Expression, purification, and characterization of gp160e, the soluble trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein,gp160

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) sh ares similar to 25% sequence identity with gp160 from the human immunodefic iency virus, type I,, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodom ain of SIV gp160. The ectodomain has been expressed in both insect cells an d Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necess ary for synthesizing complex oligosaccharides. Both the primary and a secon dary proteolytic cleavage sites between the gp120 and gp41 subunits of gp16 0 were mutated to prevent cleavage and shedding of gp120. The purified, sol uble glycoprotein is shown to be trimeric by chemical cross-linking, gel fi ltration chromatography, and analytical ultracentrifugation. It forms solub le, tight complexes with soluble CD4 and a number of Fab fragments from neu tralizing monoclonal antibodies. Soluble complexes were also produced of en zymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.