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

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.

Overview summary

Gene transfer through a CD4DAF-expressing cell line was demonstrated using an HIV-based vector. In an attempt to expand the spectrum of cells susceptible to HIV-mediated gene transfer, CD4DAF was purified from this cell line. The purified protein was able to incorporate into the membrane of native HeLa cells and bind the HIV virus. Although exposure of CD4DAF-treated HeLa cells to our HIV-derived vector produced transient neomycin resistance, incorporation of CD4DAF from outside the cell did not permit efficient transduction of virus-encoded genes. These results demonstrate that additional factors are required for HIV integration after the virion binds to CD4.