Murine cytomegalovirus m41 open reading frame encodes a Golgi-localized antiapoptotic protein.

JOURNAL OF VIROLOGY, Nov. 2003, p. 11633–11643 Vol. 77, No. 21
0022-538X/03/$08.00�0 DOI: 10.1128/JVI.77.21.11633–11643.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Murine Cytomegalovirus m41 Open Reading Frame Encodes a
Golgi-Localized Antiapoptotic Protein
Wolfram Brune,1,2* Michael Nevels,1 and Thomas Shenk1
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544,1 and Rudolf Virchow
Center for Experimental Biomedicine, University of Wu¨rzburg, D-97078 Wu¨rzburg, Germany2
Received 5 May 2003/Accepted 24 July 2003
Viruses have evolved various strategies to prevent premature apoptosis of infected host cells. Some of the
viral genes mediating antiapoptotic functions have been identified by their homology to cellular genes, but
others are structurally unrelated to genes of known function. In this study, we used a random, unbiased
approach to identify such genes in the murine cytomegalovirus genome. From a library of random transposon
insertion mutants, a mutant virus that caused premature cell death was isolated. The transposon was inserted
within open reading frame m41. An independently constructed m41 deletion mutant showed the same pheno-
type, whereas deletion mutants lacking the adjacent genes m40 and M42 did not. Apoptosis occurred in
different cell types, could be blocked by caspase inhibitors, and did not require p53. Within the murine
cytomegalovirus genome, m41, m40, and m39 form a small cluster of genes of unknown function. They are
homologous to r41, r40, and r39 of rat cytomegalovirus, but lack sequence homology to UL41, UL40, and UL37
exon 1 (UL37x1) which are located at the corresponding positions of the human cytomegalovirus genome.
Unlike UL37x1 of human cytomegalovirus, which encodes a mitochondrion-localized inhibitor of apoptosis that
is essential for virus replication, m41 encodes a protein that localizes to the Golgi apparatus. The murine
cytomegalovirus m41 product is the first example of a Golgi-localized protein that prevents premature
apoptosis and thus extends the life span of infected cells.
The programmed cell death (apoptosis) of a virus-infected
cell is a strategy of the host organism to prevent virus replica-
tion and spread. Apoptosis can occur as a direct result of virus
infection or can be triggered by immune effector cells that
recognize infected cells. As a counterstrategy, viruses have
developed mechanisms to inhibit apoptosis and extend the life
span of the infected cell (31, 44).
Herpesviruses possess large double-stranded DNA ge-
nomes. They encode up to 200 genes, many of which modulate
intrinsic functions of the host cell (38). Cytomegaloviruses
(CMVs), prototypes of the � subgroup of the Herpesviridae, are
the largest of the herpesviruses. With protracted replication
cycles of �24 h for murine cytomegalovirus (MCMV) and 48
to 72 h for human cytomegalovirus (HCMV), these viruses are
particularly vulnerable to elimination by apoptosis. Therefore,
it is not surprising that CMVs have acquired a number of genes
that prevent premature apoptosis of the infected host cell.
The immediate-early genes IE1 and IE2 of HCMV were the
first CMV genes for which an antiapoptotic function was dem-
onstrated. The IE1 and IE2 proteins both inhibit the induction
of apoptosis by tumor necrosis factor alpha or by an E1B
19-kDa protein-deficient adenovirus (50). More recent work
demonstrated that inhibition of apoptosis by IE1 and IE2 in-
volves activation of the phosphatidylinositide 3�-OH kinase
pathway and the cellular kinase Akt (49).
Two additional HCMV genes expressed at immediate-early
times after infection have been shown to inhibit apoptosis at
different levels. The product of open reading frame (ORF)
UL37 exon 1 (pUL37x1) encodes a mitochondrion-localized
protein. It forms a complex with the adenine nucleotide trans-
locator and suppresses apoptosis by blocking permeabilization
of the mitochondrial outer membrane (17). The UL37x1 pro-
tein also disrupts mitochondrial networks (28). It shows no
significant amino acid homology to Bcl-2 and does not bind
BAX or the mitochondrial voltage-dependent anion channel
VDAC, suggesting that pUL37x1 represents a separate class of
cell death inhibitors (16). The product of UL36 binds to the
prodomain of caspase 8 and prevents its activation (40). It
lacks sequence similarity to viral or cellular FLICE-inhibitory
proteins and to other known antiapoptotic proteins.
The antiapoptotic function of these HCMV genes was stud-
ied in cells expressing the isolated genes but not in the context
of viral infection. However, it has been shown that UL36 is
dispensable for virus growth in cell culture (32) and that this
gene is mutated in the most commonly used laboratory strains,
AD169 and Towne (32), owing to a missense mutation or
partial deletion that renders the UL36 protein nonfunctional
(40). In fact, the HCMV laboratory strains contain a number of
known (9, 13, 30, 40) and likely many more unknown mutations
compared to fresh clinical isolates. Unlike the clinical HCMV
strains, which infect various cell types in human patients and in
cell culture (35), the laboratory strains replicate almost exclu-
sively in cultured human fibroblasts, precluding analysis of cell
type-specific functions with virus mutants.
