Cytomegaloviruses inhibit Bak- and Bax-mediated apoptosis with two separate viral proteins

Abstract

Apoptosis of infected cells can limit virus replication and serves as an innate defense mechanism against viral infections. Consequently, viruses delay apoptosis by expressing antiapoptotic proteins, many of which structurally resemble the cellular antiapoptotic protein Bcl-2. Like Bcl-2, the viral analogs inhibit apoptosis by preventing activation and/or oligomerization of the proapoptotic mitochondrial proteins Bax and Bak. Here we show that cytomegaloviruses (CMVs) have adopted a different strategy. They encode two separate mitochondrial proteins that lack obvious sequence similarities to Bcl-2-family proteins and specifically counteract either Bax or Bak. We identified a small mitochondrion-localized protein encoded by the murine CMV open reading frame (ORF) m41.1, which functions as a viral inhibitor of Bak oligomerization (vIBO). It blocks Bak-mediated cytochrome c release and Bak-dependent induction of apoptosis. It protects cells from cell death-inducing stimuli together with the previously identified Bax-specific inhibitor viral mitochondria-localized inhibitor of apoptosis (vMIA) (encoded by ORF m38.5). Similar vIBO proteins are encoded by CMVs of rats, and possibly by other CMVs as well. These results suggest a non-redundant function of Bax and Bak during viral infection, and a benefit for CMVs derived from the ability to inhibit Bak and Bax separately with two viral proteins.

Viral Inhibition of BAK Promotes Murine Cytomegalovirus Dissemination to Salivary Glands

“…Replication of the m41.1ko virus was indistinguishable from that of the control viruses in 10.1 fibroblasts but significantly reduced in macrophages ( Fig. 3A and B), consistent with previously published results (18). The m41.1ko virus also replicated to lower titers in primary bone marrow-derived macrophages (BMDMs) of C57BL/6J but not BAK-deficient mice (B6.129-Bak1 tm1Thsn /J) (29) mice, indicating that the replication defect is BAK dependent ( Fig.…”

“…In this ⌬m157 wild-type-like (wt-l) virus genome, the m41 locus was first replaced by a galKkan cassette using kanamycin for positive selection. Using 2-deoxy-galactose for negative selection, galK-kan was then replaced by an m41 sequence in which all three m41.1 ATG codons were mutated to ACG, leaving the protein sequence of the overlapping m41 ORF unchanged (18). Using the same two-step replacement strategy (which is described in detail elsewhere [25]), the revertant (rev) was constructed (Fig.…”

“…MCMV inhibits the intrinsic apoptosis pathway at mitochondria with two separate viral proteins: a BAX-specific inhibitor, encoded by open reading frame (ORF) m38.5 (14)(15)(16)(17), and a BAK-specific inhibitor, encoded by ORF m41.1 (18). The mitochondrion-localized m41.1 protein is expressed from the m41 locus and functions as viral inhibitor of BAK oligomerization (18). In this study, we investigated the role of m41.1/viral inhibitor of BAK oligomerization (vIBO) for MCMV replication and dissemination in mice.…”

“…GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (E) Expression of m41 was analyzed by immunoblotting in virus-infected RAW264.7 macrophages using the m41-specific antibody 2A6 (18). mice (16).…”

The Human Cytomegalovirus UL36 Gene Controls Caspase-Dependent and -Independent Cell Death Programs Activated by Infection of Monocytes Differentiating to Macrophages

“…Studies with MCMV indicate that at least four genes, M36, M45, m38.5, and m41.1, are critical to prevent infection-induced cell death in macrophages (10,11,47,57). The roles of these genes during infection of differentiating cells are unclear.…”