Abstract: The HIV-1 mature capsid (CA) assumes an amorphous, fullerene conical configuration\ndue to its high flexibility. How native CA self-assembles is still unclear despite having well-defined\nstructures of its pentamer and hexamer building blocks. Here we explored the self-assembly of an\nengineered capsid protein built through artificial disulfide bonding (CA N21C/A22C) and determined\nthe structure of one fraction of the globular particles. CA N21C/A22C was found to self-assemble\ninto particles in relatively high ionic solutions. These particles contained disulfide-bonding hexamers\nas determined via non-reducing SDS-PAGE, and exhibited two major components of 57.3 S and 80.5 S\nin the sedimentation velocity assay. Particles had a globular morphology, approximately 40 nm in\ndiameter, in negative-staining TEM. Through cryo-EM 3-D reconstruction, we determined a novel\nT = 4 icosahedral structure of CA, comprising 12 pentamers and 30 hexamers at 25 Ã? resolution.\nWe engineered the HIV-1 V3 loop to the CA particles, and found the resultant particles resembled the\nmorphology of their parental particles in TEM, had a positive reaction with V3-specific neutralizing\nantibodies, and conferred neutralization immunogenicity in mice. Our results shed light on HIV CA\nassembly and provide a particulate CA for epitope display.
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