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

Cytomegaloviruses inhibit Bak- and Bax-mediated
apoptosis with two separate viral proteins
M C¸am1, W Handke1, M Picard-Maureau2 and W Brune*,1
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.
Cell Death and Differentiation (2010) 17, 655–665; doi:10.1038/cdd.2009.147; published online 9 October 2009
Programmed cell death (PCD) or apoptosis has numerous
functions in a multicellular organism. It is important during
development, for maintenance of tissue homeostasis, for
elimination of damaged or transformed cells, and as a first line
of defense against infectious agents. The cellular suicide
program can be particularly effective as an antiviral defense
mechanism, since viruses depend on the host cell for
replication.1 Viral dissemination can be severely impaired if
an infected cell executes apoptosis before the viral replication
cycle is completed. The importance of apoptosis is under-
scored by the fact that many viruses have evolved genes
encoding antiapoptotic proteins.2
Viral infection and replication exert different kinds of stress
on the host cell: depletion of nutrients, impairment of cellular
protein biosynthesis, induction of the unfolded protein
response (also called endoplasmic reticulum stress), and a
DNA-damage response. All kinds of stress can initiate
signaling pathways culminating in apoptosis.2 Caspases, a
group of aspartate-specific cysteine proteases, are pivotal
signal transducers within apoptotic signaling cascades,
whose activation is regulated by proteins of the Bcl-2 family.3
This family consists of pro- and antiapoptotic members, which
differ in the number of Bcl-2 homology (BH) domains.
According to the current model, antiapoptotic Bcl-2-family
proteins prevent caspase activation by preserving mitochon-
drial integrity.3 The activity of the antiapoptotic Bcl-2 proteins
is antagonized by the proapoptotic 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, the BH3-only proteins translocate to mitochondria
where they counteract the function of pro-survival Bcl-2
proteins and activate the proapoptotic proteins Bax and
Bak.4 The monomeric proteins undergo a conformational
change, oligomerize, and increase the permeability of the
mitochondrial outer membrane.5 This leads to release of
cytochrome c (cyt 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.4 Earlier studies have shown that Bak
and Bax function in a largely redundant manner during
development6 and in response to various apoptosis inducers.5
However, more recent results indicated that the two proteins
also fulfill some non-redundant functions.7–10
A number of viruses, particularly adenoviruses and
g-herpesviruses, express proteins homologous to the
cellular antiapoptotic proteins Bcl-2 and Bcl-xL.2,11 Poxviruses
also express apoptosis inhibitors that resemble Bcl-xL in
their three-dimensional structure, even though their
amino-acid sequences are not homologous.12,13 Like Bcl-xL,
Received 07.4.09; revised 14.8.09; accepted 10.9.09; Edited by JM Hardwick; published online 9.10.09
1Division of Viral Infections, Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany and 2Department of Virology, Institute of Medical Microbiology and Hygiene,
University Hospital Mannheim, Theodor-Kutzer-Ufer 1–3, 68167 Mannheim, Germany
*Corresponding author: Dr W Brune, Division of Viral Infections, Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany. Tel: þ 49 30 18754 2502;
Fax: þ 49 30 1810754 2502; E-mail: BruneW@rki.de
Keywords: apoptosis; cytomegalovirus; m41.1; Bak; Bax
Abbreviations: ActD, actinomycin-D; BAC, bacterial artificial chromosome; BH, Bcl-2 homology; BMH, 10,60-bismaleimidohexane; CMV, cytomegalovirus; DAPI, 40,60-
diamidino-2-phenylindole; ER, endoplasmic reticulum; FRT, FLP recombination target site; HCMV, human cytomegalovirus; hpi, hours postinfection; MCMV, murine
cytomegalovirus; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; nt, nucleotide(s); ORF, open reading frame; PCD,
programmed cell death; RCMV, rat cytomegalovirus; STS, staurosporine; TCID50, median tissue culture infective dose; TUNEL, terminal deoxynucleotidyltransferase-
mediated dUTP-biotin nick-end-labeling; vIBO, viral inhibitor of Bak oligomerization; vMIA, viral mitochondria-localized inhibitor of apoptosis; wt, wild type
Cell Death and Differentiation (2010) 17, 655–665
& 2010 Macmillan Publishers Limited All rights reserved 1350-9047/10 $32.00
www.nature.com/cdd

these viral proteins inhibit both Bax- and Bak-mediated
cell death.
A different strategy to inhibit apoptosis at the mitochondrial
checkpoint is pursued by cytomegaloviruses (CMVs). Human
CMV encodes a viral mitochondrion-localized inhibitor of
apoptosis (vMIA, encoded by open reading frame (ORF)
UL37x1) that interacts with Bax and specifically inhibits
Bax-mediated cell death.14–16 The primary structure of human
CMV (HCMV) vMIA has no obvious similarities to Bcl-2-family
proteins,14 but an in silico structural analysis predicted a fold
similar to Bcl-xL.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,20–22 and
also induces mitochondrial fragmentation in the absence of
Bak (i.e., in bak�/� cells).23 Moreover, the observation that
MCMV-infected cells are protected from Bax- and Bak-
mediated 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. In
conjunction with the m38.5/vMIA protein, it protects infected
cells from apoptosis induced by cytotoxic drugs or the viral
infection itself. Moreover, structurally and functionally similar
proteins are encoded by rat CMVs. The unusual ability of
CMVs to block Bax and Bak with two separate proteins
suggests that it might be advantageous for these viruses to
modulate Bax- and Bak-dependent pathways separately. The
identification of these two viral inhibitors should provide new
insights into the differential importance of the two proapoptotic
Bcl-2 family members in viral infection-induced cell death.
Results
Identification of the protein expressed by m41.1. We
identified ORF m41 as a region of the MCMV genome
encoding an antiapoptotic protein in a previous study.24
Deletion of the m41 ORF from the MCMV genome resulted in
premature death of infected cells that could be reduced by
addition of broad-spectrum caspase inhibitors. The m41
protein was shown to localize to the Golgi apparatus, but its
mechanism of action has remained undefined.
A recent computational re-annotation of theMCMVgenome
revealed the presence of a second ORF embedded within
m41 in a different reading frame.18 This ORF was named
m41.1 and has the potential to code for a 57-amino-acid
protein (Figure 1a and c). The putative start codon is located
10 nucleotides (nt) downstream of the m41 translational start.
A predominant transcript of approximately 0.6 kb was identi-
fied by Northern blot analysis (Figure 1b). The 50 and 30 ends
of the mRNA transcripts were determined by rapid amplifica-
tion of cDNA ends (RACE). The major transcript starts at nt
position 54 227 of the MCMV genome and terminates at
position 53 680, 93-nt downstream of the m41 ORF and 21-nt
downstream of a canonical AATAAA polyadenylation signal.
Several larger transcripts of low abundance (Figure 1b) were
also cloned and sequenced. All of them used a splice acceptor
site at position 54 217 (Figure 1a), but none of them had the
capacity to encode an N-terminally extended m41 or m41.1
protein (data not shown). Hence we concluded that the m41
and m41.1 gene products must be translated from the same
0.6-kb mRNA transcript by using the first or one of the
following start codons.
We have previously shown that the m41 ORF is expressed
during MCMV infection and translated into a protein of 138
amino acids with an apparent molecular mass of approxi-
mately 20 kDa.24 To determine whether an m41.1 protein is
also synthesized during MCMV infection, we inserted an HA
epitope tag sequence and a selectable marker (kan) at the
30 end of the m41.1 ORF (Figure 1c). This procedure leaves
the transcriptional and translational start sites of m41.1
unchanged, and is, therefore, unlikely to affect the expression
pattern of m41.1.We detected theHA-taggedm41.1 protein in
lysates of infected 10.1 fibroblasts byWestern blotting starting
about 5 hpi (Figure 1d). Further experiments showed that
m41.1 was expressed in the presence of a DNA-synthesis
inhibitor, but not after release from the cytoheximide block
(Supplementary Figure S1A). Hence m41.1 can be classified
as an early gene product. The protein had an apparent
molecular mass of approximately 7 kDa (not shown), which
corresponds to its predicted mass.
