Murine cytomegalovirus m38.5 protein inhibits Bax-mediated cell death.

JOURNAL OF VIROLOGY, May 2008, p. 4812–4822 Vol. 82, No. 10
0022-538X/08/$08.00�0 doi:10.1128/JVI.02570-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Murine Cytomegalovirus m38.5 Protein Inhibits Bax-Mediated Cell Death�
Igor Jurak,1†‡ Uwe Schumacher,1‡ Hrvoje Simic,2 Sebastian Voigt,1 and Wolfram Brune1*
Division of Viral Infections, Robert Koch Institute, 13353 Berlin, Germany,1 and Department of Histology and Embryology,
Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia2
Received 3 December 2007/Accepted 27 February 2008
Many viruses encode proteins that inhibit the induction of programmed cell death at the mitochondrial
checkpoint. Murine cytomegalovirus (MCMV) encodes the m38.5 protein, which localizes to mitochondria and
protects human HeLa cells and fibroblasts from apoptosis triggered by proteasome inhibitors but not from
Fas-induced apoptosis. However, the ability of this protein to suppress the apoptosis of murine cells and its role
during MCMV infection have not been investigated previously. Here we show that m38.5 is expressed at early
time points during MCMV infection. Cells infected with MCMVs lacking m38.5 showed increased sensitivity
to cell death induced by staurosporine, MG132, or the viral infection itself compared to the sensitivity of cells
infected with wild-type MCMV. This defect was eliminated when an m38.5 or Bcl-XL gene was inserted into the
genome of a deletion mutant. Using fibroblasts deficient in the proapoptotic Bcl-2 family proteins Bak and/or
Bax, we further demonstrated that m38.5 protected from Bax- but not Bak-mediated apoptosis and interacted
with Bax in infected cells. These results consolidate the role of m38.5 as a viral mitochondrion-localized
inhibitor of apoptosis and its functional similarity to the human cytomegalovirus UL37x1 gene product.
Although the m38.5 gene is not homologous to the UL37x1 gene at the sequence level, m38.5 is conserved among
rodent cytomegaloviruses. Moreover, the fact that MCMV-infected cells are protected from both Bak- and
Bax-mediated cell death suggests that MCMV possesses an additional, as-yet-unidentified mechanism to block
Bak-mediated apoptosis.
Programmed cell death (PCD) is a mechanism used by mul-
ticellular organisms to dispose of unwanted cells. This process
is necessary for the shaping of an organism during develop-
ment, for tissue homeostasis, and for defense against infectious
agents. The removal of infected cells during viral infections is
of particular importance, because viruses depend on the host
cell for their replication. Therefore, it is not surprising that
many viruses have evolved strategies to inhibit or delay the
onset of PCD (7, 38, 44).
One way of initiating PCD is by the stimulation of so-called
death receptors, such as the tumor necrosis factor (TNF) re-
ceptor and Fas, for instance, when immune effector cells rec-
ognize an infected cell. These death receptors can then activate
a cascade of cellular proteases (the caspase cascade), which
ultimately results in cell death (6). In addition to this extrinsic
pathway to PCD, a cell can also sense the presence of a virus
by itself and trigger a self-destruction program (7, 19). In both
extrinsic and intrinsic pathways, mitochondria play an impor-
tant role as integrators of diverse cell death-promoting and
-inhibiting factors (22).
The Bcl-2 family consists of cellular proteins that govern a
cell’s decision to live or die at the mitochondrial checkpoint
(22). These proteins are characterized by the presence of dis-
tinct Bcl-2 homology (BH) domains and can be divided into
the anti- and proapoptotic family members. The proapoptotic
family members Bax and Bak are key regulators of the apop-
totic signaling pathway and contain BH domains 1 to 3 (46). By
contrast, the antiapoptotic members of this family, such as
Bcl-2 and Bcl-XL, usually contain all four BH domains (BH1 to
BH4). The inhibition of antiapoptotic activity is mediated by
the so-called BH3-only proteins, which share only the third BH
domain with other family members. These proteins are acti-
vated as a consequence of intracellular damage, stress, or
death receptor stimulation, which subsequently leads to the
oligomerization of Bax and/or Bak at the mitochondrial outer
membrane. This oligomerization causes the permeabilization
of the membrane and the release of cytochrome c into the
cytosol, where cytochrome c forms a complex with the adaptor
protein Apaf-1 and participates in the activation of caspase-9
and caspase-3. The antiapoptotic proteins Bcl-2, Bcl-XL,
Bcl-w, Mcl-1, and A1 antagonize this process by inhibiting the
activation or the oligomerization of Bax and Bak. How exactly
the BH3-only proteins activate Bax and Bak and how the
Bcl-2-like proteins prevent this activation from happening have
not been fully resolved and are in part still controversial (17).
To inhibit premature PCD, viruses express proteins that
structurally and functionally resemble Bcl-2 (8, 47). Such pro-
teins are encoded, for instance, by adenoviruses and gamma-
herpesviruses. Poxviruses also express mitochondrial cell death
inhibitors, but these proteins show no homology in their amino
acid sequences to the cellular Bcl-2-like proteins (14, 45).
However, more recent investigations have revealed that they
closely resemble Bcl-2 family proteins in their three-dimen-
sional structures (23).
Cytomegaloviruses (CMVs), prototypes of the betaherpes-
viruses, do not encode sequence homologs of Bcl-2 in their
genomes but still inhibit apoptosis at the mitochondrial check-
point (3, 15, 37). The human CMV (HCMV) UL37x1 open
* Corresponding author. Mailing address: Division of Viral Infec-
tions, Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany.
Phone: 49 30 18754 2502. Fax: 49 30 1810754 2502. E-mail: BruneW
@rki.de.
† Present address: Department of Biological Chemistry and Molec-
ular Pharmacology, Harvard Medical School, Boston, MA 02115.
‡ I.J. and U.S. contributed equally to this work.
� Published ahead of print on 5 March 2008.
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reading frame (ORF) encodes a viral mitochondrion-localized
inhibitor of apoptosis (vMIA) which inhibits the induction of
PCD (15, 16). The UL37x1 vMIA protein was shown previ-
ously to block Bax- but not Bak-mediated apoptosis by binding
and sequestering Bax at the mitochondrial membrane (4, 34).
This finding was surprising, because many apoptotic stimuli
(such as staurosporine [STS] and the stimulation of death
receptors) can activate both Bax and Bak (46). The ability of
vMIA to protect human HeLa cells and fibroblasts from Fas-
or STS-induced PCD was proposed previously to result from
the dominance of Bax over Bak in these cells. In murine fibro-
blasts, by contrast, Bax and Bak were proposed to be codomi-
nant, because apoptosis can be induced in knockout fibroblasts
expressing either only Bax or only Bak. In these cells, vMIA
protects from apoptosis only in the absence of Bak (4). Re-
cently, Pauleau et al. demonstrated that the UL37x1 vMIA is
likely to bind Bax preferentially when Bax is inserted into the
mitochondrial membrane and that this binding function is in-
dependent of vMIA’s ability to disrupt the mitochondrial net-
work (32).
Interestingly, all primate CMVs seem to contain a vMIA
gene, but no such gene in the genomes of rodent CMVs has
been identified (29). However, a recent computational reeval-
uation of the murine CMV (MCMV) genome led to the dis-
covery of a small ORF, termed the m38.5 ORF (9), which
partially overlaps with the larger M38 ORF. The m38.5 ORF
spans the region between nucleotide positions 51780 and 52367
of the MCMV genome (GenBank accession no. NC_004065)
and was predicted previously to encode a protein of 196 amino
acids (9, 28). This protein appears to be conserved among
rodent CMVs since homologs of m38.5 are also present in both
the English and Maastricht isolates of rat CMV (see Fig. 1A).
The location of the m38.5 ORF within the MCMV genome
is analogous to that of the UL37x1 ORF within the HCMV
genome, but the m38.5 protein sequence shows little or no
similarity to the sequence of the HCMV UL37x1 protein.
Therefore, it appeared questionable whether the MCMV
m38.5 protein exerts an antiapoptotic function similar to that
of the UL37x1 vMIA protein. A study by McCormick et al.
showed that the m38.5 protein localizes to mitochondria and
protects transfected human fibroblasts and HeLa cells from
cell death induced by proteasome inhibitors (28). However,
m38.5 did not protect these cells from Fas-induced apoptosis,
even though the UL37x1 protein was protective in the same
assay. The exact nature of the antiapoptotic activity of m38.5
has remained largely unknown, particularly since the molecu-
lar mechanism by which proteasome inhibitors induce cell
death is not fully understood. Moreover, the expression kinet-
ics of m38.5 and its role during viral infection have not yet been
investigated.
In this study, we analyzed the expression and function of
m38.5 by using recombinant viruses in which the m38.5 gene
was inactivated or replaced by genes with known functions. We
showed that the m38.5 protein was expressed with early kinet-
ics during viral infection and that the protein was required to
protect MCMV-infected cells from STS- and MG132-medi-
ated cell death, as well as from premature apoptosis induced by
the viral infection itself. Using Bax- and Bak-deficient fibro-
blasts, we showed that m38.5 inhibited Bax- but not Bak-me-
diated apoptosis, likely as a result of an interaction with Bax.
Moreover, the fact that MCMV-infected cells were resistant to
both Bax- and Bak-mediated cell death indicates that MCMV
encodes an additional mechanism that inhibits apoptosis in-
duction via Bak.
MATERIALS AND METHODS
Cells and viruses. Wild-type (wt) and recombinant MCMVs were grown in
10.1 mouse fibroblasts (18) according to standard procedures (10), and virus
titers were determined using the median tissue culture infective dose method
(27). Bak�/� and Bax�/� mouse fibroblast cell lines (48) were provided by Georg
Ha¨cker (Technical University Munich, Germany) with permission from David
Huang (WEHI, Melbourne, Australia). NIH 3T3 fibroblasts (ATCC CRL-1658)
and SVEC4-10 endothelial cells (ATCC CRL-2181) were cultured in Dulbecco’s
modified Eagle’s medium supplemented with 10% fetal bovine serum with 100 U
of penicillin/ml and 0.1 mg of streptomycin/ml. For analyses of growth kinetics,
106 cells per well were seeded into six-well dishes and infected with MCMV at
the multiplicities of infection (MOIs) indicated below. Infectious medium was
removed 2 h postinfection (hpi), cells were washed with phosphate-buffered
saline, and 3 ml of fresh medium was added to each well. At the indicated time
points, 100-�l aliquots of medium were collected and replaced with fresh me-
dium. All growth kinetics experiments were done in triplicate, and the means and
standard deviations were used for the diagrams.
Plasmids. The m38.5 coding sequence (nucleotides [nt] 51780 to 52367 of the
MCMV genome; GenBank accession no. NC_004065) was amplified by PCR
and cloned with a hemagglutinin (HA) tag sequence attached to the 3� end
into pcDNA3 (Invitrogen) by using EcoRI and XhoI sites. The transfer vector
pReplacer-m38.5 was made analogously to the previously described pReplacer-
BclXL and pReplacer-UL37x1 constructs (21).
Construction of recombinant MCMVs. All recombinant viruses were based on
MCMV-GFP, a recombinant MCMV expressing the enhanced green fluorescent
protein (GFP) (11). Mutations were introduced into the MCMV genome by
using bacterial artificial chromosome (BAC) technology essentially as described
before (13). Homologous recombination was carried out with Escherichia coli
strain DY380 (49). For the tagging of the m38.5 gene, the HA tag sequence and
a kanamycin resistance gene (kan) flanked by Flp recognition target (FRT) sites
were amplified by PCR from pcDNA-m38.5HA-kan. PCR primers were de-
signed in such a way that the resulting PCR product contained a 50-bp sequence
homologous to the 3� end of the m38.5 gene sequence and a 50-bp region
homologous to the sequence downstream of the m38.5 stop codon. The kan
cassette was removed by Flp recombinase as described previously (12). For the
inactivation of the m38.5 gene, a sequence of 24 nt adjacent to the ATG start
codon (nt 52347 to 52370) was deleted and replaced with a zeocin resistance gene
(zeo) as described previously (12). Similarly, the region comprising M37, M38,
m38.5, and m39 ORFs (nt 49492 to 53150) was deleted to obtain the MCMV�
M37-m39 mutant. Rescue mutants Rm38.5, RUL37x1, and RBcl-XL were con-
structed on the basis of MCMV�M37-m39 by inserting sequences encoding
HA-tagged versions of m38.5, UL37x1, and Bcl-XL proteins driven by a phos-
phoglycerate kinase promoter into the nonessential region of MCMV between
the m02 and m06 genes by using the pReplacer system (21). Recombinant viruses
were reconstituted by transfecting 10.1 fibroblasts with MCMV BACs by using
PolyFect transfection reagent according to the protocol of the manufacturer
(Qiagen). The recombinant MCMV Rm41 expressing a HA-tagged m41 protein
(12) was constructed in our laboratory by Maren Syta (M. Syta and W. Brune,
unpublished data).
