Inhibition of programmed cell death by cytomegaloviruses

Virus Research 157 (2011) 144–150

Contents lists available at ScienceDirect

Virus Research journal homepage: www.elsevier.com/locate/virusres

Inhibition of programmed cell death by cytomegaloviruses Wolfram Brune ∗ Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Martinistr. 52, 20251 Hamburg, Germany

a r t i c l e

i n f o

Article history: Available online 20 October 2010 Keywords: Cytomegalovirus Herpesvirus Innate immunity Apoptosis Programmed necrosis Necroptosis

a b s t r a c t The elimination of infected cells by programmed cell death (PCD) is one of the most ancestral defense mechanisms against infectious agents. This mechanism should be most effective against intracellular parasites, such as viruses, which depend on the host cell for their replication. However, even large and slowly replicating viruses like the cytomegaloviruses (CMVs) can prevail and persist in face of cellular suicide programs and other innate defense mechanisms. During evolution, these viruses have developed an impressive set of countermeasures against premature demise of the host cell. In the last decade, several genes encoding suppressors of apoptosis and necrosis have been identified in the genomes of human and murine CMV (HCMV and MCMV). Curiously, most of the gene products are not homologous to cellular antiapoptotic proteins, suggesting that the CMVs did not capture the genes from the host cell genome. This review summarizes our current understanding of how the CMVs suppress PCD and which signaling pathways they target. © 2010 Elsevier B.V. All rights reserved.

1. Introduction PCD is an evolutionary conserved process of multicellular organisms to dispose of unwanted cells. It plays an important role during development, for tissue homeostasis and regeneration, and in the defense against malignancy and infection. Killing and eliminating infected cells can be an efficient means to combat viral infection, as viruses need the host cell machinery for their own proliferation. When PCD is initiated early during the viral life cycle, progeny production and dissemination can be severely impaired. Moreover, fragments of dead cells containing viral proteins can be taken up by antigen presenting cells and used to prime the adaptive immune response (Albert et al., 1998). Apoptosis is the best-characterized form of PCD and has frequently been used as a synonym for PCD. In recent years, however, other forms of PCD such as necrosis and autophagic cell death have received increasing attention. Morphologically, apoptosis involves a reduction of cell volume, chromatin condensation, membrane blebbing, and finally the disintegration of the cell into vesicles called apoptotic bodies (Kroemer et al., 2009). Apoptosis is often (but not always) associated with activation of caspases, a group of aspartate-specific cystein proteases. Apoptosis does not result in the release of intracellular enzymes. By contrast, necrosis is characterized by swelling of cytoplasmic organelles, moderate chromatin condensation, loss of membrane integrity, and leakage of cellular components into the intercellular space (Festjens et al., 2006;

∗ Corresponding author. Tel.: +49 40 48051 351; fax: +49 40 48051 352. E-mail address: [email protected] 0168-1702/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.virusres.2010.10.012

Kroemer et al., 2009). Hence necrosis is generally considered to be pro-inflammatory. For a long time, necrosis was thought to be an unorganized form of cell death, occurring after acute physical or chemical injury. However, it is meanwhile well accepted that necrosis also occurs as a form of PCD. To distinguish it from acute cell destruction, this form of PCD has been termed programmed necrosis or necroptosis (Moquin and Chan, 2010). The third, least characterized form of PCD is autophagic cell death. It is accompanied by massive autophagic vacuolization of the cytoplasm. Whether autophagic cell death really constitutes a separate form of PCD or rather represents a failed attempt to rescue the cell by autophagy is still a matter of debate. Although criteria for these three forms of PCD have been defined, it is sometimes difficult to clearly classify an observed cell death phenotype, because cells can activate different death programs at the same time, leading to intermediate form of PCD (Kroemer et al., 2009). Cytomegaloviruses (CMVs) have a protracted replication cycle and cause persistent infections in their host (Mocarski et al., 2007). Therefore, they need to keep infected cells alive and metabolically active in order to ensure their own survival. They do this by expressing a remarkable array of cell death suppressors. This review summarizes the current knowledge on how CMVs block or delay the onset of PCD. 2. Inhibition of apoptosis at the mitochondrial checkpoint Caspases are important signal transducers within apoptotic signaling cascades. Their activation is regulated by proteins of the Bcl-2 family (Youle and Strasser, 2008). This family consists of pro- and antiapoptotic members, which differ in the number of

W. Brune / Virus Research 157 (2011) 144–150

145

HCMV UL38

UL36 UL37x3

UL37x1

MCMV M36

M38 M37

UL45

UL43

m38.5

m39

UL40

m41

m41.1

UL44

M43

M45 M44

Fig. 1. Schematic view of a 12 kb region of the HCMV genome and the corresponding region in the MCMV genome. The regions encode multiple cell death suppressors, which are shown in black. MCMV genes with significant sequence homologies to HCMV genes carry a capital M.

Bcl-2 homology (BH) domains. According to the current model, antiapoptotic Bcl-2 family proteins prevent caspase activation by preserving mitochondrial integrity (Youle and Strasser, 2008). The activity of the antiapoptotic Bcl-2 proteins is antagonized by the pro-apoptotic multidomain proteins Bax and Bak and the so-called BH3-only proteins, which posses only the third out of four BH domains. In response to apoptotic stimuli from within the cell, BH3-only proteins translocate to mitochondria where they counteract the function of pro-survival Bcl-2 proteins and activate the pro-apoptotic proteins Bax and Bak (Chipuk and Green, 2008). The monomeric proteins undergo a conformational change, oligomerize, and increase the permeability of the mitochondrial outer membrane (Wei et al., 2001). This leads to a release of cytochrome c into the cytosol and subsequent activation of caspases. The release of other mitochondrial proteins such as apoptosis-inducing factor (AIF), Smac/DIABLO, Htr2A/Omi, and endonuclease G also contributes to the execution of PCD (Chipuk and Green, 2008). Early studies suggested that Bak and Bax function in a largely redundant manner during development (Lindsten et al., 2000) and in response to various apoptosis inducers (Wei et al., 2001). However, more recent results indicated that the two proteins also fulfill some non-redundant functions, particularly during infectioninduced apoptosis (Brooks et al., 2007; Cartron et al., 2003; Kepp et al., 2007; Neise et al., 2008). While gammaherpesviruses express proteins homologous to the cellular antiapoptotic proteins Bcl-2 and Bcl-xL (Galluzzi et al., 2008), no such homolog has been identified in the genomes of the CMVs or other betaherpesviruses. A random screening experiment for antiapoptotic viral genes revealed a viral mitochondrionlocalized inhibitor of apoptosis (vMIA) encoded by ORF UL37x1 of HCMV (Goldmacher et al., 1999). The UL37x1/vMIA protein contains a mitochondrial-targeting domain and an antiapoptotic domain, both of which are necessary and sufficient for vMIA’s antiapoptotic function (Hayajneh et al., 2001). The protein shows no obvious sequence similarity to Bcl-2 family proteins and was initially believed to be structurally unrelated to Bcl-2 (Goldmacher et al., 1999). However, a more recent computer-based structural analysis predicted a fold similar to Bcl-xL (Pauleau et al., 2007). Hence, vMIA might mimic Bcl-2 family proteins in its 3dimensional structure as it has been shown to be the case for two poxvirus proteins (Kvansakul et al., 2007, 2008). Since its discovery, vMIA has been studied extensively in terms of antiapoptotic activity and molecular mechanism of action. It was shown to have a strong and broad antiapoptotic activity, protecting cells from both extrinsic signals and intrinsic apoptosisinducing stimuli (reviewed in Goldmacher, 2005). Functionally, vMIA resembles the cellular antiapoptotic protein Bcl-xL . However, the molecular mechanisms of action of the two proteins show certain differences. vMIA interacts with the growth arrest and DNA damage 45␣ (GADD45␣) protein and Bcl-xL , suggesting that vMIA acts together with these cellular proteins to suppress the release of proapoptotic factors from mitochondria (Smith and Mocarski,

