MORPHOPOIETIC DETERMINANTS OF HIV-1 GAG PARTICLES ASSEMBLED IN BACULOVIRUS-INFECTED CELLS

The determinants for HIV-1 particle morphology were investigated using various deletion and insertion mutants of the Gag precursor protein ( Gag) expressed in baculovirus-infected cells and ultrastructural analy sis of membrane-enveloped Gag particles under the electron microscope. Five discrete regions were found to influence the size, the variabili ty in dimension, and the sphericity of the particles: (i) the matrix ( MA) N-terminal domain, within residues 10-21, the junctions of (ii) MA -CA (capsid), (iii) CA-spacer peptide SP1 and (iv) nucleocapsid (NC)-S P2, and (v) the p6(gag) C-terminus. Internal regions (ii), (iii), and (iv) contained HIV-1 protease cleavage sites separating major structur al domains. No particle assembly was observed for amb276, a MA-CA poly protein mutant lacking the C-terminal third of the CA domain. However, MA-CA domains including the MHR (residues 277-306), or downstream seq uence to CA residue 357 resulted in the assembly into tubular or filam entous structures, suggesting a helical symmetry of Gag packing. Mutan t amb374, derived from amb357 by further addition of the heptadecapept ide motif (358)HKARVLAEAMSQVTNSA(374), Overlapping the CA-SP1 junction and the SP1 domain, showed a drastic change in the pattern of Gag ass embly, compared to amb357, with formation of spherical particles. Thes e data suggested a novel function for the spacer domain SP1, acting as a spherical shape determinant of the Gag particle which would negativ ely affect the helical symmetry of assembly of the Gag precursor molec ules conferred by the MHR and the downstream CA sequence, within resid ues 307-357. (C) 1998 Academic Press.