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

Bing Chen, Genfa Zhou, Mikyung Kim, Yasmin Chishti, Rebecca E. Hussey, Barry Ely, John J. Skehel, Ellis L. Reinherz, Stephen C. Harrison, Don C. Wiley

Research output: Contribution to journalArticle

30 Citations (Scopus)

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 languageEnglish (US)
Pages (from-to)34946-34953
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number45
DOIs
StatePublished - Nov 10 2000
Externally publishedYes

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Simian Immunodeficiency Virus
Viruses
Purification
Glycoproteins
HIV
Virus Internalization
CD4 Antigens
Immunoglobulin Fab Fragments
Ultracentrifugation
Cricetulus
Neutralizing Antibodies
Oligosaccharides
Gel Chromatography
Insects
Ovary
Monoclonal Antibodies
Chromatography
Enzymes
Fusion reactions
Gels

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 journalArticle

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. / Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 45. pp. 34946-34953.
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