Role of murine cytomegalovirus US22 gene family members in replication in macrophages.

JOURNAL OF VIROLOGY, May 2003, p. 5557–5570 Vol. 77, No. 10
0022-538X/03/$08.00�0 DOI: 10.1128/JVI.77.10.5557–5570.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Role of Murine Cytomegalovirus US22 Gene Family Members
in Replication in Macrophages
Carine Me´nard,1 Markus Wagner,1 Zsolt Ruzsics,1 Karina Holak,1 Wolfram Brune,1†
Ann E. Campbell,2 and Ulrich H. Koszinowski1*
Department of Virology, Max von Pettenkofer Institute, Ludwig Maximilians University Munich,
80336 Munich, Germany,1 and Department of Microbiology and Molecular Cell Biology,
Eastern Virginia Medical School, Norfolk, Virginia 235072
Received 26 December 2002/Accepted 10 February 2003
The large cytomegalovirus (CMV) US22 gene family, found in all betaherpesviruses, comprises 12 members
in both human cytomegalovirus (HCMV) and murine cytomegalovirus (MCMV). Conserved sequence motifs
suggested a common ancestry and related functions for these gene products. Two members of this family, m140
and m141, were recently shown to affect MCMV replication on macrophages. To test the role of all US22
members in cell tropism, we analyzed the growth properties in different cell types of MCMV mutants carrying
transposon insertions in all 12 US22 gene family members. When necessary, additional targeted mutants with
gene deletions, ATG deletions, and ectopic gene revertants were constructed. Mutants with disruption of genes
M23, M24, m25.1, m25.2, and m128 (ie2) showed no obvious growth phenotype, whereas growth of M43
mutants was reduced in a number of cell lines. Genes m142 and m143 were shown to be essential for virus
replication. Growth of mutants with insertions into genes M36, m139, m140, and m141 in macrophages was
severely affected. The common phenotype of the m139, m140, and m141 mutants was explained by an inter-
action at the protein level. The M36-dependent macrophage growth phenotype could be explained by the
antiapoptotic function of the gene that was required for growth on macrophages but not for growth on other
cell types. Together, the comprehensive set of mutants of the US22 gene family suggests that individual family
members have diverged through evolution to serve a variety of functions for the virus.
Herpesviruses are large and complex DNA viruses, widely
found in nature. Human cytomegalovirus (HCMV), an impor-
tant human pathogen, defines the betaherpesvirus family.
Mouse CMV (MCMV) and rat CMV serve as biological model
systems for HCMV. HCMV, MCMV, and rat CMV display the
largest genomes among the herpesviruses (13, 34, 43). These
genomes are essentially colinear over the central 180 kb of the
230-kb genomes. Betaherpesviruses, which include the CMVs
as well as human herpesviruses 6 and 7, differ from alpha- and
gammaherpesviruses by the presence of additional gene fam-
ilies such as the US22 gene family, which are mainly clustered
at the ends of the genome (29, 30).
The US22 family was first described in HCMV (13). This
gene family comprises 12 members in both HCMV and
MCMV and 11 in rat CMV. Members of the US22 gene family
are characterized by stretches of hydrophobic and charged
residues as well as up to four conserved sequence motifs which
are specific for betaherpesviruses. Motif I differs between the
HCMV US and UL family members (30). In MCMV, m128
and m139 to m143 share the HCMV US-like motif I, while
M23, M24, m25.1, m25.2, M36, and M43 share UL-like motif
I. Motifs I and II have consensus sequences, while motifs III
and IV are less well defined but have stretches of nonpolar
residues (18, 24). The m139 to m141 genes contain all four of
these motifs, whereas m142 and m143 (and IRS1/TRS1 of
HCMV) lack motif II. In addition, m139, m140, m142, and
m143 each have an acidic domain, common to herpesvirus
transcriptional activators and specifically to MCMV immedi-
ate-early proteins 1 and 2 (11, 28). Several US22 gene products
represent viral tegument components (1, 33, 36).
The functions of most of the US22 genes are unknown. The
US22 genes TRS1/IRS1 and UL36 (HCMV) and m128, m142,
and m143 (MCMV) and positional homologs to UL36 to UL38
and UL43 of human herpesvirus 6 (31) are transcribed with
immediate-early kinetics. For all of these except m142 and
m143, a transcriptional transactivation function was described
(11, 14, 15, 22, 31, 37, 40). Besides studies on the ie2 gene prod-
uct of m128, which was shown to be dispensable for growth of
MCMV in vitro and in vivo (11), biological properties for some
early US22 homolog genes in MCMV have been defined (12,
19, 20). A deletion mutant encompassing genes m137 through
m143 could not be wild-type MCMV, indicating that the m142
or m143 gene or both genes might be essential for virus rep-
lication (12). Deletion of m140 and m141 had no effect on
replication of MCMV in fibroblasts but impaired the ability of
the virus to replicate in macrophages in vitro and in the spleens
of mice.
The report on the combined failure of macrophage growth
in vitro and altered tissue type distribution in vivo of mutants
RV7 (encompassing genes m137 through m141) and RV10 (en-
compassing US22 family genes m139 to m141) (12, 20) prompted
us to study all US22 gene family members with respect to this
phenotype. It was reported that the macrophage growth phe-
notype was a property not of m139 but of m140 and m141,
* Corresponding author. Mailing address: Max von Pettenkofer In-
stitut, Pettenkoferstrasse 9a, D-80336 Munich, Germany. Phone: 4989
5160 5290. Fax: 4989 5160 5292. E-mail: koszinowski@m3401.mpk
.med.uni-muenchen.de.
† Present address: Rudolf Virchow Center for Experimental Bio-
medicine, University of Wu¨rzburg, D-97078 Wu¨rzburg, Germany.
5557

whose products acted in a cooperative and interdependent
manner (19).
Furthermore, Liu and colleagues reported on an M43 mu-
tant with a transposon insertion at codon 313 of the M43 open
reading frame (ORF) (48). This mutant grew like the wild type
in fibroblasts and was attenuated in salivary glands but not in
other organs in vivo. Mutants of a number of US22 genes were
shown to grow like wild-type virus in fibroblast cells but, except
for the m139, m140, and m141 ORFs, were not tested for
growth properties in macrophages (11, 33, 42, 48).
A systematic approach to an entire herpesvirus gene family
has only been a theoretical option due to the time-consuming
labor involved in constructing, isolating, confirming, and test-
ing individual mutants. We recently pioneered the construction
of herpesvirus genomes as infectious bacterial artificial chro-
mosomes (BAC) in Escherichia coli and their targeted mu-
tagenesis (2, 5, 27, 46, 47). We further described a one-step
procedure for random insertional mutagenesis of herpesvirus
BACs, with a Tn1721-based transposon system (9). The effec-
tiveness of the latter method has been tested for MCMV (9),
HCMV (21), and the murine gammaherpesvirus 68 (O. Fuchs,
C. Menard, U. H. Koszinowski, and M. Wagner, Abstr. 26th
International Herpesvirus Workshop, abstr. 3.26, 2001). The
transposon insertion site can be determined by direct sequenc-
ing, and infectious virus can be recovered after transfection of
permissive cells with characterized mutant genomes.
We decided to start a comparative analysis of the complete
US22 gene family. For this purpose we had to limit the number
of constructs carried out for each gene of all the 12 US22 gene
family members, and we restricted testing to fibroblast, mac-
rophage, and endothelial cells. We characterized about 40 mu-
tant genomes in more detail. For two genes of the US22 family,
m142 and m143, essentiality was proven. For the other 10
genes, M23, M24, m25.1, m25.2, M36, M43, m128, m139, m140,
and m141, viable transposon insertion mutants were isolated. Our
results show that five members of the US22 gene family, M36,
M43, and m139 to m141, affect macrophage tropism. The gene
products of m139, m140, and m141 apparently interact at the
protein level, which explains their common phenotype. The
effect of M36 on viral growth in macrophages was shown to be
due to the antiapoptotic function of the gene product.
MATERIALS AND METHODS
Cells and viruses. Murine embryo fibroblasts (MEFs) from BALB/c mice were
cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal calf serum (FCS). NIH 3T3 fibroblasts (ATCC CRL1658) and J774-A1
macrophages (ATCC TIB67) were propagated in DMEM supplemented with
5% newborn calf serum. SVEC4-10 endothelial cells (EC) (ATCC CRL2181), a
simian virus 40-transformed cell line derived from lymph node vessels, were
propagated in DMEM supplemented with 10% FCS. IC-21 macrophages (ATCC
TIB186), a simian virus 40-transformed peritoneal macrophage cell line, and the
M2-10B4 bone marrow stromal cell line (ATCC CRL1972) were propagated in
RPMI medium supplemented with 10% FCS. Fresh peritoneal exudate cells
(PEC) were obtained from 8-week-old C57BL/6 mice by washing the peritoneal
cavity with medium 5 days after intraperitoneal injection of 3 ml of 3% thiogly-
colate. The exudate cells were enriched to 90% of peritoneal macrophage cells
by the thioglycolate treatment.
All MCMV mutants were generated from the parental MCMV BAC pSM3fr,
which contains the complete MCMV Smith strain genome. Virus MW97.01,
reconstituted from pSM3fr, was shown to have wild-type properties in vitro and
in vivo (46). In all experiments, we used the wild-type virus MW97.01 except for
growth on J774-A1 macrophages, PEC, and SVEC4-10 endothelial cells, where
the m152 transposon mutant was used as the control. All transposon mutants
expressed green fluorescent protein (GFP). Strong GFP expression by MCMV
may affect virus growth. m152 does not belong to the US22 gene family, and its
deletion does not affect growth in vitro (25, 44). Therefore, we used this mutant
with a transposon insertion 300 bp downstream to the native ATG (nucleotide
211077) of gene m152, which expresses the GFP, as a control. For all experi-
ments, two transposon insertion mutants for each US22 gene were tested when-
ever more than one transposon insertion mutant was found in the library of
MCMV transposon insertion mutants.
