title: Enzyme digests eliminate nonfunctional Env from HIV-1 particle surfaces, leaving native Env trimers intact and viral infectivity unaffected.
abstract: HIV-1 viruses and virus-like particles (VLPs) bear nonnative "junk" forms of envelope (Env) glycoprotein that may undermine the development of antibody responses against functional gp120/gp41 trimers, thereby blunting the ability of particles to elicit neutralizing antibodies. Here, we sought to better understand the nature of junk Env with a view to devising strategies for its removal. Initial studies revealed that native trimers were surprisingly stable in the face of harsh conditions, suggesting that junk Env is unlikely to arise by trimer dissociation or gp120 shedding. Furthermore, the limited gp120 shedding that occurs immediately after synthesis of primary HIV-1 isolate Envs is not caused by aberrant cleavage at the tandem gp120/gp41 cleavage sites, which were found to cleave in a codependent manner. A major VLP contaminant was found to consist of an early, monomeric form of gp160 that is glycosylated in the endoplasmic reticulum (gp160ER) and then bypasses protein maturation and traffics directly into particles. gp160ER was found to bind two copies of monoclonal antibody (MAb) 2G12, consistent with its exclusively high-mannose glycan profile. These findings prompted us to evaluate enzyme digests as a way to remove aberrant Env. Remarkably, sequential glycosidase-protease digests led to a complete or near-complete removal of junk Env from many viral strains, leaving trimers and viral infectivity largely intact. "Trimer VLPs" may be useful neutralizing antibody immunogens.
authors.affiliation: Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA.
authors.forename: Ema T
authors.initials: ET
authors.lastname: Crooks
authors.forename: Tommy
authors.initials: T
authors.lastname: Tong
authors.forename: Keiko
authors.initials: K
authors.lastname: Osawa
authors.forename: James M
authors.initials: JM
authors.lastname: Binley
mesh_terms: D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D000080908:Broadly Neutralizing Antibodies; D002460:Cell Line; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D006031:Glycosylation; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism; D019302:HIV Envelope Protein gp160 / Q000378:metabolism; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D006801:Humans; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D014771:Virion / Q000472:pathogenicity*; D054299:env Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*
chemical_list: C529157:2G12 monoclonal antibody; D000911:Antibodies, Monoclonal; D000080908:Broadly Neutralizing Antibodies; D015483:HIV Antibodies; D015699:HIV Envelope Protein gp120; D019302:HIV Envelope Protein gp160; D015700:HIV Envelope Protein gp41; D054299:env Gene Products, Human Immunodeficiency Virus; D006026:Glycoside Hydrolases; D010447:Peptide Hydrolases