FIG. 3.

Calcium-phosphate transfection of HPV31 and HK cells with the cloned AAV2 genome and the GFP expression vector (Clontech) as transfection control. For each experiment, nontransfected (−GFP) cells are shown in the left panels, GFP-only controls (+GFP) are shown in the middle panels, and GFP vector and AAV2 genome-transfected cells (+GFP/+AAV2) are shown in the right panels. (A) HPV31 cells transfected with the GFP expression vector and AAV2 cloned genome. AAV2-induced cell death was correlated with decreased numbers of cells adhering to plates. The membrane integrity of GFP-positive HPV31 cells in cells cotransfected with AAV2 was disrupted in comparison to the membrane integrity of cells transfected with the GFP vector alone. All images were captured with a 20× objective. (B) HK cells transfected with the GFP expression vector and cloned AAV2 genome. GFP expression is observed equally irrespective of the presence of AAV2, and cell death was not observed, as determined from membrane integrity. All images were captured with a 20× objective. (C) Transfection of AAV2 genome induced cell death, which was correlated with the expression of Rep proteins in HPV31-positive cells but not in transfected HK cells. For detecting Rep proteins in Western blots, total protein extracts were prepared as we have previously described (1). Western blotting was performed using 60 μg of total protein extracts from HPV31 and HK cells transfected with the GFP expression vector and AAV2 genome. As controls, cells were transfected with the GFP vector alone. Protein samples were resolved in a 7.5% SDS-PAGE gel and detected with an AAV2 Rep-specific antibody (Progen). For clarity of detection of individual Rep proteins, blots were exposed for 10, 20, and 45 s as indicated. Arrows point to individual Rep proteins detected in HPV31 cells transfected with AAV2. Rep52 was detected in lane 3, Rep40 was detected in lane 6, and Rep78 dimer was detected in lane 9. Rep68 expression could not be clearly established. +, present; −, absent.