Functional dissection of the m41 locus. In order to
determine whether m41, m41.1, or both gene products are
responsible for the previously described antiapoptotic
activity, we constructed mutant viruses lacking m41.1, m41,
or both ORFs (Figure 1c). In the Dm41 mutant, the m41 ORF
was replaced with a zeocin-resistance gene. To construct an
m41.1-knockout mutant (m41.1ko), we introduced three
silent mutations into the m41 ORF, which eliminated the
three potential ATG start codons of m41.1 (Figure 1a). The
m41ko virus was constructed by inserting the m41.1 ORF at
an ectopic position into the genome of the Dm41 mutant. In a
similar way, two genes encoding well-characterized
mitochondrial apoptosis inhibitors were inserted: the cellular
Bcl-xL and the myxoma virus Bcl-2 analog M11L.12,25 The
integrity of the constructed mutants was determined by
restriction-fragment length analysis and by immunoblotting
(Supplementary Figure S1B–D).
As our previous work has shown that the Dm41 mutant
induced apoptosis of infected cells,24 we first analyzed how
the newly constructedmutants affected the viability of infected
cells. Murine 10.1 fibroblasts were infected with the mutant
viruses at a highmultiplicity of infection (MOI), and cell viability
was determined using an MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide) assay. Fibroblasts infected
with Dm41 or m41.1ko showed clearly reduced viability at 72
hpi (Figure 2a). By contrast, the viability of cells infected with
m41ko or Dm41/M11L was similar to those infected with the
wild-type (wt) virus, suggesting that the m41 protein is not
required to inhibit premature PCD in fibroblasts. We also
infected RAW264.7 macrophages, which are more sensitive
to virus-induced PCD. At 18 hpi their viability was reduced
after infection with wt MCMV and even more with Dm41,
m41ko, or m41.1ko (Figure 2b and Supplementary Figure
S2). Similar results were obtained with IC-21 macrophages
CMVs inhibit Bak and Bax
M C¸am et al
656
Cell Death and Differentiation

(not shown). We also determined nuclear DNA fragmentation
as a sign of apoptosis in infected fibroblasts andmacrophages
using a terminal deoxynucleotidyltransferase-mediated
dUTP-biotin nick-end-labeling (TUNEL) assay (Figure 2c
and d). However, this assay was difficult to employ because
infected cells do not die synchronously, and dead cells
eventually disintegrate and detach, making them unavailable
for analysis. Even though the TUNEL assay considerably
underestimates the true percentage of dead cells, the results
were basically concordant with the results of the cell viability
assay (Figure 2a and b).
A strong activation of caspase-3 was detected in fibroblasts
and macrophages infected with the Dm41 or the m41.1ko
mutant (Figure 2e and f). By contrast, caspase-3 activation
was largely suppressed in cells infected with m41.1- or M11L-
expressing MCMVs. Taken together these results suggested
that m41.1 is a potent inhibitor of infection-induced apoptosis.
Under the conditions tested here, m41 was only a weak cell-
death suppressor, whose activity became apparent primarily
in macrophages.
We then infected fibroblasts and macrophages with the
different viruses and analyzed viral replication after infection
with low MOI. Multistep replication kinetics in 10.1 fibroblasts
and RAW264.7 macrophages indicated that viral replication in
fibroblasts is largely unaffected by the absence of m41 and/or
m41.1 (Figure 2g), but is severely impaired in macrophages in
the absence of m41.1 (Figure 2h).
The m41.1 protein localizes to mitochondria and
protects infected cells from drug-induced
apoptosis. The observation that the loss of m41.1 could
be compensated by inserting the Bcl-xL or the M11L gene
into the MCMV genome (Figure 2) suggested that m41.1
might function in a similar way and inhibit apoptosis at the
mitochondrial checkpoint. To determine the localization of
the m41.1 protein, we transfected cells with a plasmid
expressing m41.1 with a C-terminal HA tag and analyzed its
localization by immunofluorescence. As shown in Figure 3a,
the m41.1 protein colocalized with the mitochondrial marker
Hsp60 and with the mitochondrion-specific dye, MitoTracker,
but did not colocalize with an endoplasmic reticulum (ER)
marker. The m41.1 protein also displayed mitochondrial
localization in MCMV-infected cells (Figure 3b).
Next we tested whether m41.1 protects infected cells from
exogenous proapoptotic stimuli. To this end, fibroblasts were
infected with wt and mutant MCMVs, and treated with the
drugs staurosporine (STS) or actinomycin-D (ActD), both of
which activate the mitochondrial apoptosis pathway.5 Indeed,
GTCGTGCGCAGGTTCCTCCGAACCTTTGATGGGAGACGATGATCGTCGCGGCGATGACGGCGGCGTATATGGCTCTGGCT
54227
M G D D D R R G D D G G V Y G S G
M I V A A M T A A Y M A L A
CTGCCGACGGTGAGAAGATTGCCGCTCCCGCGAACCTTTCGTCGCGCCCTCCGCGAAGACCTAATCTCCGTCGCCTTGGC
54147
S A D G E K I A A P A N L S S R P P R R P N L R R L G
L P T V R R L P L P R T F R R A L R E D L I S V A L A
GGCCTCTTTACTATGCCTTACCGTGAGCTCGGGGGCTCTACGCCGTCGTTAGGAGACAACGTTTCCTCGGAAGGCGTC…
54067
G L F T M P Y R E L G G S T P S L G D N V S S E G V …
A S L L C L T V S S G A L R R R .
m40 m41 m42m41.1
m41 m42m41.1kan
m40 m41 m42
m40 zeo m42
m01 BclXL m07kan Ppgk
m01 M11L m07kan Ppgk
m01 m41.1 m07kan Ppgk
MCMV wt
m41.1HA
m41.1ko
Δm41
Δm41/m41.1
= m41ko
Δm41/BclXL
Δm41/M11L
actin
m41.1
E1
IE1
M44
gB
0 2.5 5 10 24 48 hpi
4
3
1
1.5
0.5
6
2
kb
m
o
ck
pc
DN
A-
m
41
M
CM
V
a
dcb
m41
m41.1
Figure 1 Transcriptional analysis and mutagenesis of the m41 locus. (a) The major transcript starts at nt position 54 227 and is cleaved and polyadenylated after nt
position 53 680. Two protein products encoded in different reading frames are translated from this transcript: the 138-amino-acid m41 (upper translation) and the 57-amino-
acid m41.1 (lower translation) proteins. Three possible m41.1 start codons are shown in bold and the predicted transmembrane domain is underlined. Note that only the first 70
amino acids of m41 are shown. (b) Northern blot analysis of m41 transcripts. In addition to the major transcript, a few larger, low-abundance transcripts are detectable, which
are spliced transcripts initiating upstream of m41. They use the splice acceptor site indicated by an arrowhead in panel a. Transcripts of similar size are detected in cells
transfected with pcDNA–m41. (c) Schematic view of the m41 locus within the MCMV genome. The m41.1 ORF was tagged within the viral genome with an HA epitope tag
sequence (grey box), resulting in disruption of the m41 ORF. In the previously described Dm41 mutant, the m41 ORF was replaced with a zeocin-resistance marker.24 The
m41.1ko mutant was obtained by re-inserting an HA-tagged version of m41, in which the m41.1 ATGs had been mutated to ACG. Based on Dm41, three additional mutants
were constructed by inserting ORFs m41.1, HA–Bcl-xL, or FlagM11L driven by a phosphoglycerate kinase promoter (Ppgk) into the nonessential m02–m06 region of the MCMV
genome. (d) The HA-tagged m41.1 protein was detected in cells infected with MCMV–m41.1HA by Western blot starting 5 hpi. The immediate-early-1 protein (IE1), the early-1
protein (E1), the M44 protein, and glycoprotein-B (gB) are shown as representatives of the immediate-early, early, early-late, and late kinetic classes of MCMV proteins
CMVs inhibit Bak and Bax
M C¸am et al
657
Cell Death and Differentiation

deletion of m41.1 sensitized infected cells to STS and ActD
(Figure 4a). We also treated infected cells with STS and
tested whether m41.1 was required to inhibit mitochondrial
cyt c release. The results in Figure 4b show that cyt c release
was inhibited in the presence of m41.1. The lack of m41.1 was
compensated by expression of Bcl-xL or M11L.
