Affinity Gradient-Based Transport of HIV Capsid Cores Through the Nuclear Pore Complex: Project Summary Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) have evolved strategies to enter the nucleus of non-dividing cells and integrate their reverse transcribed DNA into host genomes. There are several reports on the transport of HIV-1 complexes to, and import through, the Nuclear Pore Complex (NPC), a massive channel in the nuclear envelope composed of multiple copies of ~30 nucleoporins (NUPs). Recent data suggests that the supramolecular fullerene cone-shaped viral capsid (CA) core (~120x50 nm) that encapsulates the viral genome can penetrate the central channel (diameter of ~60 nm) of the NPC, at least partially, intact. It is widely accepted that at least a subset of NUPs interacts with the HIV-1 CA proteins of the viral core through their phenylalanine-glycine (FG) motifs. However, the molecular mechanisms by which a largely intact HIV-1 capsid passes through the NPC – a defining feature of lentiviruses – remains to be elucidated. Mechanistic studies of HIV-1 nuclear entry have been stymied primarily by the inherent complexity of the system. The NPC is one of the largest assemblies in the Eukaryotic cell (~120 MDa) and HIV-1 capsid core is one of the largest cargoes delivered to the cell nucleus. Based on analysis of state-of-the-art published data and pilot results, we propose a paradigm-shifting hypothesis that addresses the mechanism of transport of HIV-1 capsid core through the NPC and challenges the generalized view on nucleo-cytoplasmic transport. We propose that HIV-1 capsid core is a complex transport receptor that is translocated via an opportunistic affinity gradient within the NPC. The affinity gradient consists of various “signatures” on specific nucleoporins that are naturally distributed along the nucleo-cytoplasmic axis of the NPC. The subset of such “signatures” involves various flavors of FG motifs that are segregated in the NPC to distinct zones. We will use our multidisciplinary expertise, armamentarium of techniques, and preliminary data, to address the following specific aims: 1. Determine the structures of complexes of CA proteins with peptides of different FG motifs. 2. Biochemically and biophysically characterize the interactions between CA and various FG peptides. 3. Discriminate between various flavors of FG motif interactions with mutant capsid cores using nuclear import experiments. The scientific premise is that combined structural, biochemical, biophysical and imaging approaches will provide important mechanistic insight into interactions of HIV-1 capsid core complexes with NPC constituents and reveal the capsid core – NPC structural intermediates of translocation. Knowledge of the molecular interactions during HIV-1 nuclear import will shed light on the strategy HIV-1 deploys to deliver its genome to the nucleus; our fundamental understanding of NPC function; provide a framework for studies of the...