Engineering fluorescent imaging platforms to characterise molecular interactions with HIV-1 capsid

The HIV-1 capsid is a protein shell utilised by the virus as a binding platform to hijack host machineries to promote infection. Characterisation of novel cofactors binding to capsid demands sensitive and quantitative assays to better understand their underlying binding mechanisms. Here, I have developed three complementary fluorescence microscopy-based platforms for quantitative characterisation of capsid binding analytes using tubular, spherical or conical lattices self-assembled from recombinant capsid protein (CA) with engineered cysteine residues for cross-linking as surrogates for the authentic capsid. The first approach focused on the development of a TIRFM biosensor using CA tubes immobilised on a glass surface as the biorecognition substrate for sensitive single-molecule binding studies. The biosensor was assessed in its ability to measure binding affinity, stoichiometry and kinetics using known capsid-binding cofactors as part of its validation process. Biosensor measurements confirmed that binding of the host cell protein cyclophilin A (CypA) to the tubular lattice is substoichiometric due to steric hindrance between CypA molecules. Binding analysis of cleavage and polyadenylation specificity factor subunit 6 (CPSF6) expressed as a full-length protein in a cell-free expression system suggested a role of oligomerisation to enhance binding via avidity. In the second approach, the biosensor surface was modified with cross-linked CA spheres as an alternative substrate containing pentameric defects and highly curved regions that are lacking in CA tubes. Binding of CypA to spheres revealed a significantly higher CypA to CA binding ratio at saturation suggesting a role of curvature in accommodating specific host cofactors. The third approach utilises confocal microscopy-based single molecule spectroscopy and was developed as a medium-throughput capsid interactor screening platform that uses conical CA self-assemblies as the substrate. Fluorescence-tagged analytes are identified as capsid binders when they accumulate on the surface of the CA cones, which can be detected in the fluorescence intensity traces as the appearance of spikes in the analyte channel or as an increase in the coincidence between the analyte signal and CA signal. The assay can be adapted for competition studies by using antagonistic molecules or different CA mutants to dissect binding interfaces on the capsid. The three assays developed herein are complementary methods to accelerate characterisation of novel capsid binders.

Grantor dc:publisher

UNSW, Sydney

Year dc:date

2020

Author dc:creator

• Lau, Derrick

• spectroscopy
• single molecule
• human immunodeficiency virus
• Fluorescence microscopy
• HIV
• Capsid

Statement dc:rights

• open access
• CC BY-NC-ND 3.0
• free_to_read

Licence

• https://purl.org/coar/access_right/c_abf2
• https://creativecommons.org/licenses/by-nc-nd/3.0/au/

Language dc:language

EN

Identifier

https://doi.org/10.26190/unsworks/3967

OAI identifier oai:identifier

oai:unsworks.library.unsw.edu.au:1959.4/70143