Induction of apoptosis limits cytomegalovirus cross-species infection.

Induction of apoptosis limits cytomegalovirus
cross-species infection
Igor Jurak1,2 and Wolfram Brune1,2,*
1Rudolf Virchow Center for Experimental Biomedicine, University of
Wu¨rzburg, Wu¨rzburg, Germany and 2Division of Viral Infections,
Robert Koch Institute, Berlin, Germany
Cross-species infections are responsible for the majority
of emerging and re-emerging viral diseases. However, little
is known about the mechanisms that restrict viruses to
a certain host species, and the factors viruses need to
cross the species barrier and replicate in a different host.
Cytomegaloviruses (CMVs) are representatives of the
beta-herpesviruses that are highly species specific. They
replicate only in cells of their own or a closely related
species. In this study, the molecular mechanism under-
lying the cytomegalovirus species specificity was investi-
gated. We show that infection of human cells with the
murine cytomegalovirus (MCMV) triggers the intrinsic
apoptosis pathway involving caspase-9 activation. MCMV
can break the species barrier and replicate in human cells
if apoptosis is blocked by Bcl-2 or a functionally analogous
protein. A single gene of the human cytomegalovirus
encoding a mitochondrial inhibitor of apoptosis is suffi-
cient to allow MCMV replication in human cells. Moreover,
the same principle facilitates replication of the rat cyto-
megalovirus in human cells. Thus, induction of apoptosis
serves as an innate immune defense to inhibit cross-
species infections of rodent CMVs.
The EMBO Journal (2006) 25, 2634–2642. doi:10.1038/
sj.emboj.7601133; Published online 11 May 2006
Subject Categories: microbiology & pathogens; molecular
biology of disease
Keywords: apoptosis; caspase; cytomegalovirus; species
specificity; vMIA
Introduction
Viruses have gone through a coevolution with their hosts,
during which they have adapted to them. As a result of this
adaptation, many viruses have a limited host range.
Occasionally, a virus acquires a mutation or a new gene
that allows it to infect and replicate in a different host species.
This may lead to severe disease in the newly infected species,
to a local outbreak, or even a pandemic (Weiss, 2003) such
as the Spanish flu of 1918/19 (Taubenberger et al, 2005).
Another example of a cross-species infection is the AIDS
pandemic. In this case, simian immunodeficiency viruses
have acquired the ability to replicate and spread in humans
(Hahn et al, 2000).
The molecular mechanisms underlying the species barrier
have only begun to be understood. In some instances, the
availability of an appropriate entry receptor on the cell sur-
face limits the virus’ host range (reviewed in Baranowski
et al, 2003). In other cases, the virus is able to enter cells of
other species, but fails to block intracellular defense mechan-
isms that inhibit virus replication. It has recently been shown
for the human immunodeficiency virus that the ability of the
viral protein Vif to bind cellular cytidine deaminases limits its
host range (Mariani et al, 2003). If not blocked by Vif, these
enzymes cause a lethal hypermutation of the viral genome
(Lecossier et al, 2003). More recent work has demonstrated
that the species specificity of myxoma virus, a poxvirus of
rabbits, is a consequence of the virus’ inability to inhibit the
interferon response in cells of foreign species (Wang et al,
2004). Myxoma virus can break the species barrier and
replicate in murine cells if activation of the interferon
response is blocked by inhibitory drugs or with interferon
neutralizing antibodies. Similarly, the host range of certain
paramyxoviruses and an attenuated vaccinia virus also
depends on this innate immune defense (Parisien et al,
2002; Hornemann et al, 2003).
For the cytomegaloviruses (CMVs), the molecular basis
of their species specificity has not been determined. These
viruses belong to the b subfamily of the herpesviruses.
Representatives of this subfamily have been identified in
numerous animal species, and these viruses elicit similar
illnesses in their respective hosts (Mocarski and Courcelle,
2001). Human cytomegalovirus (HCMV) is an opportunistic
pathogen that causes generally mild infections in healthy
people, but can cause severe disease in immunocompromised
individuals such as transplant recipients or AIDS patients.
Since their first isolation in cell culture, the CMVs have been
recognized as highly species specific (Weller, 1970). They
replicate only in cells of their own or a closely related species.
For instance, simian CMV can replicate in human fibroblasts
(Lafemina and Hayward, 1988), and HCMV can replicate in
simian fibroblasts (Perot et al, 1992). Similarly, murine
cytomegalovirus (MCMV) was shown to be infectious for
rat cells (Bruggeman et al, 1982; Smith et al, 1986), but the
rat cytomegalovirus did not replicate in murine fibroblasts
(Bruggeman et al, 1982). However, cells of other more distant
species are usually nonpermissive. A number of studies have
shown that CMVs can enter cells of other species and express
a subset of viral genes (Kim and Carp, 1972; Fioretti et al,
1973; Lafemina and Hayward, 1988; Garcia-Ramirez et al,
2001). This has led to the conclusion that the restriction to
CMV replication in nonpermissive cells is associated with
a postpenetration block to viral gene expression and DNA
replication, but not due to a failure to enter the cell
(Mocarski and Courcelle, 2001). However, the molecular
mechanism for this intracellular block to viral replication
has remained elusive.
Received: 25 November 2005; accepted: 18 April 2006; published
online: 11 May 2006
*Corresponding author. 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
The EMBO Journal (2006) 25, 2634–2642 | & 2006 European Molecular Biology Organization | All Rights Reserved 0261-4189/06
www.embojournal.org
The EMBO Journal VOL 25 | NO 11 | 2006 &2006 European Molecular Biology Organization

EMBO

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In this study, we show that MCMV infection of human cells
activates programmed cell death (apoptosis). This prevents
efficient viral DNA replication and expression of late genes.
Inhibition of apoptosis by a Bcl-2-like protein of human CMV
or a different virus is sufficient to allow replication of MCMV
or the rat CMV in human cells.
Results
MCMV replicates in human 293 and 911 cells
Previous studies have shown that CMVs can enter cells of
other species but do not replicate in them (Kim and Carp,
1972; Fioretti et al, 1973; Lafemina and Hayward, 1988). To
analyze this phenomenon, we infected various human cell
types with an MCMV expressing the green fluorescent protein
(MCMV-GFP) at a low multiplicity of infection (MOI). MCMV-
GFP infected human cells as indicated by GFP expression, but
the infection did not spread from infected to uninfected
neighboring cells, and the GFP expressing cells disappeared
over time (not shown). Only in human embryonic kidney 293
and human embryonic retinoblast 911 cells, a cell-to-cell
spread of infection was noticed: groups of green cells and
occasionally even the formation of small plaques could be
observed (Figure 1A and B). A growth kinetic experiment
showed that human 293 and 911 cells can support MCMV
replication, although virus release was delayed and reached
lower titers as compared to permissive murine cells
(Figure 1C). In primary human embryonic lung fibroblasts
(MRC-5) and retinal pigment epithelial cells (RPE1) as well as
in all other human cells analyzed, MCMV replication was not
detected. RPE1 and MRC-5 cells were used for further ana-
lyses, because they are among the few cells that are permis-
sive for HCMV. When infected at a low MOI, 293 and 911 cells
did not release substantial amounts of virus into the super-
natant (data not shown). Apparently, the cells infected at
a low MOI produced enough virus to allow a limited spread
to neighboring cells, but not enough to be detectable in the
supernatant by plaque assay.
Impaired viral gene expression and apoptosis
of nonpermissive human cells
To identify a block in the cascade of viral gene expression
as a possible underlying mechanism of the species barrier,
the expression of an immediate-early (IE1), an early (E1),
an early-late (M44) and a late (gB) gene was analyzed
in permissive and nonpermissive cells (Figure 2A). In 293
cells, all kinetic classes of genes were expressed, and viral
DNA was replicated, again with delay as compared to the
murine cells. In the nonpermissive RPE1 cells, by contrast,
expression of gB was not detectable, and DNA replication
occurred only transiently. Starting at day 2 postinfection,
massive cell death was observed: cells disintegrated and
detached from the culture dish. The extent of cell death can
be seen in a time course experiment that measured cell
viability up to 120 h postinfection (Figure 2B). An apopto-
sis-specific assay showed that nonpermissive human cells
entered apoptosis upon MCMV infection (Figure 2C). The
DNA degradation associated with apoptosis probably
accounts for the loss of viral DNA seen in RPE1 cells
(Figure 2A). Interestingly, Kim and Carp (1972) had already
observed that none of the human WI-38 fibroblasts used
in their study survived an MCMV infection, but apparently
this observation was not further investigated.
A viral bcl-2 homolog is sufficient to facilitate MCMV
replication in human cells
Based on these observations, we asked which properties of
293 and 911 cells made them resistant to MCMV-induced
apoptosis and permissive for MCMV replication. These two
cell lines differ from all other human cells we have analyzed
in that they were transformed with adenovirus type 5 DNA
and express adenoviral E1A and E1B genes (Graham et al,
1977; Fallaux et al, 1996). The 293 cell line also contains
sequences from the adenovirus E4 region, but the 911 cell line
does not (Fallaux et al, 1996). E1A is a strong transactivator
of gene expression and is proapoptotic. The E1B genes are
antiapoptotic either by binding and sequestering p53 (E1B-
55k) or by functioning as a viral Bcl-2-like protein (E1B-19k)
(Shenk, 2001). Therefore, we transduced human RPE1 cells
with retroviral vectors encoding E1B-55k or E1B-19k, in order
to determine if one of these genes could make these cells
permissive for MCMV replication. Transgene expression was
tested by Western blot (Figure 3A) and immunofluorescence
(not shown). E1B-55k had no effect on the permissivity of
RPE1 cells, whereas E1B-19k inhibited MCMV-induced apop-
tosis and rendered RPE1 cells fully permissive for MCMV
replication (Figure 3B and C). Even after low MOI infection,
the virus spread across the whole monolayer and reached
remarkably high titers (Figure 3D). Similarly, overexpression
of the cellular bcl-2 or bcl-XL gene conferred permissivity, as
did the HCMV UL37x1 gene, which encodes a mitochondria-
localized inhibitor of apoptosis (vMIA), a protein with a
function similar to Bcl-2 (Goldmacher et al, 1999; Arnoult
et al, 2004) (Figure 3B and C). Thus, a single gene of HCMV
can facilitate MCMV replication in human cells.
C
10.1
911
293
RPE1
MRC-5
106
105
104
103
102
10
Ti
te
r T
CI
D 5
0/
m
l
0 4
Days p.i.
62
A B
Figure 1 MCMV replication in human 293 and 911 cells. (A, B)
Fluorescent and phase contrast images of 293 cells 6 days after
infection with MCMV-GFP at a low MOI. (C) Growth kinetic of
MCMV-GFP on murine 10.1 cells and human 293, 911, RPE1 and
MRC-5 cells. Cells were infected at an MOI of 5 TCID50/cell. Virus
titers were determined in the supernatant.
