Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis
Chantal L Márquez,
Derrick Lau,
James Walsh,
Vaibhav Shah,
Conall McGuinness,
Andrew Wong,
Anupriya Aggarwal,
Michael W Parker,
David A Jacques,
Stuart Turville,
Till Böcking
Affiliations
Chantal L Márquez
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia
Derrick Lau
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia
James Walsh
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia
Vaibhav Shah
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia
Conall McGuinness
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia
Australian Cancer Research Foundation Rational Drug Discovery Centre, St. Vincent’s Institute of Medical Research, Melbourne, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Melbourne, Australia
EMBL Australia Node in Single Molecule Science, School of Medical Sciences, UNSW, Sydney, Australia; ARC Centre of Excellence in Advanced Molecular Imaging, UNSW, Sydney, Australia
Uncoating of the metastable HIV-1 capsid is a tightly regulated disassembly process required for release of the viral cDNA prior to nuclear import. To understand the intrinsic capsid disassembly pathway and how it can be modulated, we have developed a single-particle fluorescence microscopy method to follow the real-time uncoating kinetics of authentic HIV capsids in vitro immediately after permeabilizing the viral membrane. Opening of the first defect in the lattice is the rate-limiting step of uncoating, which is followed by rapid, catastrophic collapse. The capsid-binding inhibitor PF74 accelerates capsid opening but stabilizes the remaining lattice. In contrast, binding of a polyanion to a conserved arginine cluster in the lattice strongly delays initiation of uncoating but does not prevent subsequent lattice disassembly. Our observations suggest that different stages of uncoating can be controlled independently with the interplay between different capsid-binding regulators likely to determine the overall uncoating kinetics.
