Abstract
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.
Highlights
[Background] The human immunodeficiency virus-1 (HIV-1) is a lentivirus that replicates in CD4positive immune cells
The peptidylprolyl isomerase cyclophilin A (CypA) binds to multiple CypA bindings loops that are exposed on the outside of the capsid, which may stabilize the capsid during transport from the cell periphery to the nucleus
We recently developed a novel fluorescence imaging method to follow the real-time uncoating kinetics of individual HIV capsids in vitro and applied this method to reveal how capsid disassembly is modulated by different host factors and small molecules (Mallery et al, 2018; Marquez et al, 2018)
Summary
Add 1x trypsin-EDTA (typically 1 ml for a T25 cell culture flask) and incubate for 5 min at 37 °C. b. Stop the trypsin digestion by adding new culture media and transfer the cell suspension to a 15 ml conical tube. 3. Gently add 1 ml of cell suspension to the DNA:PEI mix and incubate for 5 min at room temperature. 5. Collect the virus-containing supernatant in a 15 ml conical tube and centrifuge at 2,100 x g for 10 min at 4 °C to remove cellular debris. 2. Add the biotinylation reagent to the virus-containing medium and incubate in the dark for 90 min at 4 °C with gentle rotation. C. Purify the biotinylated HIV-1 viral particles by size exclusion chromatography (1 day) 1. Wash the column with 2 CV of purified water and with storage solution
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