m41.1 specifically inhibits Bak-mediated cell death and
synergizes with m38.5. Recently, we and others have
shown that MCMV expresses a mitochondrial protein that
specifically blocks Bax-mediated cell death.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. The results shown in Figure 5a clearly
demonstrated that m41.1 was required to inhibit Bak-
mediated cell death, whereas m38.5 was required to block
Bax-mediated cell death. In the absence of both m41.1
and m38.5, infected cells were sensitive to Bax- and
wt
Δm41
m41ko
m41.1ko
ΔBclXL
ΔM11L
tit
er
(T
CI
D 5
0/m
l)
DL
10.1 fibroblastsg h
days p.i.
101
102
103
104
105
106
107
tit
er
(T
CI
D 5
0/m
l)
101
102
103
104
105
106
DL
RAW264.7 macrophages
wt
Δm41
m41ko
m41.1ko
Δ/M11L
mock
RAW264.7
macrophages
18 hpi
RAW264.7
macrophages
%
T
UN
EL
p
os
. c
el
ls
20 hpi
0
5
10
15
10.1 fibroblasts
0
0.2
0.4
0.6
0.8
1.0
re
la
tiv
e
vi
ab
ilit
y
10.1 fibroblasts
%
T
UN
EL
p
os
. c
el
ls
48 hpi
0
5
10
15
20
25
15
kDa
25
15
0
hpi
18
hpi
m
o
ck
w
t
Δm
41
m
41
ko
m
41
.1
ko
Δ/
M
11
L
ST
S
25
15
25
15
25
15
kDa
0
hpi
24
hpi
m
o
ck
w
t Δ
m
41
m
41
ko
m
41
.1
ko
Δ/
M
11
L
ST
S
72
hpi
caspase-3
caspase-3
0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
re
la
tiv
e
vi
ab
ilit
y
0 97531
days p.i.
0 1197531
18 hpi 72 hpi
Figure 2 Phenotypic characterization of the mutant viruses. (a) 10.1 fibroblasts were infected at an MOI of 5 with wt or mutant MCMVs. Cell viability was measured using
an MTT assay and values are shown relative to the viability of wt MCMV-infected cells. (b) RAW264.7 macrophages were infected at an MOI of 10 and cell viability was
measured as above. (c, d) Nuclear DNA fragmentation was measured in infected fibroblasts and macrophages by TUNEL assay. (e, f) Caspase-3 activation was determined in
the same cells by Western blotting using an anti-caspase-3 antibody. The upper band represents the inactive procaspase-3 and the lower band represents the activated
(cleaved) form. STS-treated cells are used as positive control. Note that the effect of STS has waned at 72 hpi. (g, h) To determine the replication capacities of the mutant
viruses, 10.1 fibroblasts and RAW264.7 macrophages were infected at an MOI of 0.05 and 0.5 respectively. Viral replication was determined by titration on different days
post-infection. DL, detection limit
CMVs inhibit Bak and Bax
M C¸am et al
658
Cell Death and Differentiation

Bak-mediated apoptosis. Similar results were obtained
without STS treatment, when the infection was allowed to
proceed for a longer period of time (Figure 5b). This indicated
that the viral infection itself leads to an induction of Bak- and
Bax-mediated cell death.
The use of viral deletion mutants allowed us to determine
that m41.1 is required for inhibition of Bak-dependent
apoptosis. However, it was not clear from these results
whether m41.1 by itself is sufficient to block Bak-dependent
apoptosis. Therefore, we expressedm41.1 in Bakþ /Bax� and
Bak�/Baxþ fibroblasts by retroviral transduction and ana-
lyzed the sensitivity of the cells to drug-induced apoptosis.
Indeed, m41.1 provided protection against STS- or ActD-
induced apoptosis in Bak-only but not in Bax-only cells (Figure
5c–e). By contrast, Bcl-xL inhibited drug-induced apoptosis in
both cell types, consistent with its known ability to inhibit
Bak- and Bax-mediated cell death.26,27 In NIH-3T3 cells
(which express both Bax and Bak), transfection of an m41.1
expression plasmid was not sufficient to inhibit apoptosis
induced by Fas stimulation. Transfection of plasmids encod-
ing Bax inhibitors of MCMV (m38.5) or HCMV (UL37x1) did
also not protect from Fas-induced cell death. However, when
m41.1 was coexpressed with m38.5 or UL37x1, Fas-induced
apoptosis was inhibited to a similar extent as with Bcl-xL
(Figure 5f). These results confirmed that m41.1 and m38.5
synergize to inhibit Bak- and Bax-mediated cell death.
m41.1 interacts with Bak and inhibits Bak
oligomerization. During activation of the mitochondrial
apoptosis pathway, Bax and Bak undergo a conformational
change leading to exposure of their N-terminus. In this open
(activated) conformation, Bak and Bax molecules can
oligomerize within the mitochondrial outer membrane.4 This
leads to increased mitochondrial membrane permeability and
release of proapoptotic factors such as cyt c. As the previous
experiments had shown that m41.1 blocks cyt c release,
we wondered whether the preceding activation and
oligomerization of Bak were also inhibited.
Bak activation can be assessed by immunofluorescence
analysis using an antibody directed against the N-terminus of
Bak28 or against the Flag epitope if an N-terminally Flag-
tagged Bak is used. As shown in Figure 6a, MCMV infection
induced Bak activation regardless of the presence or absence
of m41.1. However, m41.1 itself is not responsible for Bak
activation, because Bak is activated in cells infected with the
m41.1ko virus, and m41.1 expression from a plasmid vector
does not induce the active conformation. Hence, m41.1 does
not prevent Bak activation, and neither does Bcl-xL
(Figure 6a). It is known that Bcl-xL and Mcl-1 interact with
Bak in its open (active) conformation,29 whereas VDAC2
interacts with the closed conformation.30 Thus, m41.1 might
wt
Δm41
m41ko
m41.1ko
Δ/M11L
re
la
tiv
e
vi
ab
ilit
y
0
0.2
0.4
0.6
0.8
1.0
cytc
cytc
Bak
m
o
ck
M
CM
V
wt
Δm
41
m
41
ko
Δ/
M
11
L
Δ/
Bc
lX
L
m
41
.1
ko
STS +–
SN
HM
+
m
o
ck
HM
STS ActD
+++++
Figure 4 m41.1 is required to protect MCMV-infected cells from STS- or ActD-
induced apoptosis. (a) Infected 10.1 fibroblasts were treated 6 hpi with STS or ActD
to induce apoptosis. Cell viability was determined 18 h later by MTT assay and is
shown as relative viability compared with infected cells treated with the solvent
DMSO. (b) Infected 10.1 fibroblasts were treated with STS and presence of cyt c in
the heavy membrane (HM) and supernatant (SN) fractions was determined by
immunoblotting. Bak was used as a loading control for the HM fraction
m41.1
m41.1
merge
merge
Hsp60
mergem41.1 Hsp60
m41.1
MitoTracker
PDI merge
Figure 3 Fluorescence images showing mitochondrial localization of m41.1.