2005). Moreover, vMIA interacts with Bax and sequesters activated and oligomerized Bax at mitochondria (Arnoult et al., 2004; Poncet et al., 2004). Using mouse fibroblasts lacking either Bax or Bak it was shown that vMIA protects murine cells from Bax- but not Bakmediated apoptosis (Arnoult et al., 2004). This observation raised the question of how to explain the broad antiapoptotic activity of vMIA in human cells. One possible explanation suggested that Bax is dominant over Bak in human cells, and that a potent Bax inhibitor might, therefore, be sufficient to protect human cells (Arnoult et al., 2004). Another study provided evidence that vMIA also interacts with Bak in human cells (Karbowski et al., 2006). However, the interaction of vMIA with Bak remains controversial (Arnoult et al., 2008). Murine cytomegalovirus also expresses a mitochondrial protein at a position in the viral genome analogous to HCMV UL37x1 (McCormick et al., 2005) (Fig. 1). This protein is encoded by ORF m38.5 and shows only minimal sequence similarity to HCMV vMIA. In functional studies, however, the protein behaved similar to UL37x1 in that it bound and sequestered Bax at mitochondria and inhibited Bax- but not Bak-mediated cell death (Arnoult et al., 2008; Jurak et al., 2008; Norris and Youle, 2008). The observation that MCMV-infected cells were protected from Baxand Bak-mediated apoptosis suggested that the virus encodes an additional, Bak-specific inhibitor of apoptosis (Jurak et al., 2008). This second mitochondrial inhibitor was recently identified and characterized (C¸am et al., 2010). It is the product of a small, previously unrecognized ORF named m41.1 (Fig. 1). The m41.1 protein localizes to mitochondria, interacts with Bak, and prevents Bak oligomerization. Consequently it was named viral inhibitor of Bak oligomerization (vIBO). Similar vIBO proteins are encoded by other rodent CMVs, but are missing in primate CMVs (C¸am et al., 2010). The roles of vMIA and vIBO during viral infection have been analyzed using recombinant viruses and mouse embryonic fibroblasts from bax or bak knockout mice. Deletion of m38.5 or m41.1 from the MCMV genome had only little effect on viral replication kinetics in fibroblasts (C¸am et al., 2010; Jurak et al., 2008; Manzur et al., 2009), but strongly impaired viral replication in cultured macrophages (C¸am et al., 2010; Manzur et al., 2009). An MCMV m38.5 deletion mutant also reached lower titers in the spleen of infected mice (Manzur et al., 2009), perhaps because the spleen is rich in macrophages and other antigen presenting cells. Excessive cell death was also seen in infected fibroblasts when cells were infected at a high multiplicity of infection or when infected cells were exposed to additional stress such as maintenance in exhausted medium or treatment with staurosporine (C¸am et al., 2010; Jurak et al., 2008). Somewhat different results were obtained with HCMV UL37x1 mutants. Deletion of UL37x1 in HCMV strain AD169 resulted in a virus that induced excessive apoptosis, grew poorly in normal fibroblasts, and could be grown to high titers only on complementing cells (Reboredo et al., 2004; Yu et al., 2003). Apoptosis was accompanied by caspase-9 and -3 activation and

