A multiscale approach for elucidating nuclear entry mechanisms of HIV-1 capsid

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Project Summary Human immunodeficiency virus type 1 (HIV-1) remains a major threat to global health. Therefore, it is essential that we fully understand the mechanism of viral infectivity to provide new avenues for therapeutic development. After invading a non-dividing host cell, HIV-1 must gain access to the genetic vault, the nucleus. To do this, the viral genes, packaged in a capsid assembly, need to pass through nuclear pore complexes (NPCs). NPCs are massive protein channels that function as the gatekeepers of the cell nucleus. However, how the HIV-1 capsid breaches the barrier formed by the NPC remains poorly understood. Previous studies were hampered by the complexity of the NPC structure and the lack of molecular-level details of capsid-nucleoporin interactions; there was also a general inability of conventional in vitro platforms to capture the structural complexity of the viral capsid, which presents patterns that are recognized by host factors. Therefore, unlocking more mechanistic details of HIV-1 nuclear entry calls for innovative in vitro approaches capable of recapitulating higher-order capsid assemblies and the native environment of nuclear pores. We propose to establish such a platform by leveraging our recently established DNA-origami-based NPC mimics, termed NuPODs (NucleoPorins Organized by DNA), which contain precisely controlled pore dimensions and nucleoporins grafted with programmable density and orientation, as well as the programmable capsid protein (CA) assemblies that faithfully recreate selective fragments or the entire HIV-1 capsid surface. We will further validate our in vitro findings by infectivity experiments and live-cell imaging. Our multi-investigator team will draw from our respective expertise, including HIV biochemistry, structural biology, DNA nanotechnology, nuclear transport, and live-cell imaging, to build and apply this enabling platform for the study of HIV-1 capsid nuclear transport. Specifically, we will first comprehensively study the interactions between HIV-1 capsids and an assortment of cellular factors involved in HIV-1 nuclear import (Aim 1). Using soluble high-order CA assemblies and recombinant nucleoporins, we will define the biochemical and structural basis of capsid-nucleoporin binding, laying the foundation for the rest of the study. We will then build a library of NuPODs with increasing structural and compositional complexity, to identify the key determinants of HIV-1 nuclear import and the associated remodeling of the viral capsid and the NPC (Aim 2). These NuPODs will be built with multiple types of nucleoporins positioned at designated positions on a DNA-origami channel with tunable dimensions and stiffness. Systematically varying the NuPOD design and analyzing the resultant NuPOD-capsid docking and insertion will help understand HIV-1 nuclear import with molecular-level details. Additionally, we will validate our key findings using cell-based virologic experiments (Aim 3). Overall, we expect this project to create powerful tools that will not only help define the mechanism of HIV nuclear entry, but also enable us to explore the nuclear transport of many other viruses.