RECONSTITUTION OF FLOCK HOUSE PROVIRIONS - A MODEL SYSTEM FOR STUDYING STRUCTURE AND ASSEMBLY

Assembly of Flock Douse virus in infected Drosophila cells proceeds th rough an intermediate, the provirion, which lacks infectivity until th e coat precursor protein, alpha, undergoes a spontaneous ''maturation' ' cleavage (A. Schneemann, W. Zhong, T. M. Gallagher, and R. R. Ruecke rt, J. Virol. 66:6728, 1992). We describe here methods for purifying p rovirions in a state which permitted dissociation and reassembly. Diss ociation, to monomeric alpha protein and free RNA, was accomplished by freezing at pK 9.0 in the presence of 0.5 M salt and 0.1 M urea. When dialyzed at low ionic strength and pH 6.5, the dissociation products reassembled spontaneously to form homogeneous provirions with a normal complement of RNA as judged by cosedimentation with authentic virions and by ability to undergo maturation cleavage with acquisition of sub stantial, though subnormal, infectivity. Reconstitution experiments, i .e., remixing components after separating RNA from capsid protein, gen erated abnormal particles, suggesting the presence in the unfractionat ed dissociation products of an unidentified ''nucleating'' component.