(a) NIH-3T3 cells were transfected with a plasmid expressing HA-tagged m41.1.
The protein was detected by immunofluorescence using an anti-HA antibody. The
mitochondrial Hsp60 protein and the ER marker PDI were detected with specific
antibodies. The red fluorescent MitoTracker dye was used to stain mitochondria.
(b) Colocalization of m41.1 and Hsp60 was also detected in NIH-3T3 cells 10 hpi
with MCMV–m41.1HA. Bar, 10 mm
CMVs inhibit Bak and Bax
M C¸am et al
659
Cell Death and Differentiation

vector
m41.1
Bcl-xL
m41
Bak+/Bax–
re
la
tiv
e
vi
ab
ilit
y
0.2
0.3
0.4
0.5
re
la
tiv
e
vi
ab
ilit
y
Bak–/Bax+
STSSTS ActD
0
0.2
0.4
0.6
0.8
1.0
re
la
tiv
e
vi
ab
ilit
y
pcDNA
m38.5
UL37x1
+m41.1
m38.5
+m41.1
UL37x1
Bcl-xL
m41.1
NIH-3T3
αFas+ CHX
0.2
0.4
0.6
0.8
1.0
0
Bak+/Bax+
Bak+/Bax–
Bak–/Bax+
Bak–/Bax–
fibroblasts
0
0.2
0.4
0.6
0.8
0.1
re
la
tiv
e
vi
ab
ilit
y
0
0.2
0.4
0.6
0.8
0.1
re
la
tiv
e
vi
ab
ilit
y
0
20
40
60
%
T
UN
EL
p
os
. c
el
ls
Bc
l-x
L
+RT
–RT
+RT
–RTm
41
.1
ve
ct
or
m
41
m
41
.1
c-
m
yc
Bc
l-x
L
ve
ct
or
m
41
m
41
.1
Bax
Bak
m41
tubulin
Bcl-xL
Bak+/Bax– Bak–/Bax+
Bak+/Bax– Bak–/Bax+
STS STS
wt Δm41 Δ/BclXL Δ/M11LΔm38.5m41ko m41.1ko dko
wt Δm41 Δm38.5m41ko m41.1ko dko
Figure 5 Bak-specific inhibition of apoptosis by m41.1 and synergy with the Bax-specific inhibitor vMIA. (a) Wt fibroblasts and fibroblasts lacking Bax and/or Bak were infected at
an MOI of 5 with wt and mutant MCMVs as indicated. Six hpi cells were treated with STS for 24 h. Cell viability relative to DMSO-treated control cells was determined by MTT assay.
(b) Cells were infected as described in panel a, but were not treated with STS. Cell viability at 70 hpi is shown relative to wt MCMV-infected cells. (c) Fibroblasts expressing only Bak
or only Bax were transduced with retroviral vectors encoding m41, m41.1, or Bcl-xL. Apoptosis was induced by treatment with STS or ActD. Cell viability was determined 48 h later
and is shown relative to the DMSO-treated control cells. (d) Cells were transduced and treated with STS as in panel c. Nuclear-DNA fragmentation was measured after 25 h by
TUNEL assay. (e) The presence of the relevant proteins in the cells used in panel d was determined by immunoblotting using antibodies specific to Bax, Bak, and m41. HA-tagged
Bcl-xL was detected with anti-HA antibody and tubulin served as loading control. Pilot experiments had indicated that the C-terminal HA tag reduces the antiapoptotic activity of
m41.1, and, therefore, vectors expressing untagged m41.1 were used in all cell death assays. Expression of the untagged m41.1 and c-myc (as control) was determined by RT-PCR.
Note that the m41.1 sequence is also PCR-amplified from m41 transcripts, as m41.1 lies within m41. However, the m41.1 ATGs were mutated to ACG in the m41 expression vector,
and, therefore, m41.1 is not translated from the m41 transcript. (f) NIH-3T3 fibroblasts were transfected with plasmids expressing the indicated proteins and treated with an anti-Fas
antibody and cycloheximide (CHX) to induce apoptosis. Cell viability was determined by MTT assay and is shown relative to cells transfected with a Bcl-xL expression plasmid
CMVs inhibit Bak and Bax
M C¸am et al
660
Cell Death and Differentiation

inhibit Bak in a similar way as Bcl-xL. In support of this
hypothesis, we found that m41.1 colocalized with activated
Flag–Bak in MCMV-infected cells (Figure 6b).
To test whether m41.1 interacts with Bak, we transfected
HEK 293 cells with plasmids encoding Flag–Bak and HA-
tagged m41.1 or m41, respectively. As shown in Figure 7a,
m41.1 co-immunoprecipitated with Bak and vice versa, but
m41 did not. This indicated a direct or indirect interaction of
m41.1 and Bak.
To determine the influence of m41.1 on Bak oligomeriza-
tion, we infected fibroblasts expressing Bak (but not Bax)
with wt and mutant MCMVs, stimulated the mitochondrial
apoptosis pathway with STS, and then stabilized oligomeric
complexes by adding the crosslinker 10,60-bismaleimido-
hexane (BMH). As shown in Figure 7b, Bak oligomerization
was inhibited by MCMV only if the virus expressed m41.1 or
Bcl-xL. Similarly, m41.1 inhibited Bak oligomerization in
MCMV-infected cells expressing Bak and Bax (Supplementary
Figure S3), suggesting that m41.1 inhibits both homo- and
heterooligomerization of Bak. STS-induced Bak oligomeriza-
tion was also inhibited by m41.1 or Bcl-xL alone in transduced
cells (Figure 7c). Hence we concluded that the m41.1 protein
functions as a viral inhibitor of Bak oligomerization (vIBO).
Evolutionary conservation of m41.1. HCMV expresses a
Bax-specific mitochondrial inhibitor of apoptosis (vMIA) that
is conserved in primate CMVs.31 Rodent CMVs also express
vMIA proteins, but there is no apparent sequence similarity
between primate and rodent vMIAs.19 When we searched all
available CMV sequences for the presence of putative m41.1
homologs, we identified ORFs encoding highly similar
proteins in two rat CMVs, the Maastricht and the English
isolates (Figure 8a). The ORFs are located at analogous
positions within the viral genomes and were named r41.1 and
e41.1, respectively. By contrast, we did not detect ORFs
similar to m41.1 in human or other primate CMVs.
To test whether sequence conservation correlated with
functional conservation, we analyzed the function of the e41.1
protein of the rat CMV (RCMV) English isolate. Indeed, the
e41.1 protein localized to mitochondria (Figure 8b) and
inhibited ActD-induced apoptosis in Bakþ /Bax� cells
(Figure 8c), suggesting that the function of m41.1/vIBO is
conserved among rodent CMVs.
Discussion
In this study, we identified a new viral antiapoptotic protein
that inhibits apoptosis induced by cytotoxic drugs or by the
viral infection itself in a Bak-dependent manner. We further
showed that this Bak-specific inhibitor synergizes with a
previously identified Bax-specific inhibitor to protect infected
cells from apoptosis-inducing stimuli.