146

W. Brune / Virus Research 157 (2011) 144–150

could be inhibited by the broad-spectrum caspase inhibitor, zVADfmk (Jurak and Brune, 2006; Reboredo et al., 2004). By contrast, a UL37x1 deletion mutant of the Towne strain replicated to almost the same titers as the parental wildtype virus in human fibroblasts (McCormick et al., 2005). However, cells infected with the mutant virus were more sensitive to proapoptotic stimuli (McCormick et al., 2005) and died a few days earlier than wt Towne-infected cells (McCormick et al., 2008). Cell death and fragmentation was not blocked by zVAD-fmk, but was inhibited by a serine protease inhibitor specific for HtrA2/Omi (McCormick et al., 2008). This is a protease released from mitochondria upon outer membrane permeabilization. Why infected cell demise under these conditions is caused primarily by HtrA2/Omi rather than by cytochrome c release with subsequent caspase-9 activation remains to be explored. It is also unknown why deletion of UL37x1 in AD169 has a stronger impact on cell survival and virus progeny production than the same mutation in the Towne strain. A suggested explanation is that AD169 exerts a stronger proapoptotic stress on infected cells than Towne (McCormick et al., 2005). The fact that AD169 carries an inactivating mutation in UL36 (another antiapoptotic protein described below (Skaletskaya et al., 2001)) might also be responsible. The mitochondrial cell death suppressors of HCMV and MCMV show certain species-specific differences. HCMV vMIA has a broad antiapoptotic activity in human cells, but blocks only Bax-mediated cell death in murine cells (Arnoult et al., 2004; Poncet et al., 2004). Conversely, MCMV vMIA has only a limited or transient antiapoptotic effect on human cells (Arnoult et al., 2008; McCormick et al., 2005), perhaps because Bak-mediated cell death is not inhibited. The MCMV Bak inhibitor, vIBO, also seems to have a weak activity in human cells (C¸am et al., unpublished). Consistent with these observations, MCMV infection induces apoptosis in human cells (Jurak and Brune, 2006). The early onset of apoptosis prevents efficient replication of MCMV in human cells and limits MCMV dissemination. This obstacle can be overcome by overexpressing Bcl-2 or a viral analog in infected cells (Jurak and Brune, 2006). Based on available data it can be concluded that the MCMV mitochondrial apoptosis inhibitors have insufficient activity in human cells and/or that MCMV exerts a stronger proapoptotic stress on human cells than on mouse cells (Jurak and Brune, 2006; Schumacher et al., 2010). The UL37x1 ORF encodes vMIA, but also serves as the first exon of the spliced UL37 gene transcripts (Fig. 1). The functions of the UL37 glycoproteins have not been fully elucidated, but it has been assumed that they also play a role in cell death suppression (Colberg-Poley et al., 2000). It is noteworthy that UL37x1/vMIA has additional functions that might be more indirectly involved in cell death suppression. It induces the release of Ca2+ ions from the endoplasmic reticulum into the cytosol, leading to cell rounding, swelling, and reorganization of the actin cytoskeleton (Sharon-Friling et al., 2006). These morphological changes could be inhibited by a Ca2+ chelating agent. Apart from contributing to the CMV-specific cytopathic effect, Ca2+ release likely has additional consequences such as the induction of the unfolded protein response (Sharon-Friling et al., 2006). Another study also found that the cytopathic morphology of HCMV-infected cells depended on vMIA, but attributed this effect to an impaired mitochondrial ATP production caused by vMIA (Poncet et al., 2006). Besides regulating apoptosis, Bax and Bak also promote mitochondrial fusion, thereby contributing to the maintenance of normal interconnected mitochondrial networks in healthy cells (Karbowski et al., 2006). HCMV and MCMV vMIA can both disrupt mitochondrial networks, probably by binding and sequestering Bax and Bak, resulting in a fragmented mitochondrial phenotype (McCormick et al., 2003b; Norris and Youle, 2008). The functional consequences of mitochondrial fragmentation for the viral life cycle have not been determined yet.

3. Inhibition of death receptor-dependent apoptosis Death receptors are cell surface receptors that transmit apoptotic signals initiated by specific ligands such as Fas ligand, TNF␣, or TRAIL. Stimulation of death receptors leads to the recruitment and proteolytic activation of procaspase-8, thereby initiating the extrinsic apoptosis pathway (Thorburn, 2004). This pathway is blocked by the HCMV UL36 protein, which binds to procaspase8 and prevents its activation (Skaletskaya et al., 2001). This viral inhibitor of caspase-8-induced apoptosis (vICA) is highly conserved among the mammalian betaherpesviruses, suggesting that it fulfills an important role during virus infection (McCormick et al., 2003a). Surprisingly, two HCMV laboratory strains (AD169varATCC and TownevarRIT ) carry inactivating mutations in UL36 (Skaletskaya et al., 2001). Moreover, deletion of the vICA gene in HCMV or MCMV did not impair viral replication in fibroblasts (Dunn et al., 2003; Ménard et al., 2003). However, vICA-deficient viruses induced apoptosis of macrophages and were massively impaired in their ability to replicate in macrophages (McCormick et al., 2010; Ménard et al., 2003), a cell type important for dissemination within the host organism (Hanson et al., 1999). Indeed, an MCMV lacking the vICA gene, M36, was severely growth restricted in mice and produced more apoptotic cells in infected organs (Cicin-Sain et al., 2008). Virulence was largely restored when a dominant-negative FADD was inserted in place of M36, confirming that the main function of M36 is to inhibit death receptor-dependent apoptosis (Cicin-Sain et al., 2008). Similar to the aforementioned HCMV laboratory strains, the rhesus CMV strain 68-1 also contains an inactivating mutation in UL36. Viral replication in epithelial cells was improved when this mutation was repaired (Lilja and Shenk, 2008), supporting the notion that vICA is important for replicative fitness in non-fibroblast cells. Conversely, the presence of UL36 mutations in laboratory strains of both human and rhesus CMV suggests that an intact vICA gene might even be detrimental for replication in cultured fibroblasts, on which the CMVs are usually propagated.

4. Blocking necrosis, an alternative form of programmed cell death In some cells, death receptor stimulation can also activate a caspase-independent form of programmed cell death that has been named programmed necrosis or necroptosis (Festjens et al., 2006; Moquin and Chan, 2010; Vandenabeele et al., 2010). This pathway can be initiated when caspase-dependent apoptosis is blocked by a synthetic caspase inhibitor (such as zVAD-fmk) or a viral caspase-8 inhibitor (Festjens et al., 2006). As described above, the CMVs express vICA, and thus infected cells might be sensitized to death receptor-induced programmed necrosis, unless they encode another protein that prevents this cell death program. Indeed, it has recently been shown that the MCMV M45 protein inhibits necrosis upon TNF receptor or Fas stimulation (Mack et al., 2008). M45 accomplishes this by interacting with the receptor-interacting protein 1 (RIP1) and RIP3 (Mack et al., 2008; Upton et al., 2008). RIP1 is an adaptor kinase that mediates, on the one hand, the activation of the transcription factor NF-␬B and mitogen-activated protein kinases and, on the other hand, the induction of necrosis (Festjens et al., 2007). All signaling pathways downstream of RIP1 appear to be inhibited by M45, and therefore M45 was named viral inhibitor of RIP-mediated signaling (vIRS) (Mack et al., 2008). Recent work in different laboratories has shown that TNF receptor-induced necrosis is activated by a RIP1–RIP3 complex, and that RIP3 is essential for necrosis induction (reviewed in Moquin and Chan, 2010). M45 inhibits the RIP1–RIP3 interaction with the help of a RIP homotypic interaction motif (RHIM) present near the N terminus of M45

