Expression of HIV env gene in a human T cell line for a rapid and quantifiable cell fusion assay.
Human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins present at the surface of infected cells are known to mediate fusion with CD4-positive target cells. In this study we have developed a novel Env-expressing cell line for investigating the fusion process in a biologically significant system. Cell surface expression of the HIV-1 env gene, isolated from the highly fusogenic strain SF33, was obtained in the CD4-negative T cell line A2.01. To render the system versatile and efficient, HIV-1 regulatory proteins Tat and Rev were supplied in trans. The presence of Env at the cell surface was shown by cytofluorometry and immunofluorescence and precursor processing of gp160 to gp120/gp41 was demonstrated by Western blot. The fusion capacity of A2.01-Env cells was assessed by coculture with CD4-positive T lymphocytes or the fusion indicator cell line, HeLa-CD4-LTR-beta-Gal. By coincubation with CD4-positive T cells such as SupT1, A2.01-Env cells were observed to mediate rapidly numerous well-defined syncytia in a reproducible fashion. By expressing Tat, they also had the capacity to trans-activate the LTR-linked reporter beta-Gal gene following fusion with HeLa-CD4-LTR-beta-Gal cells. The fusion-inhibiting anti-CD4 monoclonal antibodies Q425 and Q428 were used to block specifically Env-mediated fusion with CD4-positive cells and to demonstrate application of this system to the search for potential fusion-blocking agents. Our system thus offers a biologically significant model for studying fusion events with the advantages of being rapid, reproducible and versatile.
Moir
S
S
Department of Microbiology, Faculty of Medicine, Laval University, Ste-Foy, Quebec, Canada.
Poulin
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L
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001483:Base Sequence; D015704:CD4 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D002459:Cell Fusion / Q000502:physiology*; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D015752:Genes, env; D015699:HIV Envelope Protein gp120 / Q000235:genetics / Q000502:physiology; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000502:physiology; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D015459:Leukemia-Lymphoma, Adult T-Cell / Q000473:pathology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000378:metabolism*; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D015699:HIV Envelope Protein gp120; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins