title: In vitro disassembly of a parvovirus capsid and effect on capsid stability of heterologous peptide insertions in surface loops.
abstract: We have analyzed the in vitro disassembly of the capsid of the minute virus of mice, and the stability of capsid chimeras carrying heterologous epitope insertions. Upon heating in a physiological buffer, empty capsids formed by 60 copies of protein VP2 underwent first a reversible conformational change with a small enthalpy change detected by fluorescence. This change was associated with, but not limited to, externalization of the VP2 N terminus. Irreversible capsid dissociation as detected by changes in fluorescence, hemagglutination activity, and electrophoretic mobility occurred at much higher temperatures. Differential scanning calorimetry in the same conditions indicated that the dissociation/denaturation transition involved a high enthalpy change and proceeded through one or more intermediates. In contrast, in the presence of 1.5 M guanidinium chloride, heat-induced disassembly fitted a two-state irreversible process. Both thermally and chemically induced dissociation/denaturation yielded a form that had lost a part of the tertiary structure, but still retained the native secondary structure. Data from chemical dissociation indicates this form may correspond to a molten globule-like monomeric state of the capsid protein. All five antigenic peptide insertions attempted in exposed loops, despite being perhaps among the least disruptive, led to defects in folding/assembly of the capsid and, in most cases, to reduced capsid stability against thermal dissociation. The results with one of the simplest viral capsids reveal a complex pathway for disassembly, and a reduction in capsid assembly and stability upon insertion of peptides, even within the most exposed capsid loops.
authors.affiliation: Centro de Biología Molecular Severo Ochoa (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Aura
authors.initials: A
authors.lastname: Carreira
authors.forename: Margarita
authors.initials: M
authors.lastname: Menéndez
authors.forename: Juan
authors.initials: J
authors.lastname: Reguera
authors.forename: José María
authors.initials: JM
authors.lastname: Almendral
authors.forename: Mauricio G
authors.initials: MG
authors.lastname: Mateu
mesh_terms: D000818:Animals; D002152:Calorimetry, Differential Scanning; D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D006388:Hemagglutinins / Q000737:chemistry; D006358:Hot Temperature; D007313:Insecta; D051379:Mice; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017992:Parvovirus / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry; D014466:Ultraviolet Rays
chemical_list: D036022:Capsid Proteins; D006388:Hemagglutinins; D010455:Peptides; D014357:Trypsin