Wild-type and mutant viruses were reconstituted by transfection of BAC DNA
into NIH 3T3 cells with the Superfect transfection reagent (Qiagen, Hilden,
Germany) according to the manufacturer’s instructions. Briefly, 2 to 3 �g of BAC
DNA was incubated with 100 �l of medium without serum and 10 �l of Superfect
transfection reagent. The mixture was added to approximately 3 � 105 NIH 3T3
cells. Cells were washed with phosphate-buffered saline 4 h later, cultured with
fresh medium, and passaged when necessary. Plaques usually appeared 4 to 6
days after transfection. All virus stocks were prepared on M2-10B4, and virus
titers were determined on MEFs by plaque assay or on NIH 3T3 cells with the
TCID50 (median tissue culture infectious dose) method (4, 35).
Plasmids, transposon mutagenesis, and screening for transposon insertions
into US22 family genes. The pCR3 vector (Invitrogen, Karlsruhe, Germany) was
used to clone the M36 or hemagglutinin (HA)-tagged (N-terminal) M36 ORF
(M36HA). The M36 and M36HA ORFs were amplified by PCR with primers
3�-M36 (5�-GAG TCT AGA CTA TCG ATA TCC CCG TGT CA-3�) and
5�-M36 (5�-CAG GAA TTC ATG TAT GAG CAA GAG GAA CA-3�) or
5�-M36HA (5�-CAG GAA TTC ATG TAC CCA TAC GAT GTT CCA GAT
TAC GCG TAT GAG CAA GAG GAA CA-3�), respectively, and inserted
between the XbaI and EcoRI sites of the multiple cloning site in pCR3, gener-
ating the pCR3-M36 and pCR3-M36HA vectors, respectively.
Transposon mutagenesis was performed as described elsewhere (7, 9) with the
TnMax8 donor plasmid pTsTM8 (9) and the TnMax16 donor plasmid pTsTM16
(8), which is a pTsTM8 derivative containing the GFP gene under the control of
the human CMV immediate-early promoter-enhancer from plasmid pEGFP-C1
(Clontech, Palo Alto, Calif.) described in detail elsewhere (7, 9). pTsTM8 or
pTsTM16 and the MCMV BAC were maintained in E. coli DH10B (Invitrogen,
Karlsruhe, Germany). About 2,000 and 2,600 bacterial clones were established in
96-well microtiter plates as libraries of uncharacterized MCMV::TnMax8 and
MCMV::TnMax16 mutant genomes, respectively.
PCR screening of the MCMV::TnMax16 mutant library for mutants of US22
gene family members with transposon insertions at the N-terminal end of the
gene was performed essentially as described previously (21). Briefly, to detect
mutant genomes with transposon insertions at positions of interest, three rounds
of PCR were performed with two transposon-specific primers in the same reac-
tion, M13 forward (M13-for) (5�-GCC GCT GTA AAA CGA CGG CCA GT-3�)
and reverse (M13-rev) (5�-GGC CGC AGG AAA CAG CTA TGA CC-3�)
primers, which bind within the end of the transposon, and one gene-specific
search primer designed to bind about 200 bp upstream of the start of the gene.
The gene-specific search primers used in this study are listed in Table 1. The
exact transposon insertion site in identified clones was determined by direct
sequencing of the mutant BAC DNA with the M13-for and M13-rev primers as
described previously (9).
Site-directed mutagenesis of MCMV BAC. For construction of the �M36,
�m139, �ATG-m139, �m143, and �ATG-m142 mutant genomes, we used ho-
mologous recombination of linear PCR fragments with the MCMV BAC plas-
mid pSM3fr in E. coli as described in principle elsewhere (45). The linear
fragments were generated by PCR with plasmid pACYC177 (NEB, Beverly,
Mass.) (for the �m139 and �m143 mutant genomes) and plasmid pSLFRTKn
(3) (for �M36 and the �ATG mutant genomes), both of which contain the
kanamycin resistance gene as the DNA template.
The following contiguous primers were used: 5�-�M36 (5�-TTT CTC CC CTC
AC CCT CTC CGT CCC TTT CTT ATC CGT TTT CCC TCT ATC GTC GTG
GAA TGC CTT CGA ATT C-3�) and 3�-�M36 (5�-GCT CAT TCT TTC GGG
AAA GGG GTG GAG GAG GGT CGT TTG ACA GTG AAA GGA CAA
GGA CGA CGA CGA CAA GTA A-3�) for �M36-MCMV, 5�-�m139 (5�-CCT
TGC CGC CGT CGA ACA TGT CCA TGG CGC GGA CGT AAC GAC GAT
AGA AGT CGC GCG ATT TAT TCA ACA AAG CCA CG-3�) and 3�-�m139
(5�-CAG ACC GTG AGT TGA CGG CGC CGG CGC CAG ACG GAG CAG
ACA GAG AGA GAG AAG GGC CAG TGT TAC AAC CAA TTA ACC-3�)
for �m139-MCMV, 5�-�m143 (5�-CGG TCG TGT AGC GGT ACT GCC GCT
CTC GGA GGC ATT CGT GAC AAT CTC CCT CCG CCG ATT TAT TCA
ACA AAG CCA CG-3�), and 3�-�m143 (5�-AGC AGA GAG GTG GTT GCC
TCG GCT CCG CTC CGC TTC GTC CGC CCG TCT CGT GCG CGG CCA
GTG TTA CAA CCA ATT AAC C-3�) for the �m143 mutant genome; 5�-
�ATG-m139 (5�-GGG GGA AGG CTC CTC TCG TCC ACG CCG CCG TAT
5558 ME´NARD ET AL. J. VIROL.

TCT CCG AAC TTC TGG TCC GTC GTG GAA TGC CTT CGA ATT-3�) and
3�-�ATG-m139 (5�-CGT GAG TTG ACG GCG CCG GCG CCA GAC GGA
GCA GAC AGA GAG AGA GAA GGA CAA GGA CGA CGA CGA CAA
GTA A-3�) for �ATG-m139-MCMV, and 5�-�ATG-m142 (5�-CTG GTC TCT
GAA GTG ATC CGA TCG GAT CGC CGC GCA CAG GGC GTC CGT CGT
GGA ATG CCT TCG AAT TC-3�) and 3�-�ATG-m142 (5�-CCA CCC TTC
TCC ACC CGT GTT CCC GCT GCC GCC CGT CGC CCT CGC CAC AAG
GAC GAC GAC GAC AAG TAA-3�) for �ATG-m142 mutant genome.
The PCR fragments were inserted by homologous recombination via the
flanking 40- to 60-nucleotide homologies to the viral target sequences. Kanamy-
cin-resistant clones were analyzed for correct insertion. BAC DNA was isolated
from E. coli cultures with an alkaline lysis procedure (38) and purified with
NucleoBond AX100 columns (Macherey-Nagel, Du¨ren, Germany). For genera-
tion of �ATG-m139 and �ATG-m142, the kanamycin resistance cassette flanked
by the minimal FLP recombinase recognition target (FRT) sites was excised by
FLP recombinase. Here, the first two ATGs of ORF m139 (84 bp apart) were
replaced with an 86-bp extraneous sequence, and the native ATG of ORF m142
was deleted. Correct mutagenesis was confirmed by restriction pattern analysis
and sequencing.
For construction of the �ATG-m142/m142E mutant genome to ectopically
express wild-type m142, the pSM3fr BAC containing an FRT site and the GFP
gene in the position of the nonessential gene m152 (pSM3fr/GFP) was first
subjected to site-directed mutagenesis to delete the native ATG of m142 with the
same PCR fragment used for generation of the �ATG-m142 mutant genome in
principle as described previously elsewhere (6). In a second mutagenesis step, a
plasmid with a conditional origin of replication, a zeocin resistance gene, a 34-bp
FRT site (to be published elsewhere), and additionally containing the wild-type
m142 ORF under control of the human CMV immediate-early promoter-
enhancer from plasmid pEGFP-C1 (Clontech, Palo Alto, Calif.), was inserted
into the FRT site of �ATG-m142/FRT, generating �ATG-m142/m142E. The
M36-HA virus, which contains an HA tag at the C terminus of the M36 ORF, was
also constructed by homologous recombination of linear PCR fragments with the
MCMV BAC plasmid pSM3fr in E. coli by methods described previously (10).
The following primers were used for the PCR: 5�-M36-HA (5�-CTC CCC TCA
CCC TCTC CGT CCC TTT CTT ATC CGT TTT CCC TCG TCG TGG AAT
GCC TTC GAA TTC-3�) and 3�-M36-HA (5�-ATC GAG AGG AGG AGG
GTC AAG CTC TTT AAG ATG ACA CGG GGA TAT CGA TAC CCA TAC
GAT GTT CCA GAT TAC GCG TAG ACA AGG ACG ACG ACG ACA
AGT AA-3�).
Determination of MCMV replication in fibroblasts, macrophages, and endo-
thelial cells. Replication of viruses in fibroblasts, macrophages, and endothelial
cell lines was determined as follows. Approximately 1 � 105 to 5 � 105 cells in
six-well plates were infected at a multiplicity of infection (MOI) of 0.1 for end-
point titration in NIH 3T3, IC-21, and SVEC4-10 cells or with an MOI of 5 in
J774-A1 macrophages and PEC. After a 2-h incubation, the virus inoculum was
removed, the cells were washed with phosphate-buffered saline, and fresh cell
growth medium was added. At 6 days postinfection, supernatants were harvested
and virus titers were determined on MEFs by plaque assay (35) or on NIH 3T3
cells with the TCID50 method (4). Growth of each virus was quantified at least
twice and in triplicate.
Northern blot analysis. NIH 3T3 cells were infected with the wild-type and
mutant viruses at an MOI of 5 PFU/cell, and total RNA was harvested 24 h
postinfection. Total RNA was isolated with the RNeasy kit (Qiagen, Hilden,
Germany), and mRNA was harvested with the Oligotex direct mRNA kit (Qia-
gen, Hilden, Germany) according to the manufacturer’s instructions. The probe
used to analyze m139 to m141 gene expression corresponds to a DNA fragment
from nucleotide positions 196317 to 197220 of the MCMV genome. To analyze
M35 and M37 gene expression, DNA fragments from nucleotide positions 46153
to 46737 and 49680 to 50385 of the MCMV genome, respectively, were used as
probes. DNA probes were labeled with [�-32P]dCTP with the nick translation kit
from Amersham (Amersham Biosciences Europe, Freiburg, Germany) accord-
ing to the manufacturer’s instructions. To verify loading of equal amounts of viral
RNA, aliquots of the same RNA samples were hybridized with a probe specific
to the m04 gene (nucleotide positions 3419 to 3978 of the MCMV genome).
