The Human Cytomegalovirus Protein TRS1 Inhibits Autophagy via Its
Interaction with Beclin 1
Magali Chaumorcel,aMarion Lussignol,aLina Mouna,aYolaine Cavignac,aKamau Fahie,aJacqueline Cotte-Laffitte,aAdam Geballe,b
Wolfram Brune,cIsabelle Beau,aPatrice Codogno,aand Audrey Esclatinea
INSERM UMR984, Université Paris Sud, Faculté de Pharmacie, Châtenay-Malabry, Francea; Fred Hutchinson Cancer Research Center and University of Washington, Seattle,
Washington, USAb; and Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germanyc
Human cytomegalovirus modulates macroautophagy in two opposite directions. First, HCMV stimulates autophagy during the
early stages of infection, as evident by an increase in the number of autophagosomes and a rise in the autophagic flux. This stim-
ulation occurs independently of de novo viral protein synthesis since UV-inactivated HCMV recapitulates the stimulatory effect
on macroautophagy. At later time points of infection, HCMV blocks autophagy (M. Chaumorcel, S. Souquere, G. Pierron, P.
Codogno, and A. Esclatine, Autophagy 4:1–8, 2008) by a mechanism that requires de novo viral protein expression. Exploration
of the mechanisms used by HCMV to block autophagy unveiled a robust increase of the cellular form of Bcl-2 expression. Al-
though this protein has an anti-autophagy effect via its interaction with Beclin 1, it is not responsible for the inhibition induced
by HCMV, probably because of its phosphorylation by c-Jun N-terminal kinase. Here we showed that the HCMV TRS1 protein
blocks autophagosome biogenesis and that a TRS1 deletion mutant is defective in autophagy inhibition. TRS1 has previously
been shown to neutralize the PKR antiviral effector molecule. Although phosphorylation of eIF2?by PKR has been described as
a stimulatory signal to induce autophagy, the PKR-binding domain of TRS1 is dispensable to its inhibitory effect. Our results
show that TRS1 interacts with Beclin 1 to inhibit autophagy. We mapped the interaction with Beclin 1 to the N-terminal region
of TRS1, and we demonstrated that the Beclin 1-binding domain of TRS1 is essential to inhibit autophagy.
is a major cause of morbidity and mortality in immunocompro-
mised individuals, such as AIDS patients or bone marrow and
infections that compromise long-term graft function. Moreover,
HCMV congenital infection is the most common cause of virus-
Macroautophagy (here referred to as autophagy) is a vacuolar
homeostatic “self-eating” process that involves the digestion of
cytoplasmic components via the lysosomal pathway. During au-
tophagy, part of the cytoplasm is surrounded by a cisternal mem-
brane, known as the phagophore. The phagophore then closes to
The autophagosome finally fuses with the lysosome, forming the
tion of the autophagosome requires the activity of several
autophagy-related (Atg) proteins, initially identified in Saccharo-
myces cerevisiae. Beclin 1, the mammalian counterpart of Atg6
in yeast, is a keystone of this process, because it forms three
different complexes involved in both autophagosome forma-
tion and maturation. The assembly of the phagophore requires
the Beclin 1-phosphatidylinositol 3-kinase (PI3K) complex,
consisting of Beclin 1, the class III PI3K hVps34, its regulatory
protein kinase hVps15, and Atg14L. This Beclin 1 complex is
tightly modulated by the recruitment of positive and negative
regulators of autophagy (such as Bcl-2 and Bcl-xL) (48, 51, 52).
