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 immu nodeficiency virus type I (HIV-1) particles by utilizing cysteine muta genesis and cysteine-specific modifying reagents. In immature protease -minus but otherwise wild type (wt) particles, precursor pr55(Gag) pro teins did not form intermolecular cystines naturally but could be cros s-linked at cysteines, and cross-linking appeared to occur across nucl eocapsid (NC) domains, Capsid (CA) proteins in wt mature viruses posse ss cysteines near their carboxy termini at gag codons 330 and 350, but these residues are not involved in natural covalent intermolecular bo nds, nor can they be intermolecularly cross-linked by using the membra ne-permeable cross-linker bis-maleimido hexane. The cysteine at gag co don 350 (C-350) is highly reactive to thiol-specific modifying reagent s, while the one at codon 330 (C-330) appears considerably less reacti ve, even in the presence of ionic detergent. These results suggest tha t the HIV-1 CA C terminus forms an unusually stable conformation, Muta genesis of C-350 to a serine residue in the mutant C350S (C-350 change d 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 dom ain or in the C-terminal section of the major homology region. All suc h mutants appeared wt on the basis of biochemical assays but showed gr eatly reduced infectivities, indicative of a postassembly, postprocess ing replicative block. Interestingly, capsid proteins of mature major homology region mutant particles could be cysteine cross-linked, imply ing either that these mutations permit cross-linking of the native C t erminal CA cysteines or that major homology regions on neighbor capsid proteins are in close proximity in mature virions.