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

被引:4
|
作者
Costantini, Lindsey M. [1 ]
Irvin, Susan C. [2 ,3 ]
Kennedy, Steven C. [2 ]
Guo, Feng [1 ]
Goldstein, Harris [2 ,3 ]
Herold, Betsy C. [2 ,3 ]
Snapp, Erik L. [1 ]
机构
[1] Albert Einstein Coll Med, Dept Anat & Struct Biol, Bronx, NY 10461 USA
[2] Albert Einstein Coll Med, Dept Pediat, Bronx, NY 10461 USA
[3] Albert Einstein Coll Med, Dept Microbiol & Immunol, Bronx, NY 10461 USA
基金
美国国家卫生研究院;
关键词
gp120; Fluorescent protein; Superfolder GFP; HIV-1; Neutralizing antibody; Laser scanning cytometry; FRAP; Diffusion; CD4; Envelope; Inhibitory antibody; ENVELOPE GLYCOPROTEIN; ANTIBODY; 2G12; LIVING CELLS; PROTEIN; NEUTRALIZATION; BROAD; EXPRESSION; MECHANISM; MEMBRANES; SELECTION;
D O I
10.1016/j.virol.2014.12.019
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 gp120-5fGFP 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. (C) 2014 Elsevier Inc. All rights reserved.
引用
收藏
页码:240 / 248
页数:9
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