  Citation: Mouhand, A.; Zargarian, L.; Belfetmi, A.; Catala, M.; Pasi, M.; Lescop, E.; Tisné, C.; Mauffret, O. Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain. Viruses 2022, 14, 632. https://doi.org/10.3390/ v14030632 Academic Editor: Akira Ono Received: 14 February 2022 Accepted: 16 March 2022 Published: 18 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affil- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). viruses Article Investigation of the Low-Populated Excited States of the HIV-1 Nucleocapsid Domain Assia Mouhand 1 , Loussiné Zargarian 2 , Anissa Belfetmi 2 , Marjorie Catala 1 , Marco Pasi 2 , Ewen Lescop 3 , Carine Tisné 1, * and Olivier Mauffret 2, * 1 Expression Génétique Microbienne, UMR 8261, CNRS, Institut de Biologie Physico-Chimique (IBPC), Université de Paris, 75005 Paris, France; assia.mouhand@gmail.com (A.M.); marjorie.catala@ibpc.fr (M.C.) 2 Laboratoire de Biologie et de Pharmacologie Appliquée (LBPA), UMR 8113 CNRS, Institut D’Alembert, École Normale Supérieure Paris-Saclay, Université Paris-Saclay, 4, Avenue des Sciences, 91190 Gif sur Yvette, France; loussine.zargarian@ens-paris-saclay.fr (L.Z.); anissa_belfetmi@hms.harvard.edu (A.B.); marco.pasi@ens-paris-saclay.fr (M.P.) 3 Institut de Chimie des Substances Naturelles, UPR2301 CNRS, Université Paris-Saclay, 1 av. de la Terrasse, 91198 Gif-sur-Yvette, France; ewen.lescop@cnrs.fr * Correspondence: carine.tisne@cnrs.fr (C.T.); olivier.mauffret@ens-paris-saclay.fr (O.M.) Abstract: The nucleocapsid domain (NCd), located at the C-terminus of the HIV-1 Gag protein, is involved in numerous stages of the replication cycle, such as the packaging of the viral genome and reverse transcription. It exists under different forms through the viral life cycle, depending on the processing of Gag by the HIV-1 protease. NCd is constituted of two adjacent zinc knuckles (ZK1 and ZK2), separated by a flexible linker and flanked by disordered regions. Here, conformational equilibria between a major and two minor states were highlighted exclusively in ZK2, by using CPMG and CEST NMR experiments. These minor states appear to be temperature dependent, and their populations are highest at physiological temperature. These minor states are present both in NCp7, the mature form of NCd, and in NCp9 and NCp15, the precursor forms of NCd, with increased populations. The role of these minor states in the targeting of NCd by drugs and its binding properties is discussed. Keywords: HIV-1; nucleocapsid; NCp7; NCp9; NCp15; NMR; CPMG; CEST; dynamic; low-populated state; dark-state 1. Introduction HIV-1 nucleocapsid proteins are involved in many functions during the replication cycle [1–3]. The nucleocapsid domain (NCd) is a small basic protein constituted of two zinc knuckles separated by a semi-flexible short linker and flanked by N- and C-terminal disordered parts [4]. This domain is encompassed in the Pr55 Gag precursor and exists in different forms of maturation during the HIV-1 replication cycle (Figure 1A), namely NCp15, NCp9, and ultimately NCp7, the mature form of the NCd. NCd, plays a role in the protection of the viral RNA in the virion and behaves as an essential nucleic acid chaperon protein in reverse transcription in the early steps of the infection [2,3,5]. NCp7 is composed of two zinc knuckles (Figure 1A) that exhibit the same coordination motifs (CCHC) and number of residues. However, the ZKs display different roles and involvements in most of the functions of NCd [2,6,7]. It has been shown that mutations in the N-terminal zinc knuckle (ZK1) are more deleterious for the nucleic acid chaperon activity of NCp7 than mutations in the C-terminal zinc knuckle (ZK2) [8,9]. Additionally, recent data showed that mutations in ZK2 are more detrimental for the recruitment of RNA inside Gag–RNA complexes than mutations in ZK1 [7,10,11]. Additional proof of the non-equivalence of the two ZKs of the NCd relies on their different sensitivities towards NCd inhibitors based on zinc ejectors [12–14]. These families of inhibitors, notably the Viruses 2022, 14, 632. https://doi.org/10.3390/v14030632 https://www.mdpi.com/journal/viruses