The m41.1 protein is an unusual apoptosis inhibitor as it
differs in many ways from known viral Bcl-2-like proteins:
Δ/BclXL
mock
ActD
MCMV
wt
m41.1ko
DAPI
m
41
.1
Fl
ag
-B
ak
D
AP
I
m
e
rg
e
m41.1
plasmid
m41.1DAPI
activated BakGFP
activated Bak
Figure 6 Bak activation by MCMV infection. (a) NIH-3T3 cells were infected
with wt or mutant MCMVs, all of which express GFP. Activated Bak was detected by
immunofluorescence using an antibody directed against the Bak N-terminus. Bak is
inactive in mock-infected cells and in cells transfected with pcDNA-m41.1HA.
Activated Bak is detected after treatment with 10mg/ml ActD for 10 h. (b) Cells
stably expressing Flag–Bak were infected with MCMV-m41.1HA. Cells were fixed
10 hpi, stained with anti-HA and anti-Flag antibodies, and analyzed by confocal
microscopy
CMVs inhibit Bak and Bax
M C¸am et al
661
Cell Death and Differentiation

(i) With 57 amino acids and a molecular mass of approxi-
mately 7 kDa, the m41.1 protein is much smaller than cellular
and viral Bcl-2 analogs; (ii) the m41.1 protein shows no
recognizable sequence similarity to known antiapoptotic
proteins and lacks BH domains characteristic of the cellular
and viral Bcl-2-family proteins; and (iii) it is the first viral protein
that specifically inhibits Bak- but not Bax-mediated cell death.
The small size of the m41.1 protein initially suggested that
it could be an essential component of a larger viral protein
complex acting at the mitochondrial checkpoint. Additional
mitochondrial proteins of unknown function have recently
been discovered in MCMV,32 and these might be part of such
a complex. However, our subsequent results showed that the
m41.1 protein is sufficient for inhibiting Bak oligomerization
(Figure 7c) and Bak-mediated cell death (Figure 5c and d).
Although we cannot exclude a role of the other MCMV
mitochondrial proteins (e.g., for stabilizing or reinforcing the
activity of m41.1 or m38.5), these proteins are clearly not
essential for the functions of m41.1/vIBO and m38.5/vMIA.
It might seem surprising that m41.1-negative MCMVs do
not replicate in macrophages, although they grow with only
minimal defects in fibroblasts (Figure 2g and h). The most
likely reason for this is that macrophages are much more
sensitive than fibroblasts to cell death induced by infection
m
41
.1
ko
20
25
37
50
75
100
150
B
M
H
D
M
SO
m
o
ck
w
t
Δm
41
m
41
ko
Δ/
Bc
lX
L
m
o
ck
1x
2x
3x
4x
I
B
25
kDa
m
o
ck
G
FP

m
38
.5

m
41
m
41
.1
Bc
l-x
L
m
o
ck
ve
ct
or
20
25
37
50
75
100
150
25
kDa
WB: anti-HA
m
41
.1+
Ba
k
ctr
l+B
ak
m
41
+B
ak
m
41
.1+
Ba
k
ctr
l+B
ak
m
41
+B
ak
IP: anti-Flag WCL
WB: anti-Flag
10
kDa
20
25
kDa
LC
m41
m41.1
Bak
IP: anti-HA WCL
I
- STS++++++ - +++ ++++
Figure 7 Interaction with Bak and inhibition of Bak oligomerization by m41.1. (a) HEK 293 cells were co-transfected with plasmids expressing Flag-tagged Bak and
HA-tagged m41.1, m41, or a control (ctrl) plasmid. Whole-cell lysates (WCL) were subjected to immunoprecipitation (IP) using either an anti-HA or an anti-Flag antibody. The
co-immunoprecipitated proteins were detected by Western blotting (WB). LC indicates the antibody light chain. (b) Fibroblasts stably expressing Flag–Bak (but not Bax) were
infected with wt and mutant MCMVs, and treated with STS to enhance Bak oligomerization. Cell lysates were treated with DMSO or the crosslinking agent BMH for 30 min,
separated by gel electrophoresis, and probed with an anti-Flag antibody. Mono-, di-, tri-, and tetrameric complexes are indicated with arrow heads (1� to 4� ). I indicates a
faster migrating, internally crosslinked Bak isoform. B probably represents Bak crosslinked to a different cellular protein. (c) Fibroblasts expressing Flag–Bak were transduced
with retroviruses encoding the indicated proteins and treated with STS. Bak oligomerization was determined as described above
m41.1
e41.1
r41.1
e41.1 merge
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
re
la
tiv
e
vi
ab
ili
ty
Bak+/Bax–
m38.5 e41.1GFP Bcl-xL
Hsp60
Figure 8 Evolutionary conservation of m41.1. (a) ClustalW amino-acid sequence alignment of MCMV protein m41.1, rat CMV English isolate protein e41.1, and rat CMV
Maastricht isolate protein r41.1. Identical and similar amino acids are shaded in black and gray, respectively. (b) NIH-3T3 cells were transfected with a plasmid expressing
HA-tagged e41.1. Colocalization of e41.1 with the mitochondrial protein Hsp60 was determined by confocal microscopy as in Figure 3. (c) Bakþ /Bax– fibroblasts were
transduced with retroviral vectors expressing the indicated proteins and treated with ActD for 48 h as in Figure 5c. Bcl-xL and e41.1 protected from ActD-induced cell death, but
m38.5 did not
CMVs inhibit Bak and Bax
M C¸am et al
662
Cell Death and Differentiation

with m41.1-negative viruses. In macrophages, cell death
occurs 15–24h after infection with high MOI, but in fibroblasts
only after 48–72h (Figure 2a–d). As the replication cycle of
MCMV takes approximately 24 h, the early onset of apoptosis
in macrophages should massively impair virus progeny
production. An increased sensitivity to infection-induced cell
death and/or reduced viral replication in macrophages has
also been observed with MCMVs lacking other cell-death
suppressors such as m38.5,22 M36,26 or M45,33 suggesting
that macrophages are generally more sensitive to infection-
induced cell death.
Although widely used, the MTT assay has occasionally
been criticized for relying on mitochondrial respiration as a
measure of cell viability. This point was worth considering, as
it has been shown that HCMV stabilizes mitochondrial
respiration.33 Therefore, it is important to be sure that the
MTT assay really measures the viability of MCMV-infected
cells and does not merely reflect changes in mitochondrial
respiration. We have obtained results very similar to the MTT
assay results using a conventional Trypan blue exclusion
assay (data not shown) and have also published similar
results to those for the Dm41 mutant using a neutral red
inclusion assay.24 Moreover, the results of the MTT assay
were consistent with the results of two apoptosis assays
(TUNEL and caspase-3 activation; Figure 2). Thus we
concluded that the MTT assay is an adequate means to
quantify cell viability in this study.
Mammalian cells express at least six antiapoptotic Bcl-2-
family proteins: Bcl-2, Bcl-xL, Bcl-w, Bcl-B, Bfl-1/A1, and
Mcl-1. Four of them interact with and inhibit both Bax- and
Bak-, but Bcl-B predominantly blocks Bax-, and Mcl-1
predominantly blocks Bak-mediated apoptosis.27 This finding
has lent further support to the hypothesis that the numerous
Bcl-2 proteins exist because they serve at least some non-
overlapping functions and can be regulated independently in
different cells and tissues.11 If correct, this hypothesis would
imply that viral proteins with Bcl-2-like functions could also
differentially modulate the proapoptotic proteins Bax and Bak.
The present study demonstrates that this is being done by
MCMV and related viruses.
The evolutionary origin of m41.1/vIBO remains enigmatic.
