Inhibition of programmed cell death by cytomegaloviruses.

Virus Research 157 (2011) 144–150
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Inhibit eg
Wolfram
Heinrich Pette erman
a r t i c l
Article history:
Available onlin
Keywords:
Cytomegalovir
Herpesvirus
Innate immun
Apoptosis
Programmed n
Necroptosis
rogra
s. Th
end o
tome
fense
again
sis an
riousl
at the
nder
1. Introdu
PCD is an
isms 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 prolifera-
tion. When
production
fragments o
by antigen p
response (A
Apoptos
quently bee
other forms
received inc
a reduction
blebbing, a
called apop
(but not alw
of aspartate
the release
terized by s
condensatio
lar compon
∗ Correspon
E-mail add
er et
-infla
rgan
cal 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 pro-
grammed necrosis or necroptosis (Moquin and Chan, 2010). The
third, least characterized form of PCD is autophagic cell death. It
0168-1702/$ –
doi:10.1016/j.PCD is initiated early during the viral life cycle, progeny
and dissemination can be severely impaired. Moreover,
f dead cells containing viral proteins can be taken up
resenting cells and used to prime the adaptive immune
lbert et al., 1998).
is is the best-characterized form of PCD and has fre-
n used as a synonym for PCD. In recent years, however,
of PCD such as necrosis and autophagic cell death have
reasing attention. Morphologically, apoptosis involves
of cell volume, chromatin condensation, membrane
nd finally the disintegration of the cell into vesicles
totic bodies (Kroemer et al., 2009). Apoptosis is often
ays) associated with activation of caspases, a group
-specific cystein proteases. Apoptosis does not result in
of intracellular enzymes. By contrast, necrosis is charac-
welling of cytoplasmic organelles, moderate chromatin
n, loss of membrane integrity, and leakage of cellu-
ents into the intercellular space (Festjens et al., 2006;
ding author. Tel.: +49 40 48051 351; fax: +49 40 48051 352.
ress: wolfram.brune@hpi.uni-hamburg.de
is accompanied by massive autophagic vacuolization of the cyto-
plasm. 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 metaboli-
cally 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 sig-
naling 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
see front matter © 2010 Elsevier B.V. All rights reserved.
virusres.2010.10.012ion of programmed cell death by cytom
Brune ∗
Institute, Leibniz Institute for Experimental Virology, Martinistr. 52, 20251 Hamburg, G
e i n f o
e 20 October 2010
us
ity
ecrosis
a b s t r a c t
The elimination of infected cells by p
mechanisms against infectious agent
parasites, such as viruses, which dep
slowly replicating viruses like the cy
suicide programs and other innate de
an impressive set of countermeasures
genes encoding suppressors of apopto
murine CMV (HCMV and MCMV). Cu
antiapoptotic proteins, suggesting th
This review summarizes our current u
pathways they target.
ction
evolutionary conserved process of multicellular organ-
Kroem
be pro
an uno/ locate /v i rusres
aloviruses
y
mmed cell death (PCD) is one of the most ancestral defense
is mechanism should be most effective against intracellular
n the host cell for their replication. However, even large and
galoviruses (CMVs) can prevail and persist in face of cellular
mechanisms. During evolution, these viruses have developed
st premature demise of the host cell. In the last decade, several
d necrosis have been identified in the genomes of human and
y, most of the gene products are not homologous to cellular
CMVs did not capture the genes from the host cell genome.
standing of how the CMVs suppress PCD and which signaling
© 2010 Elsevier B.V. All rights reserved.
