title: Guidelines for cloning, expression, purification and functional characterization of primary HIV-1 envelope glycoproteins.
abstract: The trimeric HIV-1 envelope (Env) glycoproteins gp120 and gp41 mediate virus entry into target cells by engaging CD4 and the coreceptors CCR5 or CXCR4 at the cell surface and driving membrane fusion. Receptor/gp120 interactions regulate the virus life cycle, HIV infection transmission and pathogenesis. Env is also the target of neutralizing antibodies. Efforts have thus been made to produce soluble HIV-1 glycoproteins to develop vaccines and study the role and mechanisms of HIV/receptor interactions. However, production and purification of Env glycoproteins and their functional assessment has to cope with multiple obstacles. These include difficulties in amplifying and cloning env sequences and setting up receptor binding assays that are suitable for studies on large collections of glycoproteins, flexible enough to adapt to Env and receptor structural heterogeneities, and allow recapitulating the receptor binding properties of virion-associated Env trimers. Here we identify these difficulties and present protocols to produce primary gp120 and determination of their binding properties to receptors. The receptor binding assays confirmed that the produced glycoproteins are competent for binding CD4 and undergo proper CD4-induced conformational changes required for interaction with CCR5. These assays may help elucidate the role of gp120/receptor interactions in the pathophysiology of HIV infection and develop HIV-1 entry inhibitors.
authors.affiliation: INSERM U1108, Institut Pasteur, 75015 Paris, France; Viral Pathogenesis Unit, Department of Virology, Institut Pasteur, 75015 Paris, France. Electronic address: yann.benureau@free.fr.
authors.forename: Yann
authors.initials: Y
authors.lastname: Benureau
authors.affiliation: INSERM U1108, Institut Pasteur, 75015 Paris, France; Viral Pathogenesis Unit, Department of Virology, Institut Pasteur, 75015 Paris, France. Electronic address: philippe.colin1@gmail.com.
authors.forename: Philippe
authors.initials: P
authors.lastname: Colin
authors.affiliation: INSERM U1108, Institut Pasteur, 75015 Paris, France; Viral Pathogenesis Unit, Department of Virology, Institut Pasteur, 75015 Paris, France. Electronic address: isabelle.staropoli@pasteur.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Staropoli
authors.affiliation: AIDS Immunopathogenesis Unit, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain. Electronic address: nglez@isciii.es.
authors.forename: Nuria
authors.initials: N
authors.lastname: Gonzalez
authors.affiliation: AIDS Immunopathogenesis Unit, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain. Electronic address: eoaz@isciii.es.
authors.forename: Javier
authors.initials: J
authors.lastname: Garcia-Perez
authors.affiliation: AIDS Immunopathogenesis Unit, Instituto de Salud Carlos III, 28220, Majadahonda, Madrid, Spain. Electronic address: ppalcami@isciii.es.
authors.forename: Jose
authors.initials: J
authors.lastname: Alcami
authors.affiliation: INSERM U1108, Institut Pasteur, 75015 Paris, France; Viral Pathogenesis Unit, Department of Virology, Institut Pasteur, 75015 Paris, France. Electronic address: farenzan@pasteur.fr.
authors.forename: Fernando
authors.initials: F
authors.lastname: Arenzana-Seisdedos
authors.affiliation: INSERM U1108, Institut Pasteur, 75015 Paris, France; Viral Pathogenesis Unit, Department of Virology, Institut Pasteur, 75015 Paris, France. Electronic address: bernard.lagane@pasteur.fr.
authors.forename: Bernard
authors.initials: B
authors.lastname: Lagane
mesh_terms: D003001:Cloning, Molecular; D015870:Gene Expression; D017408:Guidelines as Topic; D008967:Molecular Biology / Q000379:methods*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D054299:env Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D054299:env Gene Products, Human Immunodeficiency Virus
keywords: AIDS; Cloning; Gene toxicity; HIV-1 gp120; Protein expression and purification; Receptor binding assays