The protein shows no apparent sequence homology to any
cellular gene and is, therefore, unlikely to be a gene captured
from the host-cell genome, as it is assumed to be the case for
the Bcl-2 homologs of other viruses. It is unclear why CMVs
have evolved their own antiapoptotic proteins instead of
capturing them. However, CMVs have also taken their own
path for inhibition of caspase-8 (a.k.a. FLICE): The UL36 and
M36 proteins of HCMV and MCMV inhibit caspase-8 activa-
tion,26,35 but share no homology with the cellular FLICE
inhibitory proteins (cFLIPs) and the vFLIPs found in
g-herpesviruses and poxviruses.36
Orthologs of m41.1/vIBO are encoded by two RCMVs
(Figure 8), and these viruses also encode orthologs of the Bax
antagonist m38.5.20 Hence, the principle of blocking Bax- and
Bak-mediated cell death with two separate viral proteins
seems to be conserved in these related viruses. HCMV
expresses vMIA from ORF UL37x1,14 which is located at a
genomic position analogous to m38.5, but shows no obvious
sequence similarity to the MCMV gene. Orthologs of HCMV
vMIA were also found in a number of primate CMVs.31 It has
been shown that HCMV vMIA interacts with Bax and inhibits
Bax- but not Bak-mediated cell death.15,16,21 This suggests
that human and primate CMVs might also express a yet to be
identified Bak-specific inhibitor. Alternatively, it has been
argued that Bax is dominant over Bak in human cells (but not
in mouse cells), and that a Bax-specific inhibitor might suffice
to prevent mitochondrial outer membrane permeabilization.15
This would explain why vMIA blocks apoptosis efficiently in
human cells expressing Bax and Bak. However, another study
challenged the Bax specificity of HCMV vMIA by presenting
evidence that vMIA also interacts with Bak.37
Future studies will elucidate the similarities and differences
in the way how primate and rodent CMVs inhibit Bax and Bak.
Particularly the rodent CMVs provide an excellent model to
study the role of Bax and Bak during viral infection and will
help to reveal non-redundant functions of these two cellular
apoptosis mediators.
Materials and Methods
Cells and viruses. IC-21 (ATCC TIB-186) and RAW264.7 (ATCC TIB-71)
macrophages were grown in RPMI 1640 or DMEM supplemented with 10% FCS,
and 10 mM HEPES. Bak�/� and Bax�/� mouse fibroblast cell lines29 were provided
by Georg Ha¨cker (Technical University Munich, Germany), with permission from
David Huang (WEHI, Melbourne, Australia). NIH-3T3 cells, 10.1 fibroblasts and
HEK 293 cells were cultured as previously described.38 Wt and recombinant
MCMVs were propagated in 10.1 mouse fibroblasts as described,38 and viral titers
were determined using the TCID50 (median tissue culture infective dose) method.
Plasmids and retroviral vectors. ORFs m41.1 and e41.1 were PCR-
amplified and cloned in pcDNA3 (Invitrogen, Karlsruhe, Germany) with and without
a C-terminal HA tag. The murine bak gene was excised from pBabe–Bak–IRES–GFP39
(kindly provided by Wei-Xing Zong, SUNY, Stony Brook, NY, USA) and inserted into
pcDNA3–Flag. The Flag-tagged bak gene was subsequently inserted into
pMSCVpuro (Clontech). A codon-optimized version of the myxoma virus M11L
gene with an N-terminal Flag tag was synthesized by GeneArt (Regensburg,
Germany). The m41.1 coding sequence and FlagM11L were inserted into pReplacer
essentially as described.38 Retroviral vector plasmids pRetroGFP, pRetro-Bcl-XL, and
pRetro-m38.5.have been described,20 and pRetro-m41, pRetro-m41.1, and pRetro-
e41.1 were constructed analogously. To exclude m41.1 protein expression from the
pRetro-m41 vector, the three potential m41.1 ATG start codons were changed to ACG.
Retroviruses were generated by transfecting retroviral vector plasmids into the
Phoenix Ampho packaging cell line as described.24 Filtered supernatants were used
for transduction of bak�/� or bax�/� fibroblasts. Transduced fibroblasts were
maintained as bulk cultures without selection. Fibroblasts stably expressing Flag-
tagged Bak were made by transducing bax�/�/bak�/� or baxþ /�/bak�/�
fibroblasts with MSCVpuro–Flag–Bak and selection with 4 mg/ml puromycin.
Recombinant MCMVs. All recombinant MCMVs are based on a GFP-
expressing MCMV, except for MCMV-m41.1HA, which does not express GFP.
The mutant viral genomes were constructed using the bacterial artificial
chromosome (BAC) technology as described previously.24 An HA epitope
sequence and a kan gene flanked by FLP recognition target (FRT) sequences
were inserted at the 30 end of the m41.1 ORF. MCMVDm41 has been described in
reference.24 The Dm41/m41.1, Dm41/Bcl-XL, and Dm41/M11L were obtained by
inserting the indicated genes into the m02–m06 region of MCMVDm41 using the
pReplacer-based recombination system.38 An m41 coding sequence with the three
potential ATG codons of m41.1 mutated to ACG was re-inserted into MCMVDm41
with an FRT–kan–FRT cassette for selection. MCMV-m41.1ko was obtained after
removal of the kan gene with FLP recombinase. Recombinant MCMV BACs were
analyzed by restriction digest and by sequencing of the mutated site(s) (data not
shown). BACs were transfected into 10.1 fibroblasts to reconstitute recombinant
viruses. MCMV virion DNA was prepared essentially as described.40
RNA analyses. For Northern blot analysis, total cellular RNA was isolated with
Trizol reagent (Invitrogen) and further purified with an RNeasy kit (Qiagen, Hilden,
CMVs inhibit Bak and Bax
M C¸am et al
663
Cell Death and Differentiation

Germany) and subsequent DNAse-I (Roche, Mannheim, Germany) digestion.
A 1-mg weight of total RNA per sample was denatured, separated on a 1.2%
denaturing agarose gel, and blotted onto a positively charged nylon membrane
(Schleicher & Schuell, Dassel, Germany). The RNA was crosslinked to the
membrane using an UV crosslinker (Peqlab, Erlangen, Germany). Digoxigenin-
labeled m41 RNA probes were synthesized with a DIG RNA Labeling kit (Roche)
using linearized pcDNA–m4124 as template for in vitro transcription with T7 or SP6
polymerase (Fermentas, St. Leon-Rot, Germany). Hybridization, washing, and
detection of the DIG-labeled probe were performed according to the manufacturer’s
protocol (Roche).
Rapid amplification of 50 and 30 cDNA ends was performed with a SMART RACE
kit (Clontech, Mountain View, CA, USA). Amplification products were cloned in
pGEM-T Easy (Promega, Mannheim, Germany) and sequenced.
For reverse transcriptase (RT)-PCR analysis of gene expression, total cellular
RNA was extracted and DNase-treated as described.41 C-myc transcripts were
PCR amplified with published primers,41 and m41.1 transcripts with primers binding
to the first and last 20 nt of the m41.1 ORF.
Cell viability and apoptosis assays. STS and ActD were purchased
from Sigma (Munich, Germany) and dissolved in DMSO at 500 mM and 100 mg/ml
concentrations, respectively. To induce cell death, MCMV-infected 10.1 cells were
treated with 200 nM STS or 250 ng/ml ActD. Infected, knockout MEFs were treated
with 450 nM STS. Retrovirus-transduced cells were treated 24 h after transduction
with 425 nM STS or 2 mg/ml ActD for 48 h. NIH-3T3 cells grown in 48-well dishes
were transfected with 0.7 mg plasmid DNA using Superfect (Qiagen). Twenty-five
hours after transfection, cells were treated with 0.5 mg/ml anti-Fas antibody (BD
Pharmingen, San Diego, CA, USA) and 2.5 mg/ml cycloheximide for 45 h. Cell
viability was measured using an MTT assay as described.38 Absorbance at 570 nm
was determined photometrically. Mean values and standard deviations of at least
four parallel experiments are shown.
