Murine cytomegalovirus m142 and m143 are both required to block protein kinase R-mediated shutdown of protein synthesis.

JOURNAL OF VIROLOGY, Oct. 2006, p. 10181–10190 Vol. 80, No. 20
0022-538X/06/$08.00�0 doi:10.1128/JVI.00908-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Murine Cytomegalovirus m142 and m143 Are both Required To Block
Protein Kinase R-Mediated Shutdown of Protein Synthesis
Ralitsa S. Valchanova,1,2 Marcus Picard-Maureau,2§ Matthias Budt,1 and Wolfram Brune1,2*
Robert Koch Institute, Division of Viral Infections, 13353 Berlin,1 and Rudolf Virchow Center for Experimental Biomedicine,
University of Wu¨rzburg, 97078 Wu¨rzburg,2 Germany
Received 3 May 2006/Accepted 21 July 2006
Cytomegaloviruses carry the US22 family of genes, which have common sequence motifs but diverse func-
tions. Only two of the 12 US22 family genes of murine cytomegalovirus (MCMV) are essential for virus
replication, but their functions have remained unknown. In the present study, we deleted the essential US22
family genes, m142 and m143, from the MCMV genome and propagated the mutant viruses on complementing
cells. The m142 and the m143 deletion mutants were both unable to replicate in noncomplementing cells at low
and high multiplicities of infection. In cells infected with the deletion mutants, viral immediate-early and early
proteins were expressed, but viral DNA replication and synthesis of the late-gene product glycoprotein B were
inhibited, even though mRNAs of late genes were present. Global protein synthesis was impaired in these cells,
which correlated with phosphorylation of the double-stranded RNA-dependent protein kinase R (PKR) and its
target protein, the eukaryotic translation initiation factor 2�, suggesting that m142 and m143 are necessary to
block the PKR-mediated shutdown of protein synthesis. Replication of the m142 and m143 knockout mutants
was partially restored by expression of the human cytomegalovirus TRS1 gene, a known double-stranded-
RNA-binding protein that inhibits PKR activation. These results indicate that m142 and m143 are both
required for inhibition of the PKR-mediated host antiviral response.
Cytomegaloviruses (CMVs) are prototypes of the � subfam-
ily of the Herpesviridae. Their genomes span 230 kb and are the
largest among the herpesviruses. Of the approximately 170
genes on a CMV genome, about 46 are conserved among the
herpesviruses (42). An additional 34 genes are characteristic of
the �-herpesviruses (12, 21, 44, 58), and the remaining genes
are unique to a particular virus.
A typical property of �-herpesvirus genomes is the presence
of gene families, which probably arose by duplication from
ancestral genes. One of the largest, the US22 gene family, was
first identified in human cytomegalovirus (HCMV) and was
later also found in other �-herpesviruses (12, 21, 44, 51, 58). It
is characterized by four conserved sequence motifs consisting
of hydrophobic residues interspersed with charged amino ac-
ids. HCMV and murine cytomegalovirus (MCMV) both in-
clude 12 members of the US22 gene family on their genomes
(12, 51). The rat CMV and human herpesviruses 6 and 7, two
other human �-herpesviruses, also possess up to 11 US22 fam-
ily genes (20, 21, 44, 58).
Little is known about the functions of US22 gene products.
However, 5 of the 12 US22 gene products of MCMV affect the
virus’ ability to replicate in macrophages: M36, M43, m139,
m140, and m141 (28, 29, 32, 41). The molecular mechanism of
action for only one of these has been elucidated. The M36 gene
encodes an antiapoptotic protein that binds to procaspase 8
and inhibits death receptor-mediated induction of apoptosis
(41). The positional and sequence homolog of M36 in HCMV,
UL36, was originally proposed to function as a transcriptional
transactivator (16). However, a later study showed that UL36
encodes a viral inhibitor of caspase 8 activation (vICA), a
function it shares with its counterpart in MCMV (40, 41, 54).
A comprehensive mutational analysis of all 12 US22 gene
family members of MCMV identified m142 and m143 as the
only two genes of this family that are essential for virus repli-
cation in cell culture (41). These two genes are transcribed with
immediate-early kinetics and lack motif II, one of the four
conserved motifs (26, 27). The functions of these two proteins,
however, remain unknown. The assumption that immediate-
early proteins are likely to function as transcriptional transac-
tivators—as has been shown for the HCMV TRS1 and IRS1
gene products (52, 55)—could not be confirmed for m142 and
m143 (26).
In the present study, we investigated the functions of m142
and m143 during MCMV infection. Viral deletion mutants of
these genes replicated only on complementing cells and were
unable to express viral late proteins and to amplify viral DNA
in normal fibroblasts. They failed to inhibit activation of the
double-stranded RNA (dsRNA)-dependent protein kinase R
(PKR) and shutdown of protein synthesis, suggesting that this
antiviral response prevents replication of the deletion mutants.
Consistent with our results, Child et al. report that m142 and
m143 form a dsRNA-binding complex and that the two pro-
teins can jointly rescue a vaccinia virus deficient for the dsRNA-
binding protein E3L (13).
