Purified GPI-anchored CD4DAF as a receptor for HIV-mediated gene transfer.
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 degrees 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.
Brodsky
R A
RA
Johns Hopkins University Oncology Center, Baltimore, MD 21205.
Jane
S M
SM
Vanin
E F
EF
Mitsuya
H
H
Peters
T R
TR
Shimada
T
T
Medof
M E
ME
Nienhuis
A W
AW
D015703:Antigens, CD / Q000378:metabolism*; D015704:CD4 Antigens / Q000378:metabolism*; D018958:CD55 Antigens; D002460:Cell Line; D018014:Gene Transfer Techniques; D017261:Glycosylphosphatidylinositols / Q000502:physiology*; D006678:HIV / Q000235:genetics*; D015699:HIV Envelope Protein gp120 / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D015703:Antigens, CD; D015704:CD4 Antigens; D018958:CD55 Antigens; D017261:Glycosylphosphatidylinositols; D015699:HIV Envelope Protein gp120; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins