title: Observed hysteresis of virus capsid disassembly is implicit in kinetic models of assembly.
abstract: For many protein multimers, association and dissociation reactions fail to reach the same end point; there is hysteresis preventing one and/or the other reaction from equilibrating. We have studied in vitro assembly of dimeric hepatitis B virus (HBV) capsid protein and dissociation of the resulting T = 4 icosahedral capsids. Empty HBV capsids composed of 120 capsid protein dimers were more resistant to dissociation by dilution or denaturants than anticipated from assembly experiments. Using intrinsic fluorescence, circular dichroism, and size exclusion chromatography, we showed that denaturants dissociate the HBV capsids without unfolding the capsid protein; unfolding of dimer only occurred at higher denaturant concentrations. The apparent energy of interaction between dimers measured in dissociation experiments was much stronger than when measured in assembly studies. Unlike assembly, capsid dissociation did not have the concentration dependence expected for a 120-subunit complex; consequently the apparent association energy systematically varied with reactant concentration. These data are evidence of hysteresis for HBV capsid dissociation. Simulations of capsid assembly and dissociation reactions recapitulate and provide an explanation for the observed behavior; these results are also applicable to oligomeric and multidomain proteins. In our calculations, we find that dissociation is impeded by temporally elevated concentrations of intermediates; this has the paradoxical effect of favoring re-assembly of those intermediates despite the global trend toward dissociation. Hysteresis masks all but the most dramatic decreases in contact energy. In contrast, assembly reactions rapidly approach equilibrium. These results provide the first rigorous explanation of how virus capsids can remain intact under extreme conditions but are still capable of "breathing." A biological implication of enhanced stability is that a triggering event may be required to initiate virus uncoating.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73190, USA.
authors.forename: Sushmita
authors.initials: S
authors.lastname: Singh
authors.forename: Adam
authors.initials: A
authors.lastname: Zlotnick
mesh_terms: D002213:Capsid / Q000737:chemistry* / Q000378:metabolism*; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006512:Hepatitis B Core Antigens / Q000737:chemistry; D006515:Hepatitis B virus / Q000737:chemistry*; D007700:Kinetics; D008027:Light; D012542:Scattering, Radiation; D013816:Thermodynamics; D013997:Time Factors; D014364:Tryptophan / Q000494:pharmacology; D014508:Urea / Q000494:pharmacology; D014758:Viral Core Proteins / Q000737:chemistry; D014771:Virion / Q000502:physiology; D019065:Virus Assembly
chemical_list: D006512:Hepatitis B Core Antigens; D014758:Viral Core Proteins; D014364:Tryptophan; D014508:Urea; D019791:Guanidine