W. Brune / Virus Research 157 (2011) 144–150

(Upton et al., 2010). Moreover, M45 also inhibits RIP3-dependent necrosis in the absence of RIP1 by interacting with RIP3, suggesting that RIP3 can also be activated by other proteins (Upton et al., 2010). Likely candidates are the adaptor protein TRIF and the cytosolic DNA sensor, DAI, as these are the only other proteins known to carry a RHIM (Kaiser et al., 2008; Rebsamen et al., 2009). M45 is a sequence homolog to ribonucleotide reductase R1 subunits, but does not possess ribonucleotide reductase activity (Lembo and Brune, 2009; Lembo et al., 2004). In addition to the R1 homology domain, which comprises roughly the C-terminal two thirds of the protein, it has a unique N terminus. The RHIM motif within the unique N terminus is necessary for inhibiting necrosis in cell culture as well as in infected mice (Upton et al., 2008, 2010). It has not been fully resolved whether or not the N-terminal part of M45 is also sufficient to block necrosis as viruses expressing a C-terminally truncated M45 protein do not suppress infectioninduced cell death (Brune et al., 2001; Lembo et al., 2004). However, the C-terminal domain is essential for the inhibition of NF-␬B activation (Mack et al., 2008). More recent work indicates that this function might involve an additional, RIP-independent mechanism (Fliss and Brune, unpublished results). M45 was originally described as a determinant of MCMV’s ability to replicate in endothelial cells (Brune et al., 2001). When M45 was mutated, infected endothelial cells died rapidly, thereby preventing a spread to neighboring cells. In contrast, fibroblasts were more resistant to infection-induced cell death, and M45 mutant viruses replicated in these cells almost to the same titers as the wildtype virus (Brune et al., 2001). More recent work strongly suggests that the level of RIP3 expression determines the sensitivity of a cell to programmed necrosis. Many murine fibroblasts express very low levels of RIP3, whereas endothelial cells (and also a particular fibroblast line) express higher amounts of RIP3 (Upton et al., 2010; Zhang et al., 2009). The RHIM motif is conserved in the R1 homologs of other rodent CMVs (rat CMV, guinea pig CMV, tupaia herpesvirus) (Lembo and Brune, 2009; Rebsamen et al., 2009), suggesting that these proteins suppress programmed necrosis like MCMV M45 does. By contrast, the human CMV R1 homolog, UL45 (Fig. 1), does not carry a RHIM motif and is not required for the inhibition of cell death or for virus replication in endothelial cells (Hahn et al., 2002; Lembo and Brune, 2009). UL45 is also devoid of ribonucleotide reductase activity (Patrone et al., 2003), and its function for the virus remains enigmatic.

5. Preservation of metabolic activity during stress The mechanism by which RIP3 triggers programmed necrosis has not been fully elucidated, but the current view is that the production of reactive oxygen species (ROS) within the cell is an important effector mechanism (Moquin and Chan, 2010; Vandenabeele et al., 2010). The mitochondria are major producers of ROS, and the function of the mitochondrial respiratory complex I can be compromised by ROS leading to mitochondrial membrane potential loss and cell death (Davis et al., 2010). Intriguingly, human CMV can stabilize mitochondrial complex I, thereby reducing ROS production and preserving mitochondrial respiration and ATP production (Reeves et al., 2007; Zhao et al., 2010). During infection, an HCMV 2.7 kb noncoding RNA (␤2.7) binds to GRIM-19, a subunit of complex I, and prevents its relocalization. Deletion of the ␤2.7 gene from the viral genome had little impact on HCMV replication in fibroblasts when the cells were supplied with glucose-rich medium. However, titers of the ␤2.7 deletion mutant dropped significantly in the presence of the mitochondrial poison rotenone or when cells were cultured in glucose-depleted media (Reeves et al., 2007).

147

While the ␤2.7 RNA stabilizes mitochondrial ATP production, HCMV UL37x1/vMIA was reported to reduce ATP synthesis, probably by compromising the function of the mitochondrial phosphate carrier, a component of the ATP synthasome (Poncet et al., 2006). How vMIA reduces the phosphate carrier’s activity has not been resolved, but it was speculated that it might be an indirect consequence of vMIA’s previously reported interaction with the adenine nucleotide transporter (ANT), another component of the ATP synthasome (Goldmacher et al., 1999; Poncet et al., 2006). On the other hand, later work from the Goldmacher laboratory suggested that the interaction with ANT might be of a nonspecific nature (Arnoult et al., 2004; Goldmacher, 2005). It also remains a matter of speculation why reduced ATP levels would be favorable for the virus. UL38 is another cell death suppressor whose function is to preserve metabolic activity under stress conditions (Moorman et al., 2008). The UL38 protein interacts with the TSC1 and TSC2 proteins of the tuberous sclerosis complex (TSC), a protein complex that integrates various stress signals. By binding to the TSC, UL38 prevents TSC-mediated activation of the mammalian target of rapamycin complex 1 (mTORC1), which responds to stress by reducing protein synthesis and cell growth (Moorman et al., 2008). Deletion of UL38 from the viral genome resulted in a virus that grew poorly in fibroblasts and induced excessive cell death (Terhune et al., 2007). However, it is not clear to which extent the cell death inhibiting activity of UL38 results from the interaction with the TSC. It appears likely that UL38 fulfills additional functions that are more directly involved in cell death suppression, particularly since it has been shown that UL38 protects cells from cell death triggered by the endoplasmic reticulum stress inducers thapsigargin and tunicamycin or by infection with an E1B-19k-deficient adenovirus, but not by death receptor stimulation (Terhune et al., 2007; Xuan et al., 2009). However, the molecular mechanism(s) of these additional functions remain unknown. 6. Activation of survival pathways The immediate-early proteins 1 and 2 (IE1 and IE2) were the first HCMV proteins for which a cell death-inhibiting activity was demonstrated (Zhu et al., 1995). Transient or stable expression of these proteins in HeLa cells protected the cells from apoptosis induced by TNF␣ + cycloheximide or by an E1B-19k-deficient adenovirus, but not by UV irradiation (Zhu et al., 1995). Later studies showed that the viral proteins activate a phosphatidylinositide 3 -OH kinase (PI3K)-dependent survival pathway involving the cellular kinase Akt (Lukac and Alwine, 1999; Yu and Alwine, 2002). This pathway is usually activated by trophic factors, and appears to be required also for CMV replication (Johnson et al., 2001). How the IE proteins promote activation of the PI3K pathway and which domains are required remains to be investigated. It would also be enlightening to study the role of IE1/2-induced PI3K activation during HCMV infection. However, this is difficult to do because IE1 and IE2 have multiple important functions in gene regulation, modulation of the cell cycle, and immune subversion (Meier and Stinski, 2006). Consequently, IE1-deficient HCMVs are severely growthrestricted (Greaves and Mocarski, 1998; Mocarski et al., 1996), and IE2 knockout mutants do not replicate at all (Heider et al., 2002; Marchini et al., 2001). 7. Summary and discussion Cytomegaloviruses are slowly replicating viruses that must keep the host cell alive for a sufficiently long time to ensure virus replication. As there are different forms of cell death and multiple pathways leading to them, it is not surprising that the CMVs had to