al., 2009). Hence necrosis is generally considered to
mmatory. For a long time, necrosis was thought to be
ized form of cell death, occurring after acute physi-

W. Brune / Virus Research 157 (2011) 144–150 145
UL36
UL37x3
UL38 UL43
UL44
UL45
UL40
UL37x1
HCMV
3
Fig. 1. Schema MCM
are shown in b pital
Bcl-2 homo
antiapoptot
preserving
activity of t
pro-apopto
BH3-only p
domains. In
BH3-only p
teract the f
pro-apopto
monomeric
ize, and in
membrane
into the cyt
of other mi
(AIF), Smac
tributes to
studies sug
dant mann
response to
ever, more
fulfill some
induced ap
et al., 2007;
While g
the cellular
2008), no su
CMVs or oth
for antiapo
localized in
of HCMV (
contains a
domain, bo
apoptotic fu
obvious seq
tially believ
et al., 199
tural analy
2007). Hen
dimensiona
poxvirus pr
Since its
of antiapop
was shown
protecting c
inducing st
vMIA resem
the molecu
tain differe
damage 45�
acts togeth
of proapop
Mor
gom
2004
own
ted a
estio
n hu
inan
ther
Ano
ak in
tion
rine
a po
rmic
and
In fu
7x1
d in
200
ation
k-m
nal,
cond
teriz
usly
caliz
gom
igom
oden
role
ed us
om b
he M
s in
2009
phag
dele
d m
in mM38 M4
M37
m39M36
m41.1m38.5
m41
MCMV
tic view of a 12 kb region of the HCMV genome and the corresponding region in the
lack. MCMV genes with significant sequence homologies to HCMV genes carry a ca
logy (BH) domains. According to the current model,
ic Bcl-2 family proteins prevent caspase activation by
mitochondrial integrity (Youle and Strasser, 2008). The
he antiapoptotic Bcl-2 proteins is antagonized by the
tic multidomain proteins Bax and Bak and the so-called
roteins, which posses only the third out of four BH
response to apoptotic stimuli from within the cell,
roteins translocate to mitochondria where they coun-
unction of pro-survival Bcl-2 proteins and activate the
tic proteins Bax and Bak (Chipuk and Green, 2008). The
proteins undergo a conformational change, oligomer-
crease the permeability of the mitochondrial outer
(Wei et al., 2001). This leads to a release of cytochrome c
osol and subsequent activation of caspases. The release
tochondrial proteins such as apoptosis-inducing factor
/DIABLO, Htr2A/Omi, and endonuclease G also con-
the execution of PCD (Chipuk and Green, 2008). Early
gested that Bak and Bax function in a largely redun-
er during development (Lindsten et al., 2000) and in
various apoptosis inducers (Wei et al., 2001). How-
recent results indicated that the two proteins also
non-redundant functions, particularly during infection-
optosis (Brooks et al., 2007; Cartron et al., 2003; Kepp
Neise et al., 2008).
ammaherpesviruses express proteins homologous to
antiapoptotic proteins Bcl-2 and Bcl-xL (Galluzzi et al.,
ch homolog has been identified in the genomes of the
er betaherpesviruses. A random screening experiment
ptotic viral genes revealed a viral mitochondrion-
hibitor of apoptosis (vMIA) encoded by ORF UL37x1
Goldmacher et al., 1999). The UL37x1/vMIA protein
mitochondrial-targeting domain and an antiapoptotic
th of which are necessary and sufficient for vMIA’s anti-
nction (Hayajneh et al., 2001). The protein shows no
uence similarity to Bcl-2 family proteins and was ini-
ed to be structurally unrelated to Bcl-2 (Goldmacher
9). However, a more recent computer-based struc-
sis predicted a fold similar to Bcl-xL (Pauleau et al.,
ce, vMIA might mimic Bcl-2 family proteins in its 3-
l structure as it has been shown to be the case for two
oteins (Kvansakul et al., 2007, 2008).
discovery, vMIA has been studied extensively in terms
2005).
and oli
et al.,
was sh
media
the qu
vMIA i
is dom
might,
2004).
with B
interac
2008).
Mu
tein at
(McCo
m38.5
vMIA.
to UL3
dria an
et al.,
observ
and Ba
additio
This se
charac
previo
tein lo
Bak oli
Bak ol
other r
2010).
