PDF(Pubmed)
Abstract：
Retroviral integration is mediated by intasome nucleoprotein complexes wherein a pair of viral DNA ends are bridged together by a multimer of integrase (IN). Atomic-resolution structures of HIV-1 intasomes provide detailed insights into the mechanism of integration and inhibition by clinical IN inhibitors. However, previously described HIV-1 intasomes are highly heterogeneous and have the tendency to form stacks, which is a limiting factor in determining high-resolution cryo-EM maps. We have assembled HIV-1 intasomes in the presence of excess IN C-terminal domain protein, which was readily incorporated into the intasomes. The purified intasomes were largely homogeneous and exhibited minimal stacking tendencies. The cryo-EM map resolution was further improved to 2.01 Å, which will greatly facilitate structural studies of IN inhibitor action and drug resistance mechanisms. The C-terminal 18 residues of HIV-1 IN, which are critical for virus replication and integration in vitro, have not been well resolved in previous intasome structures, and its function remains unclear. We show that the C-terminal tail participates in intasome assembly, resides within the intasome core, and forms a small alpha helix (residues 271-276). Mutations that disrupt alpha helix integrity impede IN activity in vitro and disrupt HIV-1 infection at the step of viral DNA integration.
摘要：
逆转录病毒整合是通过整合酶(IN)的多聚体将一对病毒DNA末端桥接在一起的整合体核蛋白复合物介导的。HIV-1肠套体的原子分辨结构为临床IN抑制剂的整合和抑制机制提供了详细的见解。然而,先前描述的HIV-1整合体是高度异质的，并且有形成堆叠的趋势，这是确定高分辨率冷冻EM图的限制因素。我们已经在过量的INC末端结构域蛋白的存在下组装了HIV-1整合体,它很容易被整合到体内。纯化的肠溶体在很大程度上是均匀的，并且表现出最小的堆积趋势。低温EM图分辨率进一步提高到2.01µ，这将极大地促进IN抑制剂作用和耐药机制的结构研究。HIV-1IN的C端18个残基，这对病毒体外复制和整合至关重要，在以前的内部结构中没有得到很好的解决，其功能尚不清楚。我们证明了C端尾部参与了完整的组装，驻留在完整的核心中，并形成一个小的α螺旋（残基271-276）。破坏α螺旋完整性的突变在体外阻碍IN活性并在病毒DNA整合步骤中破坏HIV-1感染。
