The ribonucleotide reductase R1 homolog of murine cytomegalovirus is not a functional enzyme subunit but is required for pathogenesis.

JOURNAL OF VIROLOGY, Apr. 2004, p. 4278�4288 Vol. 78, No. 8
0022-538X/04/$08.00�0 DOI: 10.1128/JVI.78.8.4278–4288.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
The Ribonucleotide Reductase R1 Homolog of Murine
Cytomegalovirus Is Not a Functional Enzyme
Subunit but Is Required for Pathogenesis†
David Lembo,1* Manuela Donalisio,1 Anders Hofer,2 Maura Cornaglia,1
Wolfram Brune,3 Ulrich Koszinowski,4 Lars Thelander,2
and Santo Landolfo1
Department of Public Health and Microbiology, University of Turin, Turin, Italy1; Department of
Medical Biochemistry and Biophysics, Umeå University, Umeå, Sweden2; and Rudolf Virchow
Center for Experimental Biomedicine, University of Wu¨rzburg, Wu¨rzburg,3 and Max von
Pettenkofer Institute, Munich,4 Germany
Received 23 September 2003/Accepted 5 December 2003
Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Alpha- and
gammaherpesviruses express a functional enzyme, since they code for both the R1 and the R2 subunits. By
contrast, betaherpesviruses contain an open reading frame (ORF) with homology to R1, but an ORF for R2 is
absent, suggesting that they do not express a functional RNR. The M45 protein of murine cytomegalovirus
(MCMV) exhibits the sequence features of a class Ia RNR R1 subunit but lacks certain amino acid residues
believed to be critical for enzymatic function. It starts to be expressed independently upon the onset of viral
DNA synthesis at 12 h after infection and accumulates at later times in the cytoplasm of the infected cells.
Moreover, it is associated with the virion particle. To investigate direct involvement of the virally encoded R1
subunit in ribonucleotide reduction, recombinant M45 was tested in enzyme activity assays together with
cellular R1 and R2. The results indicate that M45 neither is a functional equivalent of an R1 subunit nor affects
the activity or the allosteric control of the mouse enzyme. To replicate in quiescent cells, MCMV induces the
expression and activity of the cellular RNR. Mutant viruses in which the M45 gene has been inactivated are
avirulent in immunodeficient SCID mice and fail to replicate in their target organs. These results suggest that
M45 has evolved a new function that is indispensable for virus replication and pathogenesis in vivo.
Cytomegalovirus (CMV), a betaherpesvirus, is a widespread
pathogen responsible for generally asymptomatic and persis-
tent infections in healthy people. It may, however, cause severe
disease in the absence of an effective immune response, as in
immunologically immature and immunocompromised individ-
uals (41). Strict species specificity has hampered investigation
of human CMV (HCMV) in its natural host, and infection of
mice with murine CMV (MCMV) has been extensively used as
a model for studying the pathogenesis of HCMV infection (40,
52, 63). The two viruses are biologically similar in replication
and pathogenesis (34, 50, 51), and their homologous genomes
display similar genetic organizations and encode analogous
gene products with similar functions (56). However, the func-
tions of many viral gene products remain to be explored in
order to determine the interactions of CMV with the host cell
and its pathogenic mechanisms.
CMV replicates most efficiently in the absence of cellular
DNA synthesis (23, 39). Moreover, it has evolved mechanisms
to inhibit progression through the cell cycle and arrests cells
with a G1 DNA content (5, 18, 47, 53, 60, 68). The viral DNA
polymerase thus has competition-free access to the DNA pre-
cursors, and CMV replicates efficiently in vitro in quiescent
cells (18, 45) and infects terminally differentiated cells in vivo
(41, 51). However, since postmitotic cells do not replicate their
genomes, the very low levels of deoxyribonucleotides (dNTP)
and cell functions involved in DNA synthesis limit viral repli-
cation.
It has been demonstrated that CMV replication in quiescent
fibroblasts depends on its ability to stimulate expression of the
cellular enzymes involved in the biosynthesis of thymidylate,
since they are not encoded by the viral genome (9, 27, 26, 28,
44, 46). However, it is still unclear how CMV expands the pools
of all four dNTP in resting cells and provides a sufficient supply
of DNA precursors to its polymerase. The key step in dNTP
biosynthesis is carried out by the enzyme ribonucleotide reduc-
tase (RNR), which catalyzes the conversion of ribonucleoside
diphosphates to the corresponding deoxyribonucleoside
diphosphates. Both substrate specificity and overall activity are
tightly controlled by binding of nucleoside triphosphate allo-
steric effectors. The expression of RNR is cell cycle regulated;
it is very low or not detectable in resting cells and maximal in
S-phase cells (3, 10, 20, 22, 57, 66).
RNR has been divided into three classes according to its
mechanism for generation of the protein radical, metal cofac-
tor requirement, and subunit composition (38). Mammalian
cells, like most eukaryotic cells, contain a class Ia RNR. This
form also exists in some prokaryotes, e.g., the well-studied
nrdA/nrdB-encoded enzyme of Escherichia coli. Class Ia has an
�2�2 form consisting of two homodimeric subunits, proteins
R1 (�2) and R2 (�2). The R1 protein contains the active site
* Corresponding author. Mailing address: Department of Public
Health and Microbiology, University of Turin, Via Santena, 9, 10126
Turin, Italy. Phone: 39-011-6706608. Fax: 39-011-6636436. E-mail:
david.lembo@unito.it.
† This work is dedicated to the memory of Giorgio Cavallo.
4278

and the binding sites for allosteric effectors. The R2 protein is
a radical storage device containing an iron center-generated
tyrosyl free radical.
The alpha- and gammaherpesviruses (2, 14, 16, 33, 43) ex-
press a functional RNR required for virus growth in nondivid-
ing cells and for viral pathogenesis and reactivation from la-
tency in infected hosts (8, 17, 24, 25, 32, 37). The herpes
simplex virus type 1 (HSV-1) RNR is the most extensively
characterized. Like the mammalian and E. coli enzymes, it
belongs to class Ia but completely lacks allosteric regulation
(14).
Analysis of the protein-coding content of the HCMV and
MCMV genomes has revealed the presence of an open reading
frame (ORF), termed UL45 and M45, respectively (11, 56),
which shows homology to the R1 subunit of other herpesvirus.
However, like those of other betaherpesviruses, CMV ge-
nomes do not carry an ORF for the R2 subunit. It follows that
these viruses are unlikely to express a functional RNR enzyme,
and it is unclear how they can replicate in resting cells. The R1
homologs of CMV are still a biological enigma, because their
expression, cellular localization, involvement in ribonucleotide
reduction, and role in viral pathogenesis have not been de-
scribed. All these points are now addressed in the present
paper.
MATERIALS AND METHODS
Cells and culture conditions. NIH 3T3 murine fibroblasts were grown as
monolayers in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco/BRL)
supplemented with 10% calf serum (Gibco/BRL). Quiescent NIH 3T3 cells
(arrested in G0/G1 phase) were obtained by culturing the subconfluent cultures
for 48 h in DMEM plus 0.5% calf serum (low-serum medium). Flow cytometry
at this time demonstrated more than 90% of cells arrested in G0/G1.
Virus preparation and infections. MCMV (mouse salivary gland virus, strain
Smith, ATCC VR.194) was purchased from the American Type Culture Collec-
tion (Manassas, Va.). Virus stocks were first produced in salivary glands of
BALB/c mice and then propagated in vitro by infecting NIH 3T3 cells at a
virus-to-cell ratio of 0.01. Cells were incubated in DMEM supplemented with 2%
heat-inactivated calf serum, and virus was harvested, based on cytopathology, by
sonication at about 10 days postinfection and was then clarified by centrifugation.
