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

Twenty-eight amino acid residues involved in most noncovalent interactions between trimeric protein subunits in the capsid of the parvovirus minute virus of mice were truncated individually to alanine, and the effects on capsid assembly, thermostability, and conformation were analyzed. Only seven side chains were essential for protein subunit recognition. These side chains virtually corresponded with those that either buried a large hydrophobic surface on trimer association or formed buried intertrimer hydrogen bonds or salt bridges. The seven residues are evolutionarily conserved, and they define regularly spaced spots on a thin equatorial belt surrounding each trimer. Truncation of the many side chains that were dispensable for assembly, including those participating in solvent-accessible polar interactions, did not substantially affect capsid thermostability either. However, the interfacial residues located at the base of the pores delineating the capsid five-fold axes participated in a heat-induced conformational rearrangement associated with externalization of the capsid protein N terminus, and they were needed for infectivity. Thus, at the subunit interfaces of this model virus capsid, only key residues involved in the strongest interactions are critical for assembly and stability, but additional residues fulfill other important biological roles.

In vitro disassembly of a parvovirus capsid and effect on capsid stability of heterologous peptide insertions in surface loops.

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.