MUTATION-DIRECTED CHEMICAL CROSS-LINKING OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 GP41 OLIGOMERS

The human immunodeficiency virus type 1 transmembrane protein gp41 oli gomer anchors the attachment protein, gp120, to the viral envelope and mediates viral envelope-cell membrane fusion following gp120-CD4 rece ptor-chemokine coreceptor binding. We have used mutation-directed chem ical cross-linking with bis(sulfosuccinimidyl) suberate (BS3) to inves tigate the architecture of the gp41 oligomer. Treatment of gp41 with B S3 generates a ladder of four bands on sodium dodecyl sulfate-polyacry lamide gels, corresponding to monomers, dimers, trimers, and tetramers . By systematically replacing gp41 lysines with arginine and determini ng the mutant gp41 cross-linking pattern, we observed that gp41 N term ini are cross-linked. Lysine 678, which is close to the transmembrane sequence, was readily cross-linked to Lys-678 on other monomers within the oligomeric structure. This arrangement appears to be facilitated by the close packing of membrane-anchoring sequences, since the effici ency of assembly of heterooligomers between wild-type and mutant Env p roteins is improved more than twofold if the mutant contains the membr ane-anchoring sequence. We also detected close contacts between Lys-59 6 and Lys-612 in the disulfide-bonded loop/glycan cluster of one monom er and lysines in the N-terminal amphipathic alpha-helical oligomeriza tion domain (Lys-569 and Lys-583) and C-terminal alpha-helical sequenc e (Lys-650 and Lys-660) of adjacent monomers. Precursor-processing eff iciency, gp120-gp41 association, soluble recombinant CD4-induced shedd ing of gp120 from cell surface gp41 and acquisition of gp41 ectodomain conformational antibody epitopes were unaffected by the substitutions . However, the syncytium-forming function was most dependent on the co nserved Lys-569 in the N-terminal alpha-helix. These results indicate that gp160-derived gp41 expressed in mammalian cells is a tetramer and provide information about the justaposition of gp41 structural elemen ts within the oligomer.