Modifications that stabilize human immunodeficiency virus envelope glycoprotein trimers in solution

The functional unit of the human immunodeficiency virus type 1 (HIV-1) enve lope glycoproteins is a trimer composed of three gp120 exterior glycoprotei ns and three gp41 transmembrane glycoproteins. The lability of intersubunit interactions has hindered the production and characterization of soluble, homogeneous envelope glycoprotein trimers. Here we report three modificatio ns that stabilize soluble forms of HIV-1 envelope glycoprotein trimers: dis ruption of the proteolytic cleavage site between gp120 and gp41, introducti on of cysteines that form intersubunit disulfide bonds, and addition of GCN 4 trimeric helices. Characterization of these secreted glycoproteins by imm unologic and biophysical methods indicates that these stable trimers retain structural integrity. The efficacy of the GCN4 sequences in stabilizing th e trimers, the formation of intersubunit disulfide bonds between appropriat ely placed cysteines, and the ability of the trimers to interact with a hel ical, C-terminal gp41 peptide (DP178) support a model in which the N-termin al gp41 coiled coil exists in the envelope glycoprotein precursor and contr ibutes to intersubunit interactions within the trimer. The availability of stable, soluble HIV-1 envelope glycoprotein trimers should expedite progres s in understanding the structure and function of the virion envelope glycop rotein spikes.