Selective packaging of the HIV-1 genome during virus assembly is mediated by interactions between the dimeric 5ʹ-leader of the unspliced viral RNA and the nucleocapsid (NC) domains of a small number of assembling viral Gag polyproteins. Here, we show that the dimeric 5′-leader contains more than two dozen NC binding sites with affinities ranging from 40 nM to 1.4 μM, and that all high-affinity sites (K_d ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, Ψ^CES). The four initial binding sites with highest affinity reside near two symmetrically equivalent three-way junction structures. Unlike the other high-affinity sites, which bind NC with exothermic energetics, binding to these sites occurs endothermically due to concomitant unwinding of a weakly base-paired [UUUU]:[GGAG] helical element. Mutations that stabilize base pairing within this element eliminate NC binding to this site and severely impair RNA packaging into virus-like particles. NMR studies reveal that a recently discovered small-molecule inhibitor of HIV-1 RNA packaging that appears to function by stabilizing the structure of the leader binds directly to the [UUUU]:[GGAG] helix. Our findings suggest a sequential NC binding mechanism for Gag-genome assembly and identify a potential RNA Achilles’ heel to which HIV therapeutics may be targeted.

病毒组装过程中 HIV-1 基因组的选择性包装是由未剪接的病毒 RNA 的二聚体 5'-前导与少数组装病毒 Gag 多蛋白的核衣壳 (NC) 结构域之间的相互作用介导的。在这里，我们表明二聚体 5'-前导包含超过 22 个 NC 结合位点，亲和力范围从 40 nM 到 1.4 μM，并且所有高亲和力位点 ( K _d ≲ 400 nM) 都位于 ~150-nt 内前导区域足以促进 RNA 包装（核心封装信号，Ψ ^CES）。具有最高亲和力的四个初始结合位点位于两个对称等效的三向连接结构附近。与其他将 NC 与放热能量结合的高亲和力位点不同，由于弱碱基配对 [UUUU]:[GGAG] 螺旋元件的同时展开，与这些位点的结合是吸热的。稳定该元件内碱基配对的突变消除了 NC 与该位点的结合，并严重损害 RNA 包装成病毒样颗粒。核磁共振研究表明，最近发现的一种 HIV-1 RNA 包装的小分子抑制剂似乎通过稳定前导结构的结构发挥作用，直接与 [UUUU]:[GGAG] 螺旋结合。