Cytomegalovirus cross-species infection
I Jurak and W Brune
&2006 European Molecular Biology Organization The EMBO Journal VOL 25 | NO 11 | 2006 2635

If a bcl-2-like protein is sufficient to allow MCMV replica-
tion in cultured human cells, it should be possible to con-
struct a recombinant MCMV that can replicate in human
cells. To create space for the insertion of foreign genes, we
deleted the region m02–m06 of the MCMV genome. This
region contains well-characterized immune evasion genes
that are necessary for MCMV virulence and pathogenicity
in mice (Kleijnen et al, 1997; Reusch et al, 1999; Oliveira et al,
2002; Wagner et al, 2002). Therefore, the recombinant viruses
should be nonvirulent in an immunocompetent host. A total
of 12 different apoptosis-related genes and control genes,
driven by a phosphoglycerate kinase (pgk) promoter, were
inserted into the MCMV genome (Figure 4A and B). In
addition to the adenoviral and bcl-2 family genes used in
the previous experiments, a Bcl-2 homolog from Kaposi
Sarcoma associated Herpesvirus (KSBcl-2) (Cheng et al,
1997) as well as the antiapoptotic genes crmA of cowpox
virus (Miura et al, 1993; Tewari and Dixit, 1995), p35 of
baculovirus (Xue and Horvitz, 1995), E3L of vaccinia virus
(Garcia et al, 2002), and IE1 of HCMV (Zhu et al, 1995) were
included. The recombinant viruses were tested for their
ability to facilitate MCMV growth on human RPE1 and
MRC-5 cells. Figure 4 shows that only MCMVs expressing a
bcl-2-like gene were able to replicate in human cells, whereas
MCMVs expressing viral genes that inhibit different check-
points of apoptosis were not. Even after infection at a very
low MOI, the recombinant viruses containing a bcl-2-like
gene could form plaques on RPE1 cells (Figure 4E). The
infection spread across the entire monolayer and could be
passaged serially on RPE1 cells.
C
A
B
0
10
20
30
40
50
10.1 293 911 RPE1 MRC-5
%
a
po
pt
ot
ic
ce
lls
mock
24 h.p.i
48 h.p.i
72 h.p.i
%
v
ia
bi
lity
0
20
40
60
80
100
Hours p.i. Hours p.i.
10.1
911
293
RPE1
MRC-5
0 24 48 72 96 120
0 4 24 48
IE1
E1
M44
gB
βa
vDNA
10.1 293 RPE1
0 244 48 72 96 120 0 244 48 72 96 120 0 244 48 72 96 120
9672820 4 24 48 9672820 4 24 48 967282
Figure 2 Impaired MCMV gene expression and apoptosis of nonpermissive human cells. (A) Permissive murine 10.1 cells, permissive human
293 cells, and nonpermissive human RPE1 cells were infected at an MOI of 5 TCID50/cell. Viral immediate-early (IE1), early (E1), early-late
(M44) and late (gB) gene expression was analyzed by Western blot at the indicated hours postinfection. Viral DNA (vDNA) was detected by
Southern blot hybridization. Both attached and floating cells were collected for Western and Southern blot analyses. (B) Viability of cells
infected at the same MOI was analyzed by MTTassay. (C) Nuclear DNA fragmentation as a sign of apoptosis was analyzed by TUNEL assay on
adherent cells on coverslips. Since apoptotic cells tend to detach from the coverslips, the true percentage of apoptotic cells is higher than the
percentage determined by TUNEL assay.
55k
HA
βa
βa
E1B
-19K bcl-2
UL
37x1
bcl
-XL mock
E1B
-55Kmock 293 911
A
50
40
30
20
10
0
%
a
po
pt
ot
ic
ce
lls
E1B
-19k
E1B
-55k
control UL
37x1
bcl
-XL
bcl-2
mock
24 h.p.i
48 h.p.i
72 h.p.i
B
E1B
-19k
E1B
-55k
control UL
37x1
bcl
-XL
bcl-2
E1B-19k
RPE1
0
107
105
103
10
12 18
DC 2 h.p.i.
4 days p.i.
6
104
103
102
10
Ti
te
r T
CI
D 5
0/
m
l
Ti
te
r T
CI
D 5
0/
m
l
Figure 3 MCMV replication in RPE1 cells expressing E1B-19k.
(A) Western blot of transduced RPE1 cells expressing HA-tagged
E1B-19k, bcl-2, bcl-XL, UL37x1/vMIA, or untagged E1B-55k.
Proteins were detected with an anti-HA or an anti-E1B-55k anti-
body, respectively. 293 and 911 cells served as positive controls for
E1B-55k expression. ba, beta actin. (B) Apoptosis of transduced and
control RPE1 cells after high-MOI infection with MCMV-GFP was
determined by TUNEL assay as in Figure 2C. (C) Virus release after
infection of RPE1 cells at an MOI of 5 TCID50/cell. The 2 h value
represents the residual input virus after infection and washing.
(D) Virus release after infection of RPE1 cells expressing E1B-19k
at an MOI of 0.2 TCID50/cell.
Cytomegalovirus cross-species infection
I Jurak and W Brune
The EMBO Journal VOL 25 | NO 11 | 2006 &2006 European Molecular Biology Organization2636

To test if the site of insertion is important, the UL37x1/
vMIA gene was also inserted at a different location. It
replaced the immune evasion gene m152 (Ziegler et al,
1997) and was driven by the endogenous m152 promoter.
This recombinant virus also replicated in RPE1 cells, albeit to
slightly lower titers (Figure 4C). This may reflect the fact that
the m152 promoter is an early promoter and as such does not
provide the gene product immediately after infection.
An MCMV mutant expressing the adenovirus E1A gene
could not be generated. Murine fibroblasts transfected with
the recombinant genome showed morphological signs of
apoptosis, probably because E1A is proapoptotic (Shenk,
2001). The infection did not spread to neighboring cells,
and thus infectious virus could not be obtained.