The laboratory strains of murine cytomegalovirus (MCMV)
appear to be less degenerate than the HCMV laboratory
strains, as MCMV laboratory strains can replicate in various
cell types in vitro and cause disease in experimentally infected
mice. MCMV has been used to study the genetic basis for
endothelial cell tropism. A library of random transposon in-
* Corresponding author. Mailing address: Rudolf Virchow Zentrum
fu¨r Experimentelle Biomedizin, Universita¨t Wu¨rzburg, Versbacher
Str. 9, D-97078 Wu¨rzburg, Germany. Phone: 49 931 20148903. Fax: 49
931 20148123. E-mail: wolfram.brune@virchow.uni-wuerzburg.de.
11633

sertion mutants of the MCMV genome was screened for mu-
tants that had lost the ability to replicate in endothelial cells in
culture. A gene, M45, which is required for virus growth and
spread in endothelial cells by preventing rapid onset of apo-
ptosis in infected endothelial cells was identified (6). The
mechanism of action of the M45 protein has not yet been
determined. M45 shows significant sequence homology to the
ICP10 protein of herpes simplex virus type 2, including the
N-terminal protein kinase domain of ICP10. Two laboratories
have recently demonstrated that ICP10 can inhibit apoptosis
and that the protein kinase domain is required for this activity.
However, the mechanism by which apoptosis is suppressed
remains controversial (25, 33, 34).
In a different approach, systematic analysis of an entire viral
gene family revealed that another MCMV gene had antiapo-
ptotic activity, which became apparent only in a specific cell
type. Inactivation of gene M36, the genetic and functional
homolog of the HCMV gene UL36, led to a virus mutant with
a severe growth defect in macrophages but not in fibroblasts
(29).
In the present study, we identified and analyzed a previously
uncharacterized antiapoptotic gene of MCMV. We show that
the product of ORF m41 is a Golgi-resident protein that is
required to prevent premature apoptosis of infected cells and
thus extends their life span.
MATERIALS AND METHODS
Plasmids and retroviral vectors. ORFs m41 and UL37x1 were amplified by
PCR with the primers listed in Table 1, introducing an influenza virus hemag-
glutinin (HA) epitope tag at the 5� or the 3� end of the coding sequence. PCR
products were digested with BamHI and EcoRI or XhoI and EcoRI, respectively,
and cloned into pcDNA3 (Invitrogen) to generate pcDNA-m41HA, pcDNA-
HAm41, and pcDNA-UL37x1HA. The integrity of the cloned m41 and UL37x1
sequences was verified by sequence analysis.
Epitope-tagged genes were excised from pcDNA3 with the same enzymes as
above and cloned into pRetroEBNA (23) to obtain pRetro-m41HA, pRetro-
HAm41, and pRetro-UL37x1HA. Amphotropic retroviral vectors were gener-
ated by use of the Phoenix packaging cell line as described previously (23).
Mutagenesis of CMV genomes. All mutant CMV genomes were generated in
Escherichia coli based on full-length bacterial artificial chromosome (BAC)
clones of the HCMV AD169 genome (22), the MCMV genome (pSM3fr) (47),
and the MCMV-GFP genome. MCMV-GFP (kindly provided by M. Messerle)
contains the enhanced green fluorescent protein (EGFP) gene from pEGFP-C1
(Clontech) inserted into the MCMV ie2 locus. The ie2 gene has been shown to
be dispensable for virus growth in vitro and in vivo (8, 27).
A library of MCMV transposon insertion mutants has been published previ-
ously (6). The transposon insertion mutants were generated by a single-step
transposon mutagenesis protocol (5, 7) and transferred to fibroblasts by bacterial
invasion to obtain a library of mutant viruses (6). The transposon contains a kan
gene for selection in E. coli and the EGFP gene driven by an HCMV immediate-
early promoter from pEGFP-C1. The location of the transposon insertion was
determined by direct sequencing as described previously (7).
Gene deletion mutants were constructed by homologous recombination in E.
coli with linear, PCR-generated fragments. Briefly, a zeocin resistance gene (zeo)
was amplified by PCR from pEM7/Zeo (Invitrogen). The primers contained an
additional 50 nucleotides for homologous recombination. See Table 1 for a
complete list of the primers used in this study. Recombination of BACs with
linear DNA fragments was carried out in E. coli strain DY380 as described
previously in detail elsewhere (26). DY380 expresses the recombination genes
exo, bet, and gam of bacteriophage � in a temperature-dependent fashion.
Tagging of viral genes was also performed by homologous recombination with
linear fragments, essentially as described above. A kan gene flanked by FRT sites
was excised with EcoRI from plasmid pSLFRTKn (1) and inserted into pcDNA-
m41HA. The resulting plasmid, pcDNA-m41HA-FK, served as the template for
PCR amplification of the influenza virus HA epitope sequence and the FRT-
flanked kan gene. This cassette was inserted at the 3� end of the gene to be
tagged. The kan gene was subsequently removed with FLP recombinase ex-
pressed in E. coli strain DH10B from plasmid pCP20 (11). For a detailed
description of the gene tagging strategy, see the report by Uzzau et al. (45).