Mock-infected fluid was prepared from NIH 3T3 cells by the procedure used to
prepare MCMV. A virus stock solution containing approximately 107 to 108
PFU/ml (as determined by a plaque assay on the NIH 3T3 cell line) was used in
all experiments. MCMV-GFP, bd-MCMV, MCMV-REV, IIIG2, and BamX
have been described previously (6). MCMV-GFP (kindly provided by Martin
Messerle) was generated by inserting the green fluorescent protein (GFP) gene
into the ie2 region of the full-length MCMV bacterial artificial chromosome
(BAC), pSM3fr, essentially as described previously (1); the BAC-derived
MCMV (bd-MCMV) is a wild-type MCMV derived from pSM3fr (67); MCMV-
REV is a revertant virus obtained by repairing the transposon insertion in the
M45 mutant virus IID7 (6). These three viruses display a wild-type phenotype
and were used as controls. IIIG2 is an M45 mutant virus carrying a transposon
insertion at nucleotide position 62876 of the MCMV genome. BamX is a frame-
shift mutant of the M45 gene generated by replacing the transposon insertion in
mutant IID7 (nucleotide position 62547) with an M45 sequence in which the
BamHI restriction site was cut and filled in with Klenow polymerase, resulting in
a 4-bp insertion (6). The frameshift mutation leads to a missense amino acid
sequence after 188 out of 1,174 amino acids of the M45 protein. Thirty-three
missense amino acids are added before a stop codon is encountered. A schematic
map showing the transposon insertions and the BamX frameshift mutation is
available as Supplemental Figure 2, published previously (6a).
Extracellular virions were partially purified from tissue culture medium by two
rounds of centrifugation through a 15% sucrose cushion in a Kontron TST 55.5
rotor (2 h, 26,000 rpm, 4°C). The partially purified virions, resuspended in TN
(50 mM Tris [pH 7.4], 100 mM NaCl), were layered onto 9-ml potassium tar-
trate-glycerol gradients formed in TN and then centrifuged at 40,000 rpm for 15
min at 4°C in a Beckman SW41 rotor with slow acceleration and braking. Virions
were visualized with incandescent light and were removed from the gradients.
For protein level determinations, NIH 3T3 cells were infected with MCMV at
a multiplicity of infection (MOI) of 5 PFU/cell unless otherwise stated. Mock-
infected control cultures were exposed to an equal volume of mock-infecting
fluid. Virus adsorptions were carried out for 1 h at 37°C, and 0 h postinfection (0
hpi) was defined as the time immediately following this period. When quiescent
cells were used, the low-serum medium removed from the cells before infection
was returned to the plates to avoid any stimulation due to addition of fresh serum
growth factors. Inactivation of virus by UV light was performed as described
previously (44).
Preparation of protein extracts and immunoblotting. Whole-cell extracts were
prepared by resuspending pelleted cells in lysis buffer containing 125 mM Tris-Cl
(pH 6.8), 3% sodium dodecyl sulfate (SDS), 20 mM dithiotreitol, 1 mM phenyl-
methylsulfonyl fluoride (PMSF), 4 �g of leupeptin/ml, 4 �g of aprotinin/ml, and
1 �g of pepstatin/ml. After a brief sonication, soluble proteins were collected by
centrifugation at 15,000 � g. Supernatants were quantified for protein concen-
tration with a Dc protein assay kit (Bio-Rad Laboratories) and were stored at
�70° in 10% glycerol.
For immunoblotting, proteins were separated by SDS-polyacrylamide gel elec-
trophoresis (PAGE) and transferred to Immobilon-P membranes (Millipore).
Filters were then blocked with 5% nonfat dry milk in 10 mM Tris-Cl (pH
7.5)–100 mM NaCl–0.1% Tween 20 and were immunostained with anti-M45
polyclonal antibodies, the anti-R1 monoclonal antibody AD203, anti-R2 poly-
clonal antibodies (45), an anti-MCMV M44 monoclonal antibody (kindly pro-
vided by L. C. Loh, University of Saskatchewan), or an anti-actin mouse mono-
clonal antibody (Boehringer Mannheim). Immunocomplexes were then detected
by means of sheep-anti mouse immunoglobulin (Ig) or goat anti-rabbit Ig anti-
bodies, both conjugated to horseradish peroxidase (Amersham), and were visu-
alized by using enhanced chemiluminescence (Super Signal; Pierce) according to
the manufacturer’s instructions.
Immunoprecipitation. Whole-cell extracts were prepared by solubilization in
lysis buffer (150 mM NaCl, 50 mM Tris-HCl [pH 8], 1% NP-40, 1 mM phenyl-
methylsulfonyl fluoride, 4 �g of leupeptin/ml, 4 �g of aprotinin/ml, and 1 �g of
pepstatin/ml) and shaking for 20 min at 4°C. After centrifugation at 15,000 � g,
the supernatants were incubated for 1 h at 4°C on a rocking platform with either
anti-M45 antibodies or preimmune serum previously conjugated with CNBr-
activated Sepharose 4 Fast Flow (Amersham Pharmacia) according to the man-
ufacturer’s instructions. The immunocomplexes were then centrifuged (700 � g)
and washed three times with lysis buffer. The immunoprecipitated proteins were
eluted with 0.1 M glycine buffer (pH 11.5), recovered as supernatants after
centrifugation, resuspended in SDS gel sample buffer, separated by SDS-PAGE,
and subjected to immunoblotting.
Immunofluorescence microscopy. Cells grown on coverslips were infected with
MCMV at an MOI of 0.5. At 48 hpi cells were washed with phosphate-buffered
saline (PBS), fixed with 1% paraformaldehyde for 20 min at room temperature,
and washed again with PBS. They were subsequently permeabilized with 0.2%
Triton X-100 in PBS for 20 min at 4°C, washed with PBS–1% bovine serum
albumin (BSA), and incubated with the anti-M45 antibody (diluted 250-fold) in
a solution containing PBS–1% BSA and 0.2% Triton X-100 for 1 h at room
temperature. After a wash with PBS–1% BSA and 0.05% Tween 20, cells were
incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit
antibodies in PBS–1% BSA and 0.2% Triton X-100 for 1 h. Coverslips were
washed with a solution containing PBS–1% BSA and 0.05% Tween 20, and the
nuclei were counterstained with propidium iodide. After an additional wash, the
coverslips were mounted in 90% glycerol. Immunofluorescence microscopy was
performed on an Olympus IX70 inverted confocal laser scanning microscope
equipped with a krypton-argon ion laser (excitation wavelength, 488 nm; emis-
sion wavelength, 568 nm). Images derived from both channels (fluorescein and
propidium iodide) were recorded simultaneously at identical apertures. The
fluorescein-derived image was assessed with a green color, and the propidium
iodide-derived image was assessed with a red color.
Plasmid constructs. The coding sequence of the M45 gene was amplified by
PCR from total DNA of NIH 3T3 cells infected with MCMV for 48 h. The two
5� oligonucleotide primers were 5�-TCATCAACAACATATGGATCGCCAGC
CCAAAGTC-3� and 5�-TCATCAACAACATATGCACCATCATCATCATCA
TGATCGCCAGCCCAAAGTC-3�; the latter contained a sequence encoding a
six-histidine tag immediately dowstream from the ATG. The 3� oligonucleotide
primer was 5�-GCTGCTCGAGGGATCCCTTTCAGCGATAATTCACGGA-
3�. The PCR product without the histidine tag was cloned into the PCR cloning
vector pGEM-T Easy (Promega), sequenced, and then subcloned into the mam-
malian expression vector pcDNA3 (Invitrogen). The PCR product containing the
histidine tag was subcloned into the E. coli expression vector pET30a(�) (No-
vagen) and then sequenced. A 772-bp fragment encoding a C-terminal region of
M45 (from nucleotide 2563 to nucleotide 3403) was excised with the restriction
VOL. 78, 2004 CHARACTERIZATION OF THE MCMV M45 PROTEIN 4279

endonuclease FspI from the pGEM T Easy–M45 vector and subcloned in frame
in the SmaI site of the E. coli expression vector pGEX-4T3 (Novagen) in order
to express the M45 fragment as a fusion protein with glutathione S-transferase
(GST). To generate the catalytically inactive mouse R1 C429A protein, the
2.8-kb R1 cDNA fragment contained in the pET3a vector (15) was subjected to
site-directed mutagenesis with the QuikChange XL kit (Stratagene) and oligo-
nucleotide primers 5�-CCATCAAATGCAGCAACCTGGCTACAGAAATAG
TAGAGTACACC-3� and 5�-GGTGTACTCTACTATTTCTGTAGCCAGGTT
GCTGCATTTGATGG-3�. Underlining represents mutated nucleotides. The
point mutations converting cysteine 429 into an alanine were confirmed by DNA
sequence analysis. Transient transfections were performed as described previ-
ously (45).
Expression and purification of the recombinant proteins and immunizations.