Retroviral transduction. The empty retroviral vector plasmid pRetroEBNA
and the GFP-expressing derivative pRetroGFP were obtained from Tom Shenk
(Princeton University, NJ) and have been used in previous studies (12). Se-
quences encoding HA-tagged Bcl-XL and m38.5 were inserted into pRetro-
EBNA to obtain pRetro-BclXL and pRetro-m38.5, respectively. Phoenix pack-
aging cells were transfected with the plasmids. Supernatants were harvested after
48 h, passed through 0.45-�m-pore-size filters, and used for the transduction of
fibroblasts in the presence of 4 �g of Polybrene/ml or stored at �80°C for later
use.
Cell death assays. Cells were seeded at a density of approximately 5 � 105 cells
per 96-well plate and infected with MCMV at an MOI of 5. Unless stated
otherwise, cell death was induced by adding STS (250 nM; Sigma), MG132 (10
�M; Calbiochem), or TNF-� (20 ng/ml; Promokine) 4 to 8 hpi. At the time
points indicated in the figures, the medium was removed and cell viability was
assessed by measuring mitochondrial activity with an MTT [3-(4,5-dimethylthia-
zol-2-yl)-2,5-diphenyltetrazolium bromide] assay as described previously (21).
Briefly, in the mitochondria of living cells, MTT is reduced into formazan, which
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FIG. 1. Evolutionary conservation and mutagenesis of MCMV m38.5. (A) Clustal W amino acid sequence alignment of MCMV Smith strain
protein m38.5 (corresponding to nt 51780 to 52367; GenBank accession no. NC_004065), rat CMV English isolate protein e38.5 (corresponding
to GenBank accession no. EU267790), and rat CMV Maastricht isolate protein r38.5 (corresponding to nt 35738 to 36235; GenBank accession no.
NC_002512). Sequences were aligned using a Blossum matrix. Identical and similar amino acids are boxed and shaded in dark and light gray,
respectively. The consensus sequence is shown below. (B) Arrangement of the M36 through m40 ORFs of wt MCMV and construction of m38.5
mutant viruses by BAC mutagenesis. The m38.5 gene was knocked out by deleting the ATG start codon and inserting a bacterial zeo gene, resulting
in the MCMV�m38.5 deletion mutant. A HA epitope tag sequence was attached to the 3� end of the m38.5 gene by using a kan cassette flanked
by FRT sites (black ovals) as a selectable marker. The kan cassette was removed by Flp recombinase. A second m38.5 knockout mutant, MCMV�
m38.5HA, was constructed on the basis of MCMV-m38.5HA. A larger deletion comprising M37 through m39 ORFs was generated by inserting
a zeo cassette. EcoRI restriction sites are indicated by asterisks, and the sizes of the expected EcoRI fragments are listed. (C) EcoRI restriction
patterns of wt and mutant MCMV BACs separated by agarose gel electrophoresis. The 13-kb fragment of wt MCMV and the 1.4- and
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can be measured photometrically. All viability experiments were done in qua-
druplicate. The pancaspase inhibitor z-VAD-fmk and the calpain inhibitor z-
LLY-fmk were purchased from MBL International. To analyze nuclear DNA
fragmentation as a late sign of apoptosis, cells were grown and infected on
coverslips, fixed with 3% paraformaldehyde, and stained with a terminal de-
oxynucleotidyltransferase-mediated dUTP-biotin nick-end-labeling (TUNEL)
assay kit (Roche) containing tetramethylrhodamine-coupled dUTP. Nuclei were
counterstained with 4�,6�-diamidino-2-phenylindole (DAPI). Fluorescence mi-
croscopy analyses were performed with a Zeiss Axiovert 200 microscope. Images
were acquired with an AxioCamHRc camera and AxioVision software (Zeiss).
Analysis of immediate-early and early gene expression. To allow immediate-
early and early protein expression, cells were infected with MCMV in the pres-
ence of 250 �g of phosphonoacetic acid (PAA; Sigma)/ml. The selective expres-
sion of immediate-early proteins was achieved by infecting cells in the presence
of 50 �g of cycloheximide (Sigma)/ml for 4 h. The medium was subsequently
removed, and cells were incubated with medium containing 5 �g of actinomycin
D (Sigma)/ml for another 4 h.
Western blotting and immunoprecipitation. For Western blot analyses, cells
were lysed in Triton buffer (10 mM Tris-HCl [pH 8], 140 mM NaCl, 1% Triton
X-100) supplemented with a protease inhibitor cocktail (Roche). Proteins were
separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
transferred onto a nitrocellulose membrane (Amersham). We used primary
antibodies against the HA epitope (16B12; Covance Research Products), Bax
(N-20; Santa Cruz), and �-actin (A5316; Sigma); CROMA101 and CROMA103
(provided by Stipan Jonjic, University of Rijeka, Croatia) against MCMV IE1
and E1 proteins, respectively; and 2E8.12A (provided by Lambert Loh, Univer-
sity of Saskatchewan, Canada) (25) against MCMV glycoprotein B. A horserad-
ish peroxidase-conjugated secondary antibody (50447; DakoCytomation) and
enhanced chemiluminescent reagents (Amersham) were used to visualize the
proteins of interest.
For immunoprecipitations, MCMV-infected cells were lysed with a buffer
containing 140 mM NaCl, 20 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 1% Triton
X-100, and protease inhibitors. After preclearing with protein G-Sepharose (GE
Healthcare), m38.5 was precipitated with an anti-HA antibody and protein G-
Sepharose. Precipitates were washed three times with buffer B (150 mM NaCl, 1
mM Tris-HCl [pH 7.6], 2 mM EDTA, 0.2% Triton X-100), twice with buffer C
(500 mM NaCl, 1 mM Tris-HCl [pH 7.6], 2 mM EDTA, 0.2% Triton X-100), and
once with buffer D (10 mM Tris-HCl, pH 7.6). Samples were then separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to
Western blotting.
Nucleotide sequence accession number. The rat CMV (English isolate) e38.5
gene sequence determined in this study has been deposited in GenBank under
the accession number EU267790.
RESULTS
Construction of recombinant MCMVs with an altered m38.5
locus. To study the role of m38.5 during MCMV infection, we
constructed a set of recombinant viruses using the BAC tech-
nology. Specific mutagenesis of the m38.5 ORF was difficult,
because the m38.5 ORF overlaps with the adjacent M38 ORF
(Fig. 1B). First, a recombinant virus in which a HA epitope tag
sequence was attached to the 3� end of the m38.5 coding
sequence was generated. A kanamycin (kan) cassette flanked
by FRT sites was also inserted to facilitate the selection of
recombinant MCMV BACs in E. coli, and this cassette was
subsequently removed by Flp recombinase (Fig. 1B). Based on
the genome of this recombinant virus (designated MCMV-
m38.5HA) and on the wt MCMV genome, additional mutants
were generated. The ATG start codon (the only ATG codon
within the entire m38.5 ORF) was replaced with a bacterial
zeocin resistance gene (zeo). In addition, a mutant MCMV
with a larger deletion comprising the M37, M38, m38.5, and
m39 ORFs was constructed (Fig. 1B). Recombinant BACs
were digested with restriction enzymes and separated by gel
electrophoresis in order to verify the expected changes in the
restriction patterns (Fig. 1C). In addition, the mutated sites
were analyzed by sequencing (Fig. 1D).
m38.5 is expressed with early kinetics. A previous analysis of
RNA transcripts expressed from the m38.5 coding region
showed a very complex pattern with a number of overlapping
transcripts (29), making it difficult to analyze m38.5 expression
at the level of mRNA conclusively. Therefore, we decided to
use recombinant viruses expressing a tagged m38.5 protein
(Fig. 2A) in order to study the expression kinetics of the pre-
dicted m38.5 gene during viral infection. We could detect weak
m38.5 protein expression as early as 4 hpi (Fig. 2B) but not at
earlier time points, even with prolonged exposure of the film
(data not shown). By contrast, the immediate-early protein IE1
was strongly expressed at 4 hpi (Fig. 2B) and could be detected
weakly at 1 hpi upon prolonged exposure of the film (data not
shown). On Western blots, the HA-tagged m38.5 protein had
an apparent molecular mass of approximately 25 kDa, which
corresponds to its predicted mass (Fig. 2A). No differences in
the apparent molecular weights of m38.5 proteins expressed
from plasmids and those expressed from the MCMV genome
were detected (data not shown). The expression of m38.5 was
not blocked by the addition of PAA, an inhibitor of viral DNA
replication, suggesting that m38.5 is expressed with early ki-
netics (Fig. 2B). Moreover, m38.5 was not expressed at detect-
able levels after release from the cycloheximide block in the
presence of actinomycin D, whereas the immediate-early pro-
tein IE1 was readily detected under the same experimental
conditions (Fig. 2C). This finding provided additional support
for the conclusion that m38.5 falls into the category of early
proteins and thus differs from its putative analog in HCMV,
the UL37x1 protein, which is expressed with immediate-early
kinetics (16, 20, 42). In immunofluorescence experiments, we
detected the HA-tagged m38.5 protein at mitochondria (data
not shown), which is in agreement with the previously reported
intracellular localization (28).
The lack of m38.5 impairs viral replication and the survival
of infected cells. It has been shown previously for a number of
viruses that the deletion of a suppressor of PCD can lead to the
premature death of infected cells and thereby diminish viral
replication (1, 5, 11, 12, 14, 33, 36, 45). Therefore, to determine
if m38.5 has an antiapoptotic function, we tested if the lack of
m38.5 results in increased apoptosis and decreased MCMV
replication. Cells were infected with wt MCMV or the m38.5
gene deletion virus MCMV�m38.5, and nuclear DNA frag-
1.6-kb fragments of the mutant viruses are indicated by arrows. MW, molecular size standard; m38.5HA-kan, MCMV-m38.5HA-kan; m38.5HA,
MCMV-m38.5HA; �m38.5, MCMV�m38.5; �m38.5HA, MCMV�m38.5HA; �M37-m39, MCMV�M37-m39. (D) The mutated sites of all five
mutant viruses were sequenced. Three sequences are shown, and the remaining sequences contained related or combined mutations. Numbers
indicate nucleotide positions within the MCMV genome, and the inserted sequences are labeled. The FRT site is flanked by short linker sequences.
EcoRI restriction sites are shown in bold. The stop codon of the m38.5 ORF (at the end of the HA tag sequence) and a stop codon within the
FRT site that terminates the M38 ORF are shaded in gray.
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mentation (as a sign of apoptosis) at day 3 after infection at an
MOI of 0.01 was visualized by a TUNEL assay. In MCMV�
m38.5-infected foci, many cells stained positive for DNA frag-
mentation, but only a few TUNEL assay-positive cells were
found in cell populations infected with wt virus (Fig. 3). At a
higher magnification, membrane blebbing and the release of
vesicles (putative apoptotic bodies) could be observed in many
of the MCMV�m38.5-infected cells (Fig. 3, third row) but only
rarely in cells infected with the wt control virus (data not
shown). The same phenotype was seen with the independently
constructed m38.5 knockout virus MCMV�m38.5HA. This re-
sult indicated that the observed phenotype was caused by the
intended mutation at the m38.5 locus and not by an adventi-
tious mutation that might have occurred elsewhere in the viral
genome.