Southern blot analysis. Southern blot analysis was carried out to detect the
presence of the transposon within the viral genome. Briefly, genomic DNA was
digested with BamHI, separated on a 0.8% agarose gel, transferred to a nylon
membrane (Hybond-N; Roche, Mannheim, Germany), and hybridized to a DNA
probe corresponding to the NheI-PstI fragment from plasmid pEGFP-C1 (Clon-
tech, Palo Alto, Calif.) described in detail elsewhere (7, 9). The probe was
prepared with the DIG DNA labeling and detection kit from Roche (Roche,
Mannheim, Germany) according to the manufacturer’s instructions.
Immunoprecipitation and Western blot analysis of viral gene expression. The
MCMV m139 rabbit polyclonal antiserum has been described (19). For the m140
and m141 proteins, 15-amino-acid peptides from the C terminus (SVLTTRP
DRNRDTRT, amino acid positions 431 to 446) and the N terminus (ATGG-
DQNARRRAIER, amino acid positions 25 to 40), respectively, were used to
generate rabbit polyclonal antibodies (Eurogentec, Seraing, Belgium). The
m04-3 antiserum, specific for m04/gp34 (23), was used as a control for viral
infection. The anti-HA antibody (clone 3F10) directly coupled to peroxidase was
purchased from Roche (Roche, Mannheim, Germany), and the anti-caspase-3
and anti-poly(ADP-ribose) polymerase (PARP) antibodies were purchased
from Cell Signaling (Beverly, Mass.) and Transduction Laboratories (Lexington,
Ky.), respectively. The anti-caspase-8 (clone 5F7) antibody was purchased from
Upstate Biotechnology (Biozol, Eching, Germany).
Immunoprecipitation was performed as described previously (16). In brief,
subconfluent layers of cells were mock infected or infected at an MOI of 1 with
the wild-type control or the indicated MCMV mutant. At 24 h postinfection, the
cells were labeled with [35S]methionine and [35S]cysteine (Amersham Bio-
sciences, Braunschweig, Germany) for 40 min at a concentration of 350 �Ci/ml.
Cells were lysed in buffer (140 mM NaCl, 5 mM MgCl2, 20 mM Tris [pH 7.6],
1 mM phenylmethylsulfonyl fluoride) containing 1% (vol/vol) Nonidet P-40
(Sigma, St. Louis, Mo.) in the presence of a protease inhibitor mixture (Biomol,
Plymouth Meeting, Mass.). After removal of nuclei by centrifugation, lysates
were cleared with the appropriate preimmune serum and immunoprecipitated
with the anti-m141 purified antiserum and protein A-Sepharose (Amersham
Biosciences, Uppsala, Sweden).
For Western blot detection of proteins encoded from genes m139, m140, and
m141, cells were infected at an MOI of 1 (NIH 3T3) or 3 (IC-21), and lysates
were harvested 24 h postinfection in lysis buffer. Different lysis buffer contents
were used for isolation of the proteins: 1% Triton X-100–4 mM MgCl2–140 mM
NaCl–20 mM Tris (pH 7.3) to isolate the m140, m141, and M36-HA proteins; 50
mM Tris–1% sodium dodecyl sulfate (pH 7.5) for the m139 protein; and 50 mM
piperazine-N,N�-bis(2-ethanesulfonic acid) (PIPES)-NaOH (pH 6.5)–2 mM
EDTA–0.1% CHAPS (3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-
sulfonate)–5 mM dithiothreitol–20 �g of leupeptin per ml–10 �g of pepstatin per
ml–10 �g of aprotinin per ml–1 mM phenylmethylsulfonyl fluoride for caspase-3
and PARP. From 12 to 15 �g of total protein was loaded per lane. Proteins were
separated on 12.5% polyacrylamide gels for m04, m139, and m141 proteins,
caspase-3, caspase-8, HA, and PARP or on 10% polyacrylamide gels for the
m140 protein under denaturing conditions. All blots were blocked with Tris-
buffered saline–0.1% Tween 20 (TBST) and either 5% milk (for the m04 and
m139 proteins, caspase-3, caspase-8, and PARP) or 5% bovine serum albumin
(for the m140 and m141 proteins).
For immunoprecipitation and Western blot analysis, 293 cells in a 10-cm dish
were transfected with 10 �g of the pCR3-M36 or the pCR3-M36-HA vector with
TABLE 1. Search primers used for identification of transposon
insertions into pSM3fr
Primer Sequence
M23-for...........................5�-TCT TCA GCG ACT TTG CCA C-3�
M23-rev ..........................5�-CGA GCG GAT CGA TGA TGA AG-3�
M24-for...........................5�-GAG AAA GGA GGA AAA GTG GG-3�
M24-rev ..........................5�-GAT CGA CGG TTC ATC GTT CG-3�
m25-for ...........................5�-GTA ATT CTT GGG CCA GGT CAG C-3�
m25-1-rev........................5�-GCA TTG TTC TCG TCA ATC CG-3�
m25-2-rev........................5�-ACA ACC TCA CGC AGC TCA TCT C-3�
M36-for...........................5�-CTC CCG AGG AAG AGA TTG TG-3�
M36-rev ..........................5�-CTG CTT GCG TCA AAA TGC TC-3�
M43-for...........................5�-CTC GTC CGT GCA GAG AAT CAT C-3�
M43-rev ..........................5�-CTG ACA GCA TCG AGA ATC C-3�
m128-for .........................5�-GCT CGC TCG ATC CAT TCT TC-3�
m128-rev .........................5�-GGC GTG AAC ACG ATG TTC TTG-3�
m139-for .........................5�-CAG ATA CTT GGA CAG GTC CAC G-3�
m139-rev .........................5�-CTT GGA GAG GCA AGA GAG TCA C-3�
m140-for .........................5�-CAG GTG GTG GAT GCT GAA G-3�
m140-rev .........................5�-CTC CAC CTA GCT CAC TAA CAG C-3�
m141-for .........................5�-CTC GAA GCT CGA AGA GAA CAC C-3�
m141-rev .........................5�-CAT CAC ATA AAC CCC TCC CC-3�
m142-for .........................5�-ACA CAC ACA CCA ACC ACC CTT C-3�
m142-rev .........................5�-GGC GGG TGA CAT GAG AGA T-3�
m143-for .........................5�-CGA TAG ATA GAA GAT GCT GCC C-3�
m143-rev .........................5�-CAC CCG GAT GGT GTA AAA GAG-3�
m152-for .........................5�-TCC ACC TTC AAT CGT CCA CC-3�
m152-rev .........................5�-GGA TAC GGA GGA GAA TGT GGT G-3�
VOL. 77, 2003 MUTATIONAL ANALYSIS OF MCMV US22 GENES 5559

FIG. 1. Transposon insertions and analysis of US22 gene family in MCMV. (A) Locations of the transposon integration sites in US22 genes.
The top line of boxes represent HindIII fragments of the 230-kb MCMV genome. The second line of boxes represent the ORFs of the US22 genes
in MCMV. Arrows within the boxes indicate the direction of gene transcription. Designations M and m indicate ORFs with and without direct
sequence homology to individual HCMV genes, respectively (34). The HCMV genes with the greatest homology are listed above each box. The
long arrows indicate the transposon insertion sites of isolated mutants. The shorter arrows indicate additional clones that were detected by PCR
screening but not analyzed further. The exact nucleotide positions of the transposon insertions are indicated in parentheses after the names of the
mutants. (B) Structural analysis of the wild-type (wt) and mutant BAC plasmids. The ethidium bromide-stained agarose gel shows BamHI-digested
DNA from the BAC plasmids. The majority of bands remain conserved, and specific alterations occur as a result of transposon insertion. (C)
Southern blot analysis of BamHI-digested BAC plasmids hybridized with a GFP-specific probe shows a single transposon insertion for every
mutant.
5560 ME´NARD ET AL. J. VIROL.

the Superfect transfection reagent (Qiagen, Hilden, Germany) according to the
manufacturer’s instructions. After 24 h, the cells were lysed in the same buffer
used for immunoprecipitation analysis and split into two aliquots for immuno-
precipitation with either anti-HA or anti-caspase-8 antibodies. Immunoprecipi-
tates were separated on 12.5% polyacrylamide gels under denaturing conditions
for Western blot analysis as described above.
Apoptosis assay. Fas-mediated apoptosis in NIH 3T3 and IC-21 cells was
induced by exposure to the Jo2 anti-mouse Fas antibody (BD PharMingen,
Heidelberg, Germany). About 5 � 105 NIH 3T3 or 1 � 105 IC-21 cells were
mock infected or infected with wild-type or �M36 MCMV at MOIs of 1 and 5,
respectively. At 48 h postinfection, cells were exposed to 0.5 �g of anti-Fas
antibody and 12.5 �g of cycloheximide per ml or medium alone as controls. Cells
were trypsinized, and cell viability was determined by visual inspection of cells
under a phase contrast microscope with the trypan blue dye exclusion assay (41).
The results are expressed as percentage of viable cells compared to that in
nontreated controls. The caspase-8 activity in infected cells was determined with
the ApoAlert caspase colorimetric assay kit (Clontech, Palo Alto, Calif.). About
106 IC-21 macrophages were mock treated or infected at an MOI of 7. At 36 h
postinfection, cell lysates were prepared, and the assays were carried out accord-
ing to the manufacturer’s instructions.
RESULTS
Concept of US22 gene family mutant analysis. As an exten-
sion to previous work on MCMV cell type tropism (8, 12, 19,
20), in this study we tested US22 gene mutants for host cell
range effects in vitro and focused on macrophage growth. Con-
sidering that cytopathic effects in macrophages can be difficult
to observe upon virus infection, we constructed a library of
MCMV transposon insertion mutants with the pTsTM16 trans-
poson donor plasmid (8). As the TnMax16 transposon contains
the GFP gene, all mutant viruses expressed GFP from the
transposon. Altogether, 2,600 clones were screened, and 31
different clones, each with a transposon insertion within genes
of the US22 gene family, were selected (Fig. 1).