The
analyz
lasts fr
from t
kinetic
et al.,
macro
m38.5
infecte
is richtotic activity and molecular mechanism of action. It
to have a strong and broad antiapoptotic activity,
ells from both extrinsic signals and intrinsic apoptosis-
imuli (reviewed in Goldmacher, 2005). Functionally,
bles the cellular antiapoptotic protein Bcl-xL. However,
lar mechanisms of action of the two proteins show cer-
nces. vMIA interacts with the growth arrest and DNA
(GADD45�) protein and Bcl-xL, suggesting that vMIA
er with these cellular proteins to suppress the release
totic factors from mitochondria (Smith and Mocarski,
sive cell de
were infect
cells were
exhausted
2010; Jurak
with HCMV
AD169 resu
poorly in no
on complem
Apoptosis wM45
M44
V genome. The regions encode multiple cell death suppressors, which
M.
eover, vMIA interacts with Bax and sequesters activated
erized Bax at mitochondria (Arnoult et al., 2004; Poncet
). Using mouse fibroblasts lacking either Bax or Bak it
that vMIA protects murine cells from Bax- but not Bak-
poptosis (Arnoult et al., 2004). This observation raised
n of how to explain the broad antiapoptotic activity of
man cells. One possible explanation suggested that Bax
t over Bak in human cells, and that a potent Bax inhibitor
efore, be sufficient to protect human cells (Arnoult et al.,
ther study provided evidence that vMIA also interacts
human cells (Karbowski et al., 2006). However, the
of vMIA with Bak remains controversial (Arnoult et al.,
cytomegalovirus also expresses a mitochondrial pro-
sition in the viral genome analogous to HCMV UL37x1
k et al., 2005) (Fig. 1). This protein is encoded by ORF
shows only minimal sequence similarity to HCMV
nctional studies, however, the protein behaved similar
in that it bound and sequestered Bax at mitochon-
hibited Bax- but not Bak-mediated cell death (Arnoult
8; Jurak et al., 2008; Norris and Youle, 2008). The
that MCMV-infected cells were protected from Bax-
ediated apoptosis suggested that the virus encodes an
Bak-specific inhibitor of apoptosis (Jurak et al., 2008).
mitochondrial inhibitor was recently identified and
ed (C¸am et al., 2010). It is the product of a small,
unrecognized ORF named m41.1 (Fig. 1). The m41.1 pro-
es to mitochondria, interacts with Bak, and prevents
erization. Consequently it was named viral inhibitor of
erization (vIBO). Similar vIBO proteins are encoded by
t CMVs, but are missing in primate CMVs (C¸am et al.,
s of vMIA and vIBO during viral infection have been
ing recombinant viruses and mouse embryonic fibrob-
ax or bak knockout mice. Deletion of m38.5 or m41.1
CMV genome had only little effect on viral replication
fibroblasts (C¸am et al., 2010; Jurak et al., 2008; Manzur
), but strongly impaired viral replication in cultured
es (C¸am et al., 2010; Manzur et al., 2009). An MCMV
tion mutant also reached lower titers in the spleen of
ice (Manzur et al., 2009), perhaps because the spleen
acrophages and other antigen presenting cells. Exces-
ath was also seen in infected fibroblasts when cells
ed at a high multiplicity of infection or when infected
exposed to additional stress such as maintenance in
medium or treatment with staurosporine (C¸am et al.,
et al., 2008). Somewhat different results were obtained
UL37x1 mutants. Deletion of UL37x1 in HCMV strain
lted in a virus that induced excessive apoptosis, grew
rmal fibroblasts, and could be grown to high titers only
enting cells (Reboredo et al., 2004; Yu et al., 2003).
as 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, zVAD-
fmk (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
virus were
et al., 2005
cells (McCo
not blocked
inhibitor sp
protease re
meabilizati
caused prim
with subseq
also unkno
impact on
same mutat
AD169 exer
Towne (McC
tivating mu
below (Skal
The mito
show certai
antiapoptot
cell death in
Conversely,
totic effect
2005), perh
The MCMV
human cells
vations, MC
and Brune,
replication
nation. This
a viral anal
available da
apoptosis in
that MCMV
than on mo
2010).