Single-step growth analyses of wt and mutant viruses (at an
MOI of 5) showed that m38.5-deficient viruses had only mod-
est growth defects (Fig. 4A). These growth defects were not
detectable at 2 days postinfection (dpi) but became apparent at
later time points (4 and 6 dpi). A decrease in virus release
coincided with the appearance of massive cell death. Thus, we
presumed that the clear differences in virus titers at the end of
the experiment ( 10-fold at 6 dpi) were a result of premature
cell death that limited the production of viral progeny rather
than replication defects per se (Fig. 4). Multistep growth anal-
yses with a starting MOI of 0.01 50% tissue culture infective
FIG. 2. Kinetics of m38.5 expression as shown by Western blotting. (A) Lysates of cells infected with the indicated viruses were harvested 24
hpi and immunoblotted with an anti-HA antibody that recognized the tagged version of m38.5 with a size of approximately 25 kDa. The MCMV
IE1 protein was detected with an IE1-specific antibody. m38.5HA-kan, MCMV-m38.5HA-kan; m38.5HA, MCMV-m38.5HA; �m38.5HA,
MCMV�m38.5HA; �m38.5, MCMV�m38.5; �M37-m39, MCMV�M37-m39. (B) Cells infected with MCMV-m38.5HA were harvested at the
indicated hours postinfection. The MCMV proteins IE1 and E1 and the late glycoprotein B (gB) were detected with specific antibodies. The m38.5
protein was detected with an anti-HA antibody. Immediate-early and early proteins were expressed in the presence of PAA. M, mock-infected cells.
(C) Cells were mock infected (M) or infected (I) with MCMV-m38.5HA for 8 h. Viral protein expression was blocked in the presence of
cycloheximide (C) or actinomycin D (A). Immediate-early proteins were expressed selectively after release from the cycloheximide block after 4 h
and incubation with actinomycin D for another 4 h (C‹A). In all experiments, �-actin served as a loading control.
FIG. 3. MCMV�m38.5 induces nuclear DNA fragmentation. Murine 10.1 fibroblasts were infected at an MOI of 0.01 with wt MCMV or
MCMV�m38.5 (�m38.5), both of which express GFP. GFP expression and nuclear DNA fragmentation (detected using a TUNEL assay) 3 days
after infection were visualized by fluorescence microscopy. Nuclei were stained with DAPI. Cells infected with MCMV�m38.5 are also shown at
a higher magnification (third row).
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dose/ml showed even more pronounced growth defects (Fig.
4B). For these growth analyses, a 100-�l aliquot was collected
at each time point and a corresponding amount of fresh me-
dium was added. However, when the medium was exchanged
completely at each time point in a parallel experiment, wt
MCMV and the MCMV�m38.5 mutant reached similar high
titers (Fig. 4B). Fewer dead and disintegrated cells were ob-
served in the MCMV�m38.5-infected cell cultures under these
conditions than under the conditions in which only portions of
the medium were exchanged. This result suggested that cells
infected with an m38.5-deficient virus were more sensitive to
stress exerted by (partially) exhausted cell culture medium or
toxic factors released by infected cells than cells infected with
wt virus.
m38.5-deficient viruses induce cell death and do not protect
from STS- or MG132-induced apoptosis. The results from the
experiments shown in Fig. 3 and 4 indicated that m38.5 is
required to inhibit premature cell death. To further character-
ize and quantify this requirement, cells were infected at a high
MOI with different MCMV mutants, and cell viability levels at
different times after infection were measured using an MTT
assay. As shown in Fig. 5A, 10.1 fibroblasts infected with wt
MCMV or the HA-tagged virus lost only a little viability be-
tween 24 and 96 h after infection. By contrast, cells infected
with m38.5-deficient viruses showed severely reduced viability
at 96 hpi, as determined by an MTT assay. At this time point,
most cells were disintegrated and floating, whereas cells in-
fected with the control viruses showed a cytopathic effect but
were still attached to the cell culture dish and appeared to be
viable (data not shown). The induction of premature cell death
was seen not only in 10.1 fibroblasts but also in SVEC4-10
endothelial cells (Fig. 5B) and NIH 3T3 cells (Fig. 5C), sug-
gesting that it is not a cell type- or cell line-specific phenom-
enon. The loss of viability was largely blocked by PAA, an
inhibitor of viral DNA replication (Fig. 5D). This result indi-
cated that viral DNA replication itself or a process occurring
after DNA replication triggered PCD.
We also tested whether two families of proteases, caspases
and calpains, play a role in the execution of cell death triggered
by m38.5-deficient viruses. Cell death was partially inhibited by
z-VAD-fmk, a broad-spectrum caspase inhibitor (Fig. 5E).
This result may indicate that m38.5 is required for the inhibi-
tion of caspase-dependent as well as caspase-independent cell
death. By contrast, the addition of a calpain inhibitor did not
have a noticeable impact on the induction of premature cell
death (Fig. 5D).
Next, we tested if cells infected with MCMV�m38.5 would
be more sensitive to exogenously induced apoptosis than those
infected with wt MCMV. To this end, infected cells were
treated with established inducers of apoptosis. Cells infected
with MCMV�m38.5 were sensitive to STS- and MG132-in-
duced apoptosis but not to PCD induced by TNF-�, and cells
infected with wt MCMV were resistant to all three apoptotic
stimuli (Fig. 5F). By contrast, cells infected with an MCMV
mutant lacking the viral inhibitor of caspase-8 activation
(�M36) were highly sensitive to TNF-� but not to STS or
MG132 (Fig. 5F). These results strongly suggest an interfer-
ence of m38.5 with the intrinsic pathway of cell death.
The insertion of the m38.5 or Bcl-XL gene restores the wt
phenotype in a deletion mutant lacking m38.5. The deletion of
the ATG start codon of the m38.5 gene by a bacterial zeocin
resistance cassette may have an impact on the expression of the
neighboring M38 gene due to the overlapping of the two ORFs
(Fig. 1B). Hence, the observed phenotype of the MCMV�
m38.5 mutant virus may be the consequence of impaired ex-
pression of M38. However, this scenario seemed unlikely since
the HA tagging of m38.5 resulted in the disruption of M38 and
since the two HA-tagged viruses MCMV-m38.5HA and
MCMV-m38.5HA-kan behaved like wt MCMV in replication
and cell death assays (Fig. 4A and 5A to C). These findings
argued against an important role for M38 in these settings.
