Characterization of Large Conformational Changes and Autoproteolysis in the Maturation of a T=4 Virus Capsid

Abstract

ABSTRACT Nudaurelia capensis ω virus -like particles have been characterized as a 480-Å procapsid and a 410-Å capsid, both with T=4 quasisymmetry. Procapsids transition to capsids when pH is lowered from 7.6 to 5.0. Capsids undergo autoproteolysis at residue 570, generating the 74-residue C-terminal polypeptide that remains with the particle. Here we show that the particle size becomes smaller under conditions between pH 6.8 and 6.0 without activating cleavage and that the particle remains at an intermediate size when the pH is carefully maintained. At pH 5.8, cleavage is very slow, becoming detectable only after 9 h. The optimum pH for cleavage is 5.0 (half-life, ∼30 min), with a significant reduction in the cleavage rate at pH values below 5. We also show that lowering the pH is required only to make the virus particles compact and to presumably form the active site for autoproteolysis but not for the chemistry of cleavage. The cleavage reaction proceeds at pH 7.0 after ∼10% of the subunits cleave at pH 5.0. Employing the virion crystal structure for reference, we investigated the role of electrostatic repulsion of acidic residues in the pH-dependent large conformational changes. Three mutations of Glu to Gln that formed procapsids showed three different phenotypes on maturation. One, close to the threefold and quasithreefold symmetry axes and far from the cleavage site, did not mature at pH 5, and electron cryomicroscopy reconstruction showed that it was intermediate in size between those of the procapsid and capsid; one near the cleavage site exhibited a wild-type phenotype; and a third, far from the cleavage site, resulted in cleavage of 50% of the subunits after 4 h, suggesting quasiequivalent specificity of the mutation.