The UL3
exon of the
of the UL37
has been as
sion (Colber
has additio
in cell dea
from the en
rounding, s
(Sharon-Fri
be inhibite
to the CMV
tional conse
response (S
the cytopat
vMIA, but a
production
Besides
chondrial f
normal inte
(Karbowski
mitochondr
Bax and Ba
(McCormick
consequenc
have not be
3. Inhibition of death receptor-dependent apoptosis
Death receptors are cell surface receptors that transmit apop-
totic signals initiated by specific ligands such as Fas ligand, TNF�,
IL. St
oteo
ic ap
d by
prev
or of
the
imp
. Sur
wnev
001)
t imp
d et
sis o
to re
003)
sm (H
36,
popt
was
d in
hibit
ilar t
V str
eplic
as re
CA is
sely
hum
even
ich th
ckin
ath
ome
e-ind
prog
n an
n be
ynth
e-8 i
expre
ecep
r pro
ly be
n TN
plish
(RIP1
apto
tran
s and
007)
bited
med
nt lab
ctiva
rosis
s the
teraet al., 2005). However, cells infected with the mutant
more sensitive to proapoptotic stimuli (McCormick
) and died a few days earlier than wt Towne-infected
rmick et al., 2008). Cell death and fragmentation was
by zVAD-fmk, but was inhibited by a serine protease
ecific for HtrA2/Omi (McCormick et al., 2008). This is a
leased from mitochondria upon outer membrane per-
on. Why infected cell demise under these conditions is
arily by HtrA2/Omi rather than by cytochrome c release
uent caspase-9 activation remains to be explored. It is
wn why deletion of UL37x1 in AD169 has a stronger
cell survival and virus progeny production than the
ion in the Towne strain. A suggested explanation is that
ts a stronger proapoptotic stress on infected cells than
ormicket al., 2005). The fact thatAD169carries an inac-
tation in UL36 (another antiapoptotic protein described
etskaya et al., 2001)) might also be responsible.
chondrial cell death suppressors of HCMV and MCMV
n species-specific differences. HCMV vMIA has a broad
ic activity in human cells, but blocks only Bax-mediated
murine cells (Arnoult et al., 2004; Poncet et al., 2004).
MCMV vMIA has only a limited or transient antiapop-
on human cells (Arnoult et al., 2008; McCormick et al.,
aps because Bak-mediated cell death is not inhibited.
Bak inhibitor, vIBO, also seems to have a weak activity in
(C¸am et al., unpublished). Consistent with these obser-
MV infection induces apoptosis in human cells (Jurak
2006). The early onset of apoptosis prevents efficient
of MCMV in human cells and limits MCMV dissemi-
obstacle can be overcome by overexpressing Bcl-2 or
og in infected cells (Jurak and Brune, 2006). Based on
ta it can be concluded that the MCMV mitochondrial
hibitors have insufficient activity in human cells and/or
exerts a stronger proapoptotic stress on human cells
use cells (Jurak and Brune, 2006; Schumacher et al.,
7x1 ORF encodes vMIA, but also serves as the first
spliced UL37 gene transcripts (Fig. 1). The functions
glycoproteins have not been fully elucidated, but it
sumed that they also play a role in cell death suppres-
g-Poley et al., 2000). It is noteworthy that UL37x1/vMIA
nal functions that might be more indirectly involved
th suppression. It induces the release of Ca2+ ions
doplasmic reticulum into the cytosol, leading to cell
welling, and reorganization of the actin cytoskeleton
ling et al., 2006). These morphological changes could
d by a Ca2+ chelating agent. Apart from contributing
-specific cytopathic effect, Ca2+ release likely has addi-
quences such as the induction of the unfolded protein
haron-Friling et al., 2006). Another study also found that
hic morphology of HCMV-infected cells depended on
ttributed this effect to an impaired mitochondrial ATP
caused by vMIA (Poncet et al., 2006).
regulating apoptosis, Bax and Bak also promote mito-
usion, thereby contributing to the maintenance of
rconnected mitochondrial networks in healthy cells
et al., 2006). HCMV and MCMV vMIA can both disrupt
ial networks, probably by binding and sequestering
k, resulting in a fragmented mitochondrial phenotype
et al., 2003b; Norris and Youle, 2008). The functional
es of mitochondrial fragmentation for the viral life cycle
en determined yet.
or TRA
and pr
extrins
blocke
8 and
inhibit
among
fills an
2003a)
and To
et al., 2
did no
Ménar
apopto
ability
et al., 2
organi
gene, M
more a
ulence
inserte
is to in
2008).
