Structural and Biochemical Determination of the HIV-1 Capsid Nup214 Interaction

About This Grant

Abstract 40.8 million people live with human immunodeficiency virus (HIV) infection, and 1.3 million people became newly infected in 2024. To prevent acquired immunodeficiency syndrome (AIDS), patients receive a regimen of antiretroviral therapy (ART), with Lenacapavir (LEN) being an important component of this therapy. LEN is a highly potent and long-acting antiretroviral drug that targets the capsid protein (CA) and mimics phenylalanine- glycine (FG)-motifs found within host factors. CA is the building block of the capsid core, and binds at a pocket termed the FG-binding pocket (FGBP). The core is made of ~250 hexamers CA hexamers (CAHEX) and exactly 12 CA pentamers (CAPENT), with CAPENT thought to be unable to bind to FG-motifs due to steric clashing. Most CAPENT are concentrated at the narrow end of the cone, which is thought to be the first part of the core that interacts with the proteins that comprise the nuclear pore complex (NPC), nucleoporin proteins (Nups). The intact capsid core passes through the semi-selective chemical barrier of the NPC central channel like a nuclear import factor to deliver the viral genome and complete integration. To accomplish this, the core uses the crucial FG- motif found within Nups, that form a network of weak multivalent interactions assisting the transition into a distinct biomolecular phase called liquid-liquid phase separation (LLPS). Depletion of these motifs disrupts CA binding and viral infectivity. This reduction demonstrates the importance of these FG-motifs in the viral replication cycle and as a target of disruption for ART. Of particular interest is Nup214, for which there is no structural information and limited biochemical information in the context of HIV infection; it is located at the periphery of the NPC, and the C-terminal domain is enriched in FG-motifs. Nup214 has been previously reported to interact with CA in an FG-dependent manner, as Nup214 depletion impacts HIV-1 infectivity. I hypothesize that the FG repeats of Nup214 interacts with the HIV-1 capsid core to initiate the entry of the viral capsid into the nucleus. Therefore, I will characterize the Nup214-Capsid interaction by determining the structural orientation of Nup214 in the FGBP, biophysically defining the kinetic parameters of this interaction, and elucidating the biochemical properties of Nup214. Furthermore, the specificity of the Nup-CA interaction will be determined using several CA assembly morphologies with varying amounts of CAHEX vs. CAPENT. In Aim 1 the binding kinetics of this interaction and the specificity will be determined, along with the biophysical properties of Nup214 will be defined. In Aim 2 cryogenic- electronic microscopy (cryo-EM) will be used to determine structurally how Nup214 occupies the FGBP of the various CA assemblies. This project will train me in a diverse set of structural and biochemical techniques, and the data generated will increase the understanding of how the capsid core initially interacts with the NPC through the FG-motifs found in Nup214. Furthermore, the characterization of this Nup-Capsid interaction may also give rise to new strategies to interfere with this crucial step of the virus replication.