Regulation is also achieved through posttranslational modifi-
cations of Beclin 1 or Bcl-2, through interactions with BH3-
only proteins and BH3 mimetics, or by a recently discovered
dae family, is widespread in human populations, with up to
Beclin 1-targeted microRNA (65). This complex and other Atg
proteins recruit the Atg5-Atg12-Atg16L multimeric complex
and the lipidated form of LC3 (microtubule-associated protein
light chain 3) to the phagophore. These last two constituents,
which are essential for the forming autophagosome to expand,
act sequentially and result from two ubiquitin-like conjugation
systems. Two other Beclin 1 complexes regulate the maturation of
Under normal conditions, autophagy allows cells to degrade
long-lived proteins and aged organelles and has two main physi-
ological functions. The first involves recycling macromolecules
and long-lived proteins to provide cells exposed to nutrient stress
with amino acids and energy. The second function is to help the
cell eliminate damaged organelles, such as mitochondria, and
toxic aggregate-prone proteins. Autophagy has also recently been
microorganisms, such as poliovirus, use the autophagic machin-
ery for their own benefit such as enhanced replication, autophagy
can function in cellular defenses against intracellular pathogens,
nity (reviewed in references 16, 17, and 58). However, viruses
8 (HHV-8) have developed various strategies to counteract the
host’s antiviral autophagic mechanism (reviewed in references 7
Received 21 July 2011 Accepted 19 December 2011
Published ahead of print 28 December 2011
Address correspondence to Audrey Esclatine, email@example.com.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0022-538X/12/$12.00Journal of Virologyp. 2571–2584jvi.asm.org
and 17). We and others previously reported that HCMV also
modulates autophagy in infected cells (9, 40, 54).
Several viruses are able to counteract autophagy by an interac-
machinery. For example, two members of the Gammaherpesviri-
nae subfamily, HHV-8 and herpesvirus saimiri, express the viral
to regulate apoptosis from death receptors. vFLIP interacts with
emerged as the major target for the modulation of autophagy by
tively, which achieve inhibition of autophagy by their interaction
with Beclin 1 (48, 53). The infected cell protein 34.5 (ICP34.5) of
HSV-1 also interacts directly with Beclin 1 and inhibits autopha-
gosome biogenesis (44). Other viral proteins seem to block au-
tophagosome maturation rather than formation by interaction
(HIV) and the matrix protein M2 of influenza A virus interact
with Beclin 1 but are responsible for autophagosome accumula-
tion during viral infection, by blocking fusion of the autophago-
some with the lysosome (23, 33). These two viral proteins might
interact with the other Beclin 1-hVps34 complex containing
UVRAG, which is involved in autophagosome maturation.
In this paper, we have explored the modulation of autophagy
during HCMV infection and the mechanisms used by HCMV to
ral protein synthesis during the early stages of infection. Later,
HCMV actively blocks the autophagosome biogenesis by expres-
sion of viral protein(s). We observed viral-induced modifications
of Bcl-2, a negative regulator of autophagosome biogenesis. In
addition, we identified TRS1 as a new anti-autophagic protein. It
has been previously reported that TRS1 blocks the activity of the
interferon-induced double-stranded RNA-dependent protein ki-
nase PKR (10, 26). However, we found that the anti-autophagic
activity of TRS1 is independent of its interaction with PKR but is
related to its binding to Beclin 1.
MATERIALS AND METHODS
Cells and virus. Primary human embryonic lung fibroblasts MRC5 were
purchased from RD-Biotech and used between passages 23 and 28 posti-
solation. These cells were maintained in minimum essential medium
G (100 U/ml), streptomycin sulfate (100 ?g/ml), l-glutamine (1%), and
nonessential amino acids (1%). Cells were cultured at 37°C under 5%
CO2. HeLa cells were maintained at 37°C under 5% CO2in RPMI with
10% FCS. PKR?/?and PKR?/?mouse embryo fibroblasts kindly pro-
vided by B. R. G. Williams (Monash University, Victoria, Australia) were
propagated in Dulbecco MEM supplemented with 10% FCS. The AD169
strain of HCMV was obtained from ATCC and was propagated in MRC5
cells as previously described (19). The AD169?TRS1 mutant was con-
which contains the full-length HCMV AD169 genome (27). Mutagenesis
by homologous recombination was performed in Escherichia coli strain
DY380 essentially as described previously (5, 38). Briefly, a kanamycin
resistance gene (kan) flanked by FLP recombination target sites was PCR
amplified using oligonucleotide primers containing 50-nucleotide ho-
mologies immediately up- and downstream of TRS1. The PCR product
was used to replace the TRS1 gene by homologous recombination. The
kan gene was subsequently removed with FLP recombinase as described
Midi kit (Clontech). To reconstitute the ?TRS1 mutant virus, BAC DNA
was introduced by electroporation into human foreskin fibroblast (HFF)
cells essentially as described previously (38). For gradient purification of
for 10 min at 4,000 rpm. Supernatants were then ultracentrifuged for 70
min (23,000 rpm, 4°C, Beckman SW28 rotor). Pellets containing virions
and other particles were resuspended in 1 ml of MEM and transferred
onto a preformed linear glycerol-tartrate gradient (15 to 35% Na-tartrate
and 30 to 0% glycerol in 0.04% Na-phosphate), which was then ultracen-
containing band was harvested with a syringe, and the virions were
washed and pelleted by an additional ultracentrifugation for 70 min at
23,000 rpm. The pellet was resuspended in MEM and stored at ?80°C
until used for infection experiments. For UV inactivation, HCMV stocks
were diluted in MEM, placed in a thin layer in plastic dishes, and irradi-
UV lamp (Bioblock Scientific). The efficacy of inactivation was tested by
immunofluorescence staining of MRC5 cells infected with UV-HCMV
and pp65 MAb (see below), followed by a secondary fluorescein isothio-
cyanate (FITC)-conjugated goat anti-mouse antibody (data not shown).
Successful inactivation was ensured through abrogation of IE antigen ex-
pression, whereas pp65 tegument protein detection was comparable to
cell infection with nonirradiated virus by using methods previously de-
scribed (13, 34).
and 86 kDa. Antibody directed against the C-terminal region of TRS1
protein was a kind gift of T. Shenk (50). In order to detect 6?His-tagged
TRS1 constructs, we used a rabbit antibody directed against 6?His (Cell
Signaling Technology). For the detection of FLAG-tagged Beclin 1 and
FLAG-tagged ICP34.5, we used a rabbit antibody directed against FLAG
(Cell Signaling Technology). Antibody against LC3 was produced as pre-
viously described (9). Antibodies against p62 (BD Biosciences), Beclin 1
ylated Bcl-2 (Cell Signaling) were used in this study. To explore the JNK
pathway, antibody directed against total JNK was obtained from Santa
Cruz and antibodies directed against phosphorylated (Thr183/Tyr185)
JNK and against total and phosphorylated (Ser73) c-Jun were purchased
from Cell Signaling. Anti-actin antibody was supplied by Sigma. Second-
ary fluorescein isothiocyanate (FITC) and tetramethyl rhodamine iso-
purchased from Jackson. Alexa Fluor 350 donkey anti-rabbit secondary
antibody was obtained from Invitrogen.
HCMV infection of fibroblasts. MRC5 cells were grown in MEM
supplemented with 10% FCS. HCMV or UV-inactivated HCMV, diluted
in serum-free MEM, or serum-free MEM alone (mock infected) was ad-
3. After the inoculum was removed, the cells were maintained in MEM
containing 10% FCS and processed for the different assays at various
times postinfection (p.i.). Cell viability was tested by a trypan blue exclu-
sion test, and no significant cell death was observed in HCMV-infected
cells at 24 and 48 h p.i. Cells were centrifuged for 45 min at 1,400 ? g
during the adsorption period, to enhance infectivity of HCMV, in exper-
iments in which MRC5 cells were transfected with green fluorescent pro-
tein (GFP)-LC3 (21).
