using PC + DHGN monolayers, we examined membrane-bound arrays of an N-terminal His-tagged Moloney murine leukemia virus (M-MuLV) capsid (CA) protein, His-MoCA, and in vivo studies suggest that in vitro-derived His-MoCA arrays reflect some of the Gag protein interactions which occur in assembling virus particles. The His-MoCA proteins formed extensive two-dimensional (2D) protein crystals, with reflections out to 9.5 A resolution. The image-analyzed 2D projection of His-MoCA arrays revealed a distinctive cage-like network. The asymmetry of the individual building blocks of the network led to the formation of two types of hexamer rings, surrounding protein-free cage holes. These results predict that Gag hexamers constitute a retrovirus core substructure, and that cage hole sizes define an exclusion limit for entry of retrovirus envelope proteins, or other plasma membrane proteins, into virus particles. We believe that the 2D crystallization method will permit the detailed analysis of retroviral Gag proteins and other His-tagged proteins.