Inhibition of apoptosis by Bcl-xl and BALF1 in stable transfectants. (A) HeLa cells were transfected with vectors encoding GFP, GFP-BALF1 and GFP–Bcl-xl. GFP-positive transfectants were selected and then treated with IFN-γ and anti-Fas antibody or with control IFN-γ alone, using modifications of published methods (25). Transfectants were evaluated for apoptosis by flow cytometric analysis of apoptotic subdiploid nuclei (30). (B) GFP, GFP–Bcl-xl, and GFP-BALF1 clones were treated with 2.5 μM camptothecin in 2.5% DMSO. Controls were treated with 2.5% DMSO alone. The day after each series of treatments, cells were evaluated for apoptosis by flow cytometric analysis as described above. (C to F) HeLa cells were transiently cotransfected with pEGFPN1 (C and D) or with a plasmid encoding BALF1 cDNA, pcDNA3.1BALF1, and pEGFPN1 (at a pEGFPN1:BALF1 ratio of 1:2) (E and F). Following treatment with camptothecin (D and F) or DMSO (C and E) at the concentration used for panel B, the effect of BALF1 on apoptosis (E and F) was assessed by loss of fluorescence of CMXRosamine (FL2) in the GFP-cotransfected cells (FL1). Percentages indicate the percentage of cells gated in each quadrant.

Similarity of the EBV transcripts BALF1 and BHRF1 to Kaposi’s sarcoma-associated herpesvirus ORF16 (KSHVorf16) and the cellular genes bcl-x and bcl-2. Sequences were aligned via the PIMA multiple sequence alignment program, using the sequential branching clustering method (36). Areas of identity are shown in black; conserved regions are shown in gray (BOXSHADE, Isrec, Switzerland).

BALF1 associates with Bak and Bax. HeLa cell transfected with GFP, GFP-BALF1, or GFP–Bcl-xl were lysed, immunoprecipitated with anti-Bax and anti-Bak, analyzed on a 4 to 20% non-GFP-denaturing SDS-polyacrylamide gel, and scanned on a FluorImager. A 33-kDa band (indicated by the arrow) in the GFP-BALF1 and GFP–Bcl-xl lanes and a 20-kDa band in the GFP lane were recognized, in accord with other studies (31). GFP-BALF1 or GFP–Bcl-xl was present in the eluate from the immunoprecipitation on an SDS–12% polyacrylamide gel when anti-Bak (lanes 2 and 3) or anti-Bax was used (lanes 9 and 10), while GFP was not (lanes 1 and 8). MW mkrs, molecular weight markers.