To analyze nuclear DNA fragmentation, 10.1 fibroblasts were grown and infected
on coverslips. Transduced 10.1 cells were treated for 25 h with 600 nM STS.
Infected RAW264.7 were harvested from culture dishes and sedimented onto glass
slides using Cytospin (Thermo Scientific, Waltham, MA, USA). Cells were fixed with
3% paraformaldehyde and stained with a terminal TUNEL assay kit (Roche)
containing tetramethylrhodamine-coupled dUTP. Nuclei were counterstained with
40,60-diamidino-2-phenylindole (DAPI). The TUNEL assay using transduced cells
was performed in triplicate and more than 100 nuclei were evaluated per sample.
For infected fibroblasts and macrophages, an average of 500 and 280 nuclei,
respectively, in five or more randomly selected visual fields were counted.
To measure cyt c release, 106 cells were mock-infected or infected at an MOI
of 3. After 15 h, cells were treated for 5 h with 450 nM STS. Cells were harvested
and separated into a mitochondria-enriched heavy membrane fraction and a
supernatant fraction as described elsewhere.30
Bak oligomerization assay. Fibroblasts were infected at an MOI of 5 for
15 h and treated with 450 nM STS for 5 h. Bak oligomerization was determined
as described elsewhere.42 Briefly, the mitochondria-enriched fraction was
resuspended in HIM buffer (200 mM mannitol, 70 mM sucrose, 10 mM HEPES-
KOH, 1 mM EGTA, pH 7.5) and freshly prepared 10, 60-bismaleimidohexane
(from TCI Europe, Zwijndrecht, Belgium) in DMSO was added to obtain a 2-mM final
concentration. BMH-mediated crosslinking was allowed to proceed for 30 min at
room temperature. Bak oligomerization in transduced cells was induced by 15 h
incubation with 450 nM STS.
Immunoprecipitation and immunoblotting. For immunoprecipitation
experiments, 1.5� 106 HEK 293 cells were co-transfected by calcium phosphate
precipitation with pcDNAFlag–Bak and pcDNAm41.1HA, pcDNAm41HA, or a
control plasmid. After 25 h cells were lysed in a buffer containing 150 mM NaCl,
50 mM Tris-HCl (pH 7.5), 0.5% Triton-X-100, and a protease inhibitor cocktail
(Roche). Flag- or HA-tagged proteins were precipitated at 4 1C overnight with anti-
Flag M2 (Sigma) or an anti-HA (Sigma) antibodies bound to protein-G or protein-A
sepharose, respectively. Precipitates were washed six times with washing buffer
(100 mM NaCl, 20 mM Tris (pH 7.5), 100 mM EDTA, 0.05% Tween-20) and eluted
with boiling sample buffer. Cell lysates were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and transferred to nitrocellulose or PVDF
membranes by semi-dry blotting following standard protocols. Anti-HA 16B12
(Covance), anti-b-actin Ac74 (Sigma), anti-a-tubulin (Santa Cruz Biotechnology,
Santa Cruz, CA, USA), anti-Bak (Sigma), anti-Bax (Santa Cruz), anti-caspase-3
8G10 (Cell Signaling), and anti-cyt c (Santa Cruz) antibodies were purchased from
suppliers as indicated. Antibodies against MCMV IE1 and E1 were provided by
Stipan Jonjie´ (University of Rijeka, Croatia) and antibodies against M44 and
glycoprotein B were a gift from Lambert Loh (University of Saskatchewan, Canada).
Analysis of immediate-early, early, and late gene expression was performed as
described previously.20 A sequence encoding amino acids 1–116 of m41 was
cloned in pMAL-C2 (New England Biolabs, Frankfurt, Germany) that expresses
maltose-binding protein (MBP). The MBP fusion protein was expressed in
Escherichia coli and purified using an amylose resin column (New England Biolabs).
Mice were immunized with the fusion protein and monoclonal antibodies were
produced according to standard procedures. Note that the m41-specific 2A6
antibody does not recognize m41.1, since the amino-acid sequences of the two
proteins are unrelated (Figure 1a).
Immunofluorescence. Cells were seeded on coverslips at least 24 h prior to
infection or transfection. Mitochondria were stained with 100 nM MitoTracker Red
CMXRos (Invitrogen) for 20 min at 37 1C. Cells were washed with PBS and fixed
with 3% paraformaldehyde for 20 min at 4 1C, neutralized with 50 mM ammonium
chloride, permeabilized with 0.3% Triton X-100, and blocked with 0.2% gelatin.
Proteins of interest were detected with primary antibodies against HA (3F10;
Roche), Flag (M2; Sigma), Hsp60 (clone 24; BD Pharmingen), PDI (1D3; Stressgen,
Ann Arbor, MI, USA), or the Bak N-terminus (TC-102; Calbiochem, Nottingham,
UK), and secondary antibodies coupled to AlexaFluor568 or AlexaFluor488
(Invitrogen), respectively. Confocal laser-scanning microscopy was performed using
a Zeiss LSM510 META microscope.
Database accession numbers. Sequences of m41.1, r41.1, and e41.1
have been deposited at GenBank under the accession numbers FJ477245,
FJ477244, and FJ477243, respectively.
Acknowledgements. We thank Georg Ha¨cker, David Huang, and Wei-Xing
Zong for providing cells and plasmids; Bernhard Nieswandt for help with monoclonal
antibody production; Aline Pehla, Matthias Budt, and Kazimierz Madela for technical
assistance; and Uwe Schumacher, Sebastian Voigt, and Kemuel-Noel Masihi
for critically reading the manuscript. This work was supported by DFG grant
BR 1730/3-1 to WB.
1. Everett H, McFadden G. Apoptosis: an innate immune response to virus infection. Trends
Microbiol 1999; 7: 160–165.
2. Galluzzi L, Brenner C, Morselli E, Touat Z, Kroemer G. Viral control of mitochondrial
apoptosis. PLoS Pathogens 2008; 4: e1000018.
3. Youle RJ, Strasser A. The BCL-2 protein family: opposing activities that mediate cell death.
Nat Rev Mol Cell Biol 2008; 9: 47–59.
4. Chipuk JE, Green DR. How do BCL-2 proteins induce mitochondrial outer membrane
permeabilization? Trends Cell Biol 2008; 18: 157–164.
5. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ et al.
Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.
Science 2001; 292: 727–730.
6. Lindsten T, Ross AJ, King A, Zong WX, Rathmell JC, Shiels HA et al. The combined
functions of proapoptotic Bcl-2 family members bak and bax are essential for normal
development of multiple tissues. Mol Cell 2000; 6: 1389–1399.
7. Cartron PF, Juin P, Oliver L, Martin S, Meflah K, Vallette FM. Nonredundant role of Bax and
Bak in Bid-mediated apoptosis. Mol Cell Biol 2003; 23: 4701–4712.
8. Kepp O, Rajalingam K, Kimmig S, Rudel T. Bak and Bax are non-redundant during
infection- and DNA damage-induced apoptosis. EMBO J 2007; 26: 825–834.
9. Brooks C, Wei Q, Feng L, Dong G, Tao Y, Mei L et al. Bak regulates mitochondrial
morphology and pathology during apoptosis by interacting with mitofusins. Proc Natl Acad
Sci USA 2007; 104: 11649–11654.
10. Neise D, Graupner V, Gillissen BF, Daniel PT, Schulze-Osthoff K, Janicke RU et al.
Activation of the mitochondrial death pathway is commonly mediated by a preferential
engagement of Bak. Oncogene 2008; 27: 1387–1396.