Activation of caspase-9 by MCMV infection
Bcl-2 and its homologs block apoptosis by inhibiting mito-
chondrial cytochrome c release and subsequent activation of
caspase-9 (Kuwana and Newmeyer, 2003). Our observation
that bcl-2-like proteins inhibit MCMV-induced apoptosis of
human cells suggested that MCMV infection causes activation
of caspase-9 and cleavage of downstream effectors such as
caspase-3 and poly(ADP-ribose) polymerase (PARP). Indeed,
caspases-9 and -3 and PARP were cleaved in MCMV-infected
RPE1 cells (Figure 5A). Similarly, these molecules were
cleaved in cells infected with an HCMV (strain AD169)
in which UL37x1/vMIA was deleted. This is consistent
with a previous study, which has found PARP cleavage in
human fibroblasts infected with a UL37x1 mutant of AD169
(Reboredo et al, 2004). Caspase-9, caspase-3, and PARP were
not activated when viral DNA replication was inhibited by
phosphonoacetic acid (PAA), indicating that viral DNA repli-
cation or events after DNA replication were responsible for
the induction of apoptosis (Figure 5A). MCMV-induced death
of RPE1 was significantly reduced when broad-spectrum
caspase inhibitors were added after infection (Figure 5B
and C), supporting the notion that caspase-mediated cell
death inhibited viral replication. zVAD-fmk at 100 mM con-
centration was the most effective inhibitor (Figure 5B and C).
Unfortunately, Boc-D-fmk could not be used at this high
concentration, because it turned out to be toxic for cells
(not shown).
It has previously been shown that the cowpox virus CrmA
(an inhibitor of caspase-1 and -8) and the baculovirus p35
protein (a substrate inhibitor of many caspases) can also
inhibit caspase-9 in recombinant protein assays (reviewed
in Ekert et al, 1999). However, later studies have shown that
both, CrmA and p35, do not inhibit caspase-9-mediated cell
death in living cells (Ryan et al, 2002). Nevertheless, we
wanted to exclude the possibility that CrmA and p35 failed to
facilitate MCMV replication (Figure 4), because the genes
were inactivated or expressed at insufficient levels by the
recombinant MCMVs. We also wondered, whether a recently
discovered positional homolog of UL37x1 in MCMV, m38.5,
might be expressed by MCMV at levels insufficient to inhibit
MCMV-induced apoptosis of human cells. The m38.5 protein
has a low-level sequence similarity to UL37x1/vMIA, loca-
lizes to mitochondria, and inhibits proteasome inhibitor-
A E MCMV-UL37x1
MCMV∆ m02–m06
pReplacer
Not I ApaI
MCMV-GFP BAC
m05m04m03 m06m02
PGKp genekan
D
C
104
103
102
10
Ti
te
r T
CI
D 5
0/
m
l
104
105
103
102
10
Ti
te
r T
CI
D 5
0/
m
l
103
102
10
Ti
te
r T
CI
D 5
0/
m
l
2 h.p.i.
4 days p.i.
M 55
k
55
k
∆0
2-0
6
∆0
2-0
6
MG
MG MG
19
k
19
k
19
k
bcl
-2
bcl
-2
bcl
-XL
bcl
-XL
bcl
-XL
UL
37
UL
37
(2)
KS
Bc
l
Crm
A
E3
L p35 IE1
m
RF
P
UL
37
UL
37
KS
Bc
l
Crm
A
E3
L p35 p35
m
RF
P
2 h.p.i.
6 days p.i.
9 days p.i.
2 h.p.i.
9 days p.i.
FB
HA
mock mock IE1
HA
βa βa
βaβa
WT
∆02
-06
bcl
-2
bcl
-XL
UL
37
E1B
19K
E1B
55K
IE1
mock 293
55k
E1B
55K
RFP E3L
KS
Bcl
Crm
A p35
UL37
(2)
911
HCMV
Figure 4 Growth of recombinant MCMVs in human cells. (A) Construction of recombinant MCMVs using the BAC technology. Inserted genes
were driven by a murine pgk promoter (PGKp). (B) Expression of the inserted genes in cells infected with the recombinant viruses. HA-tagged
proteins were detected with an anti-HA antibody. The HCMV IE1 protein and the E1B-55k protein were detected with specific antibodies. The
wildtype MCMV-GFP (MG) and recombinant MCMVs carrying the monomeric red fluorescent protein (mRFP) gene or a deletion of m02–m06
only were used as controls. UL37(2) represents a recombinant virus, in which UL37x1/vMIA was inserted in place of the m152 gene. (C) Virus
release after infection of RPE1 cells at an MOI of 5 TCID50/cell. The 2 h values represent the residual input virus after infection and washing. M,
MCMV wild-type virus. (D) Virus release after infection of RPE1 cells at an MOI of 0.2 TCID50/cell. (E) Fluorescent and phase contrast image of
RPE1 cells 8 days after a low-MOI infection with an MCMV expressing a bcl-2-like protein (MCMV-UL37x1) or a control virus (MCMVDm02-
m06). Efficient spread of the infection and plaque formation was only seen with MCMVs expressing a bcl-2-like protein. (F) Virus release after
infection of MRC-5 cells at an MOI of 5 TCID50/cell.
Cytomegalovirus cross-species infection
I Jurak and W Brune
&2006 European Molecular Biology Organization The EMBO Journal VOL 25 | NO 11 | 2006 2637

mediated, but not Fas-mediated apoptosis (McCormick et al,
2005). However, its activity against virus-induced apoptosis
and its role for MCMV replication have not been studied yet.
