Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents.
We have examined structural interactions of Gag proteins in human immunodeficiency virus type 1 (HIV-1) particles by utilizing cysteine mutagenesis and cysteine-specific modifying reagents. In immature protease-minus but otherwise wild-type (wt) particles, precursor Pr55Gag proteins did not form intermolecular cystines naturally but could be cross-linked at cysteines, and cross-linking appeared to occur across nucleocapsid (NC) domains. Capsid (CA) proteins in wt mature viruses possess cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bonds, nor can they be intermolecularly cross-linked by using the membrane-permeable cross-linker bis-maleimido hexane. The cysteine at gag codon 350 (C-350) is highly reactive to thiol-specific modifying reagents, while the one at codon 330 (C-330) appears considerably less reactive, even in the presence of ionic detergent. These results suggest that the HIV-1 CA C terminus forms an unusually stable conformation. Mutagenesis of C-350 to a serine residue in the mutant C350S (C-350 changed to serine) virtually eliminated particle assembly, attesting to the importance of this region. We also examined a C330S mutant, as well as mutants in which cysteines were created midway through the capsid domain or in the C-terminal section of the major homology region. All such mutants appeared wt on the basis of biochemical assays but showed greatly reduced infectivities, indicative of a postassembly, postprocessing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, implying either that these mutations permit cross-linking of the native C-terminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.
McDermott
J
J
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201-3098, USA.
Farrell
L
L
Ross
R
R
Barklis
E
E
D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D004274:DNA, Recombinant; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics; D015497:HIV-1; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation
D004274:DNA, Recombinant; D015683:Gene Products, gag; D003545:Cysteine