School Colloquia Series - A/Prof Till Boecking - Single-molecule analysis of HIV-1 capsid disassembly
Abstract: HIV-1 uses its capsid to evade immune detection while copying its RNA genome into DNA en route from the cell periphery to the nucleus. The capsid is a protein nanocontainer assembled from 1000-1500 copies of a single viral protein, CA, into a conical fullerene structure composed of CA hexamers and pentamers. Disassembly or
“uncoating” of the metastable capsid releases the viral DNA prior to import into the nucleus. Current models postulate that uncoating initiates either in the cytoplasm or after the capsid has docked at the nuclear pore. In either scenario, the initiation and propagation of disassembly are thought to be controlled by host proteins and small molecules that are recruited to the capsid at different stages after cell entry. However, the experimental dissection of mechanisms controlling capsid stability is incomplete because of a lack of appropriate techniques to overcome the challenges posed by the fragility and polymorphism of the HIV-1 capsid. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we recently developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilising the viral membrane. Our assay allows us to track the dynamic interactions of proteins with the capsid, pinpoint the precise moment at which the capsid develops its first defect, and visualise the kinetics of lattice disassembly thereafter. By using single-molecule analysis we identified that dissociation of the first capsomers is the rate-limiting step of uncoating followed by catastrophic collapse of the remainder of the lattice. Small molecule drugs affect different stages of the disassembly pathway. I will discuss the implications of our findings for how the virus could regulate uncoating inside the cell.