The observation of a large number of insertions within genes
M36 and M43 and fewer insertions in other genes demon-
strated that, in this new library, the distribution of transposon
insertions was not completely random and that it differed from
that of the library generated previously (17). All the mutant
genomes used in this study were analyzed for genome integrity
by restriction enzyme digestion (Fig. 1B). There were no un-
anticipated alterations in the number or sizes of the restriction
fragments. All mutants were sequenced at the transposon in-
sertion site. Multiple transposon insertions would have become
apparent from the inability to read a clear sequence. In addi-
tion, Southern blot analysis with a GFP-specific probe was
performed (Fig. 1C). Since the restriction enzyme also cleaves
the transposon, multiple transposon insertions, even at the
same site, would have resulted in multiple bands. Collectively,
these controls indicated that only single insertions had oc-
curred, and there were no apparent alterations elsewhere in
the genome.
For comparison, fibroblasts and macrophage cells were in-
fected in parallel with individual reconstituted mutants to
screen for loss or impairment of ability to grow on macrophage
cells. Mutants with a selective macrophage phenotype were
also tested on endothelial cells. The phenotype of a single
insertion mutant cannot define a function of the targeted ORF.
Either a revertant with a wild-type phenotype or an identical
phenotype obtained with independent mutants of the same
ORF would indicate that adventitious mutations elsewhere in
the genome are probably not responsible for the observed
phenotype. To limit the work on confirmatory targeted gene
mutations but to draw conclusions on the function of specific
genes nevertheless, we took the following strategy. If a mutant
had no selective macrophage phenotype or if the phenotype
merely confirmed already published work, we limited the anal-
ysis to two independent mutants with insertions located in the
first half of the ORF whenever possible. Macrophage growth
phenotypes described for the first time or mutant phenotypes
different from published ones were retested by targeted muta-
tions. These targeted mutations were either complete deletions
of the ORF, mutations of ATG, or reinsertions of the complete
gene into an ectopic position of the viral genome.
Genes m142 and m143 are essential for MCMV replication.
Three mutant genomes with transposon insertions in m142 and
m143 (Tn-m142.A, Tn-m142.B, and Tn-m143) failed to gener-
ate infectious progeny despite numerous attempts, whereas all
other MCMV mutants in the US22 family of genes could be
reconstituted as viruses by transfection of the genomes into
NIH 3T3 cells. Genes m139 to m143 belong to a complex
transcriptional region (18). The transcripts of m142 and m143
coterminate downstream of m142 (Fig. 2A). The transposon
library contained only one m143 transposon insertion mutant
(Fig. 2B). Therefore, we constructed a targeted deletion of
m143 and confirmed the lethal phenotype of the m143 gene
(Fig. 2C). The additional 3-kb transposon insertion into m142
might destabilize the essential m143 transcript (Fig. 2A and B).
In order to minimize the possible polar effects of the trans-
poson insertion within m142 on m143 mRNA expression, we
restricted the mutagenesis to the m142 start codon. This was
carried out in a two-step procedure (44, 45). First, the ATG
codon was deleted by insertion of a kanamycin marker by PCR
based mutagenesis with linear DNA fragments as described
previously (45). To excise the kanamycin resistance marker
from the BAC, the kanamycin resistance gene was flanked by
minimal FRT sites. Expression of the FLP recombinase ex-
cised the kanamycin resistance marker, and only one FRT site
remained. (Fig. 2D). Because �ATG-m142 did not allow virus
reconstitution, we concluded that m142 is also essential for
virus replication. For confirmation, we reintroduced the m142
gene into �ATG-m142 at an ectopic position (Fig. 2E). This
genome, �ATG-m142/m142E, gave rise to progeny and defi-
nitely proved the essentiality of m142. Thus, both of the US22
family genes m142 and m143 are essential for virus replication.
Majority of US22 gene mutants have no selective macro-
phage phenotype. Transposon insertions into the other 10
US22 genes (M23, M24, m25.1, m25.2, m128, M36, M43,
m139, m140, and m141) allowed virus growth. Viable mutants
were first tested with respect to growth on NIH 3T3 fibroblasts
and on IC-21 macrophages in comparison with wild-type virus.
Figure 3 shows one representative experiment. Virus progeny
in tissue culture supernatants was quantified by plaque assay
on MEFs (Fig. 3A and C) and by the TCID50 method on NIH
3T3 fibroblasts (Fig. 3B and D). There were no significant
differences between wild-type MCMV and the transposon mu-
tants of ORFs M23, M24, m25.1, m25.2, and m128 with respect
to growth on NIH 3T3 fibroblasts and on IC-21 macrophages.
The two mutants of the M43 gene with transposon insertions
4 bp (shown) and 78 bp downstream of the ATG, showed a
growth deficit when titrated on NIH 3T3 cells (Fig. 3B) but not
when titrated on MEFs (Fig. 3A). The difference was in the
VOL. 77, 2003 MUTATIONAL ANALYSIS OF MCMV US22 GENES 5561

range of about 2 log units compared to wild-type MCMV. This
discrepancy between titration on NIH 3T3 and MEFs for the
Tn-M43 mutants was consistently observed in repeated exper-
iments. A growth reduction was also apparent when the Tn-
M43 mutants were tested in IC-21 macrophages (Fig. 3C and
D). There was no growth deficit of the Tn-M43 mutants on J774-
A1 macrophages or SVEC4-10 endothelial cells and a slight
reduction in growth on PEC (Table 2). Because the M43 mu-
tation also affected viral growth in another cell type, the mu-
tation does not define a macrophage-specific host range effect.
Mutation of genes M36, m139, m140, and m141 affects
growth on IC-21 macrophages. Although viruses with muta-
tions in M36, m139, m140, and m141 grew comparably to the
wild-type control virus on fibroblasts, growth on IC-21 macro-
phages was reduced by two to four log units (Fig. 3C and D).
This was seen with both M36 transposon mutants with trans-
poson insertions 98 bp downstream of the ATG and 363 bp
downstream of the start of exon2 (shown); for the m141 mu-
tants with insertions 241 (shown) and 972 bp downstream of
the ATG; and for the transposon insertion mutants of m139
and m140 analyzed in this study. Compared with wild-type
MCMV, the transposon mutants of genes M36 and m139 were
severely impaired for growth on IC-21 macrophages, whereas
the transposon mutants of genes m140 and m141 were less
compromised with respect to growth on this macrophage cell
line. However, in repeated experiments, the growth differences
between the M36, m139, m140, and m141 mutants were less
pronounced, and the attenuation of these mutants was in the
range of two to three log units compared to wild-type MCMV.
Since we only found one transposon mutant for m139 in the
library, we constructed a deletion mutant for m139 (�m139-
MCMV) lacking almost the complete m139 ORF except for
the last 200 bp at the C terminus. This mutant also showed an
attenuation of three log units for growth on IC-21 macro-
phages (data not shown).
Interaction among the gene products of m139, m140, and
m141 explains the common phenotype. The m139, m140, and
m141 genes belong to a complex transcriptional unit and have
3�-coterminal transcripts (Fig. 4A) (18). It is less likely that a
transposon insertion in the m141 ORF would affect the tran-
scription of the downstream m139 and m140 genes because
they have independent transcription start sites downstream of
ORF m141. Therefore, we concluded that the two independent
transposon insertion mutants for m141 probably define the
phenotype of m141 gene inactivation, which confirms similar
conclusions drawn before (19). More likely, the transposon
insertion in the m140 gene could also affect the expression of
the m141 gene and thereby cause the observed phenotype in
macrophages. A comparable m140 mutant phenotype has been
described previously with a targeted deletion mutant (19), and
this deletion reduces the stability of the m141 protein. Finally,
the transposon insertion in the m139 ORF could affect the
transcription rate or the transcript stability of the upstream
m140 and m141 genes.
To determine whether the transposon insertion in m139 had
an effect on expression of the neighboring genes, a targeted
mutation of the m139 ORF (�ATG-m139) was constructed by
site-directed mutagenesis of the wild-type MCMV BAC plas-
mid (see Fig. 4A). Since an alternative ATG in frame is located
86 bp downstream of the native ATG, we decided to delete the
first 86 bp of m139, thereby deleting both ATGs. After excision
of the kanamycin resistance marker by FLP recombination, 86
bp of noncoding sequence was left behind in the BAC, corre-
sponding exactly to the length of the deleted viral sequence. The
first two start ATGs of m139 were thus deleted, but the lengths
of the m139 to m141 mRNA transcripts were not altered.
We first analyzed transcription of the neighboring m140 and
FIG. 2. Targeted mutations of m142 and m143 genes. (A) Tran-
script map of genes m139 to m143. Black and grey arrows denote the
major transcripts of genes m142 and m143 and genes m139 to m141,
respectively, according to Hanson et al. (18). (B) transposon insertion
into the wild-type (wt) MCMV BAC pSM3fr generated the mutant
genomes Tn-m142.A, Tn-m142.B, and Tn-m143. (C) Deletion of the
entire m143 ORF and (D) deletions of the first ATG of gene m142
were achieved by homologous recombination (HR) between pSM3fr
and a linear PCR fragment containing a kanamycin resistance gene
(Kn). By flanking the kanamycin cassette with minimal FRT sites
(black boxes), the kanamycin cassette was excised by FLP recombinase
(FLP). (E) Complementation of the �ATG-m142 genome by insertion
of the m142 gene at an ectopic position. The genome of �ATG-m142/
FRT is identical to that of �ATG-m142 but carries a 48-bp FRT site
in gene m152. This FRT site was used for insertion by FLP recombi-
nase (FLP) of the m142 gene under the control of its native promoter
(P). Zeocin (Zeo) was used for selection. The resulting genome,
�ATG-m142/m142E, gave rise to virus progeny.
5562 ME´NARD ET AL. J. VIROL.

m141 genes in this mutant by Northern blot analysis. The
transcripts characteristic of this transcription unit seen in the
wild-type virus, as already shown by Hanson et al. (18), were
also present in cells infected with the �ATG-m139 mutant
(Fig. 4B). Nevertheless, �ATG-m139 MCMV showed the
same growth defect in IC-21 macrophages as the Tn-m139
mutant (Fig. 4C). These results show for the first time that
inactivation of the m139 gene of the US22 gene family also
affects MCMV growth on IC-21 macrophages.
