Ding, PengfeiKharytonchyk, SiarheiWaller, AlexisMbaekwe, UgonnaBasappa, SapnaKuo, NansenFrank, Heather M.Quasney, ChristinaKidane, AaronSwanson, CanessaVan, VernaSarkar, MitaliCannistraci, EmilyChaudhary, RidhiFlores, HanaTelesnitsky, AliceSummers, Michael F.2020-08-062020-08-062020-07-09Ding, Pengfei; Kharytonchyk, Siarhei; Waller, Alexis; Mbaekwe, Ugonna; Basappa, Sapna; Kuo, Nansen; Frank, Heather M.; Quasney, Christina; Kidane, Aaron; Swanson, Canessa; Van, Verna; Sarkar, Mitali; Cannistraci, Emily; Chaudhary, Ridhi; Flores, Hana; Telesnitsky, Alice; Summers, Michael F.; Identification of the initial nucleocapsid recognition element in the HIV-1 RNA packaging signal; PNAS July 28, 2020 117 (30) 17737-17746; https://www.pnas.org/content/117/30/17737https://doi.org/10.1073/pnas.2008519117http://hdl.handle.net/11603/19351Selective 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.10 pagesen-USThis item is likely protected under Title 17 of the U.S. Copyright Law. Unless on a Creative Commons license, for uses protected by Copyright Law, contact the copyright holder or the author.Attribution 4.0 International (CC BY 4.0)https://creativecommons.org/licenses/by/4.0/Text