Sim
sus CM
Viral r
tion w
that vI
Conver
of both
might
on wh
4. Blo
cell de
In s
caspas
named
Moqui
way ca
by a s
caspas
CMVs
death r
anothe
recent
sis upo
accom
tein 1
is an ad
of the
kinase
et al., 2
be inhi
of RIP-
differe
sis is a
for nec
inhibit
typic inimulation of death receptors leads to the recruitment
lytic activation of procaspase-8, thereby initiating the
optosis pathway (Thorburn, 2004). This pathway is
the HCMV UL36 protein, which binds to procaspase-
ents its activation (Skaletskaya et al., 2001). This viral
caspase-8-induced apoptosis (vICA) is highly conserved
mammalian betaherpesviruses, suggesting that it ful-
ortant role during virus infection (McCormick et al.,
prisingly, two HCMV laboratory strains (AD169varATCC
arRIT) carry inactivating mutations in UL36 (Skaletskaya
. Moreover, deletion of the vICA gene in HCMV or MCMV
air viral replication in fibroblasts (Dunn et al., 2003;
al., 2003). However, vICA-deficient viruses induced
f macrophages and were massively impaired in their
plicate inmacrophages (McCormicket al., 2010;Ménard
, a cell type important for dissemination within the host
anson et al., 1999). Indeed, an MCMV lacking the vICA
was severely growth restricted in mice and produced
otic cells in infected organs (Cicin-Sain et al., 2008). Vir-
largely restored when a dominant-negative FADD was
place of M36, confirming that the main function of M36
death receptor-dependent apoptosis (Cicin-Sain et al.,
o the aforementioned HCMV laboratory strains, the rhe-
ain 68-1 also contains an inactivating mutation in UL36.
ation in epithelial cells was improved when this muta-
paired (Lilja and Shenk, 2008), supporting the notion
important for replicative fitness in non-fibroblast cells.
, the presence of UL36 mutations in laboratory strains
an and rhesus CMV suggests that an intact vICA gene
be detrimental for replication in cultured fibroblasts,
e CMVs are usually propagated.
g necrosis, an alternative form of programmed
cells, death receptor stimulation can also activate a
ependent form of programmed cell death that has been
rammed necrosis or necroptosis (Festjens et al., 2006;
d Chan, 2010; Vandenabeele et al., 2010). This path-
initiated when caspase-dependent apoptosis is blocked
etic caspase inhibitor (such as zVAD-fmk) or a viral
nhibitor (Festjens et al., 2006). As described above, the
ss vICA, and thus infected cells might be sensitized to
tor-induced programmed necrosis, unless they encode
tein that prevents this cell death program. Indeed, it has
en shown that the MCMV M45 protein inhibits necro-
F receptor or Fas stimulation (Mack et al., 2008). M45
es this by interacting with the receptor-interacting pro-
) and RIP3 (Mack et al., 2008; Upton et al., 2008). RIP1
r kinase that mediates, on the one hand, the activation
scription factor NF-�B and mitogen-activated protein
, on the other hand, the induction of necrosis (Festjens
. All signaling pathways downstream of RIP1 appear to
by M45, and therefore M45 was named viral inhibitor
iated signaling (vIRS) (Mack et al., 2008). Recent work in
oratories has shown that TNF receptor-induced necro-
ted by a RIP1–RIP3 complex, and that RIP3 is essential
induction (reviewed in Moquin and Chan, 2010). M45
RIP1–RIP3 interaction with the help of a RIP homo-
ction motif (RHIM) present near the N terminus of M45

W. Brune / Virus Research 157 (2011) 144–150 147
(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 senso
carry a RHIM
M45 is
subunits, b
(Lembo and
homology d
thirds of th
within the
sis in cell c
2010). It ha
part of M45
a C-termina
induced cel
the C-termi
vation (Ma
function mi
(Fliss and B
M45 wa
ity to replic
was mutate
venting a sp
more resist
viruses rep
wildtype vi
gests that t
of a cell to p
very low le
ticular fibro
2010; Zhan
The RHIM
CMVs (rat C
Brune, 2009
suppress pr
the human
motif and i
virus replic
Brune, 2009
ity (Patrone
enigmatic.