Next, we tested whether the proteins encoded by m139,
m140, and m141 interact posttranslationally at the protein
level. The transcripts and proteins are expressed at early times
within the MCMV replication cycle (18, 42). Western blot
analysis of the �ATG-m139, �m139, Tn-m139, Tn-m140, Tn-
m141.A, and Tn-m141.B MCMV mutants was performed with
m139-, m140-, and m141-specific polyclonal antisera (Fig. 5A
and 5B). Detection of the 34-kDa m04/gp34 gene product
served as a control for comparable viral protein loading. Due
to different biochemical properties, the protein samples were
prepared in different buffers and applied on different gel sys-
tems. In wild-type MCMV-infected cells, two proteins of 72
and 61 kDa were detected with the m139-specific antiserum
(Fig. 5A), consistent with previous data (19). In cells infected
with the �m139 and the Tn-m139 MCMVs, no m139-specific
signal was seen, whereas with the �ATG-m139 MCMV, a
smaller protein of 61 kDa was still present, suggesting that this
protein is encoded by the m139 transcript, probably originating
at the third alternative start ATG of the m139 ORF at nucle-
otide position 195767. Remarkably, none or very little of the
two proteins was detectable in the Tn-m140, Tn-m141.A, and
Tn-m141.B MCMV mutants (Fig. 5A). Thus, the m140- and
m141-encoded proteins either directly or indirectly affected the
stability of the m139 proteins.
The peptide antisera to the m140 and m141 products de-
FIG. 3. Growth of US22 gene family mutants in NIH 3T3 fibroblasts and IC-21 macrophages. NIH 3T3 fibroblasts (A and B) and IC-21
macrophages (C and D) were infected in triplicate at an MOI of 0.1 with wild-type (wt) or mutant MCMV. At day 6 postinfection, supernatant
was harvested from individual wells, and infectious virus was quantified on MEF cells by plaque assay (A and C) or on NIH 3T3 fibroblasts by the
TCID50 endpoint titration method (B and D). Error bars indicate standard deviations between triplicates.
TABLE 2. In vitro growth differences between wild-type MCMV
and the US22 gene mutants at day 6 postinfection in
fibroblasts, macrophages, and endothelial cells
MCMV
mutant
In vitro growtha
NIH 3T3
fibroblasts
IC-21
macrophages
J774-A1
macrophages PEC SVEC4-10
Tn-M36 � ��� ��/��� �� �
Tn-M43 ��b ��� � � �
Tn-m139 � ��� � � �
Tn-m140 �/� ��/��� � � �/�
Tn-m141 �/� ��/��� � � �
a
�, no difference; �, ��, and ���, 1, 2, and 3 log units of difference,
respectively; �/�, �1 log unit of difference.
b The titers were at least 2 log units lower when titrated on NIH 3T3 cells.
VOL. 77, 2003 MUTATIONAL ANALYSIS OF MCMV US22 GENES 5563

tected single viral proteins with the expected sizes of 56 kDa
and 52 kDa, respectively, in wild-type MCMV-infected fibro-
blasts (Fig. 5A), consistent with previous data (19). Mutation
of m139, m140, or m141 strongly reduced or abolished the
presence of the m140 and m141 proteins.
Immunoprecipitation from wild-type MCMV-infected cells
with the peptide antiserum to m141 showed a 72- and a 61-kDa
protein coprecipitating with the 52-kDa m141 product (Fig.
5B). The 72-kDa protein most probably represents the large
m139 protein, since this protein was absent in cells infected
with �ATG-m139. The 61-kDa protein most probably repre-
sents the truncated m139 product. In cells infected with the
�ATG-m139 mutant, the m141 product was still detectable,
but not the 61-kDa protein of m139. Perhaps the 61-kDa pro-
tein requires the presence of the large m139 product in order
to be coprecipitated by the m141 peptide antiserum. However,
in cells infected with the Tn-m139 mutant, which does not
express either of these two m139 proteins, only a trace of the
m141 product was detected. This indicates that the presence of
the 61-kDa protein of m139 expressed by the �ATG-m139
mutant was required for detectable levels of the m141 product.
In cells infected with Tn-m140 MCMV and, as expected, in
cells infected with Tn-m141 MCMV, the 52-kDa m141 protein
was missing. The m141 peptide antiserum cross-reacted with
another unknown protein of about 59 kDa in infected cells
(Fig. 5B).
Altogether, these results indicated that the products of
genes m139, m140, and m141 interact at the protein level and
that lack of any of these genes affects the steady-state level of
the other proteins. These data are consistent with previous
reports that at least the m140 and m141 proteins form a stable
complex within infected cells (Z. Karabekian, L. K. Hanson,
J. S. Slater, and A. E. Campbell, Abstr. 25th International Her-
FIG. 4. Construction, analysis, and growth of mutant �ATG-m139.
(A) Transcript map of genes m139 to m143 and construction of the
ATG deletion mutant of m139. Black and grey arrows denote the
major transcripts of genes m139 to m141 and genes m142 and m143,
respectively, according to Hanson et al. (18). Deletion of the first two
ATGs of m139 was achieved by site-directed mutagenesis with a linear
PCR fragment containing a kanamycin resistance gene (Kn) flanked by
FRT sites (black boxes) and by 50 nucleotides homologous to the up-
and downstream sequences. After insertion of the kanamycin resis-
tance gene by homologous recombination (HR), the kanamycin cas-
sette was excised by FLP recombinase, generating the �ATG-m139
mutant genome. (B) Northern blot analysis of the m139 to m141
transcripts. NIH 3T3 cells were infected at 5 PFU/cell with wild-type
(wt) or �ATG-m139 MCMV, and mRNA was isolated as described in
Materials and Methods. The probe used for detection of these tran-
scripts binds to the m140 ORF. (C) Growth of the corresponding
�ATG-m139 MCMV mutant in NIH 3T3 fibroblasts and IC-21 mac-
rophages. Monolayers of NIH 3T3 fibroblasts and IC-21 macrophages
were infected at an MOI of 0.1. The titers of cell-free viruses were
determined on day 6 postinfection.
FIG. 5. Interaction of m139, m140, and m141 proteins. (A) West-
ern blot analysis. NIH 3T3 cells were infected at an MOI of 1 with
wild-type MCMV (wt) or MCMV mutants. Cell lysates were harvested
24 h postinfection and separated by polyacrylamide gel electrophore-
sis. Western blot analysis with rabbit polyclonal antisera against m139,
m140, and m141 was performed. An anti-m04 antiserum which recog-
nizes the gp34 gene product of m04 was used to control viral protein
expression. (B) Coimmunoprecipitation analysis. NIH 3T3 cells were
infected at an MOI of 1 with wild-type or mutant MCMVs. After 24 h,
cell lysates were subjected to immunoprecipitation with an anti-m141
antiserum. The anti-m141 rabbit polyclonal antiserum precipitated the
52-kDa gene product of m141 and the 72- and 61-kDa proteins of
m139 in wild-type MCMV.
5564 ME´NARD ET AL. J. VIROL.

pesvirus Workshop, abstr. 9.26, 2000; A. E. Campbell, Z. Kara-
bekian, L. K. Hanson, and J. S. Slater, Abstr. 8th Interna-
tional Cytomegalovirus Conference, p. 26, 2001). Therefore,
we concluded that the common host range phenotype detected
by deletion of each of these three genes reflects their interac-
tion at the protein level.
M36 mutants have a general macrophage phenotype. M36,
M43, m139, m140, and m141 mutants were further analyzed
for viral replication on J774-A1 macrophages and on PEC in
order to discriminate between cell type- and cell line-specific
effects (Fig. 6). On J774-A1 macrophages, which are semiper-
missive for MCMV replication (26), growth of only the Tn-
M36 MCMV mutants with transposon insertions 98 bp down-
stream of the ATG (shown) and 363 bp downstream of the
start of exon 2, was strongly reduced by three log units com-
pared to Tn-m152 MCMV (Fig. 6A). M43, m139, m140, and
m141 mutants showed no growth deficits. On PEC, the growth
of the Tn-M36 MCMV mutants (transposon insertion 363 bp
downstream of the start of exon 2 is shown) was reduced by
two log units (Fig. 6B). The Tn-m139, Tn-m140, and Tn-m141
MCMV mutants were not growth deficient in PEC. These data
are in contrast to a previous report that mutant MCMV de-
leted of m139, m140, and m141 is defective for replication in
lipopolysaccharide-activated PEC (20). The growth of the Tn-
M43 MCMV mutants in PEC (transposon insertion 4 bp down-
stream of the ATG is shown) was reduced by only one log unit
(Fig. 6B). Therefore, we concluded that only the mutation of
the M36 gene caused a robust and constant macrophage phe-
notype independently of whether primary macrophages, either
stimulated or activated, or established cell lines were used.
MCMV mutants Tn-M36, Tn-M43, Tn-m139, Tn-m140, and
Tn-m141 were further analyzed for viral replication on the
SVEC4-10 endothelial cell line (Fig. 6C). All mutants showed
no significant reduction in growth compared to Tn-m152
MCMV. This indicates that the function of genes m139, m140,
and m141 may be influenced by the state of macrophage dif-
ferentiation, while gene M36 has a more fundamental role in
MCMV replication in macrophage cells but not in endothelial
cells. The collective data for the host cell range effects of the
M36, M43, m139, m140, and m141 mutants are shown in Ta-
ble 2.
M36-encoded protein has antiapoptotic function. To prove
the phenotype-gene connection of Tn-M36 mutants, we gen-
erated a targeted deletion of the M36 ORF in which the M36
ORF was replaced by a kanamycin resistance gene (see Fig. 7A
and Materials and Methods). First, we examined whether de-
letion of the M36 ORF interfered with transcription of the
neighboring M35 and M37 genes. Northern blot analysis were
conducted with two probes specific for the first part of the M35
ORF and for the first two thirds of the M37 ORF. Transcripts
for the M35 and M37 ORFs were detected in NIH 3T3 cells
infected with wild-type MCMV or the �M36 MCMV mutant
(Fig. 7B). Identical transcripts for M35 and M37 could be
detected in wild-type and �M36 MCMV-infected cells, con-
firming that deletion of M36 did not affect transcription of
the neighboring genes. To detect the M36 gene product, an
MCMV virus containing a C-terminally HA-tagged M36 ORF
was constructed. By Western blot analysis of lysates from in-
fected NIH 3T3 cells, the anti-HA antibody detected a protein
of about 50 kDa in cells infected with the M36-HA virus but
not in cells infected with wild-type MCMV (see Fig. 7C).