11. Hardwick JM, Bellows DS. Viral versus cellular BCL-2 proteins. Cell Death Differ 2003; 10
(Suppl 1): S68–S76.
12. Kvansakul M, van Delft MF, Lee EF, Gulbis JM, Fairlie WD, Huang DC et al. A structural
viral mimic of prosurvival Bcl-2: a pivotal role for sequestering proapoptotic Bax and Bak.
Mol Cell 2007; 25: 933–942.
13. Kvansakul M, Yang H, Fairlie WD, Czabotar PE, Fischer SF, Perugini MA et al. Vaccinia
virus anti-apoptotic F1L is a novel Bcl-2-like domain-swapped dimer that binds a highly
selective subset of BH3-containing death ligands. Cell Death Differ 2008; 15: 1564–1571.
14. Goldmacher VS, Bartle LM, Skaletskaya A, Dionne CA, Kedersha NL, Vater CA et al. A
cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally
unrelated to Bcl-2. Proc Natl Acad Sci USA 1999; 96: 12536–12541.
CMVs inhibit Bak and Bax
M C¸am et al
664
Cell Death and Differentiation

15. Arnoult D, Bartle LM, Skaletskaya A, Poncet D, Zamzami N, Park PU et al.
Cytomegalovirus cell death suppressor vMIA blocks Bax- but not Bak-mediated
apoptosis by binding and sequestering Bax at mitochondria. Proc Natl Acad Sci USA
2004; 101: 7988–7993.
16. Poncet D, Larochette N, Pauleau AL, Boya P, Jalil AA, Cartron PF et al. An antiapoptotic
viral protein that recruits Bax to mitochondria. J Biol Chem 2004; 279: 22605–22614.
17. Pauleau AL, Larochette N, Giordanetto F, Scholz SR, Poncet D, Zamzami N et al. Structure
– function analysis of the interaction between Bax and the cytomegalovirus-encoded
protein vMIA. Oncogene 2007; 26: 7067–7080.
18. Brocchieri L, Kledal TN, Karlin S, Mocarski ES. Predicting coding potential from genome
sequence: application to betaherpesviruses infecting rats and mice. J Virol 2005; 79:
7570–7596.
19. McCormick AL, Meiering CD, Smith GB, Mocarski ES. Mitochondrial cell death
suppressors carried by human and murine cytomegalovirus confer resistance to
proteasome inhibitor-induced apoptosis. J Virol 2005; 79: 12205–12217.
20. Jurak I, Schumacher U, Simic H, Voigt S, Brune W. Murine cytomegalovirus m38.5 protein
inhibits Bax-mediated cell death. J Virol 2008; 82: 4812–4822.
21. Arnoult D, Skaletskaya A, Estaquier J, Dufour C, Goldmacher VS. The murine
cytomegalovirus cell death suppressor m38.5 binds Bax and blocks Bax-mediated
mitochondrial outer membrane permeabilization. Apoptosis 2008; 13: 1100–1110.
22. Manzur M, Fleming P, Huang DC, Degli-Esposti MA, Andoniou CE. Virally mediated
inhibition of Bax in leukocytes promotes dissemination of murine cytomegalovirus. Cell
Death Differ 2009; 16: 312–320.
23. Norris KL, Youle RJ. Cytomegalovirus proteins vMIA and m38.5 link mitochondrial
morphogenesis to Bcl-2 family proteins. J Virol 2008; 82: 6232–6243.
24. Brune W, Nevels M, Shenk T. Murine cytomegalovirus m41 open reading frame encodes a
Golgi-localized antiapoptotic protein. J Virol 2003; 77: 11633–11643.
25. Everett H, Barry M, Lee SF, Sun X, Graham K, Stone J et al. M11L: a novel mitochondria-
localized protein of myxoma virus that blocks apoptosis of infected leukocytes. J Exp Med
2000; 191: 1487–1498.
26. Me´nard C, Wagner M, Ruzsics Z, Holak K, Brune W, Campbell A et al. Role of murine
cytomegalovirus US22 gene family members for replication in macrophages. J Virol 2003;
77: 5557–5570.
27. Zhai D, Jin C, Huang Z, Satterthwait AC, Reed JC. Differential regulation of Bax and Bak by
antiapoptotic Bcl-2 family proteins Bcl-B and Mcl-1. J Biol Chem 2008; 283: 9580–9586.
28. Arnoult D. Apoptosis-associated mitochondrial outer membrane permeabilization assays.
Methods 2008; 44: 229–234.
29. Willis SN, Chen L, Dewson G, Wei A, Naik E, Fletcher JI et al. Proapoptotic Bak is
sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins.
Genes Dev 2005; 19: 1294–1305.
30. Cheng EH, Sheiko TV, Fisher JK, Craigen WJ, Korsmeyer SJ. VDAC2 inhibits BAK
activation and mitochondrial apoptosis. Science 2003; 301: 513–517.
31. McCormick AL, Skaletskaya A, Barry PA, Mocarski ES, Goldmacher VS. 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 2003; 316: 221–233.
32. Tang Q, Murphy EA, Maul GG. Experimental confirmation of global murine
cytomegalovirus open reading frames by transcriptional detection and partial
characterization of newly described gene products. J Virol 2006; 80: 6873–6882.
33. Brune W, Me´nard C, Heesemann J, Koszinowski UH. A ribonucleotide reductase homolog
of cytomegalovirus and endothelial cell tropism. Science 2001; 291: 303–305.
34. Reeves MB, Davies AA, McSharry BP, Wilkinson GW, Sinclair JH. Complex I binding
by a virally encoded RNA regulates mitochondria-induced cell death. Science 2007; 316:
1345–1348.
35. Skaletskaya A, Bartle LM, Chittenden T, McCormick AL, Mocarski ES, Goldmacher VS.
A cytomegalovirus-encoded inhibitor of apoptosis that suppresses caspase-8 activation.
Proc Natl Acad Sci USA 2001; 98: 7829–7834.
36. Thome M, Schneider P, Hofmann K, Fickenscher H, Meinl E, Neipel F et al. Viral FLICE-
inhibitory proteins (FLIPs) prevent apoptosis induced by death receptors. Nature 1997;
386: 517–521.
37. Karbowski M, Norris KL, Cleland MM, Jeong SY, Youle RJ. Role of Bax and Bak in
mitochondrial morphogenesis. Nature 2006; 443: 658–662.
38. Jurak I, Brune W. Induction of apoptosis limits cytomegalovirus cross-species infection.
EMBO J 2006; 25: 2634–2642.
39. Zong WX, Li C, Hatzivassiliou G, Lindsten T, Yu QC, Yuan J et al. Bax and Bak can localize
to the endoplasmic reticulum to initiate apoptosis. J Cell Biol 2003; 162: 59–69.
40. Redwood AJ, Messerle M, Harvey NL, Hardy CM, Koszinowski UH, Lawson MA et al. Use
of a murine cytomegalovirus K181-derived bacterial artificial chromosome as a vaccine
vector for immunocontraception. J Virol 2005; 79: 2998–3008.
41. Voigt S, Mesci A, Ettinger J, Fine JH, Chen P, Chou W et al. Cytomegalovirus evasion of
innate immunity by subversion of the NKR-P1B:Clr-b missing-self axis. Immunity 2007; 26:
617–627.
42. Ruffolo SC, Shore GC. BCL-2 selectively interacts with the BID-induced open conformer of
BAK, inhibiting BAK auto-oligomerization. J Biol Chem 2003; 278: 25039–25045.
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
CMVs inhibit Bak and Bax
M C¸am et al
665
Cell Death and Differentiation