5. Preserva
The mec
sis has not
the produc
is an impo
Vandenabe
of ROS, and
I can be com
potential lo
CMV can st
production
duction (Re
HCMV 2.7 k
of complex
gene from
tion in fibro
medium. H
nificantly in
when cells
2007).
While the �2.7 RNA stabilizes mitochondrial ATP production,
HCMV UL37x1/vMIA was reported to reduce ATP synthesis, proba-
bly by compromising the function of the mitochondrial phosphate
carrier, a component of the ATP synthasome (Poncet et al., 2006).
MIA
d, bu
of v
tide
e (G
and
e in
lt et
pecu
us.
8 is
ve m
2008
s of
at in
reve
myc
ng pr
n of
in fi
007)
ing a
ars li
y inv
how
dopla
in or
deat
How
ns re
ivati
imm
CMV
strat
e pr
uced
irus
wed
kinas
inase
thw
equir
prote
ns ar
tenin
MV i
e m
the
Con
ted (
ocko
ini et
ma
omeg
st cel
As
ays ler, DAI, as these are the only other proteins known to
(Kaiser et al., 2008; Rebsamen et al., 2009).
a sequence homolog to ribonucleotide reductase R1
ut does not possess ribonucleotide reductase activity
Brune, 2009; Lembo et al., 2004). In addition to the R1
omain, which comprises roughly the C-terminal two
e protein, it has a unique N terminus. The RHIM motif
unique N terminus is necessary for inhibiting necro-
ulture as well as in infected mice (Upton et al., 2008,
s not been fully resolved whether or not the N-terminal
is also sufficient to block necrosis as viruses expressing
lly truncated M45 protein do not suppress infection-
l death (Brune et al., 2001; Lembo et al., 2004). However,
nal domain is essential for the inhibition of NF-�B acti-
ck et al., 2008). More recent work indicates that this
ght involve an additional, RIP-independent mechanism
rune, unpublished results).
s originally described as a determinant of MCMV’s abil-
ate in endothelial cells (Brune et al., 2001). When M45
d, infected endothelial cells died rapidly, thereby pre-
read to neighboring cells. In contrast, fibroblasts were
ant to infection-induced cell death, and M45 mutant
licated in these cells almost to the same titers as the
rus (Brune et al., 2001). More recent work strongly sug-
he level of RIP3 expression determines the sensitivity
rogrammed necrosis. Many murine fibroblasts express
vels of RIP3, whereas endothelial cells (and also a par-
blast line) express higher amounts of RIP3 (Upton et al.,
g et al., 2009).
