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Significance Understanding the molecular determinants of retroviral genome packaging is important for drug discovery and development of vectors for gene delivery. We show that the HIV-1 leader, which contains the RNA elements necessary for genome packaging, binds approximately two dozen copies of the cognate NC protein with affinities ranging from ∼40 nM to 1.4 µM. Binding to the four highest-affinity “initial” binding sites occurs with endothermic energetics attributed to NC-induced localized RNA melting. Mutations that stabilize these sites inhibit NC binding in vitro and RNA packaging in transfected cells. A small-molecule inhibitor of RNA packaging binds specifically to the initial NC binding sites and stabilizes the RNA structure. Our findings identify a potential RNA Achilles’ heel for HIV therapeutic development., 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 (Kd ≲ 400 nM) reside within a ∼150-nt region of the leader sufficient to promote RNA packaging (core encapsidation signal, ΨCES). 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.