For molecular analysis, BACs were digested with different restriction enzymes
and separated on large 0.6% agarose gels. Southern blot hybridizations were
performed with a nonradioactive digoxigenin labeling and detection kit (Roche).
Mutations introduced were verified by PCR or sequencing.
Cells and viruses. MCMV was grown and titered on NIH 3T3 cells by standard
procedures. Virus titers were determined according to the median tissue culture
infectious dose (TCID50) method (37). NIH 3T3, M2-10B4, and SVEC4-10 cells
TABLE 1. Oligonucleotide primers used in this study
Primer or
oligonucleotide Sequence
Primersa
m41-HA 5�-aaa gga tcc acc atg gga gac gat gat cgt c-3�
5�-aaa gaa ttc aAG CGT AGT CTG GGA CGT CGT ATG GGT Atc tgt caa tga tca cga cga-3�
HA-m41 5�-aaa gga tcc acc atg TAC CCA TAC GAC GTC CCA GAC TAC GCT atg gga gac gat gat cgt c-3�
5�-aaa gaa ttc tca tct gtc aat gat cac gac-3�
UL37x1-HA 5�-aaa gaa ttc cac cat gtc tcc agt cta cgt gaa t-3�
5�-aaa ctc gag tca AGC GTA GTC TGG GAC GTC GTA TGG GTA ctg gtg aga ctg ctg ggg
Oligonucleotidesb
MCMV�m41 5�-AAT AGT CAT CCG ATG ATC GTG TCG CCG CCC GAC CGC CCT CCT CCC CCA ATt gtt gac aat taa tca tcg gca t-3�
5�-CGC CGT TTC CTC ACA TTC CGT TGT CGT GCG CAG GTT CCT CCG AAC CTT TGt cag tcc tgc tcc tcg gcc a-3�
MCMV�m40 5�-ACC ATC CCC TCG ACT CGC GTC TTC TCT GTC GAT CTG GGT GTC GGG GAG GGt cag tcc tgc tcc tcg gcc a-3�
5�-TAG CTA TCA CTT TCG AAA CGC ATA AAC GAC GCT ATG TCC CAC TCA TCT CAt gtt gac aat taa tca tcg gca t-3�
MCMV�m42 5�-ATA GAG GAC GTG GTT ATG CTA CTT TTG TGA ACG GAC GCG GTT GGG AGG ACg aat tca gtc ctg ctc ctc ctc ggc ca-3�
5�-AGA ACG AGT ATG ACC AAG AGG GAC ACA CAG ACT CCG ACC GTG ATG GCG GTt gtt gac aat taa tca tcg gca t-3�
MCMV�M45 5�-GCT AGA GAA GTT CTA CGT CGA CGT CGG GCC CCT CGT CGA GTT CGC GTG ACg aat tca gtc ctg ctc ctc ggc ca-3�
5�-TCG TGC AGG CGA TGG GGC TCG ATC TTG ACG GAG CGC ACG CAC TCA TCG AGt gtt gac aat taa tca tcg gca t-3�
AD169�UL37x1 5�-TTT CAA GAC GAC GTG AGA CCC ACA CGC GGG TTT CAC TTC TTT CTT TAA Tta ccc ata cga cgt ccc ag-3�
5�-TTT CTT AAC CAA GGC GGG AGA GGA TCT TCA AGG CGT TTT CGC TGG ATC Cag gac gac gac gac aag taa-3�
MCMV-m41HA 5�-TCC TCG CAG TGA TCT GCA TCG TCA TCC TGA TCG TCG TGA TCA TTG ACA GAt acc cat acg acg tcc cag-3�
5�-AGT CAT CCG ATG ATC GTG TCG CCG CCC GAC CGC CCT CCT CCC CCA ATT CAa gga cga cga cga caa gta a-3�
AD169-UL37x1HA 5�-GGC AAC GAG CTC GGA TGC TGC AGC ACA ACG GCC CCC AGC AGT CTC ACC AGt acc cat acg acg tcc cag-3�
5�-CCT TCT TAT ACT ATC CCG GAG TCT GTG GTT TTT TTG TTT ACC CCT GCT TAa gga cga cga cga caa gta a-3�
a The HA tag is in capital letters, and restriction sites are in italics.
b Homologous sequences are in capital letters, and introduced EcoRI sites are in italics.
11634 BRUNE ET AL. J. VIROL.

were obtained from the American Type Culture Collection. The murine 10.1 cell
line and life-extended human foreskin fibroblasts (HFFs) have been described
previously (4, 19).
To reconstitute infectious virus from mutant MCMV genomes, BAC DNA was
transfected into NIH 3T3 cells with Superfect transfection reagent (Qiagen) as
described previously (7). For HCMV, BAC DNA was transfected into HFFs by
electroporation as previously described (2). AD169�UL37x1 virus was obtained
by transfection of BAC DNA into HFFs transduced with Retro-UL37x1HA and
propagated on the same cells.
