FIG 5.
High levels of GP1,2 expression impair protein production. (A, B) Decreased GFP expression correlates with increased GP1,2 expression levels. 293T cells were transfected with 500 ng of a plasmid encoding HIVΔEnv (SG3) backbone and 100 ng of a plasmid encoding GFP, as well as various amounts of GP1,2 DNA (4 μg to 4 ng). (A) At 48 h posttransfection, cells were imaged by fluorescence microscopy to visualize GFP expression. Results are representative of those obtained in three separate experiments. (B) Cells were harvested by EDTA treatment and stained for surface GP1,2 expression using mouse anti-GP1,2 immune sera, followed by a PE-conjugated anti-mouse antibody. Cells were then analyzed by flow cytometry, and MFI was plotted for GP1,2 and GFP versus the amount of transfected GP1,2 plasmid. (C) 293T cells were transfected as described for panel A, except that instead of using GFP, cells were cotransfected with 100 ng of plasmid expressing PR8 influenza virus A nucleoprotein (NP). Cell lysate was harvested at 48 h posttransfection, normalized for β-actin, and analyzed by Western blotting using a mouse monoclonal anti-NP antibody.