148

W. Brune / Virus Research 157 (2011) 144–150

Table 1

HCMV

MCMV

Gene product

Function, mechanism

Gene product

Function, mechanism

UL36 vICA

• Viral Inhibitor of Caspase-8-induced Apoptosis • Important for virus replication in macrophages

M36 vICA

• Viral Inhibitor of Caspase-8-induced Apoptosis • Important for virus replication in macrophages and dissemination in vivo

UL37x1 vMIA

• Viral Mitochondria-localized Inhibitor of Apoptosis • Inhibits Bax-mediated apoptosis • Inhibits HtrA2/Omi-mediated cell death • Interacts with GADD45␣ • Disrupts mitochondrial networks • Induces ER Ca2+ release

m38.5 vMIA

• Viral Mitochondria-localized Inhibitor of Apoptosis • Inhibits Bax- but not Bak- mediated apoptosis • Disrupts mitochondrial networks in Bak-deficient cells • Important for replication in splenocytes in vivo

UL38

• Inhibits TSC-mediated stress response • Inhibits ER stress-induced cell death

M38

Function unknown

m41.1 vIBO

• Viral Inhibitor of Bak Oligomerization • Inhibits Bak- but not Bax-mediated apoptosis • Important for replication in macrophages

M45 vIRS

• Viral Inhibitor of RIP-mediated Signaling • Inhibits RIP1/RIP3-dependent programmed necrosis • Inhibits RIP1-dependent NF-␬B and p38 MAPK activation • Important for replication in endothelial cells and macrophages • Crucial for replication and dissemination in vivo

IE1, IE3 m41

No known effects on cell death • Golgi-localized cell death suppressor • Important for survival of infected macrophages

UL45

Function unknown (does not interact with RIP1)

␤2.7 RNA

• Binds to mitochondrial respiratory complex I • Stabilizes mitochondrial membrane potential and ATP production • Activate PI3K/Akt-dependent survival pathways

IE1, IE2

find different ways to block the induction of programmed cell death or activate survival pathways, which balance the death-promoting signals emanating from virus infection and replication. Apoptosis is the best-characterized form of programmed cell death and has frequently been used synonymously to PCD. In recent years, programmed necrosis and autophagic cell death have also been appreciated as important modes of PCD. Although criteria to discern the different forms of cell death exist (Kroemer et al., 2009), it is often difficult to keep them apart, especially when dealing with virus-infected cells. These cells are exposed to multiple death-inducing stimuli, which are largely (but perhaps not fully) antagonized by viral gene products. The CMV cell death suppressors known to date are listed and in Table 1, and their mechanisms are shown schematically in Fig. 2.

Most of the CMV cell death suppressors identified to date are clustered in a relatively small region of the large viral genome (Fig. 1). Curiously, some of the corresponding genes are spliced or overlapping with other genes. Such a complex and dense coding pattern is rather unusual for these large viruses. Moreover, the evolutionary origin of the genes remains enigmatic for most of the CMV cell death suppressors. The proteins show no apparent sequence homology to any cellular gene product. Therefore, it appears unlikely that the genes have been captured from the host genome, as it is assumed to be the case for the Bcl-2 homologs and the viral FLIPs (FLICE [=caspase-8]-like inhibitory proteins) found in gammaherpesviruses and a few other viruses (Hardwick and Bellows, 2003; Thome et al., 1997). It remains mysterious why CMVs have evolved their own cell death inhibitors instead

Fig. 2. Cell death inhibitors of HCMV and MCMV, and their cellular target proteins. The functions are summarized in table 1.

W. Brune / Virus Research 157 (2011) 144–150

of capturing them. One exception is the M45 protein, which shows obvious sequence similarity to ribonucleotide reductase R1 subunits. Here, the homology to a cellular protein is rather misleading, as M45 and its homologs in other betaherpesviruses have lost their ribonucleotide reductase activity and acquired a new function by a process termed “evolutionary tinkering” (Lembo and Brune, 2009). For a few viral proteins, the mechanism of their cell deathinhibiting activity still needs to be clarified. This is, for instance, the case for m41, a Golgi-localized protein of MCMV (Brune et al., 2003). Determining m41’s function proved to be complicated, because it had to be separated from the function of m41.1 (C¸am et al., 2010), which is encoded by a smaller ORF nested within the m41 ORF (Fig. 1). Likewise, the mechanism by which the UL38 protein inhibits ER stress-induced cell death remains unknown. The diversity of the viral cell death suppressors probably reflects the diversity of the cellular suicide pathways. Hence it is likely that the large CMV genomes harbor further antiapoptotic and anti-necrotic genes, and possibly antagonists of autophagic cell death will be found as well in future investigations.

Acknowledgment I apologize to all colleagues whose work could not be cited. This work was supported by the Deutsche Forschungsgemeinschaft (BR 1730/3-1).

