IN-VITRO ASSEMBLY OF VIRUS-LIKE PARTICLES WITH ROUS-SARCOMA VIRUS GAGDELETION MUTANTS - IDENTIFICATION OF THE P10 DOMAIN AS A MORPHOLOGICAL DETERMINANT IN THE FORMATION OF SPHERICAL-PARTICLES

Retroviruses are unusual in that expression of a single protein, Gag, leads to budding of virus-like particles into the extracellular space, We have developed conditions under which virus-like particles are for med spontaneously in vitro from fragments of Rous sarcoma virus (RSV) Gag protein purified after expression in Escherichia coli. The CA-NC f ragment of Gag was shown previously to assemble into hollow cylinders (S. Campbell and V, M. Vogt, J, Virol, 69:6487-6497, 1995). We have no w extended these studies to larger Gag proteins. In every case examine d, assembly into regular structures required RNA, A nearly full-length Gag missing only the C-terminal PR domain, as well as similar protein s missing in addition the N-terminal half of MA, the C-terminal half o f MA, the entire MA sequence, or the entire p2 sequence, all assembled into spherical particles resembling RSV in size, By contrast, protein s missing p10 assembled into cylindrical particles like those formed b y CA-NC alone, Thin section electron microscopy showed that each of th ese Gag proteins formed in the expressing E. coil cells particles simi lar in shape to those seen in vitro, We conclude from these results th at neither the sequences required for membrane binding in vivo, near t he N terminus of Gag, nor the sequences required for a late step in bu dding, in the p2 portion of Gag, are essential for formation of virus- like particles in this system. Furthermore, we postulate the existence of a shape-determining sequence in p10, which provides or facilitates interactions required for the growing particle to be constrained to a spherical shape.