Purified GPI-Anchored CD4DAF as a Receptor for HIV-Mediated Gene Transfer

Robert A. Brodsky, Stephen M. Jane, Elio F. Vanin, Hiroaki Mitsuya, Timothy R. Peters, Takashi Shimada, M. Edward Medof, Arthur W. Nienhuis

Research output: Contribution to journalArticlepeer-review

Abstract

CD4 is the major cellular receptor for the human immunodeficiency virus (HIV). A hybrid gene encoding the extracellular domains of CD4, linked to the sequence encoding the membrane attachment region of the glycosylphosphatidylinositol (GPI)-anchored protein decay accelerating factor (DAF) was stably transfected into HeLa cells. The resultant cell line (T4HD) expressed GPI-anchored CD4DAF at high levels and was susceptible to gene transfer with a recombinant HIV vector. In an effort to expand the spectrum of cells susceptible to HIV gene transfer, CD4DAF was released from the surface of the T4HD cell line by detergent lysis, purified by immunoaffinity chromatography, and reincorporated into native HeLa cells. Incorporation occurred via the GPI anchor as evidenced by cleavage with phosphatidylinositol-specific phospholipase C. More than 95% of the CD4DAF-treated HeLa cells were CD4-positive by flow cytometry, and kinetic analysis demonstrated that over 75% of the fusion protein remained anchored to the cell membrane after 90 min at 37°C. The purified protein retained its ability to bind the envelope protein of HIV. When incorporated, it bound fluorescein isothiocyanate (FITC)-conjugated gp120, and in its soluble form blocked transduction of CD4-positive cells incubated with an HIV-derived vector containing the Neo® gene. In contrast to the T4HD cells, exposure of CD4DAF-treated cells to the Neo® HIV vector yielded only transient neomycin-resistant colonies. These results suggest that endogenous synthesis of the CD4 molecule may be necessary for successful HIV genomic integration.

Original languageEnglish (US)
Pages (from-to)1231-1239
Number of pages9
JournalHuman gene therapy
Volume5
Issue number10
DOIs
StatePublished - Oct 1 1994

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics

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