Cell death assays. Cell viability was determined by a neutral red inclusion
assay essentially as described previously (24). To analyze nuclear DNA fragmen-
tation, cells were grown on coverslips, fixed with 2% paraformaldehyde, and
stained with a terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick
end labeling (TUNEL) assay kit (Roche) according to the manufacturer’s rec-
ommendations. Nuclei were counterstained with 4�,6�-diamidino-2-phenylindole
(DAPI). For detection of phosphatidylserine on the outer leaflet of the cell
membrane as an early sign of apoptosis, cells on coverslips were incubated with
labeled annexin V (Roche) and propidium iodide following the recommended
protocol. The percentage of apoptotic cells was determined by counting a total
of at least 400 cells in four or more random visual fields. The caspase inhibitors
Z-VAD-FMK and Boc-D-FMK (Calbiochem) were added to the cells 30 min
prior to infection at a final concentration of 100 �M. All assays were performed
in duplicate or triplicate.
Immunofluorescence. Cells were grown on coverslips, fixed with 4% parafor-
maldehyde, and stained with primary and secondary antibodies according to
standard procedures. HA-tagged proteins were detected with a monoclonal rat
antibody, 3F10 (Roche). A mouse monoclonal antibody against protein disulfate
isomerase (PDI) was purchased from StressGen. An antibody recognizing the
Golgi membrane protein p115 (48) was kindly provided by M. G. Waters. Sec-
ondary antibodies anti-rat immunoglobulin G-Alexa 488 and anti-mouse immu-
noglobulin G-Alexa 568 and the mitochondrion-specific dye Mitotracker Red
CMXRos were obtained from Molecular Probes.
Immunoprecipitation and Western blotting. Metabolic labeling with [35S]me-
thionine and [35S]cysteine and immunoprecipitation experiments were per-
formed essentially as described previously elsewhere (1). The 3F10 anti-HA
antibody and protein G-Sepharose were used for precipitation. Protein samples
were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE). The gels were dried and exposed to Kodak X-Omat AR films. For
Western blotting, proteins were transferred to nitrocellulose membranes and
probed with the monoclonal mouse anti-HA antibody 16B12 (Covance Research
Products). Signals where detected by enhanced chemiluminescence.
RESULTS
MCMV mutant causes apoptosis of infected cells. In a pre-
vious study, a library of mutant MCMV genomes was estab-
lished by random transposon mutagenesis of a full-length
MCMV BAC. The uncharacterized library of mutant viral ge-
nomes was subsequently converted into a library of mutant
viruses by direct transfer of the BACs from E. coli to fibroblasts
(6). Each mutant virus carries a transposon inserted at a ran-
dom position within the MCMV genome. The transposon con-
tains the EGFP gene driven by an HCMV immediate-early
promoter. Therefore, cells infected with the virus mutants can
be identified by their green fluorescence. In this library, we
found a virus mutant, IIIB11, that caused an interesting phe-
notype in infected NIH 3T3 fibroblasts: membrane blebbing
and release of vesicles reminiscent of apoptotic bodies (Fig.
1A). These phenomena were seen in only a few cells infected
with the control virus, MCMV-GFP, which contains the EGFP
FIG. 1. Apoptotic cell death induced by an MCMV transposon insertion mutant. (A) NIH 3T3 and (B) SVEC4-10 cells were infected at a
multiplicity of infection of 0.01 TCID50/cell with mutant IIIB11 and with the control virus, MCMV-GFP. The fluorescent images show virus plaques
with morphological signs of apoptosis (membrane blebbing, disintegration of cells with formation of apoptotic bodies) in individual IIIB11-infected
cells, most prominently seen around the center of the plaque. Bar, 20 �m. (C) The viability of NIH 3T3 cells infected with IIIB11 was markedly
reduced compared to that of cells infected with MCMV-GFP (MG) 36 h after infection at a multiplicity of 10 TCID50/cell. Cell death could be
largely inhibited by treating cells with caspase inhibitor I or III (CI1 or CI3, i.e., Z-VAD-FMK or Boc-D-FMK, respectively) but not by addition
of dimethyl sulfoxide (D), the solvent used for the caspase inhibitors.
VOL. 77, 2003 m41 INHIBITS APOPTOSIS 11635

gene inserted at an innocuous position. A similar phenotype
was observed upon infection of SVEC4-10 endothelial cells
(Fig. 1B) and M2-10B4 bone marrow stromal cells (not
shown), suggesting that the phenotype is not cell type specific.
Premature cell death was even more apparent when cells were
infected at a high multiplicity of infection. At 36 h after infec-
tion, the viability of cells infected with IIIB11 was markedly
lower than that of cells infected with the MCMV-GFP control
virus (Fig. 1C). This effect was inhibited when the infection was
done in the presence of broad-spectrum caspase inhibitors,
although inhibition of cell death was not complete (Fig. 1C).
Identification of ORF m41. The corresponding BAC clone
was used to determine the transposon insertion site in mutant
IIIB11. The transposon was found to be inserted at nucleotide
position 54022 of the MCMV genome, within a short ORF of
414 nucleotides (Fig. 2). This ORF, m41, is located in a group
of ORFs that are conserved between MCMV and rat CMV,
but not between the rodent CMVs and HCMV (10, 30, 36, 46)
(Fig. 2). However, HCMV contains a gene encoding a mito-
chondrion-localized inhibitor of apoptosis, UL37x1, at a simi-
lar position within its genome (17).