Yet the region surrounding the m38.5 gene has a very com-
plex transcriptional pattern (29), and it cannot be ruled out
that the phenotype of the MCMV�m38.5 mutant was
caused by a direct or indirect impact on other genes, distant
from or in close proximity to the m38.5 gene. To resolve this
question, we used a virus with a large deletion in the region
surrounding m38.5 (MCMV�M37-m39) and reinserted the
HA sequence-tagged m38.5 gene under the control of an
autonomous promoter at a distant site in the genome (be-
tween the m02 and m06 genes) known to be dispensable for
virus replication in vitro (31). In the same way, a HA se-
FIG. 4. Replication kinetics of wt and mutant MCMVs. (A) Single-
step growth curves for MCMVs in 10.1 fibroblasts infected at an MOI
of 5. m38.5HA-kan, MCMV-m38.5HA-kan; m38.5HA, MCMV-
m38.5HA; �m38.5, MCMV�m38.5; �m38.5HA, MCMV�m38.5HA;
�M37-m39, MCMV�M37-m39. (B) Multistep growth curves for
MCMVs in 10.1 fibroblasts infected at an MOI of 0.01. Infected cells
were incubated with (open symbols) or without (black symbols) the
daily exchange of the cell culture medium. TCID50, 50% tissue culture
infective dose.
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quence-tagged version of the cellular antiapoptotic Bcl-XL
gene was inserted. The resulting mutants were termed
Rm38.5 and RBcl-XL (Fig. 6A and B). The expression of the
HA-tagged proteins by the recombinant viruses was verified
by Western blotting (Fig. 6C).
The previous experiments had shown that an MCMV lack-
ing the entire region from the M37 to the m39 genes had the
same replication and cell death-inducing properties as MCMV�
m38.5 (Fig. 4A and 5A to C). The insertion of the m38.5 gene at
an ectopic location was sufficient to reverse the phenotype of the
MCMV�M37-m39 mutant: cells infected with Rm38.5 re-
tained a level of viability at 96 hpi similar to that of cells
infected with wt MCMV (Fig. 7A), and Rm38.5-infected cells
and wt MCMV-infected cells were equally resistant to STS- or
MG132-induced apoptosis (Fig. 7B). The well-characterized
cellular antiapoptotic protein Bcl-XL showed activity similar to
that of m38.5 when the Bcl-XL gene was inserted into the viral
genome, suggesting that these two mitochondrion-localized
FIG. 5. Cells infected with MCMVs lacking m38.5 show increased sensitivity to virus- and drug-induced cell death compared to cells infected
with wt MCMV. (A to C) 10.1 fibroblasts (A), SVEC4-10 endothelial cells (B), and NIH 3T3 fibroblasts (C) were infected with the indicated
viruses, and levels of cell viability at 24 and 96 hpi were measured by an MTT assay. No difference in viability among the cell types at 24 hpi was
detected (data not shown in panels B and C). m38.5HA, MCMV-m38.5HA; �m38.5HA, MCMV�m38.5HA; �M37-m39, MCMV�M37-m39.
(D) The premature death of MCMV�m38.5-infected cells was largely blocked by an inhibitor of viral DNA replication (PAA, 250 �g/ml) but not
by the calpain inhibitor z-LLY-fmk (zLLY). �, control; �m38.5, MCMV�38.5. (E) The addition of the pancaspase inhibitor z-VAD-fmk (zVAD),
but not the solvent dimethyl sulfoxide (DM), also reduced premature cell death. (F) 10.1 cells infected with wt MCMV were resistant to cell death
induced by STS, MG132, and TNF-�, but MCMV�m38.5-infected cells were sensitive to STS- and MG132-induced cell death, and �M36-infected
cells were sensitive to TNF-�-induced apoptosis. In all experiments, the 24-hpi values for untreated cells were normalized to 100%.
FIG. 6. Construction of rescue mutants expressing m38.5 or Bcl-XL. (A) Sequences expressing HA-tagged versions of m38.5 and Bcl-XL driven
by a phosphoglycerate kinase promoter (PGKp) were inserted into the nonessential region of the MCMV�M37-m39 genome between the m02
and m06 genes. m38.5HA, gene expressing HA-tagged m38.5; HABclXL, gene expressing HA-tagged Bcl-XL. (B) This insertion results in the loss
of a 2.16-kb HindIII fragment and the appearance of a new 1.25-kb fragment in the rescue mutants and an additional 0.96-kb fragment in RBcl-XL.
Band patterns for two clones of each rescue mutant are shown. MW, molecular size standard; �M37-m39, MCMV�M37-m39. (C) Detection of
HA-tagged proteins (m38.5 and Bcl-XL) and the MCMV E1 protein in infected 10.1 cells 24 hpi. m38.5HA, MCMV-m38.5HA; Rm38.5(HA) and
RBclXL(HA), Rm38.5 and RBcl-XL mutants expressing HA-tagged proteins.
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proteins have similar functions. The insertion of the m38.5
gene was also sufficient to reverse the growth defect of the
MCMV�M37-m39 mutant in a multistep growth kinetics ex-
periment (Fig. 7C).
m38.5 inhibits Bax-mediated cell death. It has been shown
previously that MCMV-infected cells are resistant to the in-
duction of apoptosis via the mitochondrial pathway (2), but the
molecular mechanism and the responsible viral gene products
have remained unknown. Many cellular and viral antiapoptotic
proteins operate at the mitochondrial membrane, inhibiting
the oligomerization and/or activation of the proapoptotic Bcl-2
family members Bax and Bak (8, 47). The HCMV UL37x1
gene product (vMIA) differs from most Bcl-2-like proteins in
that it inhibits only Bax- but not Bak-mediated cell death (4,
34). As the m38.5 gene is a positional homolog of the HCMV
UL37x1 gene, we wondered if m38.5 would have similarly
restricted activity. To test this, we infected fibroblasts lacking
Bax, Bak, or both proteins with different MCMV mutants and
stimulated the cells with STS. This drug is known to activate
Bax- as well as Bak-mediated apoptosis (46). As shown in Fig.
8A, m38.5 was required to protect Bax� but not Bax� cells
from STS-induced cell death. The insertion of an m38.5,
UL37x1, or Bcl-XL gene into the MCMV�M37-m39 virus re-
stored the ability to confer resistance to STS.