motif is conserved in the R1 homologs of other rodent
MV, guinea pig CMV, tupaia herpesvirus) (Lembo and
; Rebsamen et al., 2009), suggesting that these proteins
ogrammed necrosis like MCMV M45 does. By contrast,
CMV R1 homolog, UL45 (Fig. 1), does not carry a RHIM
s not required for the inhibition of cell death or for
ation in endothelial cells (Hahn et al., 2002; Lembo and
). UL45 is also devoid of ribonucleotide reductase activ-
et al., 2003), and its function for the virus remains
tion of metabolic activity during stress
hanism by which RIP3 triggers programmed necro-
been fully elucidated, but the current view is that
tion of reactive oxygen species (ROS) within the cell
rtant effector mechanism (Moquin and Chan, 2010;
ele et al., 2010). The mitochondria are major producers
the function of the mitochondrial respiratory complex
promised by ROS leading to mitochondrial membrane
ss and cell death (Davis et al., 2010). Intriguingly, human
abilize mitochondrial complex I, thereby reducing ROS
and preserving mitochondrial respiration and ATP pro-
eves et al., 2007; Zhao et al., 2010). During infection, an
b noncoding RNA (�2.7) binds to GRIM-19, a subunit
I, and prevents its relocalization. Deletion of the �2.7
the viral genome had little impact on HCMV replica-
blasts when the cells were supplied with glucose-rich
owever, titers of the �2.7 deletion mutant dropped sig-
the presence of the mitochondrial poison rotenone or
were cultured in glucose-depleted media (Reeves et al.,
How v
resolve
quence
nucleo
thasom
other h
that th
(Arnou
ter of s
the vir
UL3
preser
et al.,
protein
plex th
UL38 p
of rapa
reduci
Deletio
poorly
et al., 2
inhibit
It appe
directl
been s
the en
camyc
not by
2009).
functio
6. Act
The
first H
demon
of thes
sis ind
adenov
ies sho
3′-OH
lular k
This pa
to be r
the IE
domai
enligh
ing HC
IE2 hav
tion of
2006).
restric
IE2 kn
March
7. Sum
Cyt
the ho
cation.
pathwreduces the phosphate carrier’s activity has not been
t it was speculated that it might be an indirect conse-
MIA’s previously reported interaction with the adenine
transporter (ANT), another component of the ATP syn-
oldmacher et al., 1999; Poncet et al., 2006). On the
, later work from the Goldmacher laboratory suggested
teraction with ANT might be of a nonspecific nature
al., 2004; Goldmacher, 2005). It also remains a mat-
lation why reduced ATP levels would be favorable for
another cell death suppressor whose function is to
etabolic activity under stress conditions (Moorman
). The UL38 protein interacts with the TSC1 and TSC2
the tuberous sclerosis complex (TSC), a protein com-
tegrates various stress signals. By binding to the TSC,
nts TSC-mediated activation of the mammalian target
in complex 1 (mTORC1), which responds to stress by
otein synthesis and cell growth (Moorman et al., 2008).
UL38 from the viral genome resulted in a virus that grew
broblasts and induced excessive cell death (Terhune
. However, it is not clear to which extent the cell death
ctivity of UL38 results from the interaction with the TSC.
kely that UL38 fulfills additional functions that are more
olved in cell death suppression, particularly since it has
n that UL38 protects cells from cell death triggered by
smic reticulum stress inducers thapsigargin and tuni-
by infection with an E1B-19k-deficient adenovirus, but
h receptor stimulation (Terhune et al., 2007; Xuan et al.,
ever, the molecular mechanism(s) of these additional
main unknown.
on of survival pathways
ediate-early proteins 1 and 2 (IE1 and IE2) were the
proteins for which a cell death-inhibiting activity was
ed (Zhu et al., 1995). Transient or stable expression
oteins in HeLa cells protected the cells from apopto-
by TNF�+ cycloheximide or by an E1B-19k-deficient
, but not by UV irradiation (Zhu et al., 1995). Later stud-
that the viral proteins activate a phosphatidylinositide
e (PI3K)-dependent survival pathway involving the cel-
Akt (Lukac and Alwine, 1999; Yu and Alwine, 2002).
ay is usually activated by trophic factors, and appears
ed also for CMV replication (Johnson et al., 2001). How
ins promote activation of the PI3K pathway and which
e required remains to be investigated. It would also be
g to study the role of IE1/2-induced PI3K activation dur-
nfection. However, this is difficult to do because IE1 and
ultiple important functions in gene regulation, modula-
cell cycle, and immune subversion (Meier and Stinski,
sequently, IE1-deficient HCMVs are severely growth-
Greaves and Mocarski, 1998; Mocarski et al., 1996), and
ut mutants do not replicate at all (Heider et al., 2002;
al., 2001).
