Department of Medicinal Chemistry and Molecular Pharmacology, Markey Center for Structural Biology, and Purdue Cancer Center, Purdue University, West Lafayette, Indiana 47907, USA.
Biochemistry. 2010 May 18;49(19):4006-17. doi: 10.1021/bi902196e.
Assembly of retrovirus particles is promoted by interaction of the Gag polyprotein with RNA. Nonspecific RNA association with the nucleocapsid domain (NC) of Gag induces the dimerization of Gag through protein-protein contacts in the capsid domain (CA), followed by higher order assembly to form the immature virus particle. NMR relaxation studies were conducted to investigate the initial steps of Rous sarcoma virus (RSV) assembly by examining the association with nucleic acid of a fragment of Gag comprising the C-terminal domain of CA (CTD) postulated to mediate Gag dimerization, the spacer region between CA and NC (SP), and NC. This fragment, CTD-SP-NC (residues 394-577), spans the critical SP region and allows assessment of this key Gag-nucleic acid interaction in the context of the Gag polyprotein rather than the isolated domains. Main-chain amide relaxation of CTD-SP-NC was measured in the absence and presence of (GT)(4), an 8-mer DNA oligonucleotide that binds tightly to the polyprotein but is too short to promote Gag dimerization. The results show that the CTD and NC domains tumble independently. In contrast, the two zinc finger domains within NC are rotationally coupled in both the unbound and bound states, even though only the first zinc finger appears to make direct contact with (GT)(4). In addition, the NMR data indicate that SP and flanking residues undergo a conformational exchange process that is slowed in the presence of (GT)(4). This region around SP where relaxation is strongly affected by (GT)(4) binding is nearly identical to the assembly domain defined previously by mutagenesis studies. Other changes in relaxation induced by (GT)(4) implicate conformational perturbations of helices 1 and 4 in CTD. On the basis of the combined data, we propose a model for the promotion of Gag dimerization by RNA association in which NC-RNA binding disrupts an assembly inhibitory, intramolecular interaction involving SP and CTD. Disruption of this intramolecular interaction is proposed to enhance the accessibility of the Gag dimer contact surface and release the assembly domain to promote intermolecular oligomerization.
逆转录病毒颗粒的组装是通过 Gag 多聚蛋白与 RNA 的相互作用来促进的。非特异性 RNA 与 Gag 的核衣壳结构域（NC）的结合诱导 Gag 通过衣壳结构域（CA）中的蛋白-蛋白接触发生二聚化，随后进行更高阶的组装形成不成熟的病毒颗粒。通过研究 Rous 肉瘤病毒（RSV）组装的初始步骤，进行了 NMR 弛豫研究，方法是检测包含衣壳结构域（CA）的 C 端结构域（CTD）的 Gag 片段、CA 和 NC 之间的间隔区（SP）和 NC 的核酸结合情况。该片段 CTD-SP-NC（残基 394-577）跨越关键的 SP 区域，并允许在 Gag 多聚蛋白的背景下评估该关键 Gag-核酸相互作用，而不是在分离的结构域中。在不存在和存在（GT）（4）的情况下测量了 CTD-SP-NC 的主链酰胺弛豫，（GT）（4）是一种 8 聚体 DNA 寡核苷酸，与多聚蛋白紧密结合但太短而不能促进 Gag 二聚化。结果表明，CTD 和 NC 结构域独立旋转。相比之下，NC 中的两个锌指结构域在未结合和结合状态下都是旋转偶联的，尽管只有第一个锌指似乎与（GT）（4）直接接触。此外，NMR 数据表明，SP 和侧翼残基经历构象交换过程，该过程在存在（GT）（4）时会减慢。在（GT）（4）结合时强烈受影响的 SP 周围区域与先前通过诱变研究定义的组装结构域几乎相同。（GT）（4）诱导的弛豫的其他变化暗示 CTD 中螺旋 1 和 4 的构象扰动。基于综合数据，我们提出了一种模型，即 RNA 结合促进 Gag 二聚化，其中 NC-RNA 结合破坏涉及 SP 和 CTD 的组装抑制性的分子内相互作用。据推测，这种分子内相互作用的破坏会增强 Gag 二聚体接触表面的可及性，并释放组装结构域以促进分子间寡聚化。
