Binding of host factors to stabilized HIV-1 capsid tubes

Abstract

The capsid-binding assay is an in vitro experiment used to determine whether cellular proteins interact with the HIV-1 core. In vitro assembled HIV-1 capsids recapitulate the surface of the HIV-1 core. The assay involves the incubation of in vitro assembled HIV-1 capsid-nucleocapsid (CA-NC) complexes with the protein in question. Subsequently, the mixture is spun through a sucrose cushion using an ultracentrifuge, and the pellet is analyzed for the presence of the protein in question. Although this binding assay is reliable, it is labor intensive and does not contain washing steps. Here we have developed a simpler and faster assay to measure whether a cellular protein is binding to capsid. More importantly, this novel capsid-binding assay contains washing steps. In this assay, we took advantage of the HIV-1 capsid mutant A14C/E45C protein, which is stabilized by disulfide bonds, and is resistant to washing steps. We validated the reliability and specificity of this novel assay by testing the capsid binding ability of TRIMCyp, CPSF6 and MxB with their corresponding controls. Overall, this novel assay provides a reliable and fast methodology to search for novel capsid binders.

Fluorescence biosensor for real-time interaction dynamics of host proteins with HIV-1 capsid tubes

“…The appearance of the capsid tubes as lines in the fluorescence image facilitates detection and extraction of traces using automated image analysis. Tubular CA lattices are commonly used in binding studies as surrogates for the capsid 16,18,38,39 but lack CA pentamers and the variable curvature of the native conical capsid. Combining our biosensor as a tool for routine measurements with more specialised approaches using virus-like particles 32 should overcome these limitations.…”

“…We used self-assembly of recombinant CA A14C/E45C at high salt concentrations to produce tubular lattices of disulfide cross-linked CA hexamers 26 as biorecognition elements representing the HIV-1 capsid. 18 These stabilised tubes were rendered fluorescent by coassembly with a small fraction of CA K158C reacted with a fluorophore at the engineered cysteine (Supplementary Figure S1 and S2). This co-assembly reaction also yielded long tubes that aggregated into bundles in solution ( Figure 2A), a process known to be driven by interactions between hydrophobic residues (primarily located in the CypA loop of CA) on the outer tube surfaces.…”

Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating

“…Experiments using our nuclear import functional assay suggested that assembled capsids are transported into the nucleus; however, the western blots cannot distinguish between assembled or disassembled capsid within the nucleus. To directly test whether large assembled capsid complexes can be imported into the nucleus using an HIV-1 core resistant to disassembly, we produced an HIV-1 virus with a stabilized capsid, taking advantage of the capsid mutations A14C/E45C that stabilize purified capsid hexamers in vitro through disulfide bridges between monomers of hexamers (Pornillos et al, 2010;Selyutina et al, 2018). This virus lacked defects in viral budding, maturation, and reverse transcription; however, HIV-1-A14C/E45C virus was incapable of forming 2-LTR circles and productive infection ( Figure S4).…”

TRIM5α restriction of HIV-1-N74D viruses in lymphocytes is caused by a loss of cyclophilin A protection

“…If TRIM5α hu restriction occurs after cells are infected by HIV-1-N74D, it implies that Cyp A is no longer protecting the core. To test this hypothesis, we assessed the abilities of TRIM5α hu and Cyp A to bind to N74D-stabilized capsid tubes using a capsid binding assay [13]. As shown in Figure 5, TRIM5α hu bound with increased affinity to stabilized N74D capsid tubes than to wild-type tubes.…”