After infection of cells with �M36-MCMV, small cell frag-
ments reminiscent of apoptotic bodies could be observed (data
not shown). Therefore, the antiapoptotic function of M36 was
examined by studying the viability of wild-type and �M36
MCMV-infected NIH 3T3 fibroblasts and IC-21 macrophages
and of mock-infected cells after proapoptotic anti-Fas treat-
ment (Fig. 7D). In NIH 3T3 fibroblasts, there was a 4.5-fold
difference in the number of surviving cells after infection with
wild-type and �M36 MCMV, whereas the difference was 65-
fold in IC-21 macrophages. In fact, wild-type MCMV infection
in comparison to mock infection protected IC-21 macrophages
against the proapoptotic challenge (Fig. 7D). The m139, m140,
and m141 mutants were also tested for an antiapoptotic phe-
FIG. 6. Growth of M36, M43, m139, m140, and m141 mutants on
the J774-A1 macrophage cell line, on peritoneal exudate cells, and on
the SVEC4-10 endothelial cell line. J774-A1 macrophage cells (A) and
peritoneal exudate cells (B) were infected at an MOI of 5 and the
SVEC4-10 endothelial cells (C) were infected at an MOI of 1 with
either wild-type or mutant MCMV. Six days postinfection, virus titers
in the supernatant were determined on NIH 3T3 fibroblasts. Error bars
indicate standard deviations between triplicates.
VOL. 77, 2003 MUTATIONAL ANALYSIS OF MCMV US22 GENES 5565

notype, but no loss of protection against Fas-mediated apopto-
sis could be detected (Fig. 7E). After infection with some
mutants, there were more surviving cells than after infection
with wild-type MCMV. This may be due to the fact that the
m139, m140, and m141 mutants grow poorly on macrophages
and therefore had less of a lytic effect than wild-type MCMV.
To further analyze at which step of the receptor-mediated
apoptosis pathway the M36 protein acts, we examined a num-
ber of steps of the apoptotic cascade (Fig. 8). As expected, the
proteolytic processing and activation of the executioner cas-
pase-3 and the cleavage of PARP was inhibited in NIH 3T3
cells infected with the wild-type control but not in fibroblasts
infected with �M36 MCMV and not in mock-infected controls
(Fig. 8A). The total amount of cleaved and uncleaved PARP in
wild-type virus-infected cells appeared smaller, probably due
to the effect of 64 h of virus infection. However, unlike in
wild-type-infected cells, no uncleaved PARP was detectable in
�M36 MCMV-infected fibroblasts after induction of apoptosis
with anti-Fas. Next, we determined the caspase-8 activity in
IC-21 macrophages without proapoptotic stimulation. After
infection with �M36 MCMV, the caspase-8 protease activity
was 160 times higher than that after infection with wild-type
MCMV, and there was a 90-fold difference between �M36
MCMV-infected cells and uninfected cells (Fig. 8B). These
FIG. 7. �M36 mutant and antiapoptotic activity of the M36 protein. (A) Construction of the �M36 mutant genome. Deletion of the M36 gene
was achieved by homologous recombination (HR) in E. coli with a linear PCR fragment containing a kanamycin resistance gene (Kn). (B) Northern
blot analysis of M35 and M37 transcripts. NIH 3T3 cells were infected with 5 PFU of wild-type (wt) or �M36 MCMV per cell, and total RNA was
isolated 24 h postinfection. Probes used for each of the neighboring genes are indicated under the blot. An m04 probe was used to control loading
of equal amounts of viral RNA. (C) Expression of the M36-HA protein in NIH 3T3 fibroblasts infected with wild-type or M36-HA MCMV. NIH
3T3 cells were infected with 3 PFU/cell. Cell lysates were harvested 24 h postinfection, and equal volumes of lysates were separated on a 12.5%
acrylamide gel for Western blot analysis with an anti-HA monoclonal antibody. (D and E) �M36 and Tn-M36 MCMV-infected cells are susceptible
to anti-Fas-induced apoptosis. Cells were mock infected or infected with 1 or 3 PFU of virus per cell for NIH 3T3 (D and E) and IC-21 (D) cells,
respectively. At 48 h postinfection, cells were exposed for 16 h to 0.5 �g of anti-Fas plus 12.5 �g of cycloheximide per ml. Surviving cells were
counted with the trypan blue dye exclusion assay.
5566 ME´NARD ET AL. J. VIROL.

results confirmed that the M36 protein acts in the apoptosis
pathway at or above the caspase-8 level.
Recently, Skaletskaya et al. showed that the isolated gene
UL36 of HCMV has antiapoptotic function and binds to pro-
caspase-8 (39). To test if the M36 protein also binds to pro-
caspase-8, we cloned the M36 gene and an HA-tagged version
of the M36 gene (M36-HA) into the pCR3 expression vector.
These constructs and the empty vector were transfected into
293 cells, and cell lysates were subjected to immunoprecipita-
tion with either anti-caspase-8 or anti-HA antibodies. The im-
munoprecipitates were then analyzed by Western blot (Fig.
8C). The Western blot probed with anti-caspase-8 antibodies
showed procaspase-8 in all lysates immunoprecipitated with
anti-caspase-8. A faint specific signal for coimmunoprecipita-
tion with the M36-HA protein was seen only in cells trans-
fected with the pCR3-M36-HA construct, indicating that pro-
caspase-8 can be coprecipitated with the M36-HA protein. In
the Western blot probed with the anti-HA antibody, the
M36-HA protein could be detected only in cells transfected
with the pCR3-M36-HA vector after immunoprecipitation
with anti-HA, as expected. Interestingly, the M36 protein sig-
nal was also present in the lysates of pCR3-M36-HA-trans-
fected cells when first immunoprecipitated with the anti-
caspase-8 antibody. Therefore, we concluded that M36 binds
to the endogenous procaspase-8 and that the M36 antiapopto-
tic function determines the macrophage cell phenotype of the
�M36 MCMV mutant.
DISCUSSION
The data presented here are the first comprehensive muta-
tional approach to the entire US22 gene family of MCMV
based on a BAC-cloned viral genome. Random transposon
insertional mutagenesis was combined with targeted mutations
in the search for macrophage cell-specific growth differences.
We report on five genes with cell growth differences (M36,
M43, m139, m140, and m141) and two essential genes (m142
and m143). In contrast, 5 of the 12 genes conferred no detect-
able phenotype. Thus, members of this gene family are widely
divergent in function and degree of relevance to viral replica-
tion.
Random versus targeted mutagenesis of BAC-cloned her-
FIG. 8. Effect of M36 on caspase activation. (A) Caspase-3 and PARP activation in �M36 MCMV- but not in wild-type MCMV-infected cells.
NIH 3T3 (upper panel for caspase-3 and both panels for PARP) or IC-21 cells (*, lower panel for caspase-3) were mock infected or infected with
1 PFU of virus per cell. At 48 h postinfection, cells were treated for 16 h with 0.5 �g of anti-Fas plus 12.5 �g of cycloheximide per ml or left
untreated. Cell lysates were prepared, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto a nitrocellulose
membrane. The uncleaved and cleaved forms of PARP and the activated form of caspase-3 were detected by immunoblotting. (B) Caspase-8
activity in �M36- and wild-type MCMV-infected macrophages. IC-21 macrophages were mock infected or infected with 7 PFU/cell. At 36 h post-
infection, cell lysates were prepared, and the substrate of caspase-8 was added. The pNA chromophore was quantified after cleavage by activated cas-
pase-8. (C) The M36-HA protein can be coprecipitated with the endogenous procaspase-8. 293 cells were mock transfected or transfected with the empty
pCR3 vector or the pCR3 vector expressing the M36 and the M36-HA proteins. At 48 h posttransfection, cells were lysed, and protein lysates were
immunoprecipitated with either anti-procaspase-8 (C8) or anti-HA (HA) antibody. Immunoprecipitates were then used for Western blot analysis
with either anti-caspase-8 or anti-HA antibody. The procaspase-8 was slightly recognized in lysates which were immunoprecipitated with the anti-
HA antibody, and the M36-HA protein was strongly recognized in lysates which were immunoprecipitated with the anti-procaspase-8 antibody.
VOL. 77, 2003 MUTATIONAL ANALYSIS OF MCMV US22 GENES 5567

pesvirus genomes. When we started this systematic analysis, we
could only generate mutants of numerous genes by either shut-
tle mutagenesis or transposon insertion mutagenesis. Shuttle
mutagenesis results in the desired mutant, but construction of
the appropriate shuttle plasmid with its long regions of homol-
ogy often requires numerous cloning steps (47). Transposon
insertion is technically easier but requires a collection of at
least 2,000 mutants to obtain a library that covers all CMV
genes, since the distribution of insertion sites is not completely
random (17). Even with a library of 2,600 clones, it was not
possible to isolate at least two independent mutants for each of
the genes under study. We found that in more than 95% of the
mutant genomes, the transposon insertion was not associated
with deletions in the genome. When deletions were found, they
occurred at the insertion site and probably resulted from mul-
tiple transposon insertions followed by elimination of interven-
ing sequences by the transposon-specific resolvase (9). High
antibiotic concentrations during mutant selection favor selec-
tion of multiple transposon insertions and increase the number
of deletions. Analysis of transposon mutant DNA by restriction
enzyme digestion, by direct sequencing, and by Southern blot-
ting excluded these unwanted side effects for the mutants dis-
cussed here.
Whether transposon insertion mutagenesis is a method of
choice in the future depends on the number of genes to be
studied. In parallel to the work on the US22 gene mutants, we
adapted PCR-based targeted mutagenesis procedures to BAC-
cloned herpesvirus genomes (45). Presently, the targeted mu-
tagenesis of a dozen genes probably takes no more effort than
the generation of a library. Libraries, however, will keep their
value for questions of forward genetics (47).
M43 gene. The two insertion mutants independently isolated
for gene M43 showed a growth deficit that was not uniformly
seen on all cells, but the growth phenotype was not restricted
to macrophages. The group of Liu has described transposon
mutagenesis of genomic subfragments of MCMV (49). To ob-
tain mutant virus, each plasmid carrying a transposon needs to
be reinserted into the MCMV genome by homologous recom-
bination in cells. This led to the identification of a mutant with
an insertion in codon 313 of the M43 ORF which had no
phenotype in NIH 3T3 fibroblasts (48), possibly reflecting the
expression of a partial but functional product. The two mutants
studied by us had insertions in codons 2 and 26. Also, in our
hands the fibroblast phenotype was different between NIH 3T3
and MEF cells. Thus, the M43 phenotype is complex and
cannot be explained by a selective effect on macrophages.
