Characterization of a putative alpha-helix across the capsid-SP1 boundary that is critical for the multimerization of human immunodeficiency virus type 1 gag.
A 14-amino-acid spacer peptide termed SP1 that separates the capsid (CA) and nucleocapsid (NC) sequences plays an active role in the assembly of human immunodeficiency virus type 1. This activity of SP1 involves its amino-terminal residues that, together with adjacent CA residues, constitute a putative alpha-helical structure spanning Gag residues from positions 359 to 371. In this study, we have determined that the virus assembly determinants within this putative alpha-helix were residues H359, K360, A361, L364, A367, and M368, of which K360 and A367 contribute to virus production to lesser extents. Notably, changes of the two basic amino acids H359 and K360 to arginine (R) impaired virus production, whereas mutations L364I and M368I, in contrast to L364A and M368A, generated near-wild-type levels of virus particles. This suggests that within Gag complexes, amino acids H359 and K360 are involved in stricter steric interactions than L364 and M368. Since L364 and M368 are separated by four residues and thus presumably located on the same side of the helical surface, they may initiate synergistic hydrophobic interactions to stabilize Gag association. Further analysis in the context of the protease-negative mutation D185H confirmed the key roles of amino acids H359, A361, L364, and M368 in virus assembly. Importantly, when transfected cells were subjected to Dounce homogenization and the cell lysates were treated by ultracentrifugation at 100,000 x g, Gag molecules containing each of the H359A, A361V, L364A, and M368A mutations were found mainly in the supernatant fraction (S100), whereas approximately 80% of wild-type Gag proteins were found in the pellet. Therefore, these four mutations must have prevented Gag from generating large complexes.
Liang
Chen
C
McGill AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2. chen.liang@mcgill.ca
Hu
Jing
J
Russell
Rodney S
RS
Roldan
Ariel
A
Kleiman
Lawrence
L
Wainberg
Mark A
MA
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002213:Capsid / Q000737:chemistry*; D019281:Dimerization; D015967:Gene Expression Regulation, Viral; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism* / Q000472:pathogenicity; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D017433:Protein Structure, Secondary; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly
D015683:Gene Products, gag; D010455:Peptides