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J Mol Biol. 2011 Jul 22;410(4):534-52. doi: 10.1016/j.jmb.2011.04.073.

Design of in vitro symmetric complexes and analysis by hybrid methods reveal mechanisms of HIV capsid assembly.

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1
Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. yeager@virginia.edu

Abstract

Unlike the capsids of icosahedral viruses, retroviral capsids are pleomorphic, with variably curved, closed fullerene shells composed of ∼250 hexamers and exactly 12 pentamers of the viral CA protein. Structures of CA oligomers have been difficult to obtain because the subunit-subunit interactions are inherently weak, and CA tends to spontaneously assemble into capsid-like particles. Guided by a cryoEM-based model of the hexagonal lattice of HIV-1 CA, we used a two-step biochemical strategy to obtain soluble CA hexamers and pentamers for crystallization. First, each oligomer was stabilized by engineering disulfide cross-links between the N-terminal domains of adjacent subunits. Second, the cross-linked oligomers were prevented from polymerizing into hyperstable, capsid-like structures by mutations that weakened the dimeric association between the C-terminal domains that link adjacent oligomers. The X-ray structures revealed that the oligomers are comprised of a fairly rigid, central symmetric ring of N-terminal domains encircled by mobile C-terminal domains. Assembly of the quasi-equivalent oligomers requires remarkably subtle rearrangements in inter-subunit quaternary bonding interactions, and appears to be controlled by an electrostatic switch that favors hexamers over pentamers. An atomic model of the complete HIV-1 capsid was then built using the fullerene cone as a template. Rigid-body rotations around two assembly interfaces are sufficient to generate the full range of continuously varying lattice curvature in the fullerene cone. The steps in determining this HIV-1 capsid atomic model exemplify the synergy of hybrid methods in structural biology, a powerful approach for exploring the structure of pleomorphic macromolecular complexes.

PMID:
21762799
PMCID:
PMC3166646
DOI:
10.1016/j.jmb.2011.04.073
[Indexed for MEDLINE]
Free PMC Article
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