FIG. 7. The insertion of the m38.5 or Bcl-XL gene eliminates the
defects caused by the deletion of the m38.5 gene. (A and B) The levels
of viability of infected 10.1 cells at 24 and 96 hpi (A) and of infected
STS-, MG132-, or TNF-�-treated 10.1 cells at 24 hpi (B) were mea-
sured by an MTT assay. In all experiments, the 24-hpi values for
untreated cells were normalized to 100%. �m38.5, MCMV�m38.5; �,
no treatment. (C) Multistep growth kinetics of wt and mutant MCMVs
in 10.1 fibroblasts (MOI, 0.01). TCID50, 50% tissue culture infective
dose; �M37-m39, MCMV�M37-m39.
FIG. 8. m38.5 inhibits Bax- but not Bak-mediated cell death.
(A) Fibroblasts from wt, Bax knockout, Bak knockout, or double-
knockout mice were infected with the indicated viruses and treated
with STS. The percent viabilities of STS-treated cells versus dimethyl
sulfoxide-treated control cells are shown. �m38.5, MCMV�m38.5.
(B) Fibroblasts expressing only Bak were transduced with retroviral
vectors encoding m38.5, Bcl-XL, or GFP as a negative control. Bcl-XL
but not m38.5 protected cells against STS-induced cell death. Two
different concentrations of STS were used. (C) In fibroblasts express-
ing only Bax, Bcl-XL and m38.5 both protected against STS-induced
apoptosis. (D) wt and Bax�/� fibroblasts were infected with the indi-
cated viruses. Lysates of infected cells were subjected to Western
blotting and immunoprecipitation (IP) with an anti-HA antibody to
precipitate HA-tagged m38.5 and UL37x1 proteins. Bax was detected
as a coprecipitating protein, indicating an interaction between the viral
mitochondrial proteins and Bax. (E) Results of an experiment similar
to that described in the legend to panel D. A recombinant MCMV
expressing HA-tagged m41 protein (Rm41) was used as a negative
control.
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We also wondered whether m38.5 by itself (i.e., outside of
the context of viral infection) would be able to block apoptosis.
In previous studies, m38.5 did not protect human HeLa,
RPE-1, or fibroblast cells from Fas-mediated apoptosis but
provided some protection against apoptosis induced by pro-
teasome inhibitors such as MG132 (21, 28). HeLa and RPE-1
cells expressing m38.5 were also not protected from STS-in-
duced cell death (our unpublished observations). To investi-
gate if m38.5 possesses selective activity against Bax-mediated
cell death, as suggested by the results of the previous experi-
ment (Fig. 8A), we decided to use fibroblasts that expressed
either Bax or Bak. These fibroblasts were transduced with
retroviral vectors expressing m38.5, Bcl-XL, or GFP (as a neg-
ative control) and were then treated with different concentra-
tions of STS. As expected, Bcl-XL inhibited apoptosis in both
cell types. By contrast, m38.5 inhibited apoptosis only in Bax�
Bak� and not in Bax� Bak� cells (Fig. 8B and C). To substan-
tiate this finding, we examined if m38.5 interacts with Bax by
immunoprecipitation. As shown in Fig. 8D and E, Bax could be
coprecipitated with m38.5 from lysates of infected cells. These
findings reveal a specific role for m38.5 in Bax-mediated
apoptosis.
The results also show that MCMV-infected cells are pro-
tected from both Bax- and Bak-mediated cell death. As m38.5
is responsible for blocking only the Bax-mediated pathway, an
additional, hitherto-unidentified mechanism of MCMV inter-
ference with the Bak-mediated pathway must exist.
DISCUSSION
In this study, we analyzed the role of the m38.5 protein
during viral infection. Using mutant viruses, we demonstrated
that the lack of m38.5 increased sensitivity to cell death in-
duced by the virus itself, by STS, or by proteasome inhibitors
compared to the sensitivity of cells infected with the wt virus.
We further showed that m38.5 interacted with Bax and blocked
Bax- but not Bak-mediated apoptosis, suggesting the existence
of an additional, Bak-inhibiting mechanism.
By epitope tagging of the m38.5 ORF at its native position
within the viral genome, we were able to determine the expres-
sion kinetics of the m38.5 protein and classify it as an early
protein. This finding differs from that in a recent report, which
mentioned that m38.5 was transcribed only at later times (41),
but the data substantiating this conclusion were not given in
the article. It seems likely that differences in the sensitivity of
the assay systems caused this discrepancy.
In a previous study, m38.5 did not protect transfected HeLa
cells from Fas-mediated apoptosis but did reduce PCD in-
duced by proteasome inhibitors (28). This result suggested that
the role of m38.5 is to inhibit cell death induced via the intrin-
sic pathway. The extrinsic, death receptor-induced pathway is
already blocked during MCMV infection by means of the M36
gene product, which operates as a viral inhibitor of caspase-8
activation (30). When the M36 gene was deleted from the viral
genome, infected cells became sensitive to TNF-�-induced cell
death (Fig. 5F). This result indicated that the mitochondrial
inhibitors of apoptosis encoded by MCMV were unable to
protect from death receptor-derived signals, probably because
caspase-8 can directly activate downstream effector caspases
without the need to relay the signal through mitochondria.
Popkin and Virgin have shown previously that MCMV in-
hibits TNF-�-induced NF-
B activation in infected macro-
phages and have correlated this effect with the down-regula-
tion of TNF receptor 1 (TNFR1) from the cell surface (35).
Receptor down-regulation may render MCMV-infected cells
unresponsive to TNF-�. However, the down-regulation was
measured at 18 hpi and was not complete, suggesting that
TNFR1 down-regulation may not be sufficient. We have re-
cently shown that the MCMV M45 protein blocks TNFR1-
dependent NF-
B activation by interacting with the adaptor
molecule RIP1 (26). Fibroblasts infected with M45- and M36-
deficient viruses are sensitive to TNF-�-induced NF-
B acti-
vation and apoptosis, respectively (reference 26 and this
study). These results indicated that infected fibroblasts have
enough TNFR1 molecules on the surface to respond to TNF-�
stimulation, at least between 4 and 8 hpi, when TNF-� was
added. Nevertheless, TNFR1 down-regulation may contribute
to the unresponsiveness of MCMV-infected cells to TNF-� at
later times.