References Albert, M.L., Sauter, B., Bhardwaj, N., 1998. Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392 (6671), 86–89. Arnoult, D., Bartle, L.M., Skaletskaya, A., Poncet, D., Zamzami, N., Park, P.U., Sharpe, J., Youle, R.J., Goldmacher, V.S., 2004. Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated apoptosis by binding and sequestering Bax at mitochondria. Proc. Natl. Acad. Sci. U.S.A. 101 (21), 7988–7993. Arnoult, D., Skaletskaya, A., Estaquier, J., Dufour, C., Goldmacher, V.S., 2008. The murine cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Baxmediated mitochondrial outer membrane permeabilization. Apoptosis 13 (9), 1100–1110. Brooks, C., Wei, Q., Feng, L., Dong, G., Tao, Y., Mei, L., Xie, Z.J., Dong, Z., 2007. Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins. Proc. Natl. Acad. Sci. U.S.A. 104 (28), 11649–11654. Brune, W., Ménard, C., Heesemann, J., Koszinowski, U.H., 2001. A ribonucleotide reductase homolog of cytomegalovirus and endothelial cell tropism. Science 291 (5502), 303–305. Brune, W., Nevels, M., Shenk, T., 2003. Murine cytomegalovirus m41 open reading frame encodes a Golgi-localized antiapoptotic protein. J. Virol. 77 (21), 11633–11643. C¸am, M., Handke, W., Picard-Maureau, M., Brune, W., 2010. Cytomegaloviruses inhibit Bak- and Bax-mediated apoptosis with two separate viral proteins. Cell. Death Differ. 17 (4), 655–665. Cartron, P.F., Juin, P., Oliver, L., Martin, S., Meflah, K., Vallette, F.M., 2003. Nonredundant role of Bax and Bak in Bid-mediated apoptosis. Mol. Cell. Biol. 23 (13), 4701–4712. Chipuk, J.E., Green, D.R., 2008. How do BCL-2 proteins induce mitochondrial outer membrane permeabilization? Trends Cell Biol. 18 (4), 157–164. Cicin-Sain, L., Ruzsics, Z., Podlech, J., Bubic, I., Menard, C., Jonjic, S., Reddehase, M.J., Koszinowski, U.H., 2008. Dominant-negative FADD rescues the in vivo fitness of a cytomegalovirus lacking an antiapoptotic viral gene. J. Virol. 82 (5), 2056–2064. Colberg-Poley, A.M., Patel, M.B., Erezo, D.P., Slater, J.E., 2000. Human cytomegalovirus UL37 immediate-early regulatory proteins traffic through the secretory apparatus and to mitochondria. J. Gen. Virol. 81 (Pt 7), 1779–1789. Davis, C.W., Hawkins, B.J., Ramasamy, S., Irrinki, K.M., Cameron, B.A., Islam, K., Daswani, V.P., Doonan, P.J., Manevich, Y., Madesh, M., 2010. Nitration of the mitochondrial complex I subunit NDUFB8 elicits RIP1- and RIP3-mediated necrosis. Free Radic. Biol. Med. 48 (2), 306–317. Dunn, W., Chou, C., Li, H., Hai, R., Patterson, D., Stolc, V., Zhu, H., Liu, F., 2003. Functional profiling of a human cytomegalovirus genome. Proc. Natl. Acad. Sci. U.S.A. 100 (24), 14223–14228. Festjens, N., Vanden Berghe, T., Cornelis, S., Vandenabeele, P., 2007. RIP1, a kinase on the crossroads of a cell’s decision to live or die. Cell. Death Differ. 14 (3), 400–410. Festjens, N., Vanden Berghe, T., Vandenabeele, P., 2006. Necrosis, a well-orchestrated form of cell demise: signalling cascades, important mediators and concomitant immune response. Biochim. Biophys. Acta 1757 (9–10), 1371–1387. Galluzzi, L., Brenner, C., Morselli, E., Touat, Z., Kroemer, G., 2008. Viral control of mitochondrial apoptosis. PLoS Pathog. 4 (5), e1000018.

149

Goldmacher, V.S., 2005. Cell death suppression by cytomegaloviruses. Apoptosis 10 (2), 251–265. Goldmacher, V.S., Bartle, L.M., Skaletskaya, A., Dionne, C.A., Kedersha, N.L., Vater, C.A., Han, J., Lutz, R.J., Watanabe, S., McFarland, E.D., Kieff, E.D., Mocarski, E.S., Chittenden, T., 1999. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc. Natl. Acad. Sci. U.S.A. 96 (22), 12536–12541. Greaves, R.F., Mocarski, E.S., 1998. Defective growth correlates with reduced accumulation of a viral DNA replication protein after low-multiplicity infection by a human cytomegalovirus ie1 mutant. J. Virol. 72 (1), 366–379. Hahn, G., Khan, H., Baldanti, F., Koszinowski, U., Revello, M., Gerna, G., 2002. The human cytomegalovirus ribonucleotide reductase homolog UL45 is dispensable for growth in endothelial cells, as determined by a BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-type characteristics. J. Virol. 76 (18), 9551–9555. Hanson, L.K., Slater, J.S., Karabekian, Z., Virgin, H.W., Biron, C.A., Ruzek, M.C., van Rooijen, N., Ciavarra, R.P., Stenberg, R.M., Campbell, A.E., 1999. Replication of murine cytomegalovirus in differentiated macrophages as a determinant of viral pathogenesis. J. Virol. 73 (7), 5970–5980. Hardwick, J.M., Bellows, D.S., 2003. Viral versus cellular BCL-2 proteins. Cell. Death Differ. 10 (Suppl. 1), S68–S76. Hayajneh, W.A., Colberg-Poley, A.M., Skaletskaya, A., Bartle, L.M., Lesperance, M.M., Contopoulos-Ioannidis, D.G., Kedersha, N.L., Goldmacher, V.S., 2001. The sequence and antiapoptotic functional domains of the human cytomegalovirus UL37 exon 1 immediate early protein are conserved in multiple primary strains. Virology 279 (1), 233–240. Heider, J.A., Bresnahan, W.A., Shenk, T.E., 2002. Construction of a rationally designed human cytomegalovirus variant encoding a temperature-sensitive immediateearly 2 protein. Proc. Natl. Acad. Sci. U.S.A. 99 (5), 3141–3146. Johnson, R.A., Wang, X., Ma, X.L., Huong, S.M., Huang, E.S., 2001. Human cytomegalovirus up-regulates the phosphatidylinositol 3-kinase (PI3-K) pathway: inhibition of PI3-K activity inhibits viral replication and virus-induced signaling. J. Virol. 75 (13), 6022–6032. Jurak, I., Brune, W., 2006. Induction of apoptosis limits cytomegalovirus crossspecies infection. EMBO J. 25 (11), 2634–2642. Jurak, I., Schumacher, U., Simic, H., Voigt, S., Brune, W., 2008. Murine cytomegalovirus m38.5 protein inhibits Bax-mediated cell death. J. Virol. 82 (10), 4812–4822. Kaiser, W.J., Upton, J.W., Mocarski, E.S., 2008. Receptor-interacting protein homotypic interaction motif-dependent control of NF-kappa B activation via the DNA-dependent activator of IFN regulatory factors. J. Immunol. 181 (9), 6427–6434. Karbowski, M., Norris, K.L., Cleland, M.M., Jeong, S.Y., Youle, R.J., 2006. Role of Bax and Bak in mitochondrial morphogenesis. Nature 443 (7112), 658–662. Kepp, O., Rajalingam, K., Kimmig, S., Rudel, T., 2007. Bak and Bax are nonredundant during infection- and DNA damage-induced apoptosis. EMBO J. 26 (3), 825–834. Kroemer, G., Galluzzi, L., Vandenabeele, P., Abrams, J., Alnemri, E.S., Baehrecke, E.H., Blagosklonny, M.V., El-Deiry, W.S., Golstein, P., Green, D.R., Hengartner, M., Knight, R.A., Kumar, S., Lipton, S.A., Malorni, W., Nunez, G., Peter, M.E., Tschopp, J., Yuan, J., Piacentini, M., Zhivotovsky, B., Melino, G., 2009. Classification of cell death: recommendations of the Nomenclature Committee on Cell Death 2009. Cell. Death Differ. 16 (1), 3–11. Kvansakul, M., van Delft, M.F., Lee, E.F., Gulbis, J.M., Fairlie, W.D., Huang, D.C., Colman, P.M., 2007. A structural viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak. Mol. Cell 25 (6), 933–942. Kvansakul, M., Yang, H., Fairlie, W.D., Czabotar, P.E., Fischer, S.F., Perugini, M.A., Huang, D.C., Colman, P.M., 2008. Vaccinia virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly selective subset of BH3containing death ligands. Cell. Death Differ. 15 (10), 1564–1571. Lembo, D., Brune, W., 2009. Tinkering with a viral ribonucleotide reductase. Trends Biochem. Sci. 34 (1), 25–32. Lembo, D., Donalisio, M., Hofer, A., Cornaglia, M., Brune, W., Koszinowski, U., Thelander, L., Landolfo, S., 2004. The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis. J. Virol. 78 (8), 4278–4288. Lilja, A.E., Shenk, T., 2008. Efficient replication of rhesus cytomegalovirus variants in multiple rhesus and human cell types. Proc. Natl. Acad. Sci. U.S.A. 105 (50), 19950–19955. Lindsten, T., Ross, A.J., King, A., Zong, W.X., Rathmell, J.C., Shiels, H.A., Ulrich, E., Waymire, K.G., Mahar, P., Frauwirth, K., Chen, Y., Wei, M., Eng, V.M., Adelman, D.M., Simon, M.C., Ma, A., Golden, J.A., Evan, G., Korsmeyer, S.J., MacGregor, G.R., Thompson, C.B., 2000. The combined functions of proapoptotic Bcl-2 family members bak and bax are essential for normal development of multiple tissues. Mol. Cell 6 (6), 1389–1399. Lukac, D.M., Alwine, J.C., 1999. Effects of human cytomegalovirus major immediateearly proteins in controlling the cell cycle and inhibiting apoptosis: studies with ts13 cells. J. Virol. 73 (4), 2825–2831. Mack, C., Sickmann, A., Lembo, D., Brune, W., 2008. Inhibition of proinflammatory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein. Proc. Natl. Acad. Sci. U.S.A. 105 (8), 3094–3099. Manzur, M., Fleming, P., Huang, D.C., Degli-Esposti, M.A., Andoniou, C.E., 2009. Virally mediated inhibition of Bax in leukocytes promotes dissemination of murine cytomegalovirus. Cell. Death Differ. 16 (2), 312–320. Marchini, A., Liu, H., Zhu, H., 2001. Human cytomegalovirus with IE-2 (UL122) deleted fails to express early lytic genes. J. Virol. 75 (4), 1870–1878.

