Abstract
The Human Immunodeficiency virus (HIV-1) life cycle involves several highly choreographed steps during which the virus assembles at the plasma membrane (PM) of an infected cell and buds off the membrane as a viral particle. We have used conventional and superresolution imaging approaches to investigate the cell biological mechanisms underpinning three key steps in the viral assembly/budding pathway: clustering of the viral coat in the plasma membrane; recruitment of proteins into the viral bud; and virus budding off the membrane. In the first step involving viral Gag coat assembly, we show it is critically dependent on viral and/or host mRNA, which drives Gag clustering through RNA-Gag electrostatic interactions. In the second step involving protein recruitment into the viral bud, we demonstrate that Env proteins incorporate into viral buds through dynamic partitioning into a specialized microenvironment created by multimerization of Gag at the PM. In the final step involving viral abscission from the PM, we examine the 3D molecular organization of ESCRT machinery with respect to HIV bud sites using iPALM to gain critical insight. We find ESCRT-III proteins assemble within the head of the budding virion, not the base as previously proposed. This later finding prompts a reevaluation of current models for ESCRT-III scaffolding, and suggests that ESCRT abscission initiates from within the head of the budding virion.
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