Kinetics of HIV-1 capsid uncoating revealed by single-molecule analysis

Abstract

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.

Supercharged Assembly: A Broad-Spectrum Mechanism of Action for Drugs that Undermine Controlled HIV-1 Viral Capsid Formation

“…In this latter state, it is more likely for a CA dimer to dissociate from the lattice, which has been suggested as the rate-limiting step for viral uncoating. 54 The resultant defect is then the site of spontaneous dissociation of CA. In comparison, we find that one of the cores remains enclosed, which is evident from the flat profile in Fig.…”

“…These results are consistent with fluorescence experiments that have suggested both WT and PF74-treated viruses contain small populations of cores that fail to open, while the remaining PF74-treated viral cores have a greater propensity to open spontaneously. 54 Another mechanism of action for CIs may therefore be the production of eccentric cores that exhibit rapid CA disassembly due to inherent instabilities in high curvature regions with large pentamer density. Hence, despite the successful assembly of mature cores in the presence of CIs, accelerated capsid disassembly during the early stages of infection may inhibit viral infectivity.…”

Remodeling of the core leads HIV-1 pre-integration complex in the nucleus of human lymphocytes

“…The viral core is considered a fragile structure (24) that could exist only as intact or completely unbundled. However, a recent in vitro study highlighted the possibility that partial cores can be stabilized by host factors (25). Thus, HIV-1 CA can exist in different forms in infected cells: as intact cores, as monomers and probably as partial cores.…”

Atomic-scale Characterization of Mature HIV-1 Capsid Stabilization by Inositol Hexakisphosphate (IP6)

“…In particular, inositol hexakisphosphate (IP 6 ) is a cellular polyanion that is abundantly present in the cytosol and enriched in packaged viral particles that bud off from the cell 12 . The addition of IP 6 to in vitro reconstitutions of the capsid, drastically increases the stable lifetimes of the HIV-1 capsid from several minutes to over ten hours and promotes the assembly of fullerene capsids [12][13][14] . Viruses that fail to package IP 6 do not mature properly and have unstable capsids 15 .…”