To address these questions, we transduced RPE1 cells with
retroviral vectors encoding CrmA, p35, or m38.5, respec-
tively. Expression of the proteins was verified by Western
blot (not shown). The cells were then tested for resistance
against proteasome inhibitor- and Fas-mediated cell death (as
carried out by McCormick et al, 2005) and against MCMV-
induced apoptosis. Figure 6 shows that CrmA was a potent
inhibitor of Fas-meditated apoptosis, whereas m38.5 pro-
tected cells from apoptosis induced by the proteasome
inhibitor MG-132. The baculovirus p35 had only a moderate
activity against Fas- and proteasome inhibitor-induced cell
death. However, none of these three proteins protected from
MCMV-induced cell death. This confirms the results obtained
with the recombinant MCMVs (Figure 4). The inability of
CrmA and p35 to inhibit MCMV-induced apoptosis is consis-
tent with previous studies, which have shown that CrmA and
p35 expressed by recombinant Sindbis viruses conferred only
very little protection against Sindbis virus-induced neuronal
cell death in a mouse model of apoptosis (Nava et al, 1998;
Ryan et al, 2002). By contrast, Bcl-2 and a dominant-negative
caspase-9 protected mice efficiently in the same system
(Levine et al, 1996; Ryan et al, 2002). Apparently, the
inhibitory activity of p35 against effector caspases (Ekert
et al, 1999) is insufficient for blocking Sindbis virus- or
cytomegalovirus-induced apoptosis.
Replication of RCMV in human cells
To analyze whether the requirement of a bcl-2-like gene
is specific for MCMV or whether this principle is of more
general importance, we infected RPE1 cells and RPE1 cells
expressing E1B-19k with rat cytomegalovirus (RCMV,
Maastricht strain). Like MCMV, RCMV also induced cell
death in RPE1 cell, which could be suppressed by E1B-19k
(Figure 7A). RCMV grew only in the presence of a bcl-2-like
protein such as E1B-19k (Figure 7B). The amounts of virus
released from RPE1-E1B-19k cells were relatively low, but the
plaque formation seen in these cells after low-MOI infection
clearly indicated virus replication and spread (Figure 7C).
Taken together, the results suggest that the inability to inhibit
apoptosis in human cells limits cross-species infections and
represents an important determinant of MCMV’s and RCMV’s
species specificity.
Discussion
Previous analyses of the CMV species specificity have
indicated that the host cell restriction to CMV replication
observed in nonpermissive cells is the result of a postpene-
tration block to viral gene expression and not of a failure to
enter cells (Kim and Carp, 1972; Fioretti et al, 1973; Lafemina
and Hayward, 1988). However, the nature of this block has
remained elusive.
Inside the cell, viruses need to overcome several innate
immune defenses of the cell in order to replicate and spread
efficiently. These include toll-like receptor signaling, trigger-
ing of the interferon response, activation of cellular stress
responses, and induction of apoptosis. Here we show that
MCMV infection of human cells leads to activation of cas-
pase-9 and induction of apoptosis. Antiapoptotic Bcl-2 family
proteins can inhibit mitochondrial release of cytochrome c
and subsequent activation of caspase-9. This study demon-
strates that expression of such a protein inhibits apoptosis
casp-3
casp-9
βa
PARP
43
37/35
116
89
43
0
PAA
MCMV
HCMV∆
UL37x1
A
19/17
B
DMSOmock
%
v
ia
bi
lity
%
v
ia
bi
lity
0
10
30
50
70
20 1005 PAA
zVAD (µM)
C
DMSOmock 205
Boc-D (µM)
0
20
40
60
80
zVAD
100
**
*
*
**
**
h.p.i.727272 4848 2424
Figure 5 Caspase activation upon MCMV infection of human cells.
(A) Human RPE1 cells were infected at an MOI of 5 TCID50/cell.
Cleavage (activation) of caspases 9 and 3 and PARP was detected at
48 and 72 h postinfection, but not in the presence of PAA. Similarly,
an HCMV UL37x1 deletion mutant induced activation of these
molecules. (B, C) Cell death triggered by MCMV infection of RPE1
cells was inhibited by broad-spectrum caspase inhibitors zVAD-fmk
and Boc-D-fmk or by an inhibitor of viral DNA replication (PAA,
250mg/ml). Significance levels were calculated using ANOVA.
*Po0.05; **Po0.001.
A
UL
37x1
C
38.5
CrmA
%
v
ia
bi
lity
%
v
ia
bi
lity
0
30
50
70
90
p35
10
MG132
*
B
40
60
80
20
0
Anti-Fas
*
**
**
**
**
**
m UL
37x1
C
38.5
CrmA p35m
%
v
ia
bi
lity
C
0
20
40
60
80 MCMV
**
**
UL
37x1
E1B
-19
C
38.5
CrmA p35m
Figure 6 Transduced RPE1 cells stably expressing UL37x1/vMIA,
m38.5, CrmA, or p35 were treated with (A) the proteasome-inhi-
bitor MG-132 or (B) anti-Fas antibody plus cycloheximide, or
(C) infected with MCMV to induce apoptosis. Cell viability was
measured using an MTT assay. C, RPE1 control cells Significance
levels were calculated using ANOVA. *Po0.05; **Po0.001.
Cytomegalovirus cross-species infection
I Jurak and W Brune
The EMBO Journal VOL 25 | NO 11 | 2006 &2006 European Molecular Biology Organization2638

induced by cytomegalovirus infection and allows MCMV
replication in human cells. In this context, it should be
noted that Bcl-2 and the HCMV vMIA protein can also inhibit
caspase-independent cell death (Roumier et al, 2002).
