Abstract
The envelope glycoprotein, gp160, of simian immuno-deficiency virus (SIV) shares ~25% sequence identity with gp160 from the human immunodeficiency 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 ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 34946-34953 |
| Number of pages | 8 |
| Journal | Journal of Biological Chemistry |
| Volume | 275 |
| Issue number | 45 |
| DOIs | |
| State | Published - Nov 10 2000 |
| Externally published | Yes |
Fingerprint
ASJC Scopus subject areas
- Biochemistry
Cite this
Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160. / Chen, Bing; Zhou, Genfa; Kim, Mikyung; Chishti, Yasmin; Hussey, Rebecca E.; Ely, Barry; Skehel, John J.; Reinherz, Ellis L.; Harrison, Stephen C.; Wiley, Don C.
In: Journal of Biological Chemistry, Vol. 275, No. 45, 10.11.2000, p. 34946-34953.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160
AU - Chen, Bing
AU - Zhou, Genfa
AU - Kim, Mikyung
AU - Chishti, Yasmin
AU - Hussey, Rebecca E.
AU - Ely, Barry
AU - Skehel, John J.
AU - Reinherz, Ellis L.
AU - Harrison, Stephen C.
AU - Wiley, Don C.
PY - 2000/11/10
Y1 - 2000/11/10
N2 - The envelope glycoprotein, gp160, of simian immuno-deficiency virus (SIV) shares ~25% sequence identity with gp160 from the human immunodeficiency 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 ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.
AB - The envelope glycoprotein, gp160, of simian immuno-deficiency virus (SIV) shares ~25% sequence identity with gp160 from the human immunodeficiency 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 ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.
UR - http://www.scopus.com/inward/record.url?scp=0034634548&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0034634548&partnerID=8YFLogxK
U2 - 10.1074/jbc.M004905200
DO - 10.1074/jbc.M004905200
M3 - Article
C2 - 10944528
AN - SCOPUS:0034634548
VL - 275
SP - 34946
EP - 34953
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 45
ER -