Cells infected with m38.5-deficient MCMVs were more sen-
sitive than wt virus-infected cells to stress induced by viral
replication within the cells, as well as to stress exerted by
exhausted growth medium, toxic secreted factors, or cytotoxic
drugs such as STS and MG132. Premature cell death affected
the growth kinetics of the MCMV�m38.5 virus to various
degrees, dependent on the infectious dose and the growth
conditions. Surprisingly, the MCMV�m38.5 mutant grew to
almost the same titers as the wt virus when the cell culture
medium was exchanged on a daily basis. This finding parallels
results obtained previously with a UL37x1 deletion mutant of
the HCMV strain Towne, which also grew almost to wt levels
in human fibroblasts, despite the fact that increased apoptosis
of infected cells was observed (28). However, UL37x1 deletion
mutants of the HCMV strain AD169 were severely growth
defective (12, 36, 40, 50), and a similar growth defect was
observed with the HCMV strain FIX (our unpublished re-
sults). Since the UL37x1 vMIA proteins of AD169 and Towne
are almost identical, it seems likely that the two HCMV strains
differ in their propensities to induce apoptosis. The issue of
which viral processes inside the cell induce apoptosis is not
fully understood. However, the fact that the inhibition of viral
DNA replication blocks the induction of apoptosis suggests
that DNA replication itself or later processes such as virion
assembly and egress are responsible. This possibility raises the
question of why m38.5 is expressed at early times while its
antiapoptotic function becomes important only at late times.
The discrepancy is even more pronounced for HCMV, in
which UL37x1 expression starts at immediate-early times (42).
It is possible that enough vMIA protein needs to accumulate
before DNA replication starts in order to block Bax-dependent
cell death efficiently. Alternatively, m38.5 may have an addi-
tional function that is important already at early times, such as
the induction of calcium release from endoplasmatic reticulum
stores, a recently described function of the UL37x1 protein
(40).
The complex structure of the M38-m38.5 locus made it dif-
ficult to construct a mutant virus in which only the m38.5 gene
was inactivated. By deleting only 24 nt around the ATG start
codon, a 391-nt stretch upstream of the M38 start codon was
left intact. Whether this method was sufficient to preserve the
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normal expression of M38 could not be determined, because
the boundaries of the M38 promoter have not been defined
and an M38-specific antibody is not available. However, we
provide two pieces of evidence showing that M38 does not play
a significant role in the phenotype studied here. Firstly, the
MCMV-m38.5HA virus carried the HA tag sequence at the C
terminus of m38.5, which corresponds to the M38 ORF. The
insertion of the HA tag coding sequence disrupted and termi-
nated the M38 ORF (Fig. 1D). Nonetheless, the MCMV-
m38.5HA mutant virus behaved like wt MCMV in replication
and cell death assays. Secondly, the phenotype of the MCMV�
M37-m39 mutant was reversed by inserting only the m38.5
gene into the viral genome at a different location from the
original gene position (Fig. 7). Hence, none of the M37, M38,
and m39 ORFs seem to be required for the inhibition of
apoptosis under the conditions tested here. This scenario is
consistent with the results of a previous study, in which the
authors found no mutant phenotype of an M37 mutant virus in
cell culture (24). Interestingly, an antiapoptotic function was
recently attributed to the HCMV UL38 protein (43), which
shows sequence homology to M38 (27% identity and 46%
similarity). Our results suggest that the experimental condi-
tions in the present study may have been unsuitable to reveal
a presumed antiapoptotic activity of M38. Alternatively, it is
possible that M38 does not have the same antiapoptotic activ-
ity as UL38.
By contrast, m38.5 and UL37x1 proteins have little or no
sequence homology but seem to be highly similar in their
functions. Both localize to mitochondria, interact with Bax,
and inhibit Bax-mediated PCD. Cells infected with m38.5 or
UL37x1 deletion mutants show increased apoptosis compared
to that of cells infected with wt virus and are more sensitive to
stress and drugs activating the mitochondrial apoptosis path-
way than wt virus-infected cells. The antiapoptotic function of
m38.5 is probably conserved in rodent CMVs, as sequence
homologs of the m38.5 gene can be found in the genomes of
two different rat CMVs, i.e., the Maastricht and English iso-
lates (reference 9 and S. Voigt, unpublished results). The
m38.5 protein is slightly more homologous to e38.5 than to
r38.5 but differs from both rat CMV proteins by a glutamine-
rich sequence (Fig. 1A).
Mitochondrial inhibitors of apoptosis prevent the release of
cytochrome c and the activation of caspase-9. However, there
is also evidence that Bcl-2 and vMIA proteins can block certain
forms of caspase-independent cell death (39). This possibility
may explain why Bcl-XL can block the increased cell death
caused by an m38.5-deficient virus while the pancaspase inhib-
itor z-VAD-fmk blocked cell death only partially, even at high
concentrations.
The vMIAs of HCMV and MCMV differ from other viral
mitochondrial apoptosis inhibitors in that they inhibit only
Bax- and not Bak-mediated cell death (reference 4 and this
study). This difference is surprising, because Bax and Bak can
mediate apoptosis independently and many apoptosis-inducing
stimuli activate both Bax and Bak (46). We have shown here
that MCMV-infected cells are protected from PCD via Bax
and via Bak but that m38.5 is responsible for the inhibition of
the Bax-mediated pathway only. Consequently, MCMV must
either encode a separate protein that inhibits Bak or up-reg-
ulate a cellular protein that interferes with the Bak-dependent
pathway. The first possibility is supported by the recent iden-
tification of genes for four additional mitochondrion-localized
proteins in the MCMV genome, the functions of which remain
to be determined (41). By analogy, it seems likely that HCMV
also encodes a protein with a Bak-inhibiting function.
ACKNOWLEDGMENTS
We thank Kerstin Heyl for technical assistance and Georg Ha¨cker
and David Huang for making Bax/Bak knockout fibroblasts available
to us.
This work was supported by the Deutsche Forschungsgemeinschaft
grant SFB 421/TP B14 to W.B.
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