The phenotype observed in mutant IIIB11 could result from
disruption of ORF m41, an effect on the expression of one of
the adjacent genes m40 and M42, or from an additional ad-
ventitious mutation elsewhere in the viral genome. To discrim-
inate among these possibilities, we constructed targeted dele-
tion mutants MCMV�m40, MCMV�m41, MCMV�M42, and
MCMV�M45 (Fig. 3). The �M45 mutant was included as a
positive control virus, as viruses carrying mutations in this gene
have previously been shown to induce apoptosis rapidly in
infected endothelial cells (6). The mutant MCMV genomes
were constructed by homologous recombination in E. coli with
the BAC technology. The individual ORFs were deleted and
replaced with a zeocin resistance gene. For the �M42 and
�M45 mutants, short sequences at the 3� end of the ORFs
were left intact to avoid deletion of potential promoter se-
quences of downstream genes. In these two mutants, addi-
tional EcoRI restriction sites were introduced to facilitate de-
tection of the mutations by restriction digestion. Figure 3
shows a schematic representation of the mutations and the
mutant genomes in an ethidium bromide-stained gel and in a
Southern blot analysis.
The deletion mutants shown in Fig. 3 were derived from
MCMV-GFP and can thus be visualized in the same way as
IIIB11. Cells infected with MCMV�m41 displayed the same
phenotype as shown for IIIB11 in Fig. 1, whereas cells infected
with MCMV�m40 or MCMV�M42 did not (data not shown).
To compare the importance of m41 and M45 for inhibition of
virus-induced apoptosis, we used different cell death assays.
Annexin V detects phosphatidylserine on the outer leaflet of
the cell membrane, an early sign of apoptosis. At later stages of
apoptosis and during nonapoptotic cell death, cell membrane
integrity becomes compromised, allowing annexin V to enter
the cell and bind to phosphatidylserine molecules at the inner
leaflet of the cell membrane. Costaining with propidium iodide
is used to control for this. The TUNEL assay, by contrast,
detects nuclear DNA fragmentation, which occurs at late
stages of apoptosis. The neutral red inclusion assay measures
cell viability and is a very reliable test to quantitate cell survival
FIG. 2. Location of the transposon insertion in mutant IIIB11. The transposon insertion within ORF m41 of the MCMV genome results in a
change of the EcoRI restriction pattern (white arrowheads) in IIIB11 BAC (lane 2) compared to the MCMV wild-type BAC (lane 1), because the
transposon (Tn) contains EcoRI sites within its inverted repeats. The exact location of the transposon insertion was determined by sequencing.
ORFs m39, m40, and m41 show no sequence homology to the HCMV ORFs at the corresponding positions but are homologous to r39 to r41,
respectively, of rat CMV (RCMV). Genes conserved among the three CMVs are shown in gray. Lane MW, molecular size markers.
11636 BRUNE ET AL. J. VIROL.

and death. However, this assay cannot discriminate between
apoptotic and nonapoptotic cell death.
SVEC4-10 endothelial cells were infected at a high multi-
plicity of infection with the deletion mutants. At 24 h after
infection, an annexin V assay was used to detect early signs of
apoptosis in infected cells (Fig. 4A). At a later time point, cell
viability was measured by neutral red inclusion (Fig. 4B). Both
the m41 and M45 mutants induced apoptotic cell death, but
the effect of the �M45 mutant appeared to be stronger than
the effect of the m41 mutants. Similar results were obtained
when nuclear DNA fragmentation as a late sign of apoptosis
was measured with a TUNEL assay (not shown). Mutants
�m40 and �M42 did not differ significantly from the MCMV-
GFP parental virus, demonstrating that the phenotype of
IIIB11 and MCMV�m41 is caused by inactivation of m41
rather than an effect on neighboring genes. As determined by
the TUNEL assay, MCMV�m41 and MCMV�M45 also in-
duced premature apoptosis in 10.1 cells, a spontaneously im-
mortalized, p53-deficient cell line unrelated to SVEC4-10 cells
(Fig. 4C). This indicated that p53 is not required for viral
induction of apoptosis.
Growth properties of mutant viruses. In a previous study we
showed that inactivation of the M45 gene precluded growth of
MCMV in endothelial cells but allowed almost normal growth
in fibroblasts, bone marrow stromal cells, and hepatocytes (6).
Multistep growth curves, in which cells were infected with the
FIG. 3. Construction of MCMV deletion mutants. (A) ORFs m41, m40, M42, and M45 were deleted from the MCMV-GFP BAC (MG) and
replaced with a zeo gene (black box). This resulted in loss or gain of an EcoRI restriction site (E) in mutants MCMV�m40, �M42, and �M45 but
not in �m41. The calculated sizes of the EcoRI restriction fragments in the m40 to M45 region are given on the right. (B) Restriction patterns
visualized by ethidium bromide staining. Bands with sizes different from that of the parental MCMV-GFP BAC are indicated by arrowheads. An
SnaBI restriction site within m41 is lost in MCMV�m41, resulting in fusion of two SnaBI fragments of 4.8 and 3.3 kb into a new fragment of 8.1
kb (arrowheads). (C) Southern blot of EcoRI-digested BACs, hybridized with a probe specific for the zeo gene sequence. Lane MW, molecular
size markers.
