Friday, May 9, 2025
5/9/2025
Mechanism of nuclear pore passage of the HIV-1 capsid - PROJECT SUMMARY: HIV/AIDS remains a global epidemic, with 1.5 M infections per year globally, more than 40 years after the initial discovery of the virus. To find new avenues for antiviral therapies, it is important to understand the molecular and structural details of the entire process that the virus uses to enter and traffic the cell. This exploratory project is focused on the mechanisms that the HIV-1 capsid uses to traverse the nuclear pore complex (NPC), a critical step on its journey to the nucleus for uncoating, reverse transcription, and integration. A series of recent publications suggest that the capsid can translocate through the NPC and that uncoating likely takes place inside of the nucleus. This dogmatic shift came about as data showed that the NPC can, in fact, accommodate an assembled HIV-1 capsid, while it was previously perceived to be too narrow. While it is now established that the assembled capsid can translocate through an NPC, it is unclear what the driving force of that process is. To address this critical knowledge gap, we propose a new conceptual framework, in which we focus on interactions between the HIV-1 capsid and the transport barrier of the NPC, a hydrogel (or condensate) made up of phenylalanine-glycine-repeat containing NPC components (FG-NUPs). While FG- domains within several FG-NUPs are known to bind to the capsid, their role in capsid transport is still poorly understood for most. As FG-NUPs assemble into liquid-like hydrogels that fill the NPC, and as each HIV-1 capsid has >1000 potential FG-NUP interaction sites, we hypothesize that one can envision the capsid partitioning into the hydrogel, providing a thermodynamic driving force for translocation. To reveal the mechanisms behind this process, we propose an exploratory approach that combines biochemistry, biophysics, and structural biology. In Aim 1, we will perform a comprehensive biochemical characterization of the FG-capsid interaction. In a qualitative assay, we will analyze the dynamic parameters that govern the interaction of the HIV-1 capsid with all FG-Nups. Here we hypothesize that the many hundreds of FG-binding sites on an intact capsid provide the main driving force for HIV-1 capsids to enter NPCs. An important element will be to understand whether Nup153, at the nuclear side of the NPC, plays a distinct role in directing HIV-1 transport. In Aim 2, we will determine how assembled HIV-1 capsid-like particle interacts with the FG-hydrogel in vitro. Preliminary data indicates that intact ~40 nm CA spheres can rapidly enter an FG-hydrogel with NPC-like properties. This system enables us to probe the determinants that are critical for nuclear transport in a format that closely resembles the in vivo situation, taking into account avidity effects as a result of the many dynamic interactions occurring simultaneously.
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