title: Biological properties of recombinant HIV envelope synthesized in CHO glycosylation-mutant cell lines.
abstract: N-glycosylation of the human immunodeficiency virus type-1 envelope (Env) glycoprotein precursor (gp160) occurs by transfer of Glc3Man9GlcNAc2 onto the nascent protein. Maturation then occurs via cleavage of the three Glc residues, which starts during translation. These events are considered necessary to create Env functional conformation: treatment with "alpha"-glucosidase inhibitors, but not alpha-mannosidase inhibitors (i) impairs gp160 cleavage into gp120 and gp41, (ii) diminishes the accessibility of gp120 V3 region, (iii) prevents gp120 binding to its CD4 receptor, and (iv) prevents gp41-mediated membrane fusion. These inhibitors are of therapeutic interest. Here, using a collection of parent and mutant CHO cells that possess mutations in different steps of glycosylation, we reassessed the role of glycans in both the processing and the properties of recombinant gp160 expressed from a vaccinia virus vector. Mutant cells were as follows: Lec23 (which lacks alpha-glucosidase I activity) produces a collection of triglucosylated structures (Glc3Man7-9GlcNAc2); LEC10 (which has increased GlcNAc transferase III activity) produces complex glycans with a bisected GlcNAc residue; Lec1 (which lacks GlcNAc transferase I) and Lec3.2.8.1 (which lacks GlcNAc transferase I and has decreased activity of CMP-NeuNAc and UDP-Gal translocases) produce Man5GlcNAc2 glycans at complex or hybrid sites. As expected, glycosylation of Env produced from mutants was affected but, irrespective of the glycosylation phenotype, (i) similar quantities of Env were synthesized, (ii) the immunoreactivity of V3 was similar, (iii) gp160 was efficiently cleaved into gp120 and gp41, (vi) Env was exposed at the cell membrane, (v) secreted gp120 bound CD4, and (vi) membrane gp41 was able to induce membrane fusion with CD4+ cells. Thus, the glycosylation alterations examined are dispensable for Env processing and biological activity in CHO cells. In particular, removal of the three outer Glc residues was not required per se for Env folding in this system because functional Env is obtained from Lec23 cells: it appears therefore that lack of modification is not equivalent to drug inhibition of modification. These data are discussed in the light of previous reports describing the use of glycosidase inhibitors to alter glycosylation.
authors.affiliation: CNRS URA 1455, Faculté de Médecine Nord, Marseille, 13015, France.
authors.forename: E
authors.initials: E
authors.lastname: Fenouillet
authors.forename: R
authors.initials: R
authors.lastname: Miquelis
authors.forename: R
authors.initials: R
authors.lastname: Drillien
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000276:immunology; D019302:HIV Envelope Protein gp160; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D008561:Membrane Fusion; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D014616:Vaccinia virus
chemical_list: D015704:CD4 Antigens; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; C059762:HIV envelope protein gp120 (305-321); D010446:Peptide Fragments; D011498:Protein Precursors; D011994:Recombinant Proteins