We propose to prepare and purify trimeric, HIV-1 gp140 envelope glycoproteins in sufficient quantity for determination of their structure by X-ray crystallography. The research outlined in this proposal will be performed as a collaboration between John Moore's group at the Weill Medical College of Cornell University, and lan Wilson's group at the Scripps Research Institute; the areas of expertise of these groups in, respectively, envelope glycoprotein biology and structural studies are complementary. The Env complex of HIV-1 is the principal focus of neutralizing antibody-based vaccines. The native Env complex is trimeric; its six individual subunits (3 gp120's, 3 gp41's) are held in association by relatively weak, non-covalent interactions. Although some structural information on the individual gp120 and gp41 subunits is available, knowledge of the structure of the trimeric complex is lacking. This is, not least, because stable, cleaved trimers have not been available for analysis, due to the instability of their inter-subunit associations. We have now made a soluble Env protein, SOS gp140, that has gp120 stably linked to the gp41 ectodomain by an intermolecular disulfide bond. This protein is fully cleaved at the proteolysis site between gp120 and gp41 but the weakness of gp41-gp41 interactions cause it to dissociate into monomers. A variant SOS gp140 protein, designated SOSIP gp140, contains an isoleucine-to-proline substitution at position 559 in the N-terminal heptad repeat region of gp41 (SOS 1559P, or SOSIP gp140). This protein is fully cleaved, has favorable antigenic properties and is predominantly trimeric. The SOSIP gp140 trimers are non-covalently associated, can be purified by gel-filtration chromatography, dissociate into monomers after exposure to anionic detergent or mild heat, but are relatively resistant to non-ionic detergents, high salt or exposure to mildly acidic pH. They are stable at 37oC for over a week. We propose two Specific Aims: To purify the trimeric SOSIP gp140 complex or variants thereof that might be more amenable to crystallization; to crystallize the Env trimer, alone or as a CD4- or MAb-complex, then analyze its structure. Any information accruing from the structure of a native Env trimer could facilitate the rational design of Env-based vaccines.
| Kirschner, Marc; Monrose, Val; Paluch, Maciej et al. (2006) The production of cleaved, trimeric human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein vaccine antigens and infectious pseudoviruses using linear polyethylenimine as a transfection reagent. Protein Expr Purif 48:61-8 |
| Sanders, Rogier W; Busser, Els; Moore, John P et al. (2004) Evolutionary repair of HIV type 1 gp41 with a kink in the N-terminal helix leads to restoration of the six-helix bundle structure. AIDS Res Hum Retroviruses 20:742-9 |
| Sanders, Rogier W; Dankers, Martijn M; Busser, Els et al. (2004) Evolution of the HIV-1 envelope glycoproteins with a disulfide bond between gp120 and gp41. Retrovirology 1:3 |
