title: Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays.
abstract: The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFP enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs.
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Lindsey M
authors.initials: LM
authors.lastname: Costantini
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Susan C
authors.initials: SC
authors.lastname: Irvin
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Steven C
authors.initials: SC
authors.lastname: Kennedy
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Guo
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Harris
authors.initials: H
authors.lastname: Goldstein
authors.affiliation: Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Betsy C
authors.initials: BC
authors.lastname: Herold
authors.affiliation: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA. Electronic address: erik-lee.snapp@einstein.yu.edu.
authors.forename: Erik L
authors.initials: EL
authors.lastname: Snapp
mesh_terms: D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism / Q000821:virology*; D058948:Cell Tracking / Q000379:methods*; D005818:Genetic Engineering; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000378:metabolism*; D015658:HIV Infections / Q000378:metabolism / Q000821:virology*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011991:Receptors, Virus / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D015699:HIV Envelope Protein gp120; D011991:Receptors, Virus; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins
keywords: CD4; Diffusion; Envelope; FRAP; Fluorescent protein; HIV-1; Inhibitory antibody; Laser scanning cytometry; Neutralizing antibody; Superfolder GFP; gp120