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.

Viral versus cellular BCL-2 proteins

“…64,65 Although they confirmed the interaction of BHRF1 with BOK by coprecipitation of in vitro translated proteins, we and others have failed to detect dimerization of BHRF1 with BAX, BAK, BAD (BH3-only protein) and Bcl-rambo by co-precipitation of transfected cell lysates or in GST pull-downs. 28,40,66,67 However, in these same experiments, the viral BCL-2 proteins from HVS and gHV68 were found to bind BAX. 28,40 Therefore, no consistent theme prevails, possibly suggesting that viral BCL-2 proteins may specifically target only one or possibly a subset of cellular pro-death factors to inhibit cell death.…”

“…28,40,66,67 However, in these same experiments, the viral BCL-2 proteins from HVS and gHV68 were found to bind BAX. 28,40 Therefore, no consistent theme prevails, possibly suggesting that viral BCL-2 proteins may specifically target only one or possibly a subset of cellular pro-death factors to inhibit cell death. However, we favour the idea that viral BCL-2 proteins may protect cells by alternate mechanisms.…”

“…Despite the apparent inability of BHRF1 and KSBcl-2 to stably interact with BAX in transfected mammalian cells, BHRF1 and KSBcl-2 are potently protective against BAX-induced cell death in these cells, dissociating the ability to protect cells from the capacity to bind BAX. 28 This heterodimerization model was also challenged by generating mutants of cellular BCL-x L and BCL-2 that fail to bind BAX and BAK but still protect cells, 29 and by the observation that BAX and BCL-2 do not dimerize or colocalize inside cells 68,69 ( Figure 3B). Several other labs have also pointed out similar discordance between dimerization and function.…”

“…27 Surprisingly, BALF1 has little or no ability to inhibit cell death in many assay systems. 27,28 In contrast, BALF1 antagonizes the anti-apoptotic BCL-2 protein of EBV in that it suppresses the protective effects of BHRF1. 28 Interestingly, this interference activity of BALF1 is most likely not due to direct binding of BALF1 to BHRF1, similar to the actions of BAX/BAK and BCL-2/BCL-x L .…”

Epstein-Barr virus interactions with the Bcl-2 protein family and apoptosis in human tumor cells

“…Similar results were found with anti-Fas plus interferon-γ (IFN-γ) when both cycloheximide and TNF-α or chloromethyl X-rosamine (CMXRos) staining were used to induce apoptosis (Marshall et al, 1999). However, a report showed that EBV BALF1 fails to protect cells from Bax-induced apoptosis in the DG75 B cell (EBV-negative BL lymphoma) (Bellows et al, 2002), so BALF1 lacks an anti-apoptotic function. Taken together, although the function of BALF1 is not yet clear, it remains possible that BALF1 interrupts the apoptotic pathway in EBV-infected cells by interacting with pro-apoptotic proteins, such as Bax and BAK.…”