In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: Identification of the p10 domain as a morphological determinant in the formation of spherical particles
In vitro assembly of virus-like particles with Rous sarcoma virus Gag deletion mutants: Identification of the p10 domain as a morphological determinant in the formation of spherical particles
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DOI：
10.1128/jvi.71.6.4425-4435.1997
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发表时间：
1997-06-01
影响因子：
5.4
通讯作者：
Vogt, VM
中科院分区：
文献类型：
--
作者：
Campbell, S;Vogt, VM
逆转录病毒是不寻常的，在一个单一的蛋白质，Gag的表达，导致病毒样颗粒出芽到细胞外空间，我们已经开发了条件下，病毒样颗粒自发形成在体外从劳斯肉瘤病毒（RSV）Gag蛋白在大肠杆菌中表达后纯化的片段。Gag的CA-NC片段先前显示组装成中空圆柱体（S.坎贝尔和V，M.沃格特，J，Virol，69：6487-6497，1995）。我们现在已经将这些研究扩展到更大的Gag蛋白。在检查的每种情况下，组装成规则结构需要RNA，仅缺失C-末端PR结构域的几乎全长Gag，以及除了N-末端一半MA、C-末端一半MA、整个MA序列或整个p2序列之外缺失的类似蛋白质，全部组装成大小类似于RSV的球形颗粒。相比之下，缺失p10的蛋白质组装成类似于单独由CA-NC形成的圆柱形颗粒。我们从这些结果中得出结论，无论是在体内膜结合所需的序列，在N末端附近的Gag，也不是在出芽的后期步骤所需的序列，在p2部分的Gag，在这个系统中的病毒样颗粒的形成是必不可少的。此外，我们假设存在一个形状决定序列的p10，它提供或促进相互作用所需的增长粒子被约束到一个球形。
Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space, We have developed conditions under which virus-like particles are formed spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC fragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V, M. Vogt, J, Virol, 69:6487-6497, 1995). We have now extended these studies to larger Gag proteins. In every case examined, assembly into regular structures required RNA, A nearly full-length Gag missing only the C-terminal PR domain, as well as similar proteins missing in addition the N-terminal half of MA, the C-terminal half of MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size, By contrast, proteins missing p10 assembled into cylindrical particles like those formed by CA-NC alone, Thin section electron microscopy showed that each of these Gag proteins formed in the expressing E. coli cells particles similar in shape to those seen in vitro, We conclude from these results that neither the sequences required for membrane binding in vivo, near the N terminus of Gag, nor the sequences required for a late step in budding, in the p2 portion of Gag, are essential for formation of virus-like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.