MATERIALS AND METHODS
Plasmids and genes. The TRS1, m142, and m143 genes were amplified by PCR
from the HCMV AD169 and the MCMV genomes, respectively, using the fol-
lowing primers: 5�-A AAG AAT TCC ACC ATG GCC CAG CGC AAC GGC
ATG TCG-3� (TRS1-fw), 5�-AAA CTC GAG TCA AGC GTA GTC TGG GAC
GTC GTA TGG GTA TTG AGC ATT GTA ATG GTA GT-3� (TRS1-rev),
* Corresponding author. Mailing address: Robert Koch-Institut, Fach-
gebiet Virale Infektionen, Nordufer 20, 13353 Berlin, Germany. Phone:
49 30 18754 2502. Fax: 49 1810754 2502. E-mail: brunew@rki.de.
§ Present address: Universita¨tsklinik Mannheim, Institut fu¨r Mediz-
inische Mikrobiologie und Hygiene, Abteilung fu¨r Virologie, 68167
Mannheim, Germany.
10181

5�-A AAG AAT TCC ACC ATG GAC GCC CTG TGC GCG GC-3� (m142-fw),
5�-AAA CTC GAG TCA AGC GTA GTC TGG GAC GTC GTA TGG GTA
GTC GTC ATC GTC GGC GTC CGC-3� (m142-rev), 5�-AAA GGA TCC ACC
ATG TCT TGG GTG ACC GGA GAT-3� (m143-fw), and 5�-A AAG AAT TCA
AGC GTA GTC TGG GAC GTC GTA TGG GTA CGC GTC GGT CGC TCT
CTC GTC-3� (m143-rev). Suitable restriction sites (in italics) for cloning in
pcDNA3 (Invitrogen) and a hemagglutinin (HA) epitope sequence (underlined)
at the 3� end were introduced by the primers. It is noteworthy that the m143 gene
sequence differed from the published sequence (51) by the presence of an
additional guanosine residue between positions 201402 and 201403 of the
MCMV sequence (GenBank accession number NC_004065). This leads to a
frameshift compared to the published sequence analysis and a termination of the
m143 open reading frame (ORF) at nucleotide position 200963 of the MCMV
genome. The same difference was independently detected by others (13), sug-
gesting that the published sequence is incorrect.
Retroviral transduction. The genes of interest were excised from pcDNA3 and
inserted into the retroviral vector plasmid pLXSN (TRS1 and m142) or pLXRN
(m143). Production of Moloney murine leukemia virus-based retroviral vectors
using the Phoenix packaging cell line and transduction of NIH 3T3 cells was done
as in a previous study (8). Transduced cells were selected with 700 �g/ml G418
and grown as bulk cultures without clonal selection.
Mutagenesis. All mutant viruses were constructed on the basis of an MCMV
variant expressing the green fluorescent protein (MCMV-GFP), which has been
used in previous studies (7, 8, 31). The MCMV-GFP genome, cloned as a
bacterial artificial chromosome (BAC), was modified in the Escherichia coli
strain DY380 as described previously (8, 9). For the construction of the deletion
mutants, a zeocin resistance gene (zeo) was PCR amplified with primers that
contained 50-nucleotide (nt) sequences homologous to positions 199621 to
199670 and 200749 to 200798 (for �m142) or 201121 to 201170 and 202594 to
202643 (for �m143). For construction of the m142 revertant genome, the plas-
mid pBSo142 was constructed, which is based on pBluescriptII KS(�) (Strat-
agene) and contains a synthetic oligonucleotide encoding 50-nt sequences up-
and downstream of the m142 ORF, spaced by EcoRI, EcoRV, XhoI, and HpaI
restriction sites. The HA-tagged m142 gene and a kanamycin resistance gene
(kan) were inserted into pBSo142 using the EcoRI/XhoI and HpaI sites, respec-
tively. The entire cassette was excised with SacI and ApaI and used for repair of
the m142 deletion. The kan gene (which was flanked by FRT sites) was subse-
quently removed using FLP recognition target (FLP) recombinase as described
previously (8). The m143 revertant genome was constructed by the same strategy
with plasmid pBSo143, containing the 50-nt homologies up- and downstream of
the m143 ORF. To replace m142 and m143 with TRS1, we inserted the TRS1
gene into pBSo142 and pBSo143, respectively, as had been done for the rever-
tants. Alternatively, TRS1 was also inserted at an ectopic position, replacing the
nonessential ORFs m02 to m06. This was done using the pReplacer plasmid as
described previously (31). Wild-type and recombinant MCMV BAC DNAs were
isolated from E. coli using NucleoBond PC100 columns (Macherey-Nagel). The
viral genomes were digested with suitable restriction endonucleases and sepa-
rated on 0.6% agarose gels. The DNA was subsequently transferred to a nylon
membrane using a TurboBlotter device (Schleicher & Schuell). Hybridization
with a digoxigenin-labeled probe directed against the m142 and m143 genes and
chemiluminescent detection were performed using a DIG-High Prime DNA-
labeling and detection kit (Roche) according to the manufacturer’s recommen-
dations.
Cells and viruses. MCMV was propagated on NIH 3T3 cells (ATCC CRL-
1658) according to standard procedures (6). To reconstitute recombinant viruses
from mutant genomes, BAC DNA was transfected into normal or complement-
ing NIH 3T3 cells. Virus stocks were produced on the same cells and titered
using the 50% tissue culture infectious dose (TCID50) method (39). For growth
kinetics, cells were seeded in six-well plates and infected with MCMV at the
indicated MOI. Two hours after infection, the cells were washed with phosphate-
buffered saline, and fresh medium was added. The medium was replaced at the
indicated time points, and the virus content in the supernatant was determined
by titration. All growth kinetics experiments were done in triplicate. To analyze
viral-DNA replication, total DNA was extracted from infected NIH 3T3 cells,
blotted onto a nylon membrane, and detected with a digoxigenin-labeled probe
against the MCMV M45 gene as described above.
Protein detection. For Western blot analysis, cells were lysed in RIPA buffer
containing 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1% Na deoxycholate, 1%
Triton X-100, 0.1% sodium dodecyl sulfate, and a protease inhibitor cocktail
(Roche). Denatured protein samples were separated by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes
(Amersham). For immunological detection, the following antibodies were used:
CROMA101 against MCMV IE1 and CROMA103 against E1 (both provided by
Stipan Jonjic, University of Rijeka, Rijeka, Croatia), and 2E8.21A against the
MCMV envelope glycoprotein B (gB) and 3B9.22A against M44 (both provided
by Lambert Loh, University of Saskatchewan, Saskatchewan, Canada). Mono-
clonal antibodies against the HA epitope tag (16B12 [Covance Research Prod-
ucts] and 3F10 [Roche]), �-actin (A5316; Sigma), and murine PKR (B-10; Santa
Cruz) and polyclonal rabbit antibodies against phosphorylated and total murine
eIF2� (the � subunit of eukaryotic translation initiation factor 2; Cell Signaling)
were purchased from suppliers as indicated. Horseradish peroxidase-coupled
secondary antibodies (Dako and Cell Signaling) and enhanced chemilumines-
cence reagents (Amersham) were used to develop the blots. For immunofluo-
rescence, cells were grown on coverslips, fixed with 3% paraformaldehyde, and
permeabilized with 0.3% Triton X-100. Proteins were detected using an anti-HA
primary antibody and an Alexa Fluor 488- or Alexa Fluor 594-coupled secondary
antibody (Molecular Probes). Nuclei were stained with propidium iodide or
4�,6-diamidino-2-phenylindole dihydrochloride (DAPI). Fluorescent images
were generated with a Zeiss LSM 510 confocal microscope.
For metabolic labeling, NIH 3T3 cells were infected at a multiplicity of infec-
tion (MOI) of 5 TCID50/cell. Twenty-four hours after infection, the cells were
incubated for 1 h in medium containing [35S]methionine and [35S]cysteine (143
�Ci total radioactivity). Protein lysates of labeled cells were separated by poly-
acrylamide gel electrophoresis. The protein concentration for each lysate was
determined using a Bicinchoninic Acid Protein Assay kit (Pierce), and equal
amounts of total protein were loaded onto each lane. The gel was fixed, dried,
and exposed to an X-ray film (Kodak).
Real-time reverse transcription (RT)-PCR. Total RNA was isolated with
Trizol reagent (Invitrogen), purified with an RNeasy kit (QIAGEN), and sub-
sequently digested with DNAse I to remove contaminating DNA. The quality of
the RNA preparations was confirmed by denaturing agarose gel electrophoresis.
First-strand cDNA synthesis was performed with Superscript III reverse trans-
criptase (Invitrogen) using 125 ng random-hexamer-primed total RNA. The
reverse-transcribed total RNA was digested with RNAse H. For each sample, a
background control without the addition of reverse transcriptase was made.
Real-time PCR analysis was performed using a Lightcycler 1 instrument
(Roche). Each reaction mixture was made with the SYBR Green FastStart Kit
(Roche) in 15 �l total volume containing 2.5 mM MgCl2, 7.5 pmol of each
primer, and 1 ng reverse-transcribed total RNA. As a housekeeping gene control,
18S rRNA was chosen, because previous experiments had shown that its level of
expression is not significantly affected by MCMV infection. Each run was per-
formed with 45 cycles, followed by a melting-curve analysis to confirm the
identities of the PCR products. For the detection of viral- and housekeeping
gene transcripts, the following primers were used: 5�-TTA TGG TTC CTT TGG
TCG CTC G-3� (18s-fwd), 5�-CAC CGG GTT GGT TTT GAT CTG A-3�
(18s-rev), 5�-GCG ATG TCC GAG TGT GTC AAG-3� (gB-fwd), 5�-CGA CCA
GCG GTC TCG AAT AAC-3� (gB-rev), 5�-TCG TTC GTG AAC ATC GTG
GTG-3� (gM-fwd), 5�-GAT CGC GTT GTA CAT CGT CAG G-3� (gM-rev),
5�-TGC ACC AGG CGC TCT GTA AC-3� (M44-fwd), and 5�-CGC TGA GGA
AGT TCT CGA TGG-3� (M44-rev). Relative quantification was carried out
according to the method of Pfaffl (48).
RESULTS
Construction of MCMV m142 and m143 deletion mutants.
To analyze the functions of m142 and m143 in the context of an
MCMV infection, we constructed deletion mutants of these
genes. MCMV-GFP was used as a basis for the construction of
mutant viruses, because GFP expression by infected cells fa-
cilitates the monitoring of potentially growth-defective viruses.
A BAC clone of MCMV-GFP was modified by homologous
recombination in E. coli. The m142 and m143 ORFs were
replaced individually with a zeocin resistance gene (Fig. 1A).
In a second step, revertant genomes were constructed by re-
placing the zeocin resistance gene of the deletion mutants with
an HA epitope-tagged m142 or m143 gene, respectively (Fig.
1A). A kanamycin resistance gene flanked by FRT sites was
inserted downstream of the respective gene to serve as a se-
lectable marker for homologous recombination. This cassette
was subsequently removed by FLP recombinase, leaving only a
single FRT site behind. The restriction patterns of these ge-
10182 VALCHANOVA ET AL. J. VIROL.

nomes showed the expected changes (Fig. 1B). Transfection of
the wild-type and the revertant genomes into NIH 3T3 fibro-
blasts yielded replication-competent virus, but the �m142 and
�m143 deletion mutants did not grow. Individual green fluo-
rescent cells were obtained after transfection of the genomes,
but these cells disappeared after repeated passaging of the
cells.
To overcome this problem, NIH 3T3 cells were transduced
with retroviral vectors carrying m142 or m143, respectively,
and a G418 resistance gene for selection of the transduced
cells. As shown in Fig. 2A and B, the cells expressed the
HA-tagged proteins. They could be detected by Western blot-
ting and immunofluorescence. When these complementing
cells were transfected with the genomes of the MCMV dele-
tion mutants, the mutant viruses could be reconstituted. To
confirm the identities of the recombinant viruses, viral DNA
was extracted from virus preparations and analyzed by Southern
blot hybridization. All mutants showed the expected band pat-
terns (Fig. 1C). Moreover, the revertant viruses expressed HA-
tagged m142 or m143 proteins, respectively (Fig. 2C and D).
Growth properties of the �m142 and �m143 deletion mu-
tants. It is conceivable that transfection of an MCMV genome
into fibroblasts provides less favorable conditions for initiation
of the viral replication cycle than an infection. Hence, the
failure to regenerate a virus from a mutant BAC, as shown in
a previous study (41), provided only indirect evidence for es-
sential roles of the m142 and m143 genes. Moreover, it is
possible that a gene deletion prevents virus replication during
an infection at a low MOI but that this defect can be overcome
by infecting cells at a high MOI. This has been shown, for
instance, for the HCMV IE1 gene, which is required for effi-
cient growth at a low MOI but is dispensable for replication at
a high MOI (22, 43). Therefore, we infected murine fibroblasts
at low and high MOIs and analyzed virus replication. The
growth curves in Fig. 3A and B show that the �m142 and
�m143 viruses did not grow on noncomplementing fibroblasts,
either at a low or at a high MOI. However, the viruses grew to
almost normal titers on complementing cells (Fig. 3C and D).
This indicated that each of the two genes is essential for virus
growth in cell culture at both low and high MOIs.
FIG. 1. Construction of MCMV deletion mutants. (A) The m142 and m143 open reading frames were replaced by a bacterial zeocin resistance
gene (zeo), using recombinant BAC technology. To obtain the revertants, Rm142 and Rm143, the HA-tagged open reading frames, together with
an FRT-flanked kanamycin resistance gene, were inserted into the MCMV�m142 and �m143 genomes, respectively. The kan gene was
subsequently removed using FLP recombinase, leaving a single FRT site (black oval) behind. EcoRI (E) restriction sites and the expected fragment
sizes are indicated. wt, wild type. (B) EcoRI-digested MCMV BACs were separated on an ethidium bromide-stained agarose gel. The arrowheads
indicate altered fragments. (C) Southern blot of EcoRI-digested viral DNA from MCMV-infected cells hybridized with m142- and m143-specific
probes, respectively.
VOL. 80, 2006 MCMV m142 AND m143 PREVENT PROTEIN SYNTHESIS SHUTDOWN 10183

m142 and m143 are required for late-protein synthesis and
DNA replication. To identify a block in the cascade of viral-
gene expression as a potential reason for the inability of the
deletion mutants to replicate, we analyzed the expression of
immediate-early (�), early (�1), early-late (�2), and late (�)
MCMV proteins by Western blotting. To exclude the possibil-
ity that gene expression by the mutant viruses is delayed, we
analyzed the expression of these proteins not only at 24, but
also at 72 h postinfection (p.i.). Although expression of the �
gene IE1 (34) was reduced in �m142- and �m143-infected
FIG. 2. Expression of m142 and m143 in transduced and infected cells. (A) Western blot and (B) indirect immunofluorescence analysis of NIH
3T3 cells stably transduced with retroviruses encoding HA-tagged m142 and m143 proteins, respectively. (C) Detection of HA-tagged m142 and
m143 proteins expressed by the revertant MCMVs Rm142 and Rm143, respectively, by Western blotting and (D) immunofluorescent staining of
infected NIH 3T3 cells. Nuclei were stained with propidium iodide (PI) or DAPI. mock, mock infected.
FIG. 3. Growth kinetics of recombinant MCMVs. Replication of the �m142 and �m143 deletion mutants and revertants in NIH 3T3 cells was
analyzed upon infection at (A) a low or (B) a high MOI. (C) Replication of the �m142 mutant in m142-expressing NIH 3T3 cells and (D) of the
�m143 mutant in m143-expressing cells. The error bars represent the standard errors.
10184 VALCHANOVA ET AL. J. VIROL.

cells, the levels of immediate-early proteins were apparently
sufficient to activate expression of the �1 gene E1 (10) and the
�2 gene M44 (38), as their levels were not dramatically altered
compared to the wild type and the revertants. A major differ-
ence was seen in the levels of the late protein gB (37, 50),
which was barely detectable in cells infected with the deletion
mutants (Fig. 4A and B). Moreover, viral DNA was present in
comparable amounts in these cells at 24 h p.i., but an ampli-
fication to high levels at very late times (72 h p.i.) apparently
did not occur (Fig. 4C and D).
m142 and m143 are required to prevent PKR activation and
shutdown of protein synthesis. Earlier studies identified a tran-
scriptional transactivating function of the HCMV US22 gene
family proteins TRS1, IRS1, and UL36 in assays using tran-
sient transfection of expression and reporter plasmids (16, 52,
55). Although m142 and m143 failed to show similar transac-
tivating activities in a more recent study (26), we wanted to test
whether m142 and m143 are required for late-gene transcrip-
tion. The mRNA levels of the early-late gene M44 and the late
genes M55/gB and M100/gM (36) were measured by real-time
PCR. At 24 h p.i., the mRNA levels of these genes were not
significantly altered in the deletion mutants compared to wild-
type or revertant viruses (Fig. 5A). Only at very late times (72
h p.i.) were the mRNA levels reduced about 10-fold in cells
infected with �m143 (Fig. 5B). This suggested that the defect
in late-protein expression is based primarily on a posttranscrip-
tional block. Indeed, when global protein synthesis was ana-
lyzed by metabolic labeling with [35S]methionine and [35S]cys-
teine, it was found to be markedly reduced in cells infected
with the �m142 or �m143 mutant (Fig. 6A).
Global shutdown of protein synthesis is a well-known innate
defense mechanism by which infected cells can inhibit the
production of progeny virus (47, 53). This process usually in-
volves activation of the double-stranded-RNA-dependent
PKR and PKR-mediated phosphorylation of eIF2�, an essen-
tial cofactor for translation of mRNAs into polypeptides.
When PKR and eIF2� were analyzed in infected cells by West-
ern blotting, a more slowly migrating form of PKR was de-
tected only in cells infected by the deletion mutants, but not in
cells infected with the wild-type or the revertant virus (Fig. 6B).
The more slowly migrating band most probably represents
phosphorylated (i.e., activated) PKR, because treatment of
NIH 3T3 cells with poly(I · C), a known inducer of PKR
phosphorylation, resulted in a comparable band shift (data
not shown). Unfortunately, an antibody specifically recog-
nizing phosphorylated murine PKR was not available, and
thus, the identity of the more slowly migrating band could
not be formally proven. However, phosphorylation of the
PKR target protein eIF2� was clearly detected in these cells
FIG. 4. Viral-gene expression and DNA replication. NIH 3T3 cells
were infected with recombinant MCMVs at an MOI of 0.5 TCID50/
cell. Viral-gene products were detected with specific antibodies 24
(A) and 72 (B) h p.i. Detection of viral-DNA amplification in infected
cells by slot blotting and hybridization with an MCMV-specific probe
24 (C) and 72 (D) h p.i. �a, beta actin; wt, wild type; mock, mock
infected.
FIG. 5. Real-time RT-PCR quantification of MCMV late-gene transcripts. NIH 3T3 cells were infected at an MOI of 1 TCID50/cell with
wild-type (wt) and mutant MCMV. Total RNA was harvested (A) 24 and (B) 72 h p.i. and quantified by real-time RT-PCR. Two RNA preparations
from cells infected with different virus stocks were made, and from each preparation, two to four independent quantitative PCRs per sample were
done. The transcript abundance in MCMV wt-infected cells was defined as 1. The abundances of transcripts from mutant viruses are shown relative
to the wt. The error bars represent the standard errors. mock, mock infected.
VOL. 80, 2006 MCMV m142 AND m143 PREVENT PROTEIN SYNTHESIS SHUTDOWN 10185

(Fig. 6B), suggesting that the shutdown of protein synthesis
is indeed mediated by PKR-dependent phosphorylation of
eIF2�.
HCMV TRS1 can restore the growth of �m142 and �m143.
The TRS1 and IRS1 proteins of HCMV have recently been
shown to rescue a vaccinia virus (VV) lacking the dsRNA-
binding protein E3L (14, 15). Similarly, TRS1 and IRS1 were
able to reverse the PKR-mediated protein synthesis shutoff
induced by a recombinant herpes simplex virus type 1 (HSV-1)
lacking the �134.5 gene (11). In addition, TRS1 was shown to
possess dsRNA-binding activity (24). These results suggested
that TRS1 and IRS1 are responsible for inhibiting a shutdown
of protein synthesis during an HCMV infection, although this
has not yet been formally demonstrated.
TRS1 and IRS1 share a number of properties with m142 and
m143 of MCMV. They are expressed at immediate-early times,
are members of the US22 gene family, and lack one of the four
sequence motifs characteristic of this gene family (27). How-
ever, the sequence similarity of TRS1 to m142 and m143 is
noticeable, but not striking. The m143 protein has 18% iden-
tical and 30% similar amino acids to both m142 and TRS1. The
m142 protein shares only 11% identity and 21% similarity with
TRS1 (Fig. 7). Hence, we asked if TRS1 could rescue the
growth defect of the MCMV �m142 and/or �m143 virus. To
investigate this, we infected NIH 3T3 cells stably transduced
with a TRS1-expressing retrovirus and studied the growth of
the �m142 and �m143 deletion mutants. Both mutants grew
on TRS1-expressing fibroblasts (Fig. 8A), albeit to 10- to 100-
fold-lower titers than the wild-type virus. We also inserted the
TRS1 gene into the genomes of the �m142 and �m143 mu-
tants in order to test if TRS1 expression in cis would also
rescue virus growth. Surprisingly, insertion of TRS1 in place of
the deleted m142 and m143 genes (Fig. 8B) did not rescue
virus growth (data not shown). However, when TRS1 was
driven by a phosphoglycerate kinase gene (pgk) promoter and
inserted at an ectopic position, replacing the nonessential
genes m02 to m06 (Fig. 8C), virus growth on NIH 3T3 fibro-
blasts and expression of the late protein gB were restored (Fig.
8D to F). These results indicated that TRS1 can, at least in
part, compensate for a lack of m142 or m143. Since TRS1 can
block the PKR-dependent antiviral response during an HSV-1
or VV infection (11, 14), the results also support the concept
that m142 and m143 are both required to prevent PKR-medi-
ated shutdown of protein synthesis.
DISCUSSION
Replication of intracellular pathogens, such as viruses, can
be inhibited by innate immune defenses of the host cell. One of
the best characterized is the interferon-mediated antiviral re-
sponse, which proceeds in three phases (reviewed in reference
25). First, the cell senses the presence of a virus via toll-like
receptors (TLRs) that recognize pathogen-associated molecu-
lar patterns (33). TLR signaling triggers the expression and
secretion of beta interferon (IFN-�). Binding of IFN-� to type
I interferon receptors on the same and on neighboring cells
activates the Jak/STAT signaling pathway, which leads to an
upregulation of antiviral effector molecules, such as PKR, 2�-
5�-oligoadenylate synthetase, and Mx proteins. Viruses, on the
other hand, have evolved various mechanisms to counteract
the IFN-mediated antiviral response on different levels (re-
viewed in reference 25).
CMVs already trigger the activation of the IFN response
during attachment and entry into the cell. Contact of gB with
its receptor activates the IFN-responsive pathway via inter-
feron regulatory factor 3 (3, 4). CMV also activates inflamma-
tory-cytokine production by engaging the pattern recognition
receptors TLR2, TLR9, and CD14 (17, 35). However, HCMV
counteracts the induction of IFN-� expression via its tegument
protein pp65/UL83 (1, 5) and/or via a combined action of the
neighboring gene product, pp71/UL82, and the IE2 protein, as
suggested by later studies (56, 57). IFN receptor signaling is
also inhibited by both HCMV and MCMV. The HCMV IE1
protein forms a complex with the signal transducer and acti-
vator of transcription 1 (STAT1) and STAT2 and inhibits the
induction of IFN-responsive effector genes (46). In MCMV,
the M27 gene product inhibits both type I and type II IFN
signaling by selectively down-regulating STAT2 without affect-
ing STAT1 (60).
Like many other viruses, HCMV also encodes proteins that
bind dsRNA and inhibit the dsRNA-dependent activation of
FIG. 6. Shutdown of protein synthesis in �m142- and �m143-in-
fected cells. (A) NIH 3T3 cells were metabolically labeled with
[35S]methionine and [35S]cysteine 24 h p.i. at an MOI of 5 TCID50/cell.
Cell lysates (10 �g total protein per sample) were separated by gel
electrophoresis and analyzed by autoradiography. (B) Detection of
phosphorylated PKR and eIF2� (P-eIF2�) in �m142- and �m143-
infected cells by Western blotting. Total eIF2� and �-actin were used
as controls. mock, mock infected; wt, wild type.
10186 VALCHANOVA ET AL. J. VIROL.

PKR. This function is fulfilled by the TRS1 and IRS1 proteins
of HCMV (11, 14, 24). These two proteins are identical over
the N-terminal two-thirds of their amino acid sequences, be-
cause they are located in part in the short repeat region of the
HCMV genome (12). Each of the two proteins can rescue a
VV E3L or an HSV-1 �134.5 deletion mutant, suggesting at
least partly redundant functions. Unfortunately, the impor-
tance of these two proteins for the life cycle of HCMV could
not yet be analyzed, because a TRS1-IRS1 double-knockout
mutant could not be grown in normal fibroblasts, and the
generation of reliable complementing cells proved to be diffi-
cult (W. Brune, J. E. Adamo, and T. Shenk, Abstr. 27th Int.
Herpesvirus Workshop, abstr. 11.13, 2002, and W. Brune, un-
published results).
The present study shows that m142 and m143 are both
required to inhibit PKR activation and a shutdown of protein
synthesis at late times postinfection. The inability of the �m142
and �m143 mutants to prevent PKR activation is by itself not
sufficient to prove that the two proteins are directly involved in
blocking PKR activation. However, the fact that TRS1, a
known inhibitor of PKR activation, can rescue the �m142 and
�m143 deletion mutants suggests a direct role of m142 and
m143 in this process. Hence, this is the first study that dem-
onstrates the importance of the PKR-mediated antiviral re-
sponse during a cytomegalovirus infection.
The MCMV m142 and m143 proteins show significant se-
quence similarity to their presumed counterparts in HCMV,
TRS1 and IRS1 (Fig. 7). They differ, however, from TRS1 and
IRS1 in that each of them is essential for MCMV replication.
