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

Virology. 2015 Feb;476:240-248. doi: 10.1016/j.virol.2014.12.019. Epub 2014 Dec 30.

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.

Keywords: CD4; Diffusion; Envelope; FRAP; Fluorescent protein; HIV-1; Inhibitory antibody; Laser scanning cytometry; Neutralizing antibody; Superfolder GFP; gp120.

Publication types

  • Evaluation Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CD4-Positive T-Lymphocytes / metabolism
  • CD4-Positive T-Lymphocytes / virology*
  • Cell Tracking / methods*
  • Genetic Engineering
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism*
  • HIV Envelope Protein gp120 / genetics
  • HIV Envelope Protein gp120 / metabolism*
  • HIV Infections / metabolism
  • HIV Infections / virology*
  • HIV-1 / genetics
  • HIV-1 / metabolism*
  • Humans
  • Receptors, Virus / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • HIV Envelope Protein gp120
  • Receptors, Virus
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins