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.The study of the folding, association, and disassembly of large multimeric proteins is complicated by their size, the general irreversibility of the reactions involved, and the frequent occurrence of off-pathway intermediates. However, the significant advances already made hold promise for a detailed understanding of these processes (1). Spherical virus capsids are large, multimeric proteins (2-5) and constitute attractive models for the study of the association, stability, and disassembly of very large protein complexes (for reviews see Refs. 4 and 6 -13). In addition, viral capsids are highly dynamic entities and have evolved unique structural solutions in response to the diverse, sometimes conflicting functions they must perform during the virus life cycle (4, 7, 11, 14 -16). Thus, they provide good opportunities to understand finely tuned structure-function relationships in proteins and to develop new antiviral approaches based on the inhibition of assembly or uncoating (15,17, 18).The icosahedral T ϭ 1 capsids of parvoviruses (19 -24) are formed by 60 protein subunits contributed by three nonidentical polypeptide chains that show, however, identical -fold and core sequence. VP2 is the major capsid protein and can selfassemble into empty (DNA-free) capsids (viral-like particles or VLPs).1 VP1, a minor component of natural capsids, includes...

Role of interfacial amino acid residues in assembly, stability, and conformation of a spherical virus capsid

“…The fluorescence curve obtained for the parental (nonmutated) MVM capsid revealed two clearly separated sigmoidal transitions. These have been shown (47) to respectively correspond to (i) a reversible conformational change (without capsid dissociation) that occurred with a T m of 46°C (Fig. 4A), involved a change in the exposure of some capsid tryptophans to solvent, and involved also the externalization of the VP2 N terminus, as detected by the trypsin sensitivity of the exposed peptide segment; and (ii) the irreversible dissociation of the capsid with a T m of 75°C (Fig.…”

“…In a physiological buffer, the dissociation and denaturation of the capsid proceeded through an intermediate, which was apparently destabilized by the presence of a low concentration of guanidinium chloride, resulting in a two-state dissociation and denaturation transition. Chemical dissociation, followed by fluorescence spectroscopy, circulardichroism spectroscopy, and molecular-size determinations, indicated that the dissociation and denaturation product obtained corresponded to a molten-globule-like monomeric state of the capsid protein (47). The specific dissociation T m value obtained by fluorescence, which could be determined very precisely, has been used here as an indicator of the relative kinetic stability against dissociation of the mutant capsids with side-chain truncations at the intertrimer interfaces (Fig.…”

“…Site-directed mutagenesis of the VP2 gene of MVM strain p (MVMp) was carried out by using the QuikChange system (Stratagene) on recombinant plasmids pSVtk-VP1͞2 [which included the VP1͞VP2 gene of MVMp and was derived from a molecular clone of the MVMp genome (46)] and pFB1-VP2 (47). The mutations were confirmed by automated sequencing.…”

“…Recombinant bacmids BM-VP2 (parental and mutants) were used to transfect H5 insect cells essentially as described (47). The sequences of six mutant VP2 genes (R83A, T249A, F55A, D507A, L537A, and S550A) in the recombinant baculovirus DNA obtained were totally or partially determined as a final validation of the procedure.…”

“…Temperature was increased continuously from 25°C to 85°C at a rate of 1°C per min, and the intrinsic tryptophan fluorescence at an emission wavelength of 330 nm was determined at 1-min intervals. The fluorescence changes were fitted to sigmoidal cooperative transitions by using the KALEIDAGRAPH (Abelbeck Software, Reading, PA), which allowed the precise determination of the half-transition temperature (T m ) (47). For analyses at acidic pH, a 0.96 M solution of the capsid in PBS was diluted 1:1 with 50 mM phosphoric acid͞sodium phosphate buffer (pH 2), and the final pH was determined.…”