FIG. 2.
DTT induces cleavage of Pr65Gag in MMLV produced in DIBA-2. NIH 3T3 cells chronically infected with MMLV were seeded at 106 cells per 10-cm-diameter tissue culture dish. Four hours later, the medium was changed to fresh culture medium containing 100 μM DIBA-2. Control dishes lacking DIBA-2 contained the same amount of the dimethyl sulfoxide solvent as the DIBA-2-treated dishes. The culture fluid containing the virus was collected 24 h later and filtered through a 0.45-μm-pore-size filter. MMLV produced in the presence (lanes 1 and 2) or absence (lanes 3 and 4) of DIBA-2 was collected by centrifugation, resuspended in phosphate-buffered saline (PBS), and incubated with (lanes 2 and 4) or without (lanes 1 and 3) 10 mM DTT for 2 h at 37°C. It was then analyzed by immunoblotting with antiserum against p30CA. Immunoblotting was performed as described previously (13) using enhanced chemiluminescence (Amersham).