Plasmid pET30a(�) containing the six-His-tagged full-length M45 sequence was
transfected into Rosetta(DE3)pLysS bacteria (Novagen). An overnight culture
of bacteria grown in Luria-Bertani broth plus kanamycin and chloramphenicol
was used to infect a 1-liter culture of TerrificBroth containing the same antibi-
otics, and the culture was grown at 30°C to an A600 of 1.0. Then the temperature
of the incubator was decreased to 15°C, and when the temperature of the liquid
had reached 20°C, isopropyl-�-D-thiogalactopyranoside (IPTG) was added to a
final concentration of 0.05 mM. After overnight incubation at 15°C, the bacteria
were harvested by centrifugation, washed, and lysed by freeze-thawing, and the
extract was clarified by centrifugation in a Beckman 70 Ti rotor for 45 min at
45,000 rpm and 4°C. The protein extract was incubated with nickel-agarose in 50
mM Tris-Cl (pH 7.6)–0.3 m NaCl–10 mM imidazole for 1 h at 4°C and exten-
sively washed with the same buffer. Adsorbed protein was eluted in 50 mM
Tris-Cl (pH 7.6)–0.2 M imidazole and then immediately equilibrated with 50 mM
Tris-Cl (pH 7.6)–0.1 M KCl on a Sephadex G-25 column. An aliquot of this
material was further purified by chromatography on a Superdex 200 column
(Amersham Biosciences) in 50 mM Tris-Cl (pH 7.6)–0.1 M KCl. The mouse R1
C429A protein was expressed and purified as described previously (15).
The fusion of the C-terminal portion of M45 to GST was produced in E. coli
BL21(DE3) (Novagen) containing plasmid pGEX-4T3-M45. Bacteria were
grown at 37°C until they reached an optical density at 600 nm (OD600) of 0.6 and
were then induced with 1 mM IPTG for 2 h. The cells were pelleted, resuspended
in lysis buffer (50 mM Tris-HCl [pH 8], 2 mM EDTA, 1 mM dithiothreitol
[DTT], 1% Triton X-100, 500 �g of lysozyme/ml, 1 �g of pepstatin/ml, 2 �g of
leupeptin/ml) at 30°C for 15 min, and briefly sonicated. Analysis of the soluble
and insoluble fractions by SDS-PAGE followed by Coomassie blue staining
revealed that more than 90% of the recombinant protein was present in the
insoluble fraction (inclusion bodies). Inclusion bodies were washed twice with
lysis buffer supplemented with 2 M urea and then solubilized in lysis buffer
supplemented with 6 M urea. The denatured recombinant proteins were refolded
by dialysis against folding buffer (0.1% NP-40, 0.2 mM EDTA, 1 mM DTT, 200
mM PMSF, 0.5 M NaCl, 0.1 M Tris-HCl [pH 7.5]) containing decreasing con-
centrations of urea from 5 to 0.5 M. The last dialysis was performed against
folding buffer without urea. The refolded protein was concentrated through a
centrifugal filter device (Amicon), analyzed by SDS-PAGE followed by Coomas-
sie blue staining, and then used as an antigen for rabbit immunization in order
to generate specific antisera. Rabbits were injected intramuscularly with 200 �g
of protein five times and were bled after 3 months.
dNTP pool measurements. Cell cultures were extracted with ice-cold trichlo-
roacetic acid. The extracted nucleotides were separated directly by high-perfor-
mance liquid chromatography (ribonucleoside triphosphates) or first run through
a boronate affinity column (deoxyribonucleoside triphosphates) as described
previously (35). Levels of nucleotide pools are expressed as percentages of the
total nucleoside triphosphate pool (CTP � UTP � ATP � GTP � dCTP �
dTTP � dATP � dGTP) to minimize variations due to small differences in cell
number in the samples.
RNR assay. The ability of the purified M45 protein to catalyze the reduction
of [3H]CDP to [3H]dCDP at 37°C in the presence of recombinant mouse R2
protein, or to stimulate the reduction catalyzed by recombinant mouse R1 and
R2 proteins or recombinant mouse R1 C429A mutant protein and R2 protein,
was assayed as described elsewhere (21). In addition to the standard ATP-
stimulated reduction, a putative effect of the M45 protein on the allosteric
inhibition by dATP was assayed by the addition of 20 to 400 �M dATP to the
assay mixture.
In vivo growth of MCMV mutants. CB17 SCID mice (eight mice per group),
maintained under pathogen-free conditions in our animal facility, were infected
intraperitoneally with 106 PFU of either an M45 mutant virus or a control
(wild-type) virus and were observed daily for survival. At 24 days postinfection,
spleens, livers, lungs, kidneys, and salivary glands were aseptically harvested from
two mice per group, homogenized, and titrated in duplicate on NIH 3T3 cells by
plaque assay. Values given were calculated per gram of organ.
RESULTS
Generation and reactivity of an M45 antiserum. In order to
obtain protein for the generation of a specific antiserum, a
fragment of the M45 ORF encoding the hydrophilic C-termi-
nal domain was cloned into the prokaryotic expression vector
pGEX-4T3 and expressed as a GST fusion protein in E. coli.
The purified protein was then used to immunize rabbits, and
the resulting serum was tested for its specificity by Western
blot analysis. As shown in Fig. 1A, a strong signal correspond-
ing to a protein of 150 to 160 kDa was observed in lysates from
MCMV-infected NIH 3T3 cells at 48 hpi (lane 2), from cells
transiently transfected with the M45 expression vector
pcDNA3-M45 (lane 4), and from a lysate of IPTG-induced E.
coli cells transformed with the expression vector pET30-M45
(lane 5). By contrast, no signal could be observed with lysates
from mock-infected cells (lane 1) or from cells transfected with
the empty pcDNA3 vector (lane 3) or on the same blot incu-
bated with preimmune serum (data not shown). Immunopre-
cipitation followed by anti-M45 immunoblotting of lysates
from MCMV-infected cells revealed M45 (150 to 160 kDa)
and a second major polypeptide of about 116 kDa (Fig. 1B,
FIG. 1. Identification of the MCMV M45 protein by the specific
antiserum. (A) Immunoblot analysis of native or recombinant M45
expression in MCMV-infected NIH 3T3 cells, in cells transfected with
an M45 expression vector, or in E. coli. Proteins of whole-cell lysates
were separated by SDS-PAGE (5 to 15% acrylamide), transferred to a
membrane, and probed with the M45 antiserum. Lanes: 1, extracts
from mock-infected NIH 3T3 cells; 2, extracts from NIH 3T3 cells 48 h
after infection with MCMV; 3, extracts from NIH 3T3 cells transiently
transfected with the control vector pcDNA3; 4, extracts from NIH 3T3
cells transiently transfected with the M45 expression vector pcDNA3-
M45; 5, extracts from IPTG-induced E. coli containing the M45 ex-
pression vector pET30-M45. (B) Immunoprecipitation of M45 by the
specific antiserum. M45 was immunoprecipitated from cell extracts of
MCMV-infected NIH 3T3 cells prepared at 48 hpi and was analyzed by
immunoblotting with the M45 antiserum. Lanes: 1, cell extract from
infected cells; 2, cell extract immunoprecipitated with the M45 anti-
serum; 3, cell extract immunoprecipitated with preimmune serum.
Sizes of the molecular mass markers are shown on the left of each
panel. Asterisk indicates the Ig heavy chains recognized by the sec-
ondary antibody.
4280 LEMBO ET AL. J. VIROL.

lane 2) whose molecular weight corresponded to that of the
faint band observed in Fig. 1A, lane 2. The same bands could
not be detected when preimmune serum was used (Fig. 1B,
lane 3). To determine whether the lower band corresponds to
a proteolytic fragment of M45, the immunoprecipitates from
infected cells were blotted and the Coomassie-stained bands
were subjected to N-terminal sequencing. The N-terminal se-
quence of the 116-kDa polypeptide was AATMPPP, which
corresponds to cleavage between tyrosine 277 and alanine 278
of M45. Thus, the lower band represents a polypeptide gener-
ated by specific M45 proteolysis. Altogether, these results dem-
onstrate that a specific antiserum was generated for further
analysis of M45 expression.
Time course of M45 protein expression. To investigate the
kinetics of M45 expression during viral replication, cell lysates
were harvested at various time points after MCMV infection
and examined by immunoblotting with the M45 antiserum. A
specific band at approximately 150 kDa was first detected at 12
hpi and accumulated throughout the course of infection (Fig.
2A). This finding is consistent with the kinetics of M45 mRNA
accumulation, which have been reported previously (45).
Treatment with phosphonoformic acid (PFA), a specific in-
hibitor of viral DNA polymerase, did not affect M45 expression
at 18 and 24 hpi but reduced its expression at 48 hpi (Fig. 2B).
Therefore, M45 expression is not dependent on the onset of
viral DNA synthesis.
Subcellular localization of M45. Subcellular localization of
M45 was determined by indirect immunofluorescence and con-
focal microscopy of both infected and transiently transfected
cells. NIH 3T3 cells infected for 48 h with MCMV at an MOI
of 0.1 PFU/cell were incubated with the M45-specific anti-
serum and then stained with fluorescein-conjugated anti-rabbit
antibodies. Nuclei were counterstained with propidium iodide.
As shown in Fig. 2C, M45 was quite dispersed throughout the
cytoplasm, though a more intense signal was associated with
granular formations. By contrast, no specific nuclear signal
could be detected. A similar pattern of cytoplasmic staining
was observed in cells transiently transfected with the M45
FIG. 2. (A) Time course of M45 expression during MCMV replication as determined by immunoblotting. Whole-cell lysates of mock-infected
and MCMV-infected NIH 3T3 cells at various times after infection were separated by SDS-PAGE, transferred to a membrane, and probed with
the anti-M45 antiserum or with the anti-actin monoclonal antibody. Lanes: 1, mock-infected cells; 2, 6 hpi; 3, 9 hpi; 4, 12 hpi; 5, 18 hpi; 6, 24 hpi;
7, 36 hpi; 8, 48 hpi. (B) Effect of PFA on M45 expression. Whole-cell extracts were prepared at 18 hpi (lanes 1 and 2), 24 hpi (lanes 3 and 4), or
48 hpi (lanes 5 and 6) from MCMV-infected NIH 3T3 cells treated with PFA (250 �g/ml) after virus adsorption (lanes 2, 4, and 6) or left untreated
(lanes 1, 3, and 5). Protein expression was analyzed by immunoblotting with the anti-M45 antiserum or with the anti-actin monoclonal antibody.
(C) Subcellular localization of M45 in MCMV-infected NIH 3T3 cells at 48 hpi, detected by immunofluorescence and confocal microscopy. Cells
were incubated with the M45 antiserum and then with the secondary FITC-conjugated antibody. Nuclei were counterstained with propidium
iodide. (D) Subcellular localization of transiently expressed M45 protein in NIH 3T3 cells transfected with the pcDNA3-45 vector at 24 h
posttransfection. For immunofluorescence and confocal microscopy analysis, cells were incubated with the M45 antiserum and then with the
secondary FITC-conjugated antibody. Nuclei were counterstained with propidium iodide. The merged pictures are shown.
VOL. 78, 2004 CHARACTERIZATION OF THE MCMV M45 PROTEIN 4281

expression vector pcDNA3-M45, indicating that no other viral
protein is required for M45 localization (Fig. 2D). No signal
could be detected with mock-infected cells or when infected
cells were stained with preimmune serum (data not shown).
Virion association of M45. Proteins from herpesviruses
abundantly expressed at late times are often incorporated into
virus particles during assembly; therefore, we looked for M45
in the MCMV particles. Extracellular virions were purified
from tissue culture supernatants by two rounds of centrifuga-
tion through a 15% sucrose cushion or via density gradient
centrifugation and were then subjected to Western blot anal-
ysis. An extract from infected NIH 3T3 cells at 48 hpi was used
in parallel as a positive control (Fig. 3, lane 1). In the protein
extracts of purified virions, the M45 antiserum detected a pro-
tein of approximately 150 kDa as well as lower-molecular-mass
products, probably the result of proteolysis (Fig. 3, lanes 2 and
3). To assess the purity of the virion preparation, we performed
immunoblot analyses with monoclonal antibodies against the
nonstructural viral protein M44 and against the cellular pro-
teins actin and the R2 subunit of RNR, which are constitutively
expressed or highly induced by infection, respectively. M44,
actin, and R2 were detected in the cell lysate (Fig. 3, lane 1),
but not in the virion preparation (Fig. 3, lanes 2 and 3). This
observation ruled out the possibility that the M45 signal in
virions is due to contamination of the virion preparation with
cellular material and demonstrated that M45 is associated with
the virion particle.
M45 is not a functional RNR R1 subunit. Since M45 displays
the sequence features of a class Ia RNR R1 subunit, we looked
to see whether it forms an enzymatically active RNR complex
in vitro together with the cellular R2 subunit. Recombinant
His-tagged M45 protein was purified by chromatography on a
Ni-agarose column followed by chromatography on a Superdex
200 column. A 150- to 160-kDa protein band was clearly seen
in the Ni-agarose column eluate, and its identity as M45 was
confirmed in Western blots by using the M45 antiserum (data
not shown). However, many other, smaller polypeptides were
present in the eluate where the major bands react with the
antibody; these most probably represent proteolytically de-
graded M45. On the Superdex column, the material eluting as
a symmetrical peak with the void volume contains about 50%
full-length M45 and 50% degraded protein (Fig. 4A). Since
proteins with a molecular mass of around 600 kDa elute in this
position, M45 is most probably eluting as an oligomeric com-
plex.
In the RNR assay, our recombinant M45 alone or together
with an excess of mouse R2 never showed any significant re-
duction of CDP in repeated assays and with different protein
preparations. By contrast, there was a small but reproducible
stimulation of the activity of mouse R1 assayed in the presence
of an excess of R2 when M45 was added (Fig. 4B). This does
not appear to be a general protein effect, since addition of
truncated M45 protein or BSA resulted in no stimulation (data
not shown). We tested the hypothesis that, in analogy with
yeast Rnr3p (19), heterodimerization of R1 and M45 facilitates
the recruitment of M45 to the holoenzyme. Therefore, we
observed RNR activity when M45 was added to an assay sys-
tem consisting of catalytically inactive mouse R1 C429A pro-
tein together with an excess of R2. Again, no CDP reduction
was observed (Fig. 4C). Lastly, we determined whether addi-
tion of M45 to an assay system containing mouse R1 and R2
changed the allosteric regulation of the mouse RNR, and es-
pecially whether M45 made the activity more resistant to
dATP. However, addition of M45 had no effect on dATP
inhibition (Fig. 4D). This confirms an earlier observation that
M45 does not bind to dATP-Sepharose, in contrast to R1. In
conclusion, our recombinant M45 protein displays no RNR
activity.
MCMV induces dNTP pool expansion through ribonucle-
otide reduction in resting cells. The finding that MCMV does
not express a functional RNR R1 subunit prompted us to
investigate whether it relies on the cellular enzyme to obtain
dNTP in resting cells. To address this point, we first measured
the steady-state levels of the cellular RNR subunits during the
course of viral infection in serum-starved NIH 3T3 cells. Cell
extracts were prepared at different times postinfection and
analyzed by immunoblotting using anti-R1 monoclonal anti-
bodies and anti-R2 polyclonal antibodies (Fig. 5A). As ex-
pected, R1 and R2 were not expressed in uninfected cells and
were induced by serum stimulation. The expression of R2 was
upregulated in the infected cells as early as 6 hpi, and its level
increased significantly as the infection progressed. R1 was also
induced by the infection, though to a lesser extent. The ab-
sence of any signal when cells were infected with UV-inacti-
vated viruses indicates that neither potential serum contami-
nation of viral preparations nor binding and entry of the
inactivated virus particles were responsible for R1 and R2
induction.
This result led us to investigate whether the virus-induced
upregulation of the cellular RNR subunits results in increased
ribonucleotide reduction.
Therefore, we studied the effect of hydroxyurea (HU), a
specific RNR inhibitor, on the dNTP pools of quiescent
MCMV-infected cells at 48 hpi. As in an earlier study (45), we
observed that all four dNTP pools expanded after MCMV
infection compared to a sample from cells infected with a
UV-inactivated virus (Fig. 5B). When the pool measurements
FIG. 3. Detection of M45 in purified MCMV particles by immu-
noblotting. Proteins from a whole-cell lysate of MCMV-infected NIH
3T3 cells at 48 hpi (lane 1) or from virus particles purified by two
rounds of centrifugation through a 15% sucrose cushion (lane 2) or via
density gradient centrifugation (lane 3) were separated by SDS-PAGE,
blotted onto a membrane, and probed with antibodies against the viral
proteins M45 and M44 and the cellular proteins R2 and actin.
4282 LEMBO ET AL. J. VIROL.

were performed on infected cells grown in the presence of 0.5
mM HU, a significant drop in the level of the pyrimidine pools
was observed, while the purines totally disappeared, indicating
that MCMV obtains dNTP through ribonucleotide reduction.
We also measured the virus yield in the supernatants from the
cell cultures used for the dNTP pool assay. The yield in un-
treated cells was 2.3 � 104 PFU/ml, whereas no infectious virus
was found in HU-treated cells, in accordance with our previous
observations (45). Taken together, these findings demonstrate
that MCMV replication in resting cells is dependent on host
ribonucleotide reduction induced by the virus itself.
In vivo growth of M45 MCMV mutants. To determine
whether M45 is required for viral replication in its natural host,
the growth of the M45-mutant viruses IIIG2 and BamX was
compared with that of a wild-type-like MCMV expressing GFP
(MCMV-GFP), a BAC-derived wild-type MCMV (bd-
MCMV), and a revertant MCMV in immunodeficient CB17
SCID mice, which lack functional T and B lymphocytes. SCID
mice are sensitive to extremely low levels of MCMV replica-
tion and succumb within 4 weeks postinfection. Infection with
the wild-type and revertant viruses resulted in 100% mortality
within 5 weeks postinoculation. In contrast, infection with the
same dose of IIIG2 or BamX produced no mortality. Figure
6A shows results for mice infected with one of the GFP-ex-
pressing viruses MCMV-GFP and IIIG2, whereas Fig. 6B
shows results for mice infected with one of the viruses without
GFP, i.e., bd-MCMV, MCMV-REV, and BamX. Because the
M45 mutants did not cause lethal infection over the period of
observation (up to 60 days), we determined virus titers in target
organs by plaque assay 24 days after infection, which corre-
sponds roughly to the time of death for the bd-MCMV-in-
fected mice. As shown in Fig. 6C, infection with wild-type and
revertant viruses yielded virus titers in the spleen, liver, kid-
neys, lungs, and salivary glands, indicating efficient viral spread
and replication. In contrast, titers of IIIG2 and BamX were
undetectable in all target organs. Hence, M45 mutant viruses
are defective with respect to replication in their natural host.
DISCUSSION
This work addresses the long-standing question of whether
the RNR R1 homolog encoded by CMV is a functional enzyme
subunit. We carried out our study on the MCMV M45 protein
because we were also interested in assessing its role in viral
pathogenesis in vivo. The M45 reading frame has a length of
3,522 bp and encodes a 1,174-amino-acid protein with an ex-
FIG. 4. RNR assays. (A) SDS-PAGE analyses of purified recombinant M45. Lane 1, molecular mass markers at 203, 120, 90, and 51 kDa; lane
2, recombinant His-tagged M45 purified by chromatography on a nickel-agarose column followed by chromatography on a Superdex 200 column.
(B) Catalytic activity of recombinant M45 assayed in the presence of an excess of mouse R2 protein (10 �g) before and after the addition of a
constant amount of mouse R1 protein (2 �g). The increasing amounts of purified recombinant M45 are 0, 7, 14, and 28 �g. (C) Catalytic activity
of recombinant M45 assayed in the presence of an excess of mouse R2 (10 �g) together with a constant amount of mouse R1 (2 �g) or of the
catalytically inactive R1 C429A protein (9 �g). The increasing amounts of purified recombinant M45 are 0, 7, and 14 �g. (D) Allosteric inhibition
of ATP-stimulated CDP reduction by dATP. Catalytic activity of the mouse complex R1–R2 alone or together with recombinant M45 protein was
assayed in the presence of 0, 20, 80, or 400 �M dATP.
VOL. 78, 2004 CHARACTERIZATION OF THE MCMV M45 PROTEIN 4283

pected molecular mass of 125 kDa. Since no data on gene
expression from the M45 gene were available, we generated a
specific polyclonal antiserum to study its product. M45 is de-
tected as a polypeptide of 150 to 160 kDa in lysates from
MCMV-infected cells and cells transfected with an M45 ex-
pression plasmid. The same result was obtained when recom-
binant M45 expressed in E. coli was analyzed. The discrepancy
between the molecular mass estimated by SDS-PAGE and that
expected from the sequence is most probably caused by an
anomalous behavior of M45 in SDS-PAGE and not by post-
translational modifications, since these do not occur in E. coli.
Immunofluorescence and Western blot analysis revealed that
M45 is a cytoplasmic protein abundantly expressed at early and
late times in infection irrespective of the onset of viral DNA
synthesis. Moreover, we found that it is associated with puri-
fied viral particles, such as the R1 subunit of HSV-2 (61).
FIG. 5. Upregulation of cellular RNR expression and activity by MCMV. (A) Cellular R1 and R2 levels during MCMV infection. NIH 3T3 cells
were growth-arrested in 0.5% calf serum and then either infected with active or UV-irradiated MCMV (MOI, 5 PFU/cell) or mock-infected.
Whole-cell extracts were prepared at various times after infection, separated by SDS-PAGE, and analyzed by immunoblotting with the anti-R1,
anti-R2, and anti-actin antibodies. A sample from quiescent cells stimulated with 10% calf serum for 24 h was also included. Lanes: 1, mock
infection; 2, 6 hpi; 3, 12 hpi; 4, 24 hpi; 5, 36 hpi; 6, 48 hpi; 7, 60 hpi; 8, UV-inactivated MCMV 48 hpi; 9, noninfected cells grown in the presence
of 10% serum. (B) Effect of MCMV infection on dNTP pool sizes in resting cells. NIH 3T3 cells were growth arrested in 0.5% calf serum and then
infected with active or UV-inactivated MCMV (MOI, 5 PFU/cell). After virus adsorption, cells were either treated with 0.5 mM HU or left
untreated. The nucleotides were extracted at 48 hpi, and their levels were measured by high-performance liquid chromatography. Levels of
nucleotide pools are expressed as percentages of the total nucleoside triphosphate pool (CTP � UTP � ATP � GTP � dCTP � dTTP � dATP
� dGTP) to minimize variations due to small differences in cell number in the samples.
4284 LEMBO ET AL. J. VIROL.

The idea that CMV encodes an RNR R1 subunit is based on
the following arguments. Blast searches of the SwissProt pro-
tein database (http://www.ncbi.nlm.nih.gov:80/BLAST/) per-
formed with the M45 amino acid sequence identify the C-
terminal region of M45 as a homolog of a class Ia RNR R1
subunit. The closest matches occur with other herpesvirus R1
subunits (e.g., 27% amino acid identity to the HCMV R1
subunit, 24% to the human herpesvirus 6 [HHV-6] R1 subunit,
and 22% to the HSV-1 and HSV-2 R1 subunits). Interestingly,
like the HSV-1 and HSV-2 subunits, M45 possesses an N-
terminal extension that makes it the longest herpesvirus R1
subunit. Moreover, an in silico structural and functional anal-
ysis of the HCMV genome yielded complete structural identi-
fication of the UL45 protein, a homolog of M45, as a RNR R1
subunit (54). Finally, both the UL45 and the M45 gene are
positionally conserved with the gene for the RNR R1 subunit
in other herpesviruses. However, the CMV genomes lack an
apparent homolog of the RNR R2 subunit, which is position-
ally replaced by a gene encoding the processivity factor of the
viral DNA polymerase (UL44 for HCMV and M44 for
MCMV). Since M45 exhibits sequence features of a class Ia R1
subunit, it is expected to complex to an R2 subunit to form an
active enzyme; it is highly improbable that it forms a single-
subunit RNR like the class II enzymes expressed by Eubacteria
and Archaebacteria. Therefore, we raised the hypothesis that it
might usurp the place of the cellular R1 to form a hybrid
version of the enzyme together with the cellular R2 subunit,
whose expression is upregulated by MCMV infection (45; this
paper). However, the results obtained when recombinant M45
was tested in the RNR assays indicate that it is not a functional
FIG. 6. (A) Survival curves of SCID mice infected with a wild-type virus (MCMV-GFP) or with an M45 mutant virus (IIIG2) expressing GFP.
(B) Survival curves of SCID mice infected with wild-type (bd-MCMV), revertant (MCMV-REV), or M45 mutant (BamX) viruses without GFP.
SCID mice were injected intraperitoneally with 106 PFU of virus and were monitored daily for survival. (C) Accumulation of infectious viruses in
the spleen, liver, kidneys, lungs, and salivary glands. Two SCID mice for each group were infected as described above. The animals were killed 24
days after infection, and total levels of virus in homogenates of target organs were determined by plaque assays on NIH 3T3 cells. Each data point
is the average for two animals.
VOL. 78, 2004 CHARACTERIZATION OF THE MCMV M45 PROTEIN 4285

equivalent of a class Ia R1 subunit, since it has no activity
together with R2. Moreover, it has no activity alone, excluding
the remote possibility that it behaves like a homomeric RNR.
Similar results were obtained when native M45 was immuno-
purified from MCMV-infected cells and assayed for activity
(data not shown). This finding rules out the possibility that
M45 requires some kind of modifications carried out by the
infected cells for its activity and that the His tag in the recom-
binant protein could affect its function. It is noteworthy that
another betaherpesvirus R1 homolog, the U28 protein en-
coded by HHV-7, has no activity in an RNR assay (65).
The small but reproducible stimulation of the enzyme activ-
ity observed when M45 was added to an assay mixture con-
taining R1 and R2 was reminiscent of the cross talk between
the two R1 subunits encoded by Saccharomyces cerevisiae
called Rnr1 and Rnr3. It has been proposed that heterodimer-
ization of Rnr3 with Rnr1 facilitates the recruitment of Rnr3 to
the holoenzyme and results in a synergism that is more evident
when Rnr3 is allowed to form a complex with a catalytically
inactive form of Rnr1 (19). However, when we assessed
whether this model applies to M45, we found no M45 activity
in an assay containing a catalytically inactive form of R1 and an
excess of R2, indicating that M45 does not behave like the
yeast Rnr3 subunit.
Unlike the cellular enzymes, the herpesvirus RNRs com-
pletely lack allosteric regulation as well as most of the residues
involved in effector binding in the E. coli and mammalian
enzymes at both the activity and specificity sites (12, 42), indi-
cating that the function is unnecessary or perhaps detrimental
for viral replication. These considerations, along with the ob-
servation that M45 does not bind to the inhibitory effector
dATP, led us to test the hypothesis that M45 may functionally
interact with the cellular enzyme by affecting its allosteric con-
trol. However, our results demonstrated that M45 does not
make the activity of the cellular enzyme resistant to dATP
inhibition.
Finally, along with the lack of a functional interaction with
host RNR, we could not find any physical interactions between
M45 and cellular R1 and R2 in the immunoprecipitates from
infected cell extracts (data not shown). Taken together, the
results from the enzymatic assays indicate that during viral
coevolution with the host, M45 has lost its direct involvement
in ribonucleotide reduction and has become catalytically inac-
tive. Such loss of function is further highlighted by the amino
acid sequence comparison between M45 and the R1 subunit of
E. coli, which represents the prototype of class Ia, and the R1
proteins of HSV-1 and Epstein-Barr virus (EBV), chosen as
representatives of alpha- and gammaherpesviruses. As de-
picted in Fig. 7, sequence alignment analysis with the ClustalW
program (http://www.ebi.ac.uk/clustalw/index.html) revealed
that the residues shown to have a catalytic role in the E. coli R1
subunit (38) are highly conserved in the HSV-1 and EBV R1
proteins. These include E. coli cysteines 225, 439, 462, 754, and
759 and tyrosines 730 and 731. By contrast, only cysteine 814
(corresponding to cysteine 439 in E. coli) is conserved in the
M45 sequence. Moreover, the proposed GxGxxG nucleotide-
binding site (E. coli residues 514 to 519) is only partially con-
served in the M45 sequence (xxGxxG at residues 890 to 895).
When we performed sequence alignment analysis with the R1
proteins of all the betaherpesviruses for which sequencing in-
formation is available, we found that the lack of important
catalytic residues is a general feature of the subfamily. These
findings are consistent with the view that betaherpesviruses
differ from the other two subfamilies in the strategy they have
evolved to satisfy their need for DNA precursors. The alpha-
and gammaherpesvirus genomes encode a functional RNR,
thymidine kinase (TK), and dUTPase, which are essential en-
zymes for nucleotide anabolism and presumably are expressed
to provide dNTP for viral DNA synthesis in resting cells where
the cellular enzymes are not expressed (58). Although we can-
not exclude the possibility that another viral protein is required
for M45 RNR activity, betaherpesviruses seem to have aban-
doned the strategy of supplying enzymes involved in the bio-
synthesis of DNA precursors, since the genes for a TK and the
RNR R2 subunit are absent and the genes for the dUTPase
and the RNR R1 subunit are mutated and encode catalytically
inactive proteins (48, 49, 65; this work). It was previously re-
ported that CMV has evolved the ability to induce the expres-
sion of the cellular enzymes involved in the biosynthesis of
thymidylate, such as folylpolyglutamate synthase (9), dihydro-
folate reductase (44, 46), thymidylate synthase (26, 28), and
dCMP deaminase (27), in order to replicate in resting cells.
Consistent with the view that CMV relies on cellular enzymes
for dNTP biosynthesis is the finding that both MCMV (this
paper) and HCMV (62) induce expression of the R1 and R2
subunits of the cellular RNR. Moreover, we have demon-
strated that increased expression of these subunits results in
expansion of all four dNTP pools in resting fibroblasts. The
finding that treatment of infected cells with HU leads to a
specific drop in both purine and pyrimidine pools confirmed
that virus-induced pool expansion is a consequence of ribonu-
cleotide reduction and not of the triggering of a salvage path-
way. Importantly, inhibition of MCMV replication by HU re-
vealed that the virus-induced host RNR activity is essential for
a productive infection in resting cells. We propose that emer-
gence of this peculiar replicative strategy during the course of
CMV evolution has allowed many genes involved in nucleotide
metabolism to be selectively eliminated from the viral genome
or to mutate and gain new functions.
Several considerations suggest that M45 is not a vestigial
gene encoding a useless function. First, since its reading frame
FIG. 7. Line diagram representing the amino acid sequence align-
ment of M45 with the R1 proteins of E. coli, HSV-1, and EBV. The
proposed nucleotide-binding site (GxGxxG) and the five cysteines and
two tyrosines that have a direct catalytic role, as discussed in the text,
are highly conserved in the E. coli, HSV-1, and EBV R1 proteins. By
contrast, most of these residues are absent in the M45 sequence.
4286 LEMBO ET AL. J. VIROL.

has remained open, it can be presumed to encode a functional
protein. Second, its abundant expression at late times and its
association with the viral particle may point to its involvement
in virus maturation or a function immediately after virus entry,
when it is delivered to the cytoplasm of newly infected cells.
This question is the subject of current investigation. Third,
previous results have shown that MCMV mutants carrying an
inactivated M45 gene do not replicate in two different endo-
thelial cell lines and have a greatly reduced ability to grow on
macrophages, whereas they grow almost normally on fibro-
blasts (6). M45 was shown to protect cells from premature
apoptosis induced by the virus (6, 7). This antiapoptotic activity
is seen in different cell types but is most prominent in endo-
thelial cells, where it is required for virus replication and
spread. Apparently, endothelial cells are particularly sensitive
to virus-induced apoptosis. Surprisingly, the HCMV UL45
protein seems to be dispensable for HCMV growth in human
umbilical endothelial cells (29). Because macrophages and en-
dothelial cells play key roles in CMV dissemination (4, 13, 36,
55, 59, 64), viral gene products which regulate growth in these
cell types should significantly affect MCMV pathogenesis in
vivo. This has, for instance, been shown for MCMV mutants
with deletions in the m139-to-m141 region, which are growth
deficient in macrophages and attenuated in mice (30, 31). To
directly assess the role of M45 in viral pathogenesis, we inves-
tigated the virulence of two M45 mutant viruses (a transposon
insertion mutant and a frameshift mutant) in SCID mice. We
found that these viruses are avirulent in SCID mice, since they
do not kill the animals, and their replication is undetectable in
target organs. This failure to replicate in SCID mice is highly
significant, as these animals are extremely susceptible to
MCMV infection, and warrants further investigation to define
the mechanism underlying this highly attenuated phenotype. In
summary, the arguments outlined above support the view that
M45 has abandoned its function as a RNR subunit and ac-
quired a new function, probably completely unrelated to RNR
activity, that is essential for cellular tropism and viral patho-
genesis in vivo. Protecting cells from virus-induced apoptosis is
clearly a new function of M45, and it makes sense that MCMV
incorporates such a protein into the virion for immediate avail-
ability in the infected cell. Since herpesvirus proteins are often
multifunctional, it is possible that M45 has acquired an addi-
tional function, which adds to the observed phenotype. Further
studies are required to assess whether this loss and gain of
function apply to other R1 homologs of the Betaherpesvirinae
subfamily, and to understand their newly acquired functions.
ACKNOWLEDGMENTS
We thank Margareta Thelander for excellent technical assistance
and Peter Reichard for manuscript revision.
This work was supported by grants from the Associazione Italiana
per la Ricerca sul Cancro, from Ricerca Sanitaria Finalizzata (Regione
Piemonte), and from Program 40% (MIUR). U.K. and W.B. were
supported by the Deutsche Forschungsgemeinschaft (SFB 455 and
SFB 479, respectively).
REFERENCES
1. Angulo, A., P. Ghazal, and M. Messerle. 2000. The major immediate-early
gene ie3 of mouse cytomegalovirus is essential for viral growth. J. Virol.
74:11129–11136.
2. Averett, D. R., C. Lubbers, G. B. Elion, and T. Spector. 1983. Ribonucleotide
reductase induced by herpes simplex type 1 virus. Characterization of a
distinct enzyme. J. Biol. Chem. 258:9831–9838.
3. Bjorklund, S., S. Skog, B. Tribukait, and L. Thelander. 1990. S-phase-
specific expression of mammalian ribonucleotide reductase R1 and R2 sub-
unit mRNAs. Biochemistry 29:5452–5458.
4. Brautigam, A. R., F. J. Dutko, L. B. Olding, and M. B. Oldstone. 1979.
Pathogenesis of murine cytomegalovirus infection: the macrophage as a
permissive cell for cytomegalovirus infection, replication and latency. J. Gen.
Virol. 44:349–359.
5. Bresnahan, W. A., I. Boldogh, E. A. Thompson, and T. Albrecht. 1996.
Human cytomegalovirus inhibits cellular DNA synthesis and arrests produc-
tively infected cells in late G1. Virology 224:150–160.
6. 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.
6a.Brune, W., C. Menard, J. Heesemann, and U. H. Koszinowski. 12 January
2001, posting date. A ribonucleotide reductase homolog of cytomegalovirus
and endothelial cell tropism. Supplementary material. Science 291:303–305.
[Online.] http://www.sciencemag.org/cgi/content/full/291/5502/303/DC1.
7. Brune, W., M. Nevels, and T. Shenk. 2003. Murine cytomegalovirus m41
open reading frame encodes a Golgi-localized antiapoptotic protein. J. Virol.
77:11633–11643.
8. Cameron, J. M., I. McDougall, H. S. Marsden, V. G. Preston, D. M. Ryan,
and J. H. Subak-Sharpe. 1988. Ribonucleotide reductase encoded by herpes
simplex virus is a determinant of the pathogenicity of the virus in mice and
a valid antiviral target. J. Gen. Virol. 69:2607–2612.
9. Cavallo, R., D. Lembo, G. Gribaudo, and S. Landolfo. 2001. Murine cyto-
megalovirus infection induces cellular folylpolyglutamate synthetase activity
in quiescent cells. Intervirology 44:224–226.
10. Chabes, A., and L. Thelander. 2000. Controlled protein degradation regu-
lates ribonucleotide reductase activity in proliferating mammalian cells dur-
ing the normal cell cycle and in response to DNA damage and replication
blocks. J. Biol. Chem. 275:17747–17753.
11. 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.
12. Cohen, G. H. 1972. Ribonucleotide reductase activity of synchronized KB
cells infected with herpes simplex virus. J. Virol. 9:408–418.
13. Collins, T. M., M. R. Quirk, and M. C. Jordan. 1994. Biphasic viremia and
viral gene expression in leukocytes during acute cytomegalovirus infection of
mice. J. Virol. 68:6305–6311.
14. Conner, J., H. Marsden, and J. B. Clements. 1994. Ribonucleotide reductase
of herpes viruses. Rev. Med. Virol. 4:25–34.
15. Davis, R., M. Thelander, G. J. Mann, G. Behravan, F. Soucy, P. Beaulieu, P.
Lavallee, A. Graslund, and L. Thelander. 1994. Purification, characteriza-
tion, and localization of subunit interaction area of recombinant mouse
ribonucleotide reductase R1 subunit. J. Biol. Chem. 269:23171–23176.
16. Davison, A. J., and J. E. Scott. 1986. The complete DNA sequence of
varicella-zoster virus. J. Gen. Virol. 67:1759–1816.
17. de Wind, N., A. Berns, A. Gielkens, and T. Kimman. 1993. Ribonucleotide
reductase-deficient mutants of pseudorabies virus are avirulent for pigs and
induce partial protective immunity. J. Gen. Virol. 74:351–359.
18. Dittmer, D., and E. S. Mocarski. 1997. Human cytomegalovirus infection
inhibits G1/S transition. J. Virol. 71:1629–1634.
19. Domkin, V., L. Thelander, and A. Chabes. 2002. Yeast DNA damage-induc-
ible Rnr3 has a very low catalytic activity strongly stimulated after the
formation of a cross-talking Rnr1/Rnr3 complex. J. Biol. Chem. 277:18574–
18578.
20. Engstrom, Y., S. Eriksson, I. Jildevik, S. Skog, L. Thelander, and B. Tribu-
kait. 1985. Cell cycle-dependent expression of mammalian ribonucleotide
reductase. Differential regulation of the two subunits. J. Biol. Chem. 260:
9114–9116.
21. Engstrom, Y., S. Eriksson, L. Thelander, and M. Akerman. 1979. Ribonu-
cleotide reductase from calf thymus. Purification and properties. Biochem-
istry 18:2941–2948.
22. Eriksson, S., A. Graslund, S. Skog, L. Thelander, and B. Tribukait. 1984.
Cell cycle-dependent regulation of mammalian ribonucleotide reductase.
The S phase-correlated increase in subunit M2 is regulated by de novo
protein synthesis. J. Biol. Chem. 259:11695–11700.
23. Fortunato, E. A., A. K. McElroy, I. Sanchez, and D. H. Spector. 2000.
Exploitation of cellular signaling and regulatory pathways by human cyto-
megalovirus. Trends Microbiol. 8:111–119.
24. Goldstein, D. J., and S. K. Weller. 1988. Factor(s) present in herpes simplex
virus type 1-infected cells can compensate for the loss of the large subunit of
the viral ribonucleotide reductase: characterization of an ICP6 deletion
mutant. Virology 166:41–51.
25. Goldstein, D. J., and S. K. Weller. 1988. Herpes simplex virus type 1-induced
ribonucleotide reductase activity is dispensable for virus growth and DNA
synthesis: isolation and characterization of an ICP6 lacZ insertion mutant.
J. Virol. 62:196–205.
26. Gribaudo, G., L. Riera, D. Lembo, M. De Andrea, M. Gariglio, T. L. Rudge,
L. F. Johnson, and S. Landolfo. 2000. Murine cytomegalovirus stimulates
VOL. 78, 2004 CHARACTERIZATION OF THE MCMV M45 PROTEIN 4287

cellular thymidylate synthase gene expression in quiescent cells and requires
the enzyme for replication. J. Virol. 74:4979–4987.
27. Gribaudo, G., L. Riera, P. Caposio, F. Maley, and S. Landolfo. 2003. Human
cytomegalovirus requires cellular deoxycytidylate deaminase for replication
in quiescent cells. J. Gen. Virol. 84:1437–1441.
28. Gribaudo, G., L. Riera, T. L. Rudge, P. Caposio, L. F. Johnson, and S.
Landolfo. 2002. Human cytomegalovirus infection induces cellular thymidy-
late synthase gene expression in quiescent fibroblasts. J. Gen. Virol. 83:2983–
2993.
29. Hahn, G., H. Khan, F. Baldanti, U. H. Koszinowski, M. G. Revello, and G.
Gerna. 2002. The human cytomegalovirus ribonucleotide reductase homolog
UL45 is dispensable for growth in endothelial cells, as determined by a
BAC-cloned clinical isolate of human cytomegalovirus with preserved wild-
type characteristics. J. Virol. 76:9551–9555.
30. Hanson, L. K., J. S. Slater, Z. Karabekian, H. W. Virgin IV, 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.
31. Hanson, L. K., J. S. Slater, Z. Karabekian, G. Ciocco-Schmitt, and A. E.
Campbell. 2001. Products of US22 genes M140 and M141 confer efficient
replication of murine cytomegalovirus in macrophages and spleen. J. Virol.
75:6292–6302.
32. Heineman, T. C., and J. I. Cohen. 1994. Deletion of the varicella-zoster virus
large subunit of ribonucleotide reductase impairs growth of virus in vitro.
J. Virol. 68:3317–3323.
33. Henry, B. E., R. Glaser, J. Hewetson, and D. J. O’Callaghan. 1978. Expres-
sion of altered ribonucleotide reductase activity associated with the replica-
tion of the Epstein-Barr virus. Virology 89:262–271.
34. Ho, M. 1991. Cytomegalovirus: biology and infection, 2nd ed. Plenum Pub-
lishing Corp., New York, N.Y.
35. Hofer, A., J. T. Ekanem, and L. Thelander. 1998. Allosteric regulation of
Trypanosoma brucei ribonucleotide reductase studied in vitro and in vivo.
J. Biol. Chem. 273:34098–34104.
36. Ibanez, C. E., R. Schrier, P. Ghazal, C. Wiley, and J. A. Nelson. 1991. Human
cytomegalovirus productively infects primary differentiated macrophages.
J. Virol. 65:6581–6588.
37. Jacobson, J. G., D. A. Leib, D. J. Goldstein, C. L. Bogard, P. A. Schaffer,
S. K. Weller, and D. M. Coen. 1989. A herpes simplex virus ribonucleotide
reductase deletion mutant is defective for productive acute and reactivatable
latent infections of mice and for replication in mouse cells. Virology 173:
276–283.
38. Jordan, A., and P. Reichard. 1998. Ribonucleotide reductases. Annu. Rev.
Biochem. 67:71–98.
39. Kalejta, R. F., and T. Shenk. 2002. Manipulation of the cell cycle by human
cytomegalovirus. Front. Biosci. 7:d295–d306.
40. Koszinowski, U. H., M. Del Val, and M. J. Reddehase. 1990. Cellular and
molecular basis of the protective immune response to cytomegalovirus in-
fection. Curr. Top. Microbiol. Immunol. 154:189–220.
41. Landolfo, S., M. Gariglio, G. Gribaudo, and D. Lembo. 2003. The human
cytomegalovirus. Pharmacol. Ther. 98:269–297.
42. Langelier, Y., and G. Buttin. 1981. Characterization of ribonucleotide re-
ductase induction in BHK-21/C13 Syrian hamster cell line upon infection by
herpes simplex virus (HSV). J. Gen. Virol. 57:21–31.
43. Lankinen, H., A. Graslund, and L. Thelander. 1982. Induction of a new
ribonucleotide reductase after infection of mouse L cells with pseudorabies
virus. J. Virol. 41:893–900.
44. Lembo, D., A. Angeretti, M. Gariglio, and S. Landolfo. 1998. Murine cyto-
megalovirus induces expression and enzyme activity of dihydrofolate reduc-
tase in quiescent cells. J. Gen. Virol. 78:2803–2808.
45. Lembo, D., G. Gribaudo, A. Hofer, L. Riera, M. Cornaglia, A. Mondo, A.
Angeretti, M. Gariglio, L. Thelander, and S. Landolfo. 2000. Expression of
an altered ribonucleotide reductase activity associated with the replication of
murine cytomegalovirus in quiescent fibroblasts. J. Virol. 74:11557–11565.
46. Lembo, D., G. Gribaudo, R. Cavallo, L. Riera, A. Angeretti, L. Hertel, and S.
Landolfo. 1999. Human cytomegalovirus stimulates cellular dihydrofolate
reductase activity in quiescent cells. Intervirology 42:30–36.
47. Lu, M., and T. Shenk. 1996. Human cytomegalovirus infection inhibits cell
cycle progression at multiple points, including the transition from G1 to S. J.
Virol. 70:8850–8857.
48. McGeehan, J. E., N. W. Depledge, and D. J. McGeoch. 2001. Evolution of the
dUTPase gene of mammalian and avian herpesviruses. Curr. Protein Peptide
Sci. 2:325–333.
49. McGeoch, D. J., and A. J. Davison. 1999. The molecular evolutionary history
of herpesviruses, p. 441�465. In E. Domingo, R. Webster, and J. Holland
(ed.), Origin and evolution of viruses. Academic Press, London, United
Kingdom.
50. Mocarski, E. S. 1993. Cytomegalovirus biology and replication, p. 173–226.
In B. Roizman, R. Whitley, and C. Lopez (ed.), The human herpesviruses.
Raven Press, New York, N.Y.
51. Mocarski, E. S., and C. T. Courcelle. 2001. Cytomegaloviruses and their
replication, p. 2629–2673. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A.
Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology.
Lippincott Williams and Wilkins, Philadelphia, Pa.
52. Mocarski, E. S., G. B. Abenes, W. C. Manning, L. C. Sambucetti, and J. M.
Cherrington. 1990. Molecular genetic analysis of cytomegalovirus gene reg-
ulation in growth, persistence and latency. Curr. Top. Microbiol. Immunol.
154:47–74.
53. Noris, E., C. Zannetti, A. Demurtas, J. Sinclair, M. De Andrea, M. Gariglio,
and S. Landolfo. 2002. Cell cycle arrest by human cytomegalovirus 86-kDa
IE2 protein resembles premature senescence. J. Virol. 76:12135–12148.
54. Novotny, J., I. Rigoutsos, D. Coleman, and T. Shenk. 2001. In silico struc-
tural and functional analysis of the human cytomegalovirus (HHV5) ge-
nome. J. Mol. Biol. 310:1151–1166.
55. Percivalle, E., M. G. Revello, L. Vago, F. Morini, and G. Gerna. 1993.
Circulating endothelial giant cells permissive for human cytomegalovirus
(HCMV) are detected in disseminated HCMV infections with organ involve-
ment. J. Clin. Investig. 92:663–670.
56. 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.
57. Reichard, P. 1988. Interactions between deoxyribonucleotide and DNA syn-
thesis. Annu. Rev. Biochem. 57:349–374.
58. Roizman, B., and D. M. Knipe. 2001. Herpes simplex viruses and their
replication, p. 2399–2459. In D. M. Knipe, P. M. Howley, D. E. Griffin, R. A.
Lamb, M. A. Martin, B. Roizman, and S. E. Straus (ed.), Fields virology.
Lippincott Williams and Wilkins, Philadelphia, Pa.
59. Saltzman, R. L., M. R. Quirk, and M. C. Jordan. 1988. Disseminated cyto-
megalovirus infection. Molecular analysis of virus and leukocyte interactions
in viremia. J. Clin. Investig. 81:75–81.
60. Salvant, B. S., E. A. Fortunato, and D. H. Spector. 1998. Cell cycle dysregu-
lation by human cytomegalovirus: influence of the cell cycle phase at the time
of infection and effects on cyclin transcription. J. Virol. 72:3729–3741.
61. Smith, C. C., and L. Aurelian. 1997. The large subunit of herpes simplex
virus type 2 ribonucleotide reductase (ICP10) is associated with the virion
tegument and has PK activity. Virology 234:235–242.
62. Song, Y. J., and M. F. Stinski. 2002. Effect of the human cytomegalovirus
IE86 protein on expression of E2F-responsive genes: a DNA microarray
analysis. Proc. Natl. Acad. Sci. USA 99:2836–2841.
63. Staczek, J. 1990. Animal cytomegaloviruses. Microbiol. Rev. 54:247–265.
64. Stoddart, C. A., R. D. Cardin, J. M. Boname, W. C. Manning, G. B. Abene,
and E. S. Mocarski. 1994. Peripheral blood mononuclear phagocytes medi-
ate dissemination of murine cytomegalovirus. J. Virol. 68:6243–6253.
65. Sun, Y., and J. Conner. 1999. The U28 ORF of human herpesvirus-7 does
not encode a functional ribonucleotide reductase R1 subunit. J. Gen. Virol.
80:2713–2718.
66. Thelander, L., and P. Reichard. 1979. Reduction of ribonucleotides. Annu.
Rev. Biochem. 48:133–158.
67. Wagner, M., S. Jonjic, U. H. Koszinowski, and M. Messerle. 1999. Systematic
excision of vector sequences from the BAC-cloned herpesvirus genome
during virus reconstitution. J. Virol. 73:7056–7060.
68. Wiebusch, L., and C. Hagemeier. 1999. Human cytomegalovirus 86-kilodal-
ton IE2 protein blocks cell cycle progression in G1. J. Virol. 73:9274–9283.
4288 LEMBO ET AL. J. VIROL.