Therefore, we cannot exclude that this activity contributed
to the inhibition of MCMV-induced apoptosis. However, the
fact that two broad-spectrum caspase inhibitors, zVAD-fmk
and Boc-D-fmk, were capable of inhibiting virus-induced cell
death argues for an important role of caspases in this process.
At high concentrations, zVAD-fmk can also inhibit calpains.
These molecules are activated upon genotoxic stress and act
upstream of caspases (Waterhouse et al, 1998). This suggests
a possible role of calpains in MCMV-induced apoptosis and
could explain why zVAD-fmk is a somewhat more potent
inhibitor than Boc-D-fmk.
Our finding that the HCMV vMIA protein can facilitate
MCMV replication in human cells is in agreement with
previous results showing that this protein is required for
efficient replication of the HCMV laboratory strain
AD169varATCC (Brune et al, 2003; Yu et al, 2003; Reboredo
et al, 2004) and the clinical strain VR1814/FIX (S Ho¨lzer
and W Brune, unpublished). However, it was recently
reported that vMIA is not required for efficient replication
of the HCMV strain Towne, although a UL37x1 deletion
mutant caused increased apoptosis of infected fibroblasts
(McCormick et al, 2005). Since Towne and FIX both encode
a functional copy of the inhibitor of caspase-8 activation
(vICA), a gene product of UL36, whereas AD169 does not,
the requirement of vMIA appears to be strain-dependent, but
not dependent on the function of vICA.
Insertion of the UL37x1 gene into the MCMV genome is
sufficient to facilitate MCMV growth in human cells. This
leaves two possible explanations: either MCMV does not
encode an analogous protein and does not need it for
replication in murine cells, or the virus does encode such a
protein, but it functions in a species-specific manner (i.e. in
murine but not in human cells). A recent reevaluation of
the MCMV genome sequence has identified a previously
unrecognized ORF, m38.5, which is a positional homolog
of UL37x1/vMIA and shows a low-level sequence similarity
to this HCMV protein (McCormick et al, 2003). The m38.5
protein localizes to mitochondria and inhibits cell death
induced by a proteasome inhibitor, but does not inhibit Fas-
induced apoptosis of human HeLa cells like vMIA does
(McCormick et al, 2005). Our results confirm this activity
and show that m38.5 cannot block MCMV-induced cell death
in human cells. It remains to be determined whether the
m38.5 protein is responsible for inhibiting virus-induced
apoptosis in murine cells, which would suggest a species-
specific function. Another recent study has detected
increased levels of the cellular antiapoptotic bcl-2 family
protein Bfl-1/A1, but also of the proapoptotic protein Bim
in MCMV-infected dendritic cells (Andoniou et al, 2004).
Although the study did not resolve whether the increased
Bfl-1/A1 levels were responsible for the observed resistance
of infected cells against apoptotic stimuli, it points out the
possibility that MCMV could compensate for a lack of a Bcl-2-
like protein by upregulating a cellular antiapoptotic gene.
The present study shows that inhibition of apoptosis
enables not only the murine but also the rat cytomegalovirus
to cross the species barrier and replicate in human cells. This
indicates that the mechanism identified is not unique to
MCMV. It also raises the question, whether—conversely—
HCMV triggers apoptosis upon infection of rodent cells.
Preliminary data from our laboratory indicate that infection
of murine NIH-3T3 and 10.1 fibroblasts with HCMV strains
AD169 or FIX does not induce significant levels of apoptosis,
even when the cells were infected at a high MOI. A possible
explanation for this can be found in a previous study, which
has shown that HCMV does not reach the stage of viral DNA
replication in mouse cells (Lafemina and Hayward, 1988).
This is consistent with the observation that an inhibitor of
viral DNA replication prevents the induction of apoptosis
(Reboredo et al, 2004; and this study). It further suggests that
the species specificity of HCMV depends on an additional
intracellular mechanism, which prevents the onset of DNA
replication. To date, it is not known whether viral DNA
replication itself induces apoptosis, or whether later pro-
cesses are responsible: viral DNA replication can activate
the DNA damage response, which is known to be proapopto-
tic (Smith and Mocarski, 2005; Sinclair et al, 2006).
Subsequently, DNA-filled capsids have to leave the nucleus,
which is associated with dissolution of the nuclear lamina
(Muranyi et al, 2002). This process is also likely to trigger
apoptosis, if it does not occur at an appropriate time during
the cell cycle. Viral glycoproteins traveling through the ER
and Golgi could cause ‘ER stress’. This process, also known
as ‘unfolded protein response’, has recently been shown to
be activated during HCMV replication (Isler et al, 2005).
Considering the large size and the protracted replication
cycle of the CMVs, it seems likely that these viruses have
found ways to subvert many if not all aspects of innate
immunity. CMV proteins inhibiting apoptosis (Zhu et al,
1995; Goldmacher et al, 1999; Brune et al, 2001, 2003;
Skaletskaya et al, 2001; Me´nard et al, 2003) and the interferon
C
0 4 6
Days p.i.
REF
E1B-19K
RPE1
106
104
102
B
Ti
te
r T
CI
D 5
0/
m
l
A
%
v
ia
bi
lity
0
20
40
60
80
100
Days p.i.
E1B-19K
RPE1
1 5 23
Figure 7 Growth of rat cytomegalovirus in human cells. (A) RPE1
cells died after infection with RCMV at an MOI of 5 TCID50/cell, but
RPE1 cells expressing E1B-19k were mostly protected from RCMV-
induced cell death. (B) RPE1 cells, RPE1-E1B-19k cells, and rat
embryo fibroblasts (REF) were infected with RCMV at an MOI of
5 TCID50/cell, and titers in the supernatant were determined.
(C) Phase contrast image of RPE1 cells expressing E1B-19k 7 days
after low-MOI infection with RCMV. Plaque formation as an indica-
tion of virus replication and spread was only observed in RPE1-E1B-
19k cells, but not in normal RPE1 cells.
