Murine Cytomegalovirus m38.5 Protein Inhibits Bax-Mediated Cell Death

Abstract

Many viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes to mitochondria and protects human HeLa cells and fibroblasts from apoptosis triggered by proteasome inhibitors but not from Fas-induced apoptosis. However, the ability of this protein to suppress the apoptosis of murine cells and its role during MCMV infection have not been investigated previously. Here we show that m38.5 is expressed at early time points during MCMV infection. Cells infected with MCMVs lacking m38.5 showed increased sensitivity to cell death induced by staurosporine, MG132, or the viral infection itself compared to the sensitivity of cells infected with wild-type MCMV. This defect was eliminated when an m38.5 or Bcl-X L gene was inserted into the genome of a deletion mutant. Using fibroblasts deficient in the proapoptotic Bcl-2 family proteins Bak and/or Bax, we further demonstrated that m38.5 protected from Bax-but not Bak-mediated apoptosis and interacted with Bax in infected cells. These results consolidate the role of m38.5 as a viral mitochondrion-localized inhibitor of apoptosis and its functional similarity to the human cytomegalovirus UL37x1 gene product. Although the m38.5 gene is not homologous to the UL37x1 gene at the sequence level, m38.5 is conserved among rodent cytomegaloviruses. Moreover, the fact that MCMV-infected cells are protected from both Bak-and Bax-mediated cell death suggests that MCMV possesses an additional, as-yet-unidentified mechanism to block Bak-mediated apoptosis.

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

“…20,21,23 We further showed that Bax-knockout cells infected with an m38.5-deficient MCMV mutant (Dm38.5) were resistant to STS-induced apoptosis, suggesting that Bak-mediated apoptosis was inhibited by a different MCMV protein. 20 To test whether m41.1 inhibits apoptosis in a Bak-specific manner, we infected fibroblasts deficient for Bax, Bak, or both proteins with different MCMV mutants and analyzed their sensitivity to STS. For comparison, we also included the previously described Dm38.5 mutant and constructed a double-knockout (dko) mutant lacking both m41.1 and m38.5.…”

“…23 Moreover, the observation that MCMV-infected cells are protected from Bax-and Bakmediated apoptosis suggested that the virus encodes an additional, Bak-specific antiapoptotic protein. 20 In the present study, we identified the product of MCMV ORF m41.1 as a Bak-specific inhibitor of apoptosis. The m41.1 protein localizes to mitochondria and prevents Bak oligomerization, cyt c release, and execution of PCD.…”

“…17 Murine CMV (MCMV) also expresses a vMIA protein, which is encoded at an analogous position within the viral genome by ORF m38.5, but displays little sequence similarity to HCMV vMIA. 18,19 The MCMV m38.5 protein inhibits Bax-but not Bak-mediated apoptosis, [20][21][22] and also induces mitochondrial fragmentation in the absence of Bak (i.e., in bak À/À cells). 23 Moreover, the observation that MCMV-infected cells are protected from Bax-and Bakmediated apoptosis suggested that the virus encodes an additional, Bak-specific antiapoptotic protein.…”

Sheeppox Virus SPPV14 Encodes a Bcl-2-Like Cell Death Inhibitor That Counters a Distinct Set of Mammalian Proapoptotic Proteins

“…They include myxoma virus-encoded M11L (19,20,25), human cytomegalovirus-encoded vMIA (24), murine cytomegalovirus-encoded m38 (38), and vaccinia virus-encoded F1L (21,48,49,61,63) and N1L (2,7,12). Despite the lack of sequence similarity to the Bcl-2 protein family, both F1L and M11L adopt a Bcl-2 fold (16,40,42), and the structure of M11L in complex with the BH3 peptide from Bak revealed that M11L binds BH3 ligands via the canonical BH3 binding groove (40).…”

Viral Inhibition of BAK Promotes Murine Cytomegalovirus Dissemination to Salivary Glands

“…Cytochrome c then induces the formation of a so-called apoptosome complex and subsequent activation of caspases (13). 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).…”