Zinc ions trigger conformational change and oligomerization of hepatitis B virus capsid protein.
Assembly of virus particles in infected cells is likely to be a tightly regulated process. Previously, we found that in vitro assembly of hepatitis B virus (HBV) capsid protein is highly dependent on protein and NaCl concentration. Here we show that micromolar concentrations of Zn2+ are sufficient to initiate assembly of capsid protein, whereas other mono- and divalent cations elicited assembly only at millimolar concentrations, similar to those required for NaCl-induced assembly. Altered intrinsic protein fluorescence and highly cooperative binding of at least four Zn2+ ions (KD approximately 7 microM) indicated that binding induced a conformational change in capsid protein. At 37 degrees C, Zn2+ enhanced the initial rate of assembly and produced normal capsids, but it did not alter the extent of assembly at equilibrium. Assembly mediated by high zinc concentrations (> or =300 microM) yielded few capsids but produced a population of oligomers recognized by capsid-specific antibodies, suggesting a kinetically trapped assembly reaction. Comparison of kinetic simulations to in vitro assembly reactions leads us to suggest that kinetic trapping was due to the enhancement of the nucleation rate relative to the elongation rate. Zinc-induced HBV assembly has hallmarks of an allosterically regulated process: ligand binding at one site influences binding at other sites (cooperativity) indicating that binding is associated with conformational change, and binding of ligand alters the biological activity of assembly. We conclude that zinc binding enhances the kinetics of assembly by promoting formation of an intermediate that is readily consumed in the reaction. Free zinc ions may not be the true in vivo activator of assembly, but they provide a model for regulation of assembly.
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, P.O. Box 26901, Oklahoma City, Oklahoma 73190, USA.
Stephen J
SJ
Stray
Pablo
P
Ceres
Adam
A
Zlotnick
D036022:Capsid Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D002413:Cations, Divalent / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006512:Hepatitis B Core Antigens / Q000032:analysis; D006515:Hepatitis B virus / Q000737:chemistry* / Q000276:immunology / Q000502:physiology; D007700:Kinetics; D008027:Light; D010441:Peptide Chain Elongation, Translational; D011485:Protein Binding; D011487:Protein Conformation; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D019065:Virus Assembly; D015032:Zinc / Q000737:chemistry* / Q000378:metabolism; D017967:Zinc Compounds / Q000737:chemistry
D036022:Capsid Proteins; D002413:Cations, Divalent; D002712:Chlorides; D006512:Hepatitis B Core Antigens; D017967:Zinc Compounds; C016837:zinc chloride; D015032:Zinc