ry and discussion
aloviruses are slowly replicatingviruses thatmustkeep
l alive for a sufficiently long time to ensure virus repli-
there are different forms of cell death and multiple
ading 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 • Viral Inhibitor of Caspase-8-induced Apoptosis M36 • Viral Inhibitor of Caspase-8-induced Apoptosis
vICA • Important for virus replication in macrophages vICA • Important for virus replication in macrophages and
dissemination in vivo
UL37x1 • Viral Mitochondria-localized Inhibitor of Apoptosis m38.5 • Viral Mitochondria-localized Inhibitor of Apoptosis
vMIA • Inhibits Bax-mediated apoptosis vMIA • Inhibits Bax- but not Bak- mediated apoptosis
• Inhibits HtrA2/Omi-mediated cell death • Disrupts mitochondrial networks in Bak-deficient cells
• Interacts with GADD45� • Important for replication in splenocytes in vivo
• Disrupts mitochondrial networks
• Induces ER Ca2+ release
UL38 • Inhibits TSC-mediated stress response M38 Function unknown
• Inhibits ER stress-induced cell death
m41.1 • Viral Inhibitor of Bak Oligomerization
vIBO • Inhibits Bak- but not Bax-mediated apoptosis
• Important for replication in macrophages
UL45 Function unknown M45 • Viral Inhibitor of RIP-mediated Signaling
(does not interact with RIP1) vIRS • 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
�2.7 RNA • Binds to mitochondrial respiratory complex I
• Stabilizes mitochondrial membrane potential and ATP
production
IE1, IE2 • Activate PI3K/Akt-dependent survival pathways IE1, IE3 No known effects on cell death
m41 • Golgi-localized cell death suppressor
• Important for survival of infected macrophages
find differen
or activate s
signals ema
Apoptos
death and h
years, prog
been appre
to discern t
2009), it is o
ing with vi
death-indu
antagonized
sors known
are shown s
st of
ed in
). Cu
rlapp
ttern
oluti
CMV
uen
s un
e, as
e vir
in gat ways to block the induction of programmed cell death
urvival pathways, which balance the death-promoting
nating from virus infection and replication.
is is the best-characterized form of programmed cell
as frequently been used synonymously to PCD. In recent
rammed necrosis and autophagic cell death have also
ciated as important modes of PCD. Although criteria
he different forms of cell death exist (Kroemer et al.,
ften difficult to keep them apart, especially when deal-
rus-infected cells. These cells are exposed to multiple
cing stimuli, which are largely (but perhaps not fully)
by viral gene products. The CMV cell death suppres-
Mo
cluster
(Fig. 1
or ove
ing pa
the ev
of the
ent seq
appear
genom
and th
foundto date are listed and in Table 1, and their mechanisms
chematically in Fig. 2.
and Bellow
why CMVs
Fig. 2. Cell death inhibitors of HCMV and MCMV, and their cellular target protethe CMV cell death suppressors identified to date are
a relatively small region of the large viral genome
riously, some of the corresponding genes are spliced
ing with other genes. Such a complex and dense cod-
is rather unusual for these large viruses. Moreover,
onary origin of the genes remains enigmatic for most
cell death suppressors. The proteins show no appar-
ce homology to any cellular gene product. Therefore, it
likely that the genes have been captured from the host
it is assumed to be the case for the Bcl-2 homologs
al FLIPs (FLICE [=caspase-8]-like inhibitory proteins)
mmaherpesviruses and a few other viruses (Hardwick
s, 2003; Thome et al., 1997). It remains mysterious
have evolved their own cell death inhibitors instead
ins. The functions are summarized in table 1.

W. Brune / Virus Research 157 (2011) 144–150 149
of capturing them. One exception is the M45 protein, which shows
obvious sequence similarity to ribonucleotide reductase R1 sub-
units. Here, the homology to a cellular protein is rather misleading,
as M45 and its homologs in other betaherpesviruses have lost their
ribonucleot
process term
For a fe
inhibiting a
case form4
Determinin
it had to b
2010), whic
ORF (Fig. 1)
inhibits ER
sityof thevi
of the cellul
genomes ha
possibly an
in future in
Acknowled
I apologi
work was s
1730/3-1).
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