Quantum mechanics

Molecular biology

Biology

Förster resonance energy transfer

Virology

Biochemistry

Physics

In vitro

Human immunodeficiency virus (HIV)

Fluorescence

Kinetics

Group-specific antigen

Chemistry

Capsid

Fluorescence microscope

Virus

Biophysics

Cyclophilin A

BIO-PROTOCOL

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.

Fluorescence Microscopy Assay to Measure HIV-1 Capsid Uncoating Kinetics in vitro