VOL. 77, 2003 m41 INHIBITS APOPTOSIS 11637

mutant viruses at a multiplicity of 0.1 TCID50/cell, revealed
that, in comparison to the wild-type virus, the m41 mutant
viruses grew to a slightly reduced level in fibroblasts but were
diminished by up to 50-fold in endothelial cells (Fig. 5A and
B), probably because SVEC4-10 endothelial cells are more
prone to apoptosis than NIH 3T3 fibroblasts (unpublished
observation). Single-step growth curves in which NIH 3T3 cells
were infected at a multiplicity of 5 TCID50/cell showed a more
rapid decline in viral titers for the m41 and M45 deletion
mutants 3 to 5 days after infection (Fig. 5C). The decline in
virus titer was probably caused by premature cell death, as seen
in a time course analysis of cell viability (Fig. 5D). When
SVEC4-10 cells were used instead of NIH 3T3 cells, cell via-
bility was even more compromised (not shown), consistent
with our previous observation that SVEC4-10 cells are more
sensitive to virus-induced apoptosis. However, the titer drop
was delayed compared to the decline in cell viability. Virus
release by dying cells and stability of previously released virus
particles could account for this delay. Taken together, the
results suggests that the antiapoptotic function of the m41 gene
product became apparent predominantly at late stages after
infection and when cells were infected at a high multiplicity of
infection (see also Fig. 1 and 4).
Subcellular localization of the m41 proteins. According to
the published MCMV sequence (36), ORF m41 encodes a
polypeptide of 138 amino acids. It contains a hydrophobic
stretch close to the C terminus and is predicted to be a type II
transmembrane protein of 14.6 kDa.
To determine the subcellular distribution of the protein en-
coded by the m41 ORF, the m41 sequence was cloned by PCR
and fused to an HA epitope tag sequence at either the 5� or the
3� end. In addition, the HCMV UL37x1 gene was cloned and
HA tagged at the 3� end. Proteins HA-m41, m41-HA, and
UL37x1-HA were expressed in NIH 3T3 cells by using retro-
FIG. 4. MCMV deletion mutations cause apoptosis. SVEC4-10 endothelial cells were infected at a multiplicity of 5 TCID50/cell with MCMV-
GFP (MG) or deletion mutants. (A) Phosphatidylserine on the outer leaflet of the cell membrane was detected with annexin V at 24 h after
infection. The percentage of cells staining positive with both annexin V and propidium iodide (indicating late-stage apoptosis or nonapoptotic cell
death) is shown in black. (B) Cell viability was determined 32 h after infection by the neutral red inclusion assay. The viability of cells infected with
the �m41 or the �M45 mutant was markedly reduced, whereas cells infected with MCMV�m40 or MCMV�M42 were as viable as cells infected
with MCMV-GFP. (C) Cells of p53-deficient fibroblast cell line 10.1 also underwent apoptosis after infection with the �m41 or the �M45 mutant.
Cells were stained on coverslips by the TUNEL assay and counterstained with DAPI. Fewer cells are seen in the photomicrographs of cells infected
with the �m41 or �M45 mutant virus because dead and disintegrated cells detached from the coverslips.
11638 BRUNE ET AL. J. VIROL.

viral expression vectors and visualized by immunofluorescence
with an antibody directed against the HA epitope. Figure 6
shows that the m41 protein colocalized with a marker for the
Golgi apparatus but not with markers for the endoplasmic
reticulum or mitochondria. This localization was observed for
both the N- and C-terminally tagged m41 proteins. By contrast,
UL37x1-HA localized to the mitochondria (not shown), con-
sistent with previous reports and its function as a mitochon-
drion-localized inhibitor of apoptosis (12, 17).
Similar protein distributions were seen when m41 and
UL37x1 were expressed in NIH 3T3 cells by transient trans-
fection with pcDNA3 plasmid expression vectors. However,
the extremely high expression levels obtained with these vec-
tors sometimes resulted in cell toxicity and partly aberrant
subcellular distribution patterns, which were interpreted as
overexpression artifacts. Therefore, retroviral expression vec-
tors were preferred for colocalization studies.
To test the hypothesis that the m41 gene product requires
interactions with other viral proteins to acquire its proper
structure or to be transported to a specific compartment, we
analyzed its intracellular localization during MCMV infection.
As an m41-specific antibody was not available, we created a
mutant MCMV BAC in which the m41 sequence was tagged in
situ (Fig. 7A). For comparison, we also created recombinant
HCMV AD169 BACs, in which the UL37x1 gene was tagged
or inactivated (Fig. 7B and C). As the UL36-38 region contains
spliced genes (41), we wanted to minimize the sequence alter-
ations in this region. Therefore, only a short sequence encod-
ing amino acids 5 through 34 was deleted and replaced with a
prokaryotic kan gene for inactivation of UL37x1. This short
sequence has previously been shown to be essential for both
mitochondrial localization and antiapoptotic activity of the
UL37x1 protein (20).
