Figure 1

Construction and characterization of an HIV derivative carrying the β-Gal alpha peptide. (A) The coding sequence for amino acids 1-51 of β-Gal (gray box) was inserted into the gag open reading frame of plasmid pCHIV. Amino acids displayed in bold represent authentic sequences from HIV Gag or β-Gal, respectively, while introduced linker sequences are displayed in italics. Arrowheads indicate cleavage sites for HIV PR. (B) Immunoblot analysis of HIV.MAα particles. 293T cells transfected with the indicated constructs were grown in the absence (-) or presence (+) of 2 μM LPV. At 44 h post transfection, particles were purified by ultracentrifugation and analyzed by immunoblotting using the indicated antisera. Molecular mass standards (in kDa) are shown on the left, specific protein products are identified on the right. (C) β-Gal activity in lysates of transfected 293T cells dependent on HIV PR activity. Cell lysates from untransfected 293T cells (filled circles), or from 293T cells transfected with a mixture of pCMVω and pCHIV.MAα and incubated in the presence of DMSO (filled triangles) or 2 μM LPV (open triangles, respectively, were prepared at 48 h post transfection and β-Gal activity was determined in vitro through cleavage of the colorimetric substrate CPRG by measuring changes in OD592 over time. The graph shows mean values and standard deviations from five independent experiments. Relative rates of CPRG cleavage were determined by linear regression, yielding an average value of 0.109 min^-1 for the DMSO controls and 0.054 min^-1 for the LPV treated samples, respectively