Essentiality of genes m142 and m143. Transposon insertions
leading to lethal phenotypes in fibroblasts were controlled by
targeted mutations. Here we identified the US22 gene family
members m142 and m143 as being essential for virus replica-
tion. The essential nature of these genes is consistent with a
previous report that mutant MCMV with a deletion spanning
genes m137 through m143 (RV9) could not be purified from
wild-type virus, while mutants deleted of m137 through m141
were independently replication competent (12). To date, no
one has successfully constructed viruses with mutations in
these two genes. Clearly, no conclusion with regard to the
essentiality of individual genes can be made from this type of
negative recombination experiment in cells. However, the data
suggested that either m142 or m143 as individual genes, as a
tandem, or in combination with other genes deleted in RV9
might be essential for growth.
Transcript mapping of the m142 to m144 region revealed
that the transcripts derived from this region use a common
polyadenylation signal downstream of m142 (18). Our Tn-
m142 and Tn-m143 mutant genomes failed to produce prog-
eny, as did a deletion mutant of the complete m143 ORF.
Transcription of m142 is most probably not affected by trans-
poson insertion into m143, since m142 and m143 have inde-
pendent transcripts and the transposon insertion in the m143
mutant was located 1,493 bp upstream of the m142 start ATG.
Although this suggested that m143 is essential for MCMV
replication, the transposon insertion within m142 could have
resulted in destabilization of the m143 transcript. However, a
start ATG mutant of the m142 ORF, which should not affect
the m143 transcript, also failed to produce progeny. Further-
more, the m142 revertant, which contained the m142 gene in
an ectopic position, rescued virus growth. Therefore, we con-
clude that both m142 and m143 are essential for virus replica-
tion and form a new subfamily of essential US22 family genes
expressed at immediate-early times after infection. It will be of
interest to study their functions and to see whether this pre-
diction can be confirmed by studying the homologous genes of
HCMV.
Genes m139 to m141. A recent publication demonstrated an
interaction between m140 and m141 at the protein level, since
a reduction in the m141 protein was observed after m140
deletion (19). The m141 (and m140) deletion also resulted in
reduced levels of the m139 proteins within the nuclear fraction
of IC-21 macrophages, whereas deletion of m139 did not lead
to a detectable difference in the steady-state levels of the other
two proteins (19). Thus, there was an effect of m140 and m141
on m139 but not vice versa. With regard to replication in
macrophages, the deletion of either m140 or m141 caused the
same phenotype, whereas deletion of m139 had no effect.
Our studies confirm the relevant aspects of that paper,
namely, the cooperativity of the gene products. We also con-
clude that the macrophage phenotype is defined at the level of
protein stability. However, there are differences. Different
from the previous report, we found a comparable macrophage
growth phenotype regardless of which of the three genes was
inactivated. When we analyzed the interaction of the protein
products, we found, in accordance with the biological proper-
ties, that mutagenesis of one of the genes affected the stability
of the other two gene products. When we deleted the first two
ATGs in the m139 ORF, we observed that the mutants still
produced a smaller 61-kDa m139 product. Our immunopre-
cipitation studies revealed that the m141 protein was synthe-
sized in �ATG-m139-infected cells (Fig. 5B); nevertheless, the
expression of the 61-kDa product did not suffice to stabilize the
steady-state expression of m140 and m141 (Fig. 5A). Alto-
gether, these data suggest that m139, m140, and m141 interact
at the protein level in the Smith strain genome that we used
and that this is the cause of the common phenotype. The
differences are not explained by technical differences because
the phenotype of the m139 mutant was confirmed in two lab-
oratories.
The most likely explanation is that the Smith substrains kept
in the two laboratories differ in this genomic region. Fresh
HCMV isolates differ in biological properties and gene content
5568 ME´NARD ET AL. J. VIROL.

and individual gene sequences from laboratory strains as a
result of continuous cultivation in cell lines in vitro without the
physiological selection pressure found in vivo. Resequencing of
the MCMV Smith substrains in that region should reveal the
differences. Whether interdependence or independence of the
m139, m140, and m141 genes represents the wild-type situation
will only be solved by BAC cloning and mutagenesis of a new
wild-type isolate of MCMV. We are in the process of BAC
cloning new virus isolates from mice. An alternative explana-
tion for the differences is that BAC cloning requires fewer
passages of progeny virus in cell culture than does the former
method of cloning. It is possible that the repeated passages
required to purify recombinant progeny from wild-type paren-
tal virus may select, by way of a growth advantage, viruses with
a compensatory mutation elsewhere in the genome.
M36, antiapoptotic gene required for growth on macro-
phages. In a previous study, we identified the transposon in-
sertion into gene M45 with a forward genetic approach (8).
The disruption of M45 prevented MCMV replication in endo-
thelial cells, and this host cell range restriction could be ex-
plained by an antiapoptotic function of M45 that is particularly
relevant in infected endothelial cells. Therefore, we checked
for all host range mutants whether the growth reduction could
be due to an antiapoptotic function. Among the US22 gene
family mutants, only the deletion of M36 resulted in apoptosis
of infected macrophages. In addition, it enhanced apoptosis
following proapoptotic stimulation of infected fibroblasts (Fig.
7D). This is consistent with a lack of the Tn-M36 MCMV
growth defect in fibroblasts without extrinsic proapoptotic
stimulation (Fig. 3A).
While we were studying the antiapoptotic activity of M36 in
more detail, Goldmacher and colleagues reported the con-
structing and screening of an HCMV genomic DNA library for
genes with antiapoptotic function (39). They found that the
isolated UL36 gene product formed a complex with pro-
caspase-8. Our data allowed analysis of M36 function in the
viral context and showed that the MCMV M36 protein also
forms a complex with procaspase-8. Our data contribute the
observation that murine macrophages need the function of
M36 even in absence of proapoptotic stimuli for highly pro-
ductive MCMV infection. Further studies in vitro and in vivo
will show whether this phenotype extends to other cell types
that MCMV can infect.
Altogether, we could segregate insertion mutations in all
members of the US22 gene family of MCMV with regard to
essentiality and growth properties in fibroblasts and macro-
phages. We were able to associate the macrophage growth
defect of the M36 mutants with the antiapoptotic function of
this gene product, whereas the function of the m139 to m141
genes was difficult to resolve due to the interaction at the
protein level. Although US22 family genes share sequence
motifs, perhaps indicative of a common origin, evolution and
gene duplications created new functions that apparently dom-
inate the unknown original function, delineated by the motif
signature. Whether the definition of related ORFs as protein
families is still valid remains a matter of debate (32). There-
fore, we expect that the comparison between positional ho-
mologs of US22 family genes in HCMV and MCMV will reveal
more common features than the comparison of gene families
in general. This assumption has held true for the M36 and
UL36 genes.
ACKNOWLEDGMENTS
We thank L. K. Hanson for technical controls. We thank L. Cicin-
Sain for providing primary cells and S. Mathys for the pSM3fr/GFP
BAC. We also thank A. Bubeck for comments on the manuscript.
This work was supported by grants from the Boehringer Ingelheim
Fonds (C.M.), the Deutsche Forschungsgemeinschaft through DFG
SFB 455 (U.H.K.) and SFB 479 (W.B.), and PHS grant CA41451
(A.E.C.).
REFERENCES
1. Adair, R., E. R. Douglas, J. B. Maclean, S. Y. Graham, J. D. Aitken, F. E.
Jamieson, and D. J. Dargan. 2002. The products of human cytomegalovirus
genes UL23, UL24, UL43 and US22 are tegument components. J. Gen.
Virol. 83:1315–1324.
2. Adler, H., M. Messerle, M. Wagner, and U. H. Koszinowski. 2000. Cloning
and mutagenesis of the murine gammaherpesvirus 68 genome as an infec-
tious bacterial artificial chromosome. J. Virol. 74:6964–6974.
3. Atalay, R., A. Zimmermann, M. Wagner, E. Borst, C. Benz, M. Messerle, and
H. Hengel. 2002. Identification and expression of human cytomegalovirus
transcription units coding for two distinct Fc receptor homologs. J. Virol.
76:8596–8608.
4. Blacklaws, B. A., P. Bird, D. Allen, D. J. Roy, I. C. MacLennan, J. Hopkins,
D. R. Sargan, and I. McConnell. 1995. Initial lentivirus-host interactions
within lymph nodes: a study of maedi-visna virus infection in sheep. J. Virol.
69:1400–1407.
5. Borst, E. M., G. Hahn, U. H. Koszinowski, and M. Messerle. 1999. Cloning
of the human cytomegalovirus (HCMV) genome as an infectious bacterial
artificial chromosome in Escherichia coli: a new approach for construction of
HCMV mutants. J. Virol. 73:8320–8329.
6. Borst, E. M., S. Mathys, M. Wagner, W. Muranyi, and M. Messerle. 2001.
Genetic evidence of an essential role for cytomegalovirus small capsid pro-
tein in viral growth. J. Virol. 75:1450–1458.
7. Brune, W. 2002. Random transposon mutagenesis of large DNA molecules
in Escherichia coli. Methods Mol. Biol. 182:165–171.
8. Brune, W., C. Menard, J. Heesemann, and U. H. Koszinowski. 2001. A
ribonucleotide reductase homolog of cytomegalovirus and endothelial cell
tropism. Science 291:303–305.
9. Brune, W., C. Menard, U. Hobom, S. Odenbreit, M. Messerle, and U. H.
Koszinowski. 1999. Rapid identification of essential and nonessential her-
pesvirus genes by direct transposon mutagenesis. Nat. Biotechnol. 17:360–
364.
10. Bubeck, A., U. Reusch, M. Wagner, T. Ruppert, W. Muranyi, P. M. Kloetzel,
and U. H. Koszinowski. 2002. The glycoprotein gp48 of murine cytomega-
lovirus L proteasome-dependent cytosolic dislocation and degradation.
