title: Design of in vitro symmetric complexes and analysis by hybrid methods reveal mechanisms of HIV capsid assembly.
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.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, Charlottesville, VA 22908, USA. yeager@virginia.edu
authors.forename: Mark
authors.initials: M
authors.lastname: Yeager
mesh_terms: D002213:Capsid / Q000502:physiology* / Q000648:ultrastructure; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D004220:Disulfides / Q000378:metabolism; D015497:HIV-1 / Q000502:physiology* / Q000648:ultrastructure; D006860:Hydrogen Bonding; D008958:Models, Molecular; D008967:Molecular Biology / Q000379:methods*; D055503:Protein Multimerization; D012399:Rotation; D055672:Static Electricity; D019065:Virus Assembly / Q000502:physiology*; D054301:gag Gene Products, Human Immunodeficiency Virus
chemical_list: D036022:Capsid Proteins; D004220:Disulfides; D054301:gag Gene Products, Human Immunodeficiency Virus