150

W. Brune / Virus Research 157 (2011) 144–150

McCormick, A.L., Meiering, C.D., Smith, G.B., Mocarski, E.S., 2005. Mitochondrial cell death suppressors carried by human and murine cytomegalovirus confer resistance to proteasome inhibitor-induced apoptosis. J. Virol. 79 (19), 12205–12217. McCormick, A.L., Roback, L., Livingston-Rosanoff, D., St Clair, C., 2010. The human cytomegalovirus UL36 gene controls caspase-dependent and -independent cell death programs activated by infection of monocytes differentiating to macrophages. J. Virol. 84 (10), 5108–5123. McCormick, A.L., Roback, L., Mocarski, E.S., 2008. HtrA2/Omi terminates cytomegalovirus infection and is controlled by the viral mitochondrial inhibitor of apoptosis (vMIA). PLoS Pathog. 4 (5), e1000063. McCormick, A.L., Skaletskaya, A., Barry, P.A., Mocarski, E.S., Goldmacher, V.S., 2003a. Differential function and expression of the viral inhibitor of caspase 8-induced apoptosis (vICA) and the viral mitochondria-localized inhibitor of apoptosis (vMIA) cell death suppressors conserved in primate and rodent cytomegaloviruses. Virology 316 (2), 221–233. McCormick, A.L., Smith, V.L., Chow, D., Mocarski, E.S., 2003b. Disruption of mitochondrial networks by the human cytomegalovirus UL37 gene product viral mitochondrion-localized inhibitor of apoptosis. J. Virol. 77 (1), 631–641. Meier, J.L., Stinski, M.F., 2006. Major immediate-early enhancer and its gene products. In: Reddehase, M.J. (Ed.), Cytomegaloviruses: Molecular Biology and Immunology. Caister, Wymondham, UK, pp. 150–166. Ménard, C., Wagner, M., Ruzsics, Z., Holak, K., Brune, W., Campbell, A., Koszinowski, U., 2003. Role of murine cytomegalovirus US22 gene family members for replication in macrophages. J. Virol. 77 (10), 5557–5570. Mocarski, E.S., Kemble, G.W., Lyle, J.M., Greaves, R.F., 1996. A deletion mutant in the human cytomegalovirus gene encoding IE1(491aa) is replication defective due to a failure in autoregulation. Proc. Natl. Acad. Sci. U.S.A. 93 (21), 11321–11326. Mocarski, E.S., Shenk, T., Pass, R.F., 2007. Cytomegaloviruses. In: Knipe, D.M., Howley, P.M. (Eds.), Fields Virology, 5th ed. Lippincott, Williams and Wilkins, Philadelphia, pp. 2701–2772. Moorman, N.J., Cristea, I.M., Terhune, S.S., Rout, M.P., Chait, B.T., Shenk, T., 2008. Human cytomegalovirus protein UL38 inhibits host cell stress responses by antagonizing the tuberous sclerosis protein complex. Cell Host Microbe 3 (4), 253–262. Moquin, D., Chan, F.K., 2010. The molecular regulation of programmed necrotic cell injury. Trends Biochem. Sci. 35 (8), 434–441. Neise, D., Graupner, V., Gillissen, B.F., Daniel, P.T., Schulze-Osthoff, K., Janicke, R.U., Essmann, F., 2008. Activation of the mitochondrial death pathway is commonly mediated by a preferential engagement of Bak. Oncogene 27 (10), 1387–1396. Norris, K.L., Youle, R.J., 2008. Cytomegalovirus proteins vMIA and m38.5 link mitochondrial morphogenesis to Bcl-2 family proteins. J. Virol. 82 (13), 6232–6243. Patrone, M., Percivalle, E., Secchi, M., Fiorina, L., Pedrali-Noy, G., Zoppe, M., Baldanti, F., Hahn, G., Koszinowski, U.H., Milanesi, G., Gallina, A., 2003. The human cytomegalovirus UL45 gene product is a late, virion-associated protein and influences virus growth at low multiplicities of infection. J. Gen. Virol. 84 (Pt 12), 3359–3370. Pauleau, A.L., Larochette, N., Giordanetto, F., Scholz, S.R., Poncet, D., Zamzami, N., Goldmacher, V.S., Kroemer, G., 2007. Structure–function analysis of the interaction between Bax and the cytomegalovirus-encoded protein vMIA. Oncogene 26 (50), 7067–7080. Poncet, D., Larochette, N., Pauleau, A.L., Boya, P., Jalil, A.A., Cartron, P.F., Vallette, F., Schnebelen, C., Bartle, L.M., Skaletskaya, A., Boutolleau, D., Martinou, J.C., Goldmacher, V.S., Kroemer, G., Zamzami, N., 2004. An anti-apoptotic viral protein that recruits Bax to mitochondria. J. Biol. Chem. 279 (21), 22605–22614. Poncet, D., Pauleau, A.L., Szabadkai, G., Vozza, A., Scholz, S.R., Le Bras, M., Briere, J.J., Jalil, A., Le Moigne, R., Brenner, C., Hahn, G., Wittig, I., Schagger, H., Lemaire, C., Bianchi, K., Souquere, S., Pierron, G., Rustin, P., Goldmacher, V.S., Rizzuto, R., Palmieri, F., Kroemer, G., 2006. Cytopathic effects of the cytomegalovirusencoded apoptosis inhibitory protein vMIA. J. Cell Biol. 174 (7), 985– 996. Reboredo, M., Greaves, R.F., Hahn, G., 2004. Human cytomegalovirus proteins encoded by UL37 exon 1 protect infected fibroblasts against virus-induced apop-