Immunoblot analysis. MRC5 and HeLa cells were lysed in 65 mM
Tris, pH 6.8, 4% SDS, 1.5% ?-mercaptoethanol and held at 100°C for 5
min. Proteins were separated on gels containing different percentages of
SDS-polyacrylamide: 10% to resolve Beclin 1, His-tagged TRS1, or TRS1,
12% for Bcl-2 and p62, and 15% to resolve LC3 forms I and II. For im-
munoblot analysis, the proteins were electrotransferred onto a polyvi-
Chaumorcel et al.
jvi.asm.org Journal of Virology
nylidene difluoride membrane (Perkin Elmer, Les Ulis, France). The
and then incubated with primary antibodies at an appropriate dilution.
After three washes with Tris-buffered saline, a goat anti-rabbit or anti-
mouse horseradish peroxidase-labeled antibody (Amersham, Orsay,
France) was used at 1/10,000 dilution as a secondary antibody. Bound
immunoglobulins were revealed using the ECL? detection system under
conditions recommended by the manufacturer (Immobilon, Orsay,
France). Anti-actin was used to ensure equal loadings. The effect of
and confirmed by comparing untreated samples with those treated with
radation of autolysosome content.
Coimmunoprecipitation assays. To immunoprecipitate Bcl-2 and
endogenous Beclin 1 in MRC5 cells, cells were lysed in lysis buffer (20
mM Tris HCl, pH 7.4, 140 mM NaCl, 2 mM EDTA, 0.2% CHAPS, 10%
glycerol) for 1 h at 4°C, followed by centrifugation to remove cell
debris. Immunoprecipitation was performed overnight at 4°C with a
goat polyclonal anti-Beclin 1 antibody (Santa Cruz Biologicals, Santa
Cruz, CA) or a control goat serum. Protein G-Sepharose beads (Sigma-
Aldrich, St. Louis, MO) were added for 1 h at 4°C and were washed three
1-propanesulfonate) buffer (20 mM Tris HCl, pH 7.4, 140 mM NaCl, 2
tates were eluted in Laemmli buffer and subjected to SDS-PAGE, and
Bcl-2 was detected by immunoblot analysis. To immunoprecipitate en-
transfected with different TRS1 constructs and then treated as described
HCl, 50 mM NaCl, 0.5% Triton, 0.2% bovine serum albumin [BSA],
25 mM NaPPi, 50 mM Na3VO4) for 1 h at 4°C, followed by ultracentrifu-
gation at 120,000 ? g to remove cell debris. Immunoprecipitation was per-
formed overnight at 4°C with a goat polyclonal anti-Beclin 1 antibody or a
were washed three times with buffer (50 mM Tris HCl, 50 mM NaCl, 0.5%
Triton, 0.2% BSA, 25 mM NaPPi, 50 mM Na3VO4) and two times with an-
other buffer (20 mM Tris HCl, 50 mM NaCl, 0.2% BSA). Anti-Beclin 1 im-
munoprecipitates were eluted in Laemmli buffer and subjected to SDS-
Immunofluorescence analysis. Cell monolayers prepared on glass
dehyde (PFA) in phosphate-buffered saline (PBS) and then washed three
times with PBS. Different staining protocols were used for different anti-
bodies. In some experiments, cell permeabilization was performed by in-
cubating the coverslips for 4 min with PBS–0.2% Triton X-100. The cells
were stained with a mouse antibody against HCMV IE antigens (1:50;
Argene Biosoft) followed by a TRITC-conjugated goat anti-mouse anti-
body (1:200). To detect 6?His TRS1 construct expression, cells were
stained with anti 6?His antibody (1:100; Cell Signaling) followed by
antibodies. Cells were also stained overnight with an antibody against
phosphorylated Bcl-2 (1:100; Cell Signaling), followed by FITC-
conjugated secondary antibody. Coverslips were mounted with Glycergel
(Dako) before being analyzed on a Nikon Eclipse 80i epifluorescence mi-
experiments. Photographic images were resized, organized, and labeled
using Adobe Photoshop software.