Cytomegalovirus cross-species infection
I Jurak and W Brune
&2006 European Molecular Biology Organization The EMBO Journal VOL 25 | NO 11 | 2006 2639

response (reviewed in Hengel et al, 2005) have already been
identified, and viral proteins blocking TLR signaling or the
stress response are likely to follow. Analyses of viral inhibi-
tors of the adaptive immune response encoded by HCMV and
MCMV, for example, proteins downregulating MHC class I
surface expression, have shown that some of the proteins do
not function properly with target molecules of other species
(Machold et al, 1997). Thus, it can be assumed that some of
the viral inhibitors of the innate immune response will also
operate in a species-specific manner. Inhibiting apoptosis
of the infected cell is clearly a crucial task for the virus
(Andoniou and Degli-Esposti, 2006) and can restrict the
virus’ cell tropism (Brune et al, 2001) and host range, as we
show here. However, the fact that MCMV replicates slower
and spreads less efficiently in human cells even in the
presence of an antiapoptotic protein suggests that inhibition
of apoptosis represents an important, but not the only limit-
ing factor for efficient replication and spread.
We are only beginning to understand the molecular me-
chanisms underlying the species barrier of different viruses,
and only few mechanisms have been identified to date.
Failure to inhibit the interferon response was identified as
a limiting factor for cross-species infections of certain para-
myxo- and poxviruses (Parisien et al, 2002; Hornemann et al,
2003; Wang et al, 2004), which replicate in the cytoplasm.
The present study shows that certain b-herpesviruses—large
DNA viruses that replicate their genomes in the nucleus—
induce apoptosis in cells of a foreign species, even though
they can inhibit premature apoptosis in cells of their own
species. A number of other viruses also depend on inhibition
of apoptosis for normal replication, and consequently they
encode potent cell death suppressors: Adenoviruses and
g-herpesviruses are two prominent examples (Pilder et al,
1984; White et al, 1984; Altmann and Hammerschmidt,
2005). It is possible that the species restriction of these
viruses also depends (in part) on their ability to inhibit cell
death in cells of a foreign species, even if in the end more
than one mechanism should turn out to be involved, as it
appears to be the case for human CMV.
Studies on viral species specificity teach us, how viruses
counteract innate immune defenses, how these innate
immune defenses operate, and how they differ from one
species to another. These insights should lead to a better
understanding of zoonotic infections in general.
Materials and methods
Cells
MRC-5 cells (ATCC CCL-171) are primary human embryonic lung
fibroblasts (Jacobs et al, 1970). hTERT RPE1 cell (ATCC CRL-4000)
are telomerase-immortalized retinal RPE1 (Bodnar et al, 1998). 293
cells (ATCC CRL-1573) are human embryonic kidney cells
transformed with adenovirus 5 DNA (Graham et al, 1977). For
the experiments in this study, the 293A subclone was used, which
was selected for a flattened morphology (Invitrogen). 911 cells are
human embryonic retinoblasts transformed with adenovirus 5 E1
genes (Fallaux et al, 1996). 10.1 and REF cells are spontaneously
immortalized mouse and rat embryo fibroblasts, respectively
(Burns et al, 1988; Harvey and Levine, 1991).
Plasmids and genes
The human bcl-2 and bcl-XL genes, adenovirus E1B-19k, cowpox
virus crmA, baculovirus p35, and vaccinia virus E3L were cloned by
PCR in pcDNA3 (Invitrogen), adding an HA tag to the 50 end.
Plasmids pcDNA-UL37x1HA and pcDNA-m38.5 contain the HCMV
UL37x1 and the MCMV m38.5, respectively, tagged with an HA
epitope at the 30 end. pcDNA3-IE1 containing the HCMV IE1 gene
was provided by Michael Nevels (University of Regensburg,
Germany). Plasmid pRC-RSV-E1A containing the adenovirus E1A
gene was provided by Thorsten Stiewe (University of Wu¨rzburg,
Germany). The pSTK146 plasmid (Schiedner et al, 2000) containing
the murine pgk promoter and the adenovirus 5 E1 region was
provided by Stefan Kochanek (University of Ulm, Germany). The
mRFP gene was excised from pRSET-mRFP1 (Campbell et al, 2002).
The kanamycin resistance gene flanked by FRT sites was taken from
pSLFRTkn (Atalay et al, 2002).
Retroviral transduction
The E1B-19k, E1B-55k, bcl-2, bcl-XL, and UL37x1 genes were
inserted into the murine leukemia virus-based retroviral plasmid
pLXSN (Clontech). Production of retroviral vectors using the
Phoenix packaging cell line, and transduction of RPE1 cells was
done as in previous studies (Brune et al, 2003). Transduced cells
were selected with 700 mg/ml G418 and grown as bulk cultures
without clonal selection.
Western blotting and immunofluorescence
For Western blot analysis, cells were lysed with lysis buffer
containing 1% Triton X-100. Protein samples were separated by
sodium dodecyl sulfate polyacrylamide gel electrophoresis and
transferred onto nitrocellulose membranes. For immunological
detection, the following monoclonal antibodies were used: CRO-
MA101 against MCMV IE1 and CROMA103 against E1 (both
provided by Stipan Jonjic, University of Rijeka, Croatia), 2E8.21A
against MCMV gB and 3B9.22A against M44 (both provided by
Lambert Loh, University of Saskatchewan, Canada), 2A6 against
E1B-55k and 1B12 against HCMV IE1 (both provided by Tom Shenk,
Princeton University, USA). Antibodies against the HA epitope tag
(16B12, Covance Research Products), b-actin (A5316, Sigma),
caspases-3 and -9 (8G10 and 9502, Cell Signaling), and PARP
(7D3–6, BD Biosciences) were purchased from suppliers as
indicated. For immunofluorescence, cells were grown on coverslips,
fixed with 3% paraformaldehyde, and permeabilized with 0.3%
Triton X-100. Proteins were detected using the primary antibodies
listed above and an AlexaFluor 594-coupled secondary antibody
(Molecular Probes).