Recombinant viruses were obtained by transfecting the
MCMV-m41HA and AD169-UL37x1HA BACs (Fig. 7D) into
fibroblasts. In infected cells, the tagged proteins were detected
by Western blotting (not shown) and by immunofluorescence
microscopy (Fig. 7E). Both proteins, UL37x1 and m41, exhib-
Vi
ru
s
tit
er
(lo
g 1
0
TC
ID
50
/m
l)
days after infection
1
2
3
4
5
6
SVEC4-10
MOI 0.1
0 1 2 3 4 5 6
MCMV-GFP
∆M45
IIIB11
∆m41
∆m40
∆M42
Vi
ru
s
tit
er
(lo
g 1
0
TC
ID
50
/m
l)
NIH/3T3
MOI 0.1
1
2
3
4
5
6
0 1 2 3 4 5 6
0 1 2 3 4 5
NIH/3T3
MOI 5
days after infection
0 1 2 3 4 5
NIH/3T3
MOI 5
100
80
60
40
20
%
v
ia
bi
lit
y
A B
C D
FIG. 5. Growth properties of MCMV mutants. (A) NIH 3T3 fibroblasts and (B) SVEC4-10 endothelial cells were infected at a multiplicity of
0.1 TCID50/cell for multistep growth curves. Deletion and insertion mutagenesis of the m41 ORF (mutants MCMV�m41 and IIIB11) had no
obvious effect on virus growth in NIH 3T3 fibroblasts and resulted in only moderately reduced titers in SVEC4-10 endothelial cells. By contrast,
the �M45 mutant failed to grow on SVEC4-10 cells, consistent with previous results (6). (C) After infection at a multiplicity of 5 TCID50/cell, the
titers of the �m41 and the �M45 mutants declined faster than the titers of the control viruses. All titers represent mean values of two or three
parallel experiments. Dotted line, detection limit. (D) A time course analysis showed a faster decline of cell viability for the m41 mutant viruses
(�m41 and IIIB11) compared to MCMV-GFP. Values were determined in triplicate with standard deviations (error bars).
VOL. 77, 2003 m41 INHIBITS APOPTOSIS 11639

11640 BRUNE ET AL. J. VIROL.

ited the same localization within CMV-infected cells as was
observed for cells that received the individual protein in the
absence of CMV infection (Fig. 6).
Transfection of human fibroblasts with the AD169-�UL37x1
BAC did not yield recombinant virus. However, when fibro-
blasts transduced with a retroviral vector expressing UL37x1
were used, AD169-�UL37x1 virus could be grown. The mutant
virus grew slowly on complementing cells, with obvious signs of
apoptosis in infected cells, suggesting that the fibroblasts ex-
pressed UL37x1 at levels insufficient for full complementation
of the defect (not shown). Recombinant virus generated on the
complementing cells could not be propagated in noncomple-
menting fibroblasts. Although the same results were obtained
with two separate AD169-�UL37x1 BAC clones, a second-site
mutation that contributes to the observed phenotype cannot be
entirely excluded. However, results recently presented by oth-
ers also indicated that UL37x1 is essential for AD169 replica-
tion in human fibroblasts (G. Hahn, S. T. Eichhorst, B. Korn,
P. H. Krammer, and R. Greaves, presentation at the 26th
International Herpesvirus Workshop, abstr. 7.06, 2001). Our
findings are in accordance with these results.
ORF m41 encodes at least two distinct protein products. As
we analyzed the protein products of the m41 ORF by immuno-
precipitation and in Western blot experiments with the tagged
MCMV-m41HA virus, we identified two distinct protein products
with apparent molecular masses of approximately 19 and 21 kDa
(Fig. 7F). When the HA-tagged ORF was expressed by a retro-
viral or a plasmid expression vector, only the smaller product of
19 kDa was detected (Fig. 7F). Moreover, the apparently larger
product was also absent in cells transduced with Retro-m41HA
and superinfected with MCMV (not shown), ruling out the pos-
sibility that the larger product resulted from a posttranslational
modification by MCMV proteins.
DISCUSSION
In this study, we provide genetic evidence that the m41 ORF
is required to prevent premature apoptosis of MCMV-infected
cells. Apoptosis of cells infected with m41 mutant viruses was
shown by morphology and by apoptosis-specific cell death as-
says (annexin V, TUNEL, and caspase inhibitors). Systematic
deletion mutagenesis of the region surrounding the m41 ORF
confirmed that the neighboring ORFs, m40 and M42, did not
encode the function. This function of an m41 gene product
could not be predicted by sequence analysis, as m41 shows no
apparent homology with any protein of known function. This
demonstrates that random unbiased mutagenesis and screen-
ing procedures are valuable tools with which to identify viral
gene functions.
The m41 ORF encodes at least two distinct protein prod-
ucts. Only one of them could be detected by heterologous
expression of the m41 ORF with retroviral or plasmid vectors.
The second gene product, which accumulated to a similar level,
was only seen when m41 expression was analyzed during virus
infection. In the absence of a specific antibody, this was
achieved by tagging the coding sequence within the viral ge-
nome. Such an in situ tagging procedure, which has previously
been used to tag bacterial and yeast genes (39, 45), should be
very useful for herpesvirus genetics as well. Although we have
not yet been able to determine the origin of the second m41
gene product, preliminary results suggest that it derives from a
spliced transcript (W. Brune, unpublished results). A more
detailed analysis of transcription in this region will be neces-
sary to identify all spliced and unspliced genes. At present it is
not known whether the antiapoptotic function is mediated by
the smaller or the larger m41 gene product or both.
