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Title: Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

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. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.
Authors:
 [1] ;  [2] ;  [3] ;  [2] ;  [1] ; ;  [2] ;  [3] ;  [1]
  1. Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)
  2. Department of Pediatrics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461 (United States)
  3. (United States)
Publication Date:
OSTI Identifier:
22470151
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 476; Other Information: Copyright (c) 2014 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; AIDS VIRUS; ANIMAL CELLS; ANTIBODIES; BIOLOGICAL RECOVERY; CELLULOSE; DRUGS; ENDOPLASMIC RETICULUM; FLUORESCENCE; GLYCOPROTEINS; LASERS; MATERIALS RECOVERY; QUALITY CONTROL; RECEPTORS; SACCHAROSE; THERAPY