title: Oligomeric and conformational properties of a proteolytically mature, disulfide-stabilized human immunodeficiency virus type 1 gp140 envelope glycoprotein.
abstract: We describe the further properties of a protein, designated SOS gp140, wherein the association of the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein is stabilized by an intersubunit disulfide bond. HIV-1(JR-FL) SOS gp140, proteolytically uncleaved gp140 (gp140(UNC)), and gp120 were expressed in stably transfected Chinese hamster ovary cells and analyzed for antigenic and structural properties before and after purification. Compared with gp140(UNC), SOS gp140 reacted more strongly in surface plasmon resonance and radioimmunoprecipitation assays with the neutralizing monoclonal antibodies (MAbs) 2G12 (anti-gp120), 2F5 (anti-gp41), and 17b (to a CD4-induced epitope that overlaps the CCR5-binding site). In contrast, gp140(UNC) displayed the greater reactivity with nonneutralizing anti-gp120 and anti-gp41 MAbs. Immunoelectron microscopy studies suggested a model for SOS gp140 wherein the gp41 ectodomain (gp41(ECTO)) occludes the "nonneutralizing" face of gp120, consistent with the antigenic properties of this protein. We also report the application of Blue Native polyacrylamide gel electrophoresis (BN-PAGE), a high-resolution molecular sizing method, to the study of viral envelope proteins. BN-PAGE and other biophysical studies demonstrated that SOS gp140 was monomeric, whereas gp140(UNC) comprised a mixture of noncovalently associated and disulfide-linked dimers, trimers, and tetramers. The oligomeric and conformational properties of SOS gp140 and gp140(UNC) were largely unaffected by purification. An uncleaved gp140 protein containing the SOS cysteine mutations (SOS gp140(UNC)) was also oligomeric. Surprisingly, variable-loop-deleted SOS gp140 proteins were expressed (although not yet purified) as cleaved, noncovalently associated oligomers that were significantly more stable than the full-length protein. Overall, our findings have relevance for rational vaccine design.
authors.affiliation: Progenics Pharmaceuticals Inc., Tarrytown, New York 10591, USA.
authors.forename: Norbert
authors.initials: N
authors.lastname: Schülke
authors.forename: Mika S
authors.initials: MS
authors.lastname: Vesanen
authors.forename: Rogier W
authors.initials: RW
authors.lastname: Sanders
authors.forename: Ping
authors.initials: P
authors.lastname: Zhu
authors.forename: Min
authors.initials: M
authors.lastname: Lu
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Anselma
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Villa
authors.forename: Paul W H I
authors.initials: PW
authors.lastname: Parren
authors.forename: James M
authors.initials: JM
authors.lastname: Binley
authors.forename: Kenneth H
authors.initials: KH
authors.lastname: Roux
authors.forename: Paul J
authors.initials: PJ
authors.lastname: Maddon
authors.forename: John P
authors.initials: JP
authors.lastname: Moore
authors.forename: William C
authors.initials: WC
authors.lastname: Olson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron; D008958:Models, Molecular; D011487:Protein Conformation; D015531:Radioimmunoprecipitation Assay; D020349:Surface Plasmon Resonance; D014162:Transfection; D054299:env Gene Products, Human Immunodeficiency Virus
chemical_list: D000911:Antibodies, Monoclonal; D004220:Disulfides; D015686:Gene Products, env; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D054299:env Gene Products, Human Immunodeficiency Virus; C097729:gp140 envelope protein, Human immunodeficiency virus 1