tosis and are required for efficient virus replication. J. Gen. Virol. 85 (Pt 12), 3555–3567. Rebsamen, M., Heinz, L.X., Meylan, E., Michallet, M.C., Schroder, K., Hofmann, K., Vazquez, J., Benedict, C.A., Tschopp, J., 2009. DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB. EMBO Rep. 10 (8), 916–922. Reeves, M.B., Davies, A.A., McSharry, B.P., Wilkinson, G.W., Sinclair, J.H., 2007. Complex I binding by a virally encoded RNA regulates mitochondria-induced cell death. Science 316 (5829), 1345–1348. Schumacher, U., Handke, W., Jurak, I., Brune, W., 2010. Mutations in the M112/M113coding region facilitate murine cytomegalovirus replication in human cells. J. Virol. 84 (16), 7994–8006. Sharon-Friling, R., Goodhouse, J., Colberg-Poley, A.M., Shenk, T., 2006. Human cytomegalovirus pUL37 × 1 induces the release of endoplasmic reticulum calcium stores. Proc. Natl. Acad. Sci. U.S.A. 103 (50), 19117–19122. Skaletskaya, A., Bartle, L.M., Chittenden, T., McCormick, A.L., Mocarski, E.S., Goldmacher, V.S., 2001. A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation. Proc. Natl. Acad. Sci. U.S.A. 98 (14), 7829–7834. Smith, G.B., Mocarski, E.S., 2005. Contribution of GADD45 family members to cell death suppression by cellular Bcl-xL and cytomegalovirus vMIA. J. Virol. 79 (23), 14923–14932. Terhune, S., Torigoi, E., Moorman, N., Silva, M., Qian, Z., Shenk, T., Yu, D., 2007. Human cytomegalovirus UL38 protein blocks apoptosis. J. Virol. 81 (7), 3109–3123. Thome, M., Schneider, P., Hofmann, K., Fickenscher, H., Meinl, E., Neipel, F., Mattmann, C., Burns, K., Bodmer, J.L., Schroter, M., Scaffidi, C., Krammer, P.H., Peter, M.E., Tschopp, J., 1997. Viral FLICE-inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 386 (6624), 517–521. Thorburn, A., 2004. Death receptor-induced cell killing. Cell. Signal. 16 (2), 139–144. Upton, J.W., Kaiser, W.J., Mocarski, E.S., 2008. Cytomegalovirus M45 Cell Death Suppression Requires Receptor-interacting Protein (RIP) Homotypic Interaction Motif (RHIM)-dependent Interaction with RIP1. J. Biol. Chem. 283 (25), 16966–16970. Upton, J.W., Kaiser, W.J., Mocarski, E.S., 2010. Virus inhibition of RIP3-dependent necrosis. Cell Host Microbe 7 (4), 302–313. Vandenabeele, P., Declercq, W., Van Herreweghe, F., Vanden Berghe, T., 2010. The role of the kinases RIP1 and RIP3 in TNF-induced necrosis. Sci. Signal. 3 (115), re4. Wei, M.C., Zong, W.X., Cheng, E.H., Lindsten, T., Panoutsakopoulou, V., Ross, A.J., Roth, K.A., MacGregor, G.R., Thompson, C.B., Korsmeyer, S.J., 2001. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292 (5517), 727–730. Xuan, B., Qian, Z., Torigoi, E., Yu, D., 2009. Human cytomegalovirus protein pUL38 induces ATF4 expression, inhibits persistent JNK phosphorylation, and suppresses endoplasmic reticulum stress-induced cell death. J. Virol. 83 (8), 3463–3474. Youle, R.J., Strasser, A., 2008. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell. Biol. 9 (1), 47–59. Yu, D., Silva, M.C., Shenk, T., 2003. Functional map of human cytomegalovirus AD169 defined by global mutational analysis. Proc. Natl. Acad. Sci. U.S.A. 100 (21), 12396–12401. Yu, Y., Alwine, J.C., 2002. Human cytomegalovirus major immediate-early proteins and simian virus 40 large T antigen can inhibit apoptosis through activation of the phosphatidylinositide 3’-OH kinase pathway and the cellular kinase Akt. J. Virol. 76 (8), 3731–3738. Zhang, D.W., Shao, J., Lin, J., Zhang, N., Lu, B.J., Lin, S.C., Dong, M.Q., Han, J., 2009. RIP3, an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis. Science 325 (5938), 332–336. Zhao, J., Sinclair, J., Houghton, J., Bolton, E., Bradley, A., Lever, A., 2010. Cytomegalovirus ␤2.7 RNA transcript protects endothelial cells against apoptosis during ischemia/reperfusion injury. J. Heart Lung Transplant. 29 (3), 342–345. Zhu, H., Shen, Y., Shenk, T., 1995. Human cytomegalovirus IE1 and IE2 proteins block apoptosis. J. Virol. 69 (12), 7960–7970.