Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro
Chantal L Marquez, Derrick Lau, James Walsh, KM Rifat Faysal, Michael W Parker, Stuart G Turville, Till Bocking
BIO-PROTOCOL | BIO-PROTOCOL | Published : 2019
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 o..View full abstract
Awarded by National Health and Medical Research Council of Australia
The construct for expression of CypA was supplied by Nick Dixon (University of Wollongong, Australia). Recombinant PFO was purified by Sara Lawrence (St. Vincent's Institute of Medical Research and University of Melbourne, Australia). The TIRF microscope was designed and built by Philip Nicovich (current address: Allen Institute for Brain Science, Seattle, USA). CM and DL received an Australian Government Research Training Program Scholarship. TB received funding from the Australian Centre for HIV and Hepatitis Virology Research and from the National Health and Medical Research Council of Australia (NHMRC APP1100771). Funding from the Victorian Government Operational Infrastructure Support Scheme to St Vincent's Institute is acknowledged. MWP is a National Health and Medical Research Council of Australia Research Fellow.