J. Biol. Chem. 277:2216–2224.
11. Cardin, R. D., G. B. Abenes, C. A. Stoddart, and E. S. Mocarski. 1995.
Murine cytomegalovirus IE2, an activator of gene expression, is dispensable
for growth and latency in mice. Virology 209:236–241.
12. Cavanaugh, V. J., R. M. Stenberg, T. L. Staley, H. W. Virgin, M. R. Mac-
Donald, S. Paetzold, H. E. Farrell, W. D. Rawlinson, and A. E. Campbell.
1996. Murine cytomegalovirus with a deletion of genes spanning HindIII-J
and -I displays altered cell and tissue tropism. J. Virol. 70:1365–1374.
13. Chee, M. S., A. T. Bankier, S. Beck, R. Bohni, C. M. Brown, R. Cerny, T.
Horsnell, C. A. Hutchison, T. Kouzarides, J. A. Martignetti, et al. 1990.
Analysis of the protein-coding content of the sequence of human cytomeg-
alovirus strain AD169. Curr. Top. Microbiol. Immunol. 154:125–169.
14. Colberg, P. A. 1996. Functional roles of immediate early proteins encoded by
the human cytomegalovirus UL36–38, UL115–119, TRS1/IRS1 and US3
loci. Intervirology 39:350–360.
15. Colberg, P. A., L. D. Santomenna, P. P. Harlow, P. A. Benfield, and D. J.
Tenney. 1992. Human cytomegalovirus US3 and UL36–38 immediate-early
proteins regulate gene expression. J. Virol. 66:95–105.
16. del Val, M., H. Hengel, H. Hacker, U. Hartlaub, T. Ruppert, P. Lucin, and
U. H. Koszinowski. 1992. Cytomegalovirus prevents antigen presentation by
blocking the transport of peptide-loaded major histocompatibility complex
class I molecules into the medial-Golgi compartment. J. Exp. Med. 176:729–
738.
17. Gutermann, A., A. Bubeck, M. Wagner, U. Reusch, C. Menard, and U.
Koszinowski. 2002. Strategies for the identification and analysis of viral
immune evasion genes—cytomegalovirus as example. Curr. Top. Microbiol.
Immunol. 269:1–22.
18. Hanson, L. K., B. L. Dalton, Z. Karabekian, H. E. Farrell, W. D. Rawlinson,
R. M. Stenberg, and A. E. Campbell. 1999. Transcriptional analysis of the
murine cytomegalovirus HindIII-I region: identification of a novel immedi-
ate-early gene region. Virology 260:156–164.
19. Hanson, L. K., J. S. Slater, Z. Karabekian, G. Ciocco-Schmitt, and A. E.
VOL. 77, 2003 MUTATIONAL ANALYSIS OF MCMV US22 GENES 5569

Campbell. 2001. Products of US22 genes M140 and M141 confer efficient
replication of murine cytomegalovirus in macrophages and spleen. J. Virol.
75:6292–6302.
20. Hanson, L. K., J. S. Slater, Z. Karabekian, H. W. Virgin, C. A. Biron, M. C.
Ruzek, N. van Rooijen, R. P. Ciavarra, R. M. Stenberg, and A. E. Campbell.
1999. Replication of murine cytomegalovirus in differentiated macrophages
as a determinant of viral pathogenesis. J. Virol. 73:5970–5980.
21. Hobom, U., W. Brune, M. Messerle, G. Hahn, and U. H. Koszinowski. 2000.
Fast screening procedures for random transposon libraries of cloned her-
pesvirus genomes: mutational analysis of human cytomegalovirus envelope
glycoprotein genes. J. Virol. 74:7720–7729.
22. Iskenderian, A. C., L. Huang, A. Reilly, R. M. Stenberg, and D. G. Anders.
1996. Four of eleven loci required for transient complementation of human
cytomegalovirus DNA replication cooperate to activate expression of repli-
cation genes. J. Virol. 70:383–392.
23. Kavanagh, D. G., M. C. Gold, M. Wagner, U. H. Koszinowski, and A. B. Hill.
2001. The multiple immune-evasion genes of murine cytomegalovirus are
not redundant: m4 and m152 inhibit antigen presentation in a complemen-
tary and cooperative fashion. J. Exp. Med. 194:967–978.
24. Kouzarides, T., A. T. Bankier, S. C. Satchwell, E. Preddy, and B. G. Barrell.
1988. An immediate early gene of human cytomegalovirus encodes a poten-
tial membrane glycoprotein. Virology 165:151–164.
25. Krmpotic, A., M. Messerle, M. I. Crnkovic, B. Polic, S. Jonjic, and U. H.
Koszinowski. 1999. The immunoevasive function encoded by the mouse
cytomegalovirus gene m152 protects the virus against T cell control in vivo.
J. Exp. Med. 190:1285–1296.
26. Lewis, M. A., J. S. Slater, L. I. Leverone, and A. E. Campbell. 1990. En-
hancement of interleukin-1 activity by murine cytomegalovirus infection of a
macrophage cell line. Virology 178:452–460.
27. Messerle, M., I. Crnkovic, W. Hammerschmidt, H. Ziegler, and U. H. Kos-
zinowski. 1997. Cloning and mutagenesis of a herpesvirus genome as an
infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 94:
14759–14763.
28. Munch, K., M. Messerle, B. Plachter, and U. H. Koszinowski. 1992. An
acidic region of the 89K murine cytomegalovirus immediate early protein
interacts with DNA. J. Gen. Virol. 73:499–506.
29. Neipel, F., K. Ellinger, and B. Fleckenstein. 1991. The unique region of the
human herpesvirus 6 genome is essentially collinear with the UL segment of
human cytomegalovirus. J. Gen. Virol. 72:2293–2297.
30. Nicholas, J. 1996. Determination and analysis of the complete nucleotide
sequence of human herpesvirus. J. Virol. 70:5975–5989.
31. Nicholas, J., and M. E. Martin. 1994. Nucleotide sequence analysis of a
38.5-kilobase-pair region of the genome of human herpesvirus 6 encoding
human cytomegalovirus immediate-early gene homologs and transactivating
functions. J. Virol. 68:597–610.
32. Novotny, J., I. Rigoutsos, D. Colemann, and T. S. Shenk. 2001. In silico
structural and functional analysis of the human cytomegalovirus (human
herpesvirus5) genome. J. Mol. Biol. 310:1151–1166.
33. Patterson, C. E., and T. Shenk. 1999. Human cytomegalovirus UL36 protein
is dispensable for viral replication in cultured cells. J. Virol. 73:7126–7131.
34. Rawlinson, W. D., H. E. Farrell, and B. G. Barrell. 1996. Analysis of the
complete DNA sequence of murine cytomegalovirus. J. Virol. 70:8833–8849.
35. Reddehase, M. J., F. Weiland, K. Munch, S. Jonjic, A. Luske, and U. H.
Koszinowski. 1985. Interstitial murine cytomegalovirus pneumonia after ir-
radiation: characterization of cells that limit viral replication during estab-
lished infection of the lungs. J. Virol. 55:264–273.
36. Romanowski, M. J., G. E. Garrido, and T. Shenk. 1997. pIRS1 and pTRS1
are present in human cytomegalovirus virions. J. Virol. 71:5703–5705.
37. Romanowski, M. J., and T. Shenk. 1997. Characterization of the human
cytomegalovirus irs1 and trs1 genes: a second immediate-early transcription
unit within irs1 whose product antagonizes transcriptional activation. J. Vi-
rol. 71:1485–1496.
38. Sambrook, J. T., and D. Russel.2001. Molecular cloning: a laboratory man-
ual, 3rd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
39. Skaletskaya, A., L. M. Bartle, T. Chittenden, A. L. McCormick, E. S. Mo-
carski, and V. S. Goldmacher. 2001. A cytomegalovirus-encoded inhibitor of
apoptosis that suppresses caspase-8 activation. Proc. Natl. Acad. Sci. USA
98:7829–7834.
40. Stasiak, P. C., and E. S. Mocarski. 1992. Transactivation of the cytomega-
lovirus ICP36 gene promoter requires the alpha gene product TRS1 in
addition to IE1 and IE2. J. Virol. 66:1050–1058.
41. Tzung, S. P., K. M. Kim, G. Basanez, C. D. Giedt, J. Simon, J. Zimmerberg,
K. Y. Zhang, and D. M. Hockenbery. 2001. Antimycin A mimics a cell-death-
inducing Bcl-2 homology domain 3. Nat. Cell Biol. 3:183–191.
42. Vieira, J., H. E. Farrell, W. D. Rawlinson, and E. S. Mocarski. 1994. Genes
in the HindIII J fragment of the murine cytomegalovirus genome are dis-
pensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt
cassette. J. Virol. 68:4837–4846.
43. Vink, C., E. Beuken, and C. A. Bruggeman. 2000. Complete DNA sequence
of the rat cytomegalovirus genome. J. Virol. 74:7656–7665.
44. Wagner, M., and U. H. Koszinowski. Mutagenesis of viral BACs with linear
PCR fragments (ET recombination). Methods Mol. Biol., in press.
45. Wagner, M., A. Gutermann, J. Podlech, M. J. Reddehase, and U. H. Koszi-
nowski. 2002. Major histocompatibility complex class I allele-specific coop-
erative and competitive interactions between immune evasion proteins of
cytomegalovirus. J. Exp. Med. 196:805–816.
46. Wagner, M., S. Jonjic, U. H. Koszinowski, and M. Messerle. 1999. Syst.
excision of vector sequences from the BAC-cloned herpesvirus genome
during virus reconstitution. J. Virol. 73:7056–7060.
47. Wagner, M., Z. Ruzsics, and U. H. Koszinowski. 2002. Herpesvirus genetics
has come of age. Trends Microbiol. 10:318–324.
48. Xiao, J., T. Tong, X. Zhan, E. Haghjoo, and F. Liu. 2000. In vitro and in vivo
characterization of a murine cytomegalovirus with a transposon insertional
mutation at open reading frame M43. J. Virol. 74:9488–9497.
49. Zhan, X., M. Lee, G. Abenes, R. Von, I., C. Kittinunvorakoon, P. Ross-
Macdonald, M. Snyder, and F. Liu. 2000. Mutagenesis of murine cytomeg-
alovirus with a Tn3-based transposon. Virology 266:264–274.
5570 ME´NARD ET AL. J. VIROL.