STRUCTURAL INTERACTIONS BETWEEN RETROVIRAL GAG PROTEINS EXAMINED BY CYSTEINE CROSS-LINKING

We have examined structural interactions between Gag proteins within M oloney murine leukemia virus (M-MuLV) particles by making use of the c ysteine-specific cross-linking agents iodine and bis-maleimido hexane. Virion-associated wild-type M-MuLV Pr65(Gag) proteins in immature par ticles were intermolecularly cross-linked at cysteines to form pr65(Ga g) oligomers, from dimers to pentamers or hexamers. Following a system atic approach of cysteine-to-serine mutagenesis, we have shown that cr oss-linking of pr65(Gag) occurred at cysteines of the nucleocapsid (NC ) Cys-His motif, suggesting that the Cys-His motifs within virus parti cles are packed in close proximity. The M-MuLV pr65(Gag) protein did n ot cross-link to the human immunodeficiency virus pr55(Gag) protein wh en the two molecules were coexpressed, indicating either that they did not coassemble or that heterologous Gag proteins were not in close en ough proximity to be cross linked. Using an assembly-competent, protea se-minus, cysteine-minus pr65(Gag) protein as a template, novel cystei ne residues were generated in the M-MuLV capsid domain major homology region (MHR). Cross-linking of proteins containing MHR cysteines showe d above-background levels of Gag Gag dimers but also identified a nove l cellular factor, present in virions, that cross-linked to MHR residu es. Although the NC cysteine mutation was compatible with M-MuLV parti cle assembly, deletions of the NC domain were not tolerated. These res ults suggest that the Cys-His motif is held in close proximity within immature M-MuLV particles by interactions between CA domains and/or no n-Cys-His motif domains of the NC.