Abstract Antiretroviral therapies (ART) have transformed the once deadly HIV/AIDS disease into a manageable, chronic infection. Yet, there are still a number of pressing problems associated with current ARTs, including the necessity of daily administration of HIV-1 medications, suboptimal treatment adherence, and the emergence of drug-resistant viral phenotypes. Therefore, there is a need for developing long-acting antiretroviral agents targeting clinically unexploited viral proteins to mitigate the above problems. HIV-1 capsid protein is a novel, attractive target as its plays multiple essential roles during the virus life cycle. GS-6207 (Lenacapavir, Gilead Sciences) is a recently discovered, first-in-class, long-acting, and ultra- potent HIV-1 capsid inhibitor. Recently completed phase 1 clinical trials (NCT03739866) have suggested advancement of GS-6207 into phase 2/3 clinical trials (NCT04143594/NCT04150068) with a six-month dosing interval. Our research objective is to elucidate structural and mechanistic bases for a highly potent antiviral activity of GS-6207 and exploit the knowledge obtained to develop second-generation inhibitors. For this, we have synthesized and examined the antiviral activities of GS-6207. Consistent with the multifaceted role of capsid in HIV-1 biology, the inhibitor potently (EC50 of ~55 pM) impaired incoming virus and exhibited a second, slightly reduced (EC50 of ~314 pM) antiviral activity during virus egress. Mode-of- action studies of GS-6207 revealed that the inhibitor blocks post-entry steps of infection by stabilizing and thereby preventing functional disassembly of the capsid shell in the cytoplasm of infected cells. In addition, GS-6207 interfered with capsid binding to the cellular HIV-1 cofactors Nup153 and CPSF6 that mediate viral nuclear import and direct integration into gene-rich regions of chromatin. Our x-ray crystallography, cryo-electron microscopy, and hydrogen-deuterium exchange experiments have revealed that GS-6207 tightly binds two adjoining capsid subunits and promotes distal intra- and inter-hexamer interactions that strikingly stabilize the curved capsid lattice. Furthermore, our high-resolution x-ray structure of GS-6207 bound to a capsid hexamer enabled us to map drug-resistant variants in close proximity to the GS-6207 binding site. This information will be critical for rational design of second-generation inhibitors. We propose to extend these studies to better understand the multimodal, exceptionally potent antiviral activity of GS- 6207 during both early and late steps of HIV-1 replication. For this, we will pursue the following three specific aims: Aim 1 will elucidate structural and mechanistic bases for inhibition of post-entry steps of HIV- 1 infection by GS-6207; Aim 2 will dissect underlying mechanisms of inhibition of virus production and maturation by GS-6207; and Aim 3 will investigate the structural basis for viral drug-resistance to GS-6207 and rationally develop second-g...