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To better understand the specific cascade of protein interactions that lead to the pathological aggregation of α-synuclein (wild-type and mutant forms), Leitão et al. present a method to measure interactions of monomeric, oligomeric, and fibrillar forms of α-syn with 65 proteins that were previously shown to be components of Lewy bodies. This approach is useful to understand the sequence of protein-binding events that lead to α-syn aggregation.

The human immunodeficiency virus 1 (HIV-1) consists of a viral membrane surrounding the conical capsid. The capsid is a protein container assembled from approximately 1,500 copies of the viral capsid protein (CA), functioning as a reaction and transport chamber for the viral genome after cell entry. Transmission electron microscopy (TEM) is a widely used technique for characterizing the ultrastructure of isolated viral capsids after removal of the viral membrane, which otherwise hinders negative staining of structures inside the viral particle for TEM. Here, we provide a protocol to permeabilize the membrane of HIV-1 particles using a pore-forming toxin for negative staining of capsids, which are stabilized with inositol hexakisphosphate to prevent premature capsid disassembly. This approach revealed the pleomorphic nature of capsids with a partially intact membrane surrounding them. The permeabilization strategy using pore-forming toxins can be readily applied to visualize the internal architecture of other enveloped viruses using TEM.Graphical abstract:

Abstract Background Efficient HIV-1 replication depends on interaction of the viral capsid with the host protein cyclophilin A (CypA). CypA, a peptidylprolyl isomerase, binds to an exposed loop in the viral CA protein via the enzyme’s active site. Recent structural analysis of CypA in complex with CA tubes in conjunction with molecular dynamics simulations identified a secondary CA binding site on CypA that allows a bridging interaction with two hexameric subunits of the assembled CA lattice, leading to capsid stabilization (Liu et al. in Nat Commun 7:10714, 2016). Results We performed mutational analysis of residues that have been proposed to mediate CA binding at the secondary binding site on CypA (A25, K27, P29 and K30) and tested the effects of the amino acid substitutions using interaction assays and HIV-1 infection assays in cells. The binding of recombinant CypA to self-assembled CA tubes or native HIV-1 capsids was measured in vitro using a quantitative fluorescence microscopy binding assay revealing that affinity and stoichiometry of CypA to the CA lattice was not affected by the substitutions. To test for functionality of the CypA secondary CA-binding site in HIV-1 infection, mutant CypA proteins were expressed in cells in which endogenous CypA was deleted, and the effects on HIV-1 infection were assayed. In normal HeLa-P4 cells, infection with HIV-1 bearing the A92E substitution in CA is inhibited by endogenous CypA and was inhibited to the same extent by expression of CypA mutants in CypA-null HeLa-P4 cells. Expression of the mutant CypA proteins in CypA-null Jurkat cells restored their permissiveness to infection by wild type HIV-1. Conclusions The amino acid changes at A25, K27, P29 and K30 did not affect the affinity of CypA for the CA lattice and did not impair CypA function in infection assays suggesting that these residues are not part of a secondary CA binding site on CypA.

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.

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.