Real-time PCR. Total RNA was extracted with the aid of TRIzol re-
DNase (Life Technologies), extracted with phenol-chloroform, and pre-
cipitated with ethanol to remove contaminating DNA. Total RNA (2.5
?g) from mock-infected and HCMV-infected cell samples was reverse
transcribed with random hexamers and SuperScript reverse transcriptase
(Invitrogen). cDNA quantities were normalized to 18S rRNA quantities.
The following primers were used: Bcl-2 forward, 5=-ATGTGTGTGGAG
AGCGTCAA-3=, and reverse, 5=-ACAGTTCCACAAAGGCATCC-3=.
The primers for Beclin 1 were previously validated and described (18).
sample, primers (0.5 ?M), and LightCycler DNA Master SYBR green I
cler 2.0 detection system (Roche). To compare mock-infected and in-
fected cell samples, relative changes in gene expression were determined
using the 2???CTmethod.
Transfection. The GFP-LC3 expression vector and the GFP and red
fluorescent protein (RFP) tandemly tagged LC3 construct (mRFP-GFP-
LC3) were kindly provided by T. Yoshimori (Research Institute for Mi-
crobial Diseases, Osaka University, Osaka, Japan) (30). The pEQ1180
construct, containing a full-length HCMV TRS1 protein cDNA and the
pEQ979 (1-738), pEQ1001 (1-679), pEQ1013 (45-795), and pEQ1000
(93-795) constructs, containing the indicated lengths of TRS1, were de-
scribed previously (25). These constructs were tagged with 6?His. Plas-
Cultures of HeLa cells at 50 to 80% confluence were transiently trans-
fected with empty vector, GFP-LC3, or mRFP-GFP-LC3 plasmids or var-
ious TRS1 constructs using FuGENE HD transfection reagent (Roche),
according to the manufacturer’s instructions. After 4 h, the medium was
replaced by MEM-10% FCS, and the cells were incubated for 24 to 48 h.
Amino acid starvation by EBSS (starvation medium) was carried out 4 h
before fixation. The cells were then fixed in PFA for 10 min at room
temperature and washed three times with PBS. Data were statistically
compared by using a global Student test (P ? 0.01) as indicated in the
Proteolysis. This assay was adapted from that of Bauvy et al. (1).
MRC5 cells were incubated for 24 h at 37°C with 0.2 ?Ci/ml L-[14C]
valine. At the end of the radiolabeling period, the cells were washed three
times with PBS, pH 7.4. Cells were then incubated in complete medium
time short-lived proteins are degraded, the medium was replaced with
fresh medium (MEM plus 10% bovine serum albumin and 10 mM cold
valine), and the incubation was continued for an additional 4 h. Wort-
mannin (200 nM) was added to inhibit de novo formation of autophagic
precipitated in trichloroacetic acid at a final concentration of 10% (vol/
vol) at 4°C. The precipitated proteins were separated from the soluble
0.5 ml of 0.2 N NaOH. Radioactivity was determined by liquid scintil-
lation counting. Protein degradation was calculated by dividing the
acid-soluble radioactivity recovered from both cells and medium by
the radioactivity contained in precipitated proteins from both cells
(SEM) of at least three experiments. The statistical significance was as-
sessed by a Student’s t test.
We previously demonstrated that HCMV inhibits autophagy in
fibroblasts 24 h postinfection (9). During the first 24 h of infec-
tion, HCMV binds to the cell surface, penetrates into the cyto-
plasm, and targets its genome to the nucleus, and viral immediate
early proteins are synthesized. In order to guide our research into
first investigated at what time this inhibition started. To measure
autophagy, we transfected fibroblasts with GFP-LC3, a specific
marker of mammalian autophagy. Upon autophagy induction,
the lipidated form of LC3 associates with autophagosomal mem-
HCMV Bidirectionally Modulates Autophagy
March 2012 Volume 86 Number 5 jvi.asm.org 2573
be visualized by fluorescence microscopy. MRC5 cells were trans-
fected with GFP-LC3 and then infected with HCMV (MOI ? 1)
for 4 to 24 h, and the number of puncta of GFP-LC3 per cell was
by density gradient centrifugation because we observed a stimu-
lation of autophagy when cells were exposed only to supernatant
of HCMV-infected cells, probably via soluble factors (Fig. 2A). In
purified HCMV-infected cells, we observed an increase in the
number of puncta per cell from 4 to 8 h p.i. but a clear decrease at
24 h p.i. (Fig. 1A and B), confirming our previous results (9).
