Estudio del desensamblaje in vitro y análisis estructura-función de cavidades de la cápsida del virus diminuto del ratón

Abstract

Sphencal virus capsids are large, multimeric proteins and constitute attractive models for the study of the assoeiation, stabiity and disassembly of very large protein complexes. In addition, they provide good oppominities to understand finely tuned shucturehinction relationships in viral particles, and to develop more stable vaccines and new antiviral approaches based on the inhibition of assembly or uncoating. In this doctoral thesis we have analyzed in viíro the disassembly of the MVM capsid, and carried out a shucture-function analysis of the role of cavities and a bwied charge located within the hydrophobic core of each capsid subunit on capsid assembly and stabiiity and viral function. The specific results obtained can be summarized as fouows. 1. Biophysical and biochemical Vi viíro analyses of the disassembly of the MVM capsid reveal a complex pathway that may involve several steps: a reversible conformational rearrangement associated, but not limited to the externalization of the N-terminal segment of a fraction of the capsid subunits; the irreversible dissociation of the capsid into an intermediate; and the dissociatioddenaturation of the latter to yield a molten globulelike monomeric form. 2. Determination by intrinsic Trp fluorescence of the transition temperature of the specific capsid dissociation step has been validated as a reliable and acauate procedure to analyze the effect of mutations or conditions on the relative stability of the MVM capsid against thermal dissociation. 3. Insertion of heterologous epitopes in four solvent-exposed loops at the MVM capsid surface led to senous defects in capsid assembly en4 in most cases, to a reduction in capsid stability, even though the insertions were made in pooitions that are, presumably, the most tolerant from a structural point of view. Two out of nine conservative point replacements of residues located within surface loops, and involved in very few or no interactions, led also to defe& in capsid assembly. 4. The region surrounding an ensemble of conspicuous cavities within the hydrophobic core of each MVM capsid subunit is able to accept the introduction of as many as three extra methylenes or equivalent-sized groups, or the removal of as many as fow groups, without any signiscant effect on capsid assembly or stability against thermal dissociation. Assembly was impaired only when fow or more groups were introduced, or six or more groups removed 5. Such shucairal tolerance to mutation around cavities within the protein core may no1 imply a similar functional tolerance. A combinatorial mutagenesis approach suggests that MVM requires, for optimum biological fítness, the presence of very specific residues that preserve the size and shape of the largest cavities within the core of each subunit This obsmration may explain the high degree of conservation of those residues in MVM and evolutionarily related pawoviruses. 6. Assembly of the MVM capsid requires aiso the presence of the negative charge of a carboxylate buried within the largest caviiy in the protein core, and speciñcaily located at position 115. Viral DNA encapsidation specifically requires, in addition, the presence of aspartate, and not glutamate, at position 115. This justifies the absolute conservation of AspllS in MVM and other parvoviruses.