Epstein-Barr virus BALF1 is a BCL-2-like antagonist of the herpesvirus antiapoptotic BCL-2 proteins

Abstract

Cellular BCL-2 family proteins can inhibit or induce programmed cell death in part by counteracting the activity of other BCL-2 family members. All sequenced gammaherpesviruses encode a BCL-2 homologue that potently inhibits apoptosis and apparently escapes some of the regulatory mechanisms that govern the functions of their cellular counterparts. Examples of these protective proteins include BHRF1 of Epstein-Barr virus (EBV) and KSBcl-2 of Kaposi's sarcoma-associated herpesvirus, also known as human herpesvirus 8. The gamma-1 subgroup of these viruses, such as EBV, encodes a second BCL-2 homologue. We have now found that this second BCL-2 homologue encoded by EBV, BALF1, inhibits the antiapoptotic activity of EBV BHRF1 and of KSBcl-2 in several transfected cell lines. However, BALF1 failed to inhibit the cellular BCL-2 family member, BCL-x(L). Thus, BALF1 acts as a negative regulator of the survival function of BHRF1, similar to the counterbalance observed between cellular BCL-2 family members. Unlike the cellular BCL-2 family antagonists, BALF1 lacked proapoptotic activity and could not be converted into a proapoptotic factor in a manner similar to cellular BCL-2 proteins by caspase cleavage or truncation of the N terminus. Coimmunoprecipitation experiments and immunofluorescence assays suggest that a minimal amount, if any, of the BHRF1 and BALF1 proteins colocalizes inside cells, suggesting that mechanisms other than direct interaction explain the suppressive function of BALF1.

FIG. 1.

Human and primate herpesvirus BALF1. (A) Diagram of EBV genome region containing BALF1. Boxes that indicate the direction of transcription represent open reading frames. BALF1 (shaded) results from an internal initiation within the BALF0 reading frame. (B) Diagram of the EBV genome segment containing putative BALF1 transcripts (drawn in reverse of the usual orientation). Nucleotide sequences for putative TATA boxes (bent arrows), initiation codons, stop codon (underlined), and poly(A) signal (underlined) are shown. Numbers of intervening nucleotides (nt) are shown. Calculated transcript sizes [excluding poly(A)] are shown. (C) Alignment of the DNA sequences of BALF1 from EBV (V01555), pan herpesvirus (GenBank accession no. AF306944), herpesvirus papio (GenBank accession no. AF306943), and pongine herpesvirus 3 (GenBank accession no. AY034056). (D) ClustalW alignment of amino acid sequences of the indicated human and viral BCL-2 family members, including callitrichine herpesvirus 3 (GenBank accession no. AF319782). Identical (dark shading) and similar (light shading) amino acids occurring in 6 of 11 entries are marked. Homology domains BH1 to BH4 and the transmembrane domain (TM) of cellular BCL-x_L are marked with horizontal lines. The BALF1 consensus caspase cleavage recognition site is marked by an arrowhead.

FIG. 2.

BALF1 does not inhibit Sindbis virus-induced apoptosis in a variety of cell lines. (A) Rat-1 cells were infected with recombinant Sindbis viruses expressing the indicated constructs and cells were evaluated for apoptosis by flow cytometry to detect cell shrinkage (forward scatter) and membrane permeability (propidium iodide uptake). (B to D) Infected cells were harvested at 24 h post infection for Rat-1 cells (B), the indicated times for HeLa cells (C), and 40 h for 308 epidermal cells (D), treated with propidium iodide, and analyzed by flow cytometry as in the results shown in panel A. Means ± the standard error of the mean (SEM) are shown for three independent experiments. All proteins had N-terminal HA tags. Corresponding immunoblot analyses with anti-HA antibody are shown for each cell type.

FIG. 3.

BALF1 does not protect a lymphocyte cell line against BAX-induced death. (A) DG75 cells were transiently cotransfected with plasmids encoding the indicated proteins with N-terminal HA tags as well as 0.5 μg of EGFP to mark transfected cells. Apoptosis was assessed at 18 h posttransfection by measuring the loss of mitochondrial membrane potential (CMX-Ros fluorescence) in GFP-expressing cells. (B) Graphic presentation of the mean ± SEM for three independent experiments as shown in panel A. Shown below is the amount (in micrograms) of plasmid DNA transfected (with all samples receiving equal amounts of DNA balanced with empty vector) and a corresponding immunoblot analysis.

FIG. 4.

BALF0-BALF1 protein is resistant to cleavage with apoptotic cell extracts. (A) In vitro-translated, ³⁵S-labeled proteins (without HA tags) were digested with apoptotic 293 cell extracts plus dATP to activate the extract as previously described (5, 24). Proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. (B and C) Deletion of the N terminus of BALF1 (ΔN50) does not induce proapoptotic activity. Apoptosis was determined by flow cytometry (forward scatter and propidium iodide staining as described for Fig. 2 at 24 h postinfection of CHO cells (B) and mouse epidermal 308 cells (C) with recombinant Sindbis viruses encoding the indicated proteins or a control virus containing KSBcl-2 in the reverse orientation. Means ± SEM are shown for three independent experiments. All proteins contain an N-terminal HA tag. Corresponding immunoblot analyses with anti-HA antibody are shown for each cell type.

FIG. 5.

BALF1 and BALFO antagonize the antiapoptotic activity of BHRF1. (A) Cell viability of CHO cells transfected with the indicated plasmids (plus 0.05 μg of a β-galactosidase plasmid to mark transfected cells) was determined at 18 h posttransfection by scoring the percentage of transfected cells that were live and/or nonapoptotic (counting >200 lacZ-positive cells per sample). All samples received equal amounts of DNA. The data presented are the means ± SEM for at least three independent experiments. (B) BHK cells were transfected with the indicated plasmids and cell viability was determined as described for panel A. A representative immunoblot is shown. (C) Viability of 308 epidermal cells was determined 40 h after coinfection with recombinant Sindbis viruses expressing the indicated proteins (with N-terminal HA tags) as described in the legend to Fig. 2.

FIG. 6.

BALF1 binds to itself and BHRF1 in vitro. (A) COS-1 cells were transfected with plasmids expressing the indicated HA-tagged BCL-2 family members plus either FLAG-BALF1 or FLAG-BCL-x_L as a positive control. Anti-HA immunoprecipitates and total cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the antibodies indicated on the right of each panel. A representative immunoprecipitation is shown (n = 3). (B, C, and D) Coimmunoprecipitation experiments were performed with lysates of COS-1 cells transfected with the indicated plasmids, as described for panel A.

FIG. 7.

BALF1 does not colocalize with BHRF1 in cells. (A) Fluorescence microscopy of CHO cells transfected with N-terminal HA-tagged BALF1 (top), HA-tagged BALF0 (middle), or N-terminal FLAG-tagged BHRF1 (bottom). At 24 h posttransfection, cells were treated with CMX-Ros for 1 h (red, center panels) and stained with monoclonal anti-HA or FLAG antibodies (fluorescein isothiocyanate secondary antibody, left panels). The images were merged using SPOT software (yellow, right panels), but the merge failed to detect colocalized proteins. (B) CHO cells were cotransfected with plasmids containing FLAG-tagged BHRF1 (green, fluorescein isothiocyanate) plus either HA-tagged BALF1 (red, top row) or HA-tagged BALF0 (M39G) (red, bottom row). The two images were merged to reveal any colocalization (yellow, third column). (C) The experiment described in panel B was also performed with COS-1 cells.