Therefore, these results suggest that autophagy is induced early in
To assess whether the inhibition of autophagy observed in
HCMV-infected cells requires expression of viral proteins, we
receptors and to enter the cells but does not exhibit any viral gene
expression. The number of puncta per cell in UV-inactivated in-
fected cells (detected by identification of virion-associated tegu-
ment protein pp65 inside the cells) was constantly increased (to
around 40 per cell) regardless of the time of infection, compared
to that in mock-infected cells (Fig. 1C). Twenty-four hours after
infection, a large number of MRC5 cells infected with UV-
inactivated HCMV (visualized by pp65 staining) showed a punc-
tuate staining of GFP-LC3 compared to the results in mock- and
HCMV-infected cells (Fig. 1A).
However, an increased accumulation of autophagosomes in
cells can also correspond to an inhibition of their fusion with
lysosomes and to an impaired autophagy (41). Therefore, it is not
sufficient to monitor static levels of autophagy, and three addi-
tional independent assays were performed to quantify the au-
tophagic flux: the use of a tandem mRFP-GFP LC3 probe, the
degradation of p62, and the degradation of [14C]valine-labeled
long-lived proteins. We did not use the alternative method of
measuring LC3-II by Western blot analysis, because we noticed a
constant accumulation of LC3-II, independent of the autophagic
level (see Fig. 2B).
differentiate autophagosomes (GFP?RFP?or yellow puncta)
from autolysosomes (GFP?RFP?or red puncta), as the GFP sig-
nal is quenched in acidic compartments (31). The number of
measured in MRC5 cells infected with HCMV or UV-inactivated
HCMV (Fig. 3A and B). We used amino acid-starved cells as a
cell, confirming a constant increase in the number of autolyso-
of the autophagy substrate p62 in UV-inactivated HCMV- and
HCMV-infected cells by immunoblotting (Fig. 3C) (2). Whereas
p62 protein levels were decreased in HCMV-infected cells com-
18 and 24 h p.i. Moreover, the presence of lysosomal inhibitors
FIG 1 Autophagosome formation at early and late time points after
HCMV infection. MRC5 cells were transfected with GFP-LC3 for 48 h and
then mock-infected or infected with HCMV or with UV-inactivated
HCMV (UV-HCMV) for 4, 6, 8, and 24 h. Cells were fixed and immuno-
(B and C) Autophagy was quantified in pp65-positive cells by counting the
number of GFP-LC3 puncta per cell. The results are the means of three
independent experiments. Between 50 and 100 cells were analyzed per
assay. *, P ? 0.05; **, P ? 0.01 (t test).
Chaumorcel et al.
jvi.asm.org Journal of Virology
the autophagy flux is blocked at later stages of infection. In con-
trast, in UV-inactivated HCMV-treated cells, p62 protein levels
were clearly increased in the presence of lysosomal inhibitors at 4
time during the treatment (Fig. 3C). Finally, the effects of HCMV
of long-lived proteins. As illustrated in Fig. 3D, UV-inactivated
teins degradation sensitive to wortmannin, an inhibitor of au-
tophagic sequestration (4). However, the stimulation of protein
degradation sensitive to wortmannin was no longer observed in
cells infected with HCMV at 24 h p.i. Together these results indi-
cate that HCMV stimulates autophagy at early times of infection
but then inhibits it by 24 h p.i. The later inhibitory effect requires
de novo viral gene expression, since UV-inactivated virus is no
longer able to thwart autophagy, consistent with our previous
Expression of Beclin 1 and Bcl-2 during HCMV infection.