Viruses and growth kinetics
MCMV-GFP, a recombinant MCMV expressing the enhanced GFP,
was constructed by Martin Messerle (Medical School Hannover,
Germany) and has been used in previous studies (Brune et al, 2001,
2003). The Maastricht strain of RCMV (Bruggeman et al, 1982) was
provided to us by Sebastian Voigt (Charite´, Berlin, Germany). All
recombinant viruses were constructed using bacterial artificial
chromosome (BAC) technology (Brune et al, 2000) and are based on
the MCMV-GFP BAC. Construction of these recombinant MCMVs
has been approved by the Central Commision for Biological Safety
(ZKBS) of the Federal Republic of Germany. To delete the immune
evasion genes m02–m06 and insert foreign genes, a plasmid named
pReplacer was constructed on the basis of pBluescriptII KSþ
(Stratagene). It contains 50 nucleotide homologies to sequences
upstream of m02 and downstream of m06, a kanamycin resistance
gene, a pgk promoter, and a multiple cloning site as shown in
Figure 4A. Foreign genes were inserted using the multiple cloning
site. The mutagenesis cassette can be excised from the backbone of
pReplacer with restriction enzymes NotI or SacII at the 50 and ApaI
or KpnI at the 30 end. The linear recombination substrates were
used for homologous recombination in Escherichia coli strain DY380
containing the MCMV-GFP BAC as previously described (Brune
et al, 2003). Recombinant MCMV genomes were analyzed by
restriction digest and Southern blot. Wild-type and recombinant
MCMV were grown on murine 10.1 fibroblasts essentially as
described (Brune et al, 1999), and RCMV was propagated of REFs.
Titrations were performed on the same cells using the median tissue
culture infectious dose (TCID50) method (Mahy and Kangro, 1996).
For growth kinetics, cells were seeded in six-well plates and infected
with MCMV at the indicated MOI. At 2 h after infection, cells were
washed with PBS, and fresh medium was added. Medium was
replaced at the indicated time points, and the content of virus in
the supernatant was determined by titration. All growth kinetic
experiments were performed in triplicate.
Cytomegalovirus cross-species infection
I Jurak and W Brune
The EMBO Journal VOL 25 | NO 11 | 2006 &2006 European Molecular Biology Organization2640

The HCMV mutant AD169DUL37x1 and the clinical HCMV strain
VR1814 (FIX) have been described previously (Hahn et al, 2002;
Brune et al, 2003).
Southern blot analysis
Adherent and floating (dead) cells were collected at the indicated
time points, and DNA was extracted by standard procedures. Three
micrograms of each DNA sample was digested with HindIII,
separated on a 0.8% agarose gel, and transferred onto a nylon
membrane. Hybridization with a digoxigenin-labeled probe directed
against the GFP gene and chemiluminescent detection was
performed using a DIG-High Prime DNA labeling and detection
kit (Roche), according to the manufacturer’s recommendations.
Apoptosis assays
Cell viability was determined by MTT assay, which measures
mitochondrial activity, according to standard protocols. Briefly,
cells were seeded in 96-well plates at 5�103 cells per well and
treated with proapoptotic reagents or infected with MCMV. At
appropriate time points after treatment, cells were incubated for 4 h
with 100 ml medium containing 500mg/ml 3-(4,5-dimethylthiazole-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The formazan
crystals were solubilized with 100ml of a 1:1 DMSO:ethanol
mixture. The formazan concentration was measured at 570 nm
using an ELISA plate reader. Every test was carried out with at least
four replicates of each sample. Statistical analyses were carried out
using the analysis of variance (ANOVA, F-test). 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 deoxynucleotidyltransferase-mediated
dUTP nick end labeling (TUNEL) assay kit (Roche). Nuclei were
counterstained with 40,60-diamidino-2-phenylindole (DAPI). The
percentage of apoptotic cells was determined by counting 500 cells
in about 20 random visual fields.
For induction of apoptosis, cells were treated with 10mM of
the proteasome inhibitor MG-132 (Calbiochem) or 0.2mg/ml
anti-Fas antibody (clone 7C11, Coulter) and 10 mg/ml cycloheximide
(AppliChem). To analyze caspase and PARP cleavage, cells were
infected at an MOI of 5 TCID50/ml, washed with PBS 2 h p.i., and
harvested at the indicated time points. Lysates were prepared as
described above, and proteins were detected by Western blot. The
broad-spectrum caspase inhibitors zVAD-fmk and Boc-D-fmk were
purchased from MBL International as 100 and 20 mM stock
solutions in DMSO, respectively. RPE1 cells were infected at an
MOI of 5. After 6 h, cells were washed and incubated with medium
containing zVAD-fmk, Boc-D-fmk, DMSO, or PAA.
Acknowledgements
We thank S Erhard for technical assistance, Ed Mocarski, Ulrich
Koszinowski, Tom Shenk, Sebastian Voigt, and Matthias Budt for a
critical reading of the manuscript, and numerous colleagues for
providing reagents. This study was supported by the Emmy Noether
Program of the Deutsche Forschungsgemeinschaft (BR 1730/2) and
SFB 479, TP B6.
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