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 Gap precursor protein (Gag) expressed in baculovirus-infected cells and ultrastructural analysis of membrane-enveloped Gag particles under the electron microscope. Five discrete regions were found to influence the size, the variability 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)-SP2, and (v) the p6gag C-terminus. Internal regions (ii), (iii), and (iv) contained HIV-1 protease cleavage sites separating major structural domains. No particle assembly was observed for am276, a MA-CA polyprotein mutant lacking the C-terminal third of the CA domain. However, MA-CA domains including the MHR (residues 277-306), or downstream sequence to CA residue 357, resulted in the assembly into tubular or filamentous structures, suggesting a helical symmetry of Gag packing. Mutant amb374, derived from amb 357 by further addition of the heptadecapeptide motif HKARVLAEAMSQVTNSA, overlapping the CA-SP1 junction and the SP1 domain, showed a drastic change in the pattern of Gag assembly, compared to amb357, with formation of spherical particles. These data suggested a novel function for the spacer domain SP1, acting as a spherical shape determinant of the Gag particle which would negatively affect the helical symmetry of assembly of the Gag precursor molecules conferred by the MHR and the downstream CA sequence, within residues 307-357.
Gay
B
B
Laboratoire de Virologie et Pathogénèse Moléculaire (CNRS UMR 5812), Faculté de Médecine, Montpellier, France.
Tournier
J
J
Chazal
N
N
Carrière
C
C
Boulanger
P
P
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D002460:Cell Line; D015683:Gene Products, gag / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D015497:HIV-1 / Q000502:physiology* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D018411:Spodoptera
D015683:Gene Products, gag; D011498:Protein Precursors; C059767:p55 gag precursor protein, Human immunodeficiency virus 1