Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
Chantal Márquez,
Derrick Lau,
James Walsh,
K. M. Rifat Faysal,
Michael Parker,
Stuart Turville,
Till Böcking
Affiliations
Chantal Márquez
EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, UNSW, Sydney, Australia
Derrick Lau
EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, UNSW, Sydney, Australia
James Walsh
EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, UNSW, Sydney, Australia
K. M. Rifat Faysal
EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, UNSW, Sydney, Australia
Michael Parker
St. Vincent’s Institute of Medical Research, Australia; Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Australia
Stuart Turville
The Kirby Institute, UNSW, Sydney, Australia
Till Böcking
EMBL Australia Node in Single Molecule Science and ARC Centre of Excellence in Advanced Molecular Imaging, School of Medical Sciences, UNSW, Sydney, Australia
The stability of the HIV-1 capsid and the spatiotemporal control of its disassembly, a process called uncoating, need to be finely tuned for infection to proceed. Biochemical methods for measuring capsid lattice disassembly in bulk are unable to resolve intermediates in the uncoating reaction. We have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro. The assay utilizes immobilized viral particles that are permeabilized with the a pore-former protein, and is designed to (1) detect the first defect of the capsid by the release of a solution phase marker (GFP) and (2) visualize the disassembly of the capsid over time by “painting” the capsid lattice with labeled cyclophilin A (CypA), a protein that binds weakly to the outside of the capsid. This novel assay allows the study of dynamic interactions of molecules with hundreds of individual capsids as well as to determine their effect on viral capsid stability, which provides a powerful tool for dissecting uncoating mechanisms and for the development of capsid-binding drugs.