The HCMV proteins, by contrast, are together essential only
for HCMV replication (Brune et al., Abstr. 27th Int. Herpes-
virus Workshop), consistent with redundant functions of the
two proteins. These findings suggest either that the m142 and
m143 proteins block different steps toward PKR activation or
that they form an inhibitory complex. The latter possibility is
supported by a report which shows that m142 and m143 can
bind dsRNA and rescue a VV deficient for the dsRNA-binding
protein E3L only when expressed together (13).
Inhibition of the dsRNA-dependent antiviral response is
obviously a crucial task for RNA viruses, as their replication
inevitably results in the generation of dsRNA molecules. How-
ever, a recent study showed that DNA viruses, such as herpes-
viruses, vaccinia virus, or adenoviruses, also produce dsRNA
(59). One possible explanation is that DNA viruses carry
1 10 20 30 40 50 60 70 80
m142 FL G L G P GS V P LT A A L DF AQ S V ES L L WYL QMDALCAAIR DHFRDQRP GIR ...RSRY A AVT V Q ADI NDHT VENV RK R S A T CLRSAD VE
m143 FL G L G P GV P A L DF Q RS K V RES T W L V PR EMSW TGD AWRPEALIRP DREQI R KSF T TSE LK T K K CV Y P W A V G LV
TRS1 FL G L G P GS LT A AN R K RD TL L Y V PR Q...GAPPSSGNN NFWHGPERLLLSQIPVERQA ELEYQ MGAVWRAA STG AMR WSQ A P R Y F AR T S MN
Cons FL G L G P GS V P LT A A L DF A RS K V R S TL L WYL V PR......... ........ ... ....... . ... . . ... .... .. . . . . . . . . ....
90 100 110 120 130 140 150 160
m142 P G DE R GG G L V F L DA L SFP YPVWETYGGA A RYTL GQV ..............RRFRGVVPTEELRW VY PQGQLLCHQERT VFVMSLSI LA R V
m143 P G DE R GG DLR G W V I LYLAM R FVY DA LAA D L EMD R PI...L T E V VLGE ..................RSPMVQHTS KES M SAEE IA N L FS I L
TRS1 P G DE R GDLR W VL V LYLAL R FVY N LAA DGVGAT QL...S RDA I VAT VHEVDPAADPTLGDKAGHPEGLCAQDG AGF V DLAN TLI R A WF H AG
Cons P G DE R GG DLR G W VL L LA R FVY A LAA D L ... . ...... . . . ............................... . . ... . ..... .. . . .. . .
170 180 190 200 210 220 230 240
m142 Y P L TPG P L PE L L T P V E L E G TI LV G E L W F MNF P AERH....L T AV VKL A FSA DPRDGTAVA RALMCQ R L H EGD.R C TD C RRW CR SD.GEFA
m143 Y P L TPG P L PVE R L L V D L L R L I LI L E YW M ST FI .......V SIP KKDP W AIG CVTGYD N H ST.YRNSV E R KQETN DRL LCCK LVA DK.. RI
TRS1 Y P L TPG P L PV L R T LP Q L REL G TV MV LG Q L Y F SE VR CNRLGVGT RA L QPALR TLLRAEEATA G RRRWA T A Q RR.LQ AW E AQ E ASAPHPA LL
Cons Y P L TPG P L PVE L R L T LP V L L REL G T LG L YW F M S. . ........ . . ... ..... ...... . ..... . .. . ..... .. .... .. .. .... ..
250 260 270 280 290 300 310 320 330
m142 R W G V A G G S G F L G G IF WR D EL RM L K P RR DRG RLEKFTD VTRNLKT H KHF V LRVGQDP RVESV SVI N A HTDPDMDVT I NVD F T LYL K L TM RA
m143 R W G V A G G S L G R G F G L GSIF SG WR D AEL RM L K RR DR A RLEDGLI YASRKMS R YT L VY DT V H AQL VFDDS YLSLI EL L SA R L IFTAG V DL SV
TRS1 R W G V A G G L R G G G S SG A P G ATAVR HLNQRLCCG LA A LPA WL CAA PATGTAA TT P...PAA TETEA GGD PCAIA AVGSAV VPPQPY A GGAIC
Cons R W G V A G G S L G R G F G L G GSIF SG WR D AEL RM L K P RR DRG A RLE.... ....... . .. . .. .. . . ... ... . ..... .. . .. . . ... . . . ..
340 350 360 370 380 390 400 410
m142 P A A A V A V A Q W R GR S DA AE K VR V A AL VA GC... H A A WEETY IFGCRIVRDF S LG LM E FDES GTWDEMD..... MRKTFD S EL RD EV D ND D T
m143 P A A A V V A A Q W R GR LS E AV A LK V V PP PDQELWFHSRP THLC D APPGE QL Q FA IS P AEI E ET ....... DAFLRLSR VT TEENSSRVLCTH A
TRS1 P A A A V A A V A L E AV D AEL R V A AL PV NADAH V G D AAAAPT MVGST MAGPAASGTVPRAMLVV LD LG FGYCPL GHVYPLA SHFL AG LG L GRES
Cons P A A A V A A V V A A Q W R GR LS E AV DA AELK VR V A AL V P. ..... . . . .... . ..... .. . .. .. . ... . .. ...... ...... .. .. .. . .. .
420 430
m142 R SA DDFRGCP NPR DADA DD
m143 RA RL SAL R ED W D PR P GR A ARRRA APEP T V L D C RPESA KADIV FKEKPPP SPEA...... YDTDQSPA V............... H ASSQTG
TRS1 RA RL L R Q W D PR P GR A ARRRA AAAE A LPE D E .. ERPRW ALHLH AALWARE HGQLAFLLRP GEAEVLTL TKHPAICANVEDYLQD D QALGLD
Cons RA RL SAL R D W D PR P GR A ARRRA A... . . . .. . ..... ..... ....... .......... ........ ................ . ......
m142
m143 AT G E L T PP P R D TV RHHAPILEPPRSLRSEAE DCH C RERQYRYT DDSP AP STPNSTGSSPSPRPIPAP RRRPPPPR ERA DA
TRS1 AT G E L T PP P R D TL VVMEAGGQMIHKKTKKPK KED S MKGKHSRY RPTE LT QASLGRALRRDDEDWKPS LPGEDSWY LDE FW...........
Cons AT G E L T PP P R D T. .................. ... . ........ .... .. .................. ........ ... ..
motif IV
motif III
motif I
FIG. 7. Alignment of the m142, m143, and TRS1 proteins. Amino acid residues conserved between the three proteins are indicated by black
boxes; similar residues are marked with open boxes. The alignment was generated with Multalign software (18). The TRS1 sequence was truncated
at both ends, and only the overlapping part is shown. US22 gene family motifs I, III, and IV are marked. Periods represent gaps in gene sequences
and nonconserved residues in the consensus sequence.
VOL. 80, 2006 MCMV m142 AND m143 PREVENT PROTEIN SYNTHESIS SHUTDOWN 10187

genes on both strands of their genomes. This can lead to the
formation of dsRNA by hybridization of (partly) comple-
mentary mRNAs transcribed from opposing strands. An-
other possibility is that these viruses need to prevent PKR
activation through viral microRNAs. Recent studies have
shown that HCMV, like many other herpesviruses, have
such microRNAs (19, 23, 49).
The essential roles of m142, m143, and TRS1/IRS1 may not
rely exclusively on their abilities to block the PKR-mediated
antiviral response. Many herpesvirus proteins are multifunc-
tional, and this may also hold true for the proteins studied
here. For instance, previous studies have demonstrated that
TRS1 or IRS1 is required for transient complementation of
HCMV DNA replication (30, 45). This is consistent with our
observation that MCMV DNA is not replicated to high levels
in the absence of m142 or m143 (Fig. 4). However, it is possible
that MCMV DNA replication is not blocked completely but
only inhibited at late times postinfection as an indirect conse-
quence of the antiviral response. In fact, the detection of late-
gene transcripts argues for at least limited viral-DNA replica-
tion, as �-gene transcription should occur only after DNA
replication.
A recent study has revealed that TRS1 (but not IRS1) is
required for efficient assembly of DNA-containing capsids (2),
and this function is apparently not secondary to a reduced
transcription or translation of viral genes. The molecular
mechanism for this additional function of TRS1 has not yet
been determined, nor is it known whether m142, m143, or any
other MCMV protein serves a similar function.
Considering the multiple functions of the HCMV TRS1
protein, it may appear surprising that TRS1 rescued gB ex-
pression and replication in the �m142 and �m143 mutants
only partly. Insertion of the TRS1 gene into the genomes of the
deletion mutants restored virus replication only when TRS1
expression was driven by an extraneous pgk promoter. Since
the endogenous promoters of m142 and m143 are known to be
weak during the initial phase of infection (26), artificial over-
expression of TRS1 appears to be required to compensate for
the lack of m142 or m143. This could indicate that these
MCMV and HCMV proteins are not fully capable of blocking
the antiviral response in cells of a heterologous host species.
The genes of the US22 family probably arose from a com-
mon ancestral gene but have diverged in their specific func-
tions. However, it is remarkable that all of the US22 family
genes for which a function has been determined are involved in
the subversion of innate or adaptive immune functions. At
least in some cases, the functions were conserved between the
human and the murine cytomegaloviruses: UL36 and M36
FIG. 8. TRS1 rescues m142 and m143 deletion mutants. (A) Multistep growth kinetics of the wild type (wt) and deletion mutants on NIH 3T3
cells stably transduced with a TRS1-expressing retroviral vector. (B) Introduction of the HA-tagged TRS1 gene into the MCMV genome, either
by replacing the deleted gene or (C) by insertion at a distant position together with a pgk promoter. (D) Multistep growth curves of the T�m142
and T�m143 mutants on NIH 3T3 fibroblasts. (E) Viral-protein expression 24 and (F) 72 h p.i. at an MOI of 0.5 TCID50/cell. The error bars
represent the standard errors.
10188 VALCHANOVA ET AL. J. VIROL.

inhibit tumor necrosis factor alpha- and Fas-mediated signal-
ing by blocking caspase 8 activation (41, 54), and TRS1/IRS1
and m142/m143 inhibit the IFN-dependent antiviral response
by preventing PKR activation (references 11, 13, 14, and 24
and this study). Thus, it should not be surprising if other US22
family genes, whose functions are as yet unknown, are also
engaged in immune evasion.
ACKNOWLEDGMENTS
We thank Katharina Wolf for valuable help with confocal micros-
copy and Stipan Jonjic and Lambert Loh for providing antibodies. We
also thank Adam Geballe for communicating unpublished results and
Michael Nevels for critical reading of the manuscript.
This work was supported by the Emmy Noether Program of the
DFG (Br 1730/2-3), SFB 421, and the Georg und Agnes Blumenthal
Stiftung.
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