Even though MCMV m41 and HCMV UL37x1 show no
sequence homology, their similar positions within the two viral
genomes suggested a potential functional homology. Both pro-
teins are involved in inhibition of apoptosis induced by virus
infection. However, the antiapoptotic effect of UL37x1 appears
to be stronger, as UL37x1 has been shown to be essential for
replication of the HCMV AD169 strain. By contrast, expres-
sion of m41 improves the virus yield but is not essential for
virus replication in cell culture. On the other hand, one has to
take into consideration that an AD169 strain with a mutant
UL37x1 is in fact mutant for two antiapoptotic genes, as UL36
has previously been shown to be mutant in this strain (40). It is
possible that UL36 and UL37x1 have additive effects and that
only one of the genes is required to allow survival of the
infected cell and virus replication. This question can only be
resolved by use of an HCMV strain that contains a wild-type
UL36 gene or by repairing the UL36 mutation in AD169.
The different subcellular distributions of the UL37x1 and
m41 proteins suggest quite divergent mechanisms of action.
The UL37x1 protein localizes to mitochondria, where it fulfills
a Bcl-2-like function even though it has no structural homology
FIG. 6 and 7. Figure 6 (top panels) shows subcellular distribution of the m41 protein. ORF m41 was tagged with an HA epitope sequence at
the 3� or the 5� end and expressed in NIH 3T3 cells with a retroviral vector (panels A and B, respectively). The m41 protein colocalized with a
marker for the Golgi (p115) but not with markers for the endoplasmic reticulum (PDI) or mitochondria (Mitotracker). Colocalizations are shown
in yellow in the merged pictures.
Figure 7 (bottom panels) shows construction of epitope-tagged CMV mutants. (A) An HA epitope tag sequence (hatched box) was fused to the
3� end of the MCMV m41 ORF by homologous recombination. A kan gene flanked by FRT sites (black ovals) was inserted for selection of
recombinant BACs in E. coli. The kan gene was subsequently removed with FLP recombinase, leaving a single FRT site behind. (B) In an analogous
fashion, an AD169-UL37x1 HA-tagged mutant was constructed. (C) In mutant AD169�UL37x1, a domain essential for UL37x1 protein function
was deleted and replaced with a kan gene. EcoRI restriction sites are indicated by arrows. (D) EcoRI restriction patterns of the MCMV wild-type
BAC (lane 1), MCMV-m41HA (lane 2), the AD169 wild-type BAC (lane 3), AD169�UL37x1 (lane 4), and AD169-UL37x1HA (lane 5). Changes
in the restriction pattern are indicated by arrowheads. Lane MW, molecular size markers. (E) The m41 protein expressed in NIH 3T3 cells by a
tagged MCMV mutant colocalizes with a marker for the Golgi apparatus (p115), whereas the HA-tagged pUL37x1 expressed in HFF cells by
AD169-UL37x1HA colocalizes with a mitochondrial marker (Mitotracker). (F) NIH 3T3 cells were infected with wild-type MCMV (M) or
MCMV-m41HA (clones T1 and T2) or transduced with Retro-m41HA (R). After metabolic labeling with [35S]methionine and [35S]cysteine,
HA-tagged proteins were immunoprecipitated and separated by SDS-PAGE. Two m41 protein products were expressed by MCMV-m41HA but
only one by Retro-m41HA. Similar results were obtained in Western blot experiments (not shown).
VOL. 77, 2003 m41 INHIBITS APOPTOSIS 11641

to Bcl-2 (16, 17). By contrast, m41 encodes a protein product
that localizes to the Golgi apparatus and is thus not likely to be
a functional homolog of UL37x1.
How could a protein found in the secretory pathway inter-
fere with induction of apoptosis? It is conceivable that m41
interacts with death receptors or their ligands to downmodu-
late their presentation on the cell surface. Such a function has
been demonstrated for the RID protein complex (also known
as E3-10.4K/14.5K) of adenovirus type 5 (14, 18, 42, 43). How-
ever, the RID complex does not accumulate in the Golgi as
m41 does, but binds to death receptors on the cell surface and
targets them to lysosomes for degradation. To our knowledge,
m41 represents the first example of a Golgi-resident inhibitor
of apoptosis. Interestingly, an endoplasmic reticulum-resident
protein, M-T4, that is required to prevent premature apoptosis
of infected lymphocytes (3, 21) was recently identified in myxo-
mavirus, a poxvirus with numerous antiapoptotic genes (15).
Although the mechanisms by which the m41 and M-T4 pro-
teins interfere with apoptosis are still unknown, these examples
highlight the use of multiple strategies by large DNA viruses
like the herpesviruses and poxviruses to ensure their survival in
different host cells. Identifying the mechanism of m41 action
will enhance our understanding of cellular defenses against
virus infection.
ACKNOWLEDGMENTS
We thank J. Goodhouse for excellent help with confocal microscopy
and S. Erhard for technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft
(Emmy Noether Fellowships to W.B. and M.N. and SFB 479) and by
grant CA85786 from the National Cancer Institute.
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