FIG. 3.
Effect of JCV agnoprotein on Tat-mediated activation of the HIV-1 LTR. Primary human fetal astrocytes were prepared according to a modified procedure based on the methods of Cole and de Vellis (16) and Yong and Antel (79). Astrocytes were plated (2 × 105 in 60-mm plate) and maintained in regular growth medium (Dulbecco's modified Eagle's medium-F-12 supplemented with 15% fetal bovine serum). Cells were then transfected using FuGENE 6 transfection reagent with 1 μg of luciferase-based reporter constructs containing full-length (−450 to +80) HIV-LTR (A) or a deletion mutant of the LTR lacking the TAR sequence (+3 to +80) (B). Transfections were carried out in the absence or presence of plasmids (1 μg) expressing either Tat or agnoprotein under control of the cytomegalovirus (CMV) promoter (58). The concentration of DNA in each transfection mixture remained constant by adding pCDNA3. Forty hours after transfection, cells were harvested and luciferase enzymatic activity was measured. The average values of multiple experiments are presented as n-fold effects with a baseline remaining as an arbitrary unit of 1. (C) Western blot analysis of protein extract from the transfected cells (as indicated in panel A) for detection of Tat and agnoprotein.