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J. Biol. Chem., Vol. 279, Issue 8, 6517-6525, February 20, 2004

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In Vitro Disassembly of a Parvovirus Capsid and Effect on Capsid Stability of Heterologous Peptide Insertions in Surface Loops^*

Aura Carreira, Margarita Menéndez, Juan Reguera, José María Almendral, and Mauricio G. Mateu

From the Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain and the Instituto de Química Física "Rocasolano" (CSIC), Serrano 119, 28006 Madrid, Spain

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.

Received for publication, July 16, 2003 , and in revised form, December 1, 2003.

^* This work was supported by Ministerio de Ciencia y Tecnología (MCyT) Grant BIO2000-0408) and Comunidad Autónoma de Madrid (CAM) Grant 08.2/0008.2/2000 (to M. G. M.), MCyT Grant BIO2000-1307 (to M. M.), MCyT Grants SAF2001-1325 and CAM 07B/0020/2002 (to J. M. A.), and by an institutional grant from Fundación Ramón Areces. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 34-91-4978462; Fax: 34-91-4974799; E-mail: mgarcia{at}cbm.uam.es.

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