title: A New Approach to Produce HIV-1 Envelope Trimers: BOTH CLEAVAGE AND PROPER GLYCOSYLATION ARE ESSENTIAL TO GENERATE AUTHENTIC TRIMERS.
abstract: The trimeric envelope spike of HIV-1 mediates virus entry into human cells. The exposed part of the trimer, gp140, consists of two noncovalently associated subunits, gp120 and gp41 ectodomain. A recombinant vaccine that mimics the native trimer might elicit entry-blocking antibodies and prevent virus infection. However, preparation of authentic HIV-1 trimers has been challenging. Recently, an affinity column containing the broadly neutralizing antibody 2G12 has been used to capture recombinant gp140 and prepare trimers from clade A BG505 that naturally produces stable trimers. However, this antibody-based approach may not be as effective for the diverse HIV-1 strains with different epitope signatures. Here, we report a new and simple approach to produce HIV-1 envelope trimers. The C terminus of gp140 was attached to Strep-tag II with a long linker separating the tag from the massive trimer base and glycan shield. This allowed capture of nearly homogeneous gp140 directly from the culture medium. Cleaved, uncleaved, and fully or partially glycosylated trimers from different clade viruses were produced. Extensive biochemical characterizations showed that cleavage of gp140 was not essential for trimerization, but it triggered a conformational change that channels trimers into correct glycosylation pathways, generating compact three-blade propeller-shaped trimers. Uncleaved trimers entered aberrant pathways, resulting in hyperglycosylation, nonspecific cross-linking, and conformational heterogeneity. Even the cleaved trimers showed microheterogeneity in gp41 glycosylation. These studies established a broadly applicable HIV-1 trimer production system as well as generating new insights into their assembly and maturation that collectively bear on the HIV-1 vaccine design.
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Wadad
authors.initials: W
authors.lastname: AlSalmi
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Marthandan
authors.initials: M
authors.lastname: Mahalingam
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Neeti
authors.initials: N
authors.lastname: Ananthaswamy
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Christopher
authors.initials: C
authors.lastname: Hamlin
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Dalia
authors.initials: D
authors.lastname: Flores
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064.
authors.forename: Guofen
authors.initials: G
authors.lastname: Gao
authors.affiliation: From the Department of Biology, The Catholic University of America, Washington, D. C. 20064 rao@cua.edu.
authors.forename: Venigalla B
authors.initials: VB
authors.lastname: Rao
mesh_terms: D000595:Amino Acid Sequence; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000956:Antigens, Viral / Q000032:analysis* / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006031:Glycosylation; D015699:HIV Envelope Protein gp120 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D000906:Antibodies; D000956:Antigens, Viral; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C583458:Trp-Ser- His-Pro-Gln-Phe-Glu-Lys; D054299:env Gene Products, Human Immunodeficiency Virus; C514793:gp120 protein, Human immunodeficiency virus 1; C097729:gp140 envelope protein, Human immunodeficiency virus 1; C514787:gp41 protein, Human immunodeficiency virus 1
keywords: AIDS; envelope protein; glycosylation; gp140 trimer; human immunodeficiency virus (HIV); recombinant protein expression; vaccine