and to modulate its autophagy activity (23, 33, 44). Moreover, it
has been reported in the context of HIV infection that the Env-
mediated autophagy triggered in T cells correlates with Beclin 1
protein accumulation (22). In a first series of experiments in
MRC5 cells, neither Beclin 1 protein expression nor Beclin 1
mRNA level was modified during HCMV infection (data not
shown). We then analyzed the impact of HCMV infection on the
expression of Bcl-2 protein, a negative regulator of the Beclin 1
complex (48). A significant increase in the amount of Bcl-2 pro-
tein was detected in HCMV-infected cells from 1 to 3 days p.i.,
of Bcl-2 in HCMV-infected cells (Fig. 4A). The amount of Bcl-2
mRNA rapidly increased, as early as 6 h p.i. At 24 and 48 h p.i.,
corresponding to the time when HCMV inhibited autophagy (9),
the level of Bcl-2 mRNA was 3-fold higher than in mock-infected
cells. We did not observe any modification of Bcl-2 mRNA levels
in UV-inactivated HCMV-infected cells compared to mock-
sary for this induction.
of Bcl-2 inhibits starvation-induced autophagy and that Bcl-2 is a
negative regulator of autophagy which interacts with Beclin 1 to
avoid stimulation of autophagy (48). We therefore hypothesized
that HCMV increases cellular Bcl-2 to potentiate its interaction
with Beclin 1 and thereby inhibit autophagy. Fibroblasts were
grown 4 h in amino acid excess (rich medium [RM]) to inhibit
we observed that Bcl-2 bound to Beclin 1 in normal conditions
and that Bcl-2 coimmunoprecipitated more with Beclin 1 during
munoprecipitation of Beclin 1 and Bcl-2 in fibroblasts infected
with HCMV showed that, whereas Bcl-2 was overexpressed com-
interacted with Beclin 1 in these cells (Fig. 4B). These results sug-
overexpressed Bcl-2 binding to Beclin 1.
disruption of Beclin 1-Bcl-2 interaction during HCMV infection:
phosphorylation of either Bcl-2 or Beclin 1 (61, 63), competitive
ics (36), or, potentially, effects of membrane-anchored receptors
we observed an increase of Bcl-2 phosphorylation in HCMV-
infected cells by immunoblotting as early as 24 h p.i. (Fig. 4C).
Moreover, there was no detectable signal of the phosphorylation
of Bcl-2 in mock-infected cells but HCMV-infected cells, identi-
fied by staining for viral IE antigens, showed strong staining for
phosphorylated Bcl-2, visualized by a clear juxtanuclear signal af-
ter 24 h of infection (Fig. 4D). Taken together, these results sug-
gest that posttranslational modifications of Bcl-2 might explain
why the interaction between Beclin 1 and Bcl-2 is reduced in
It has been previously reported that the c-Jun N-terminal ki-
nase (JNK) disrupts the Bcl-2/Beclin 1 complex after nutrient de-
privation and ceramide-induced autophagy, by phosphorylating
Bcl-2 (46, 61). We observed that the JNK pathway was activated
during HCMV infection based on accumulation of phosphory-
lated JNK during the first 24 h p.i. and of its phosphorylated sub-
MRC5 cells were treated during 4 h with supernatant of HCMV-infected cells
clarified of viral particles and cellular debris by ultracentrifugation (see Mate-
rials and Methods) or infected with UV-inactivated HCMV, followed by im-
munoblotting with anti-LC3, anti-p62, or anti-actin antibodies. Lysosomal
inhibitors E64 and pepstatin A (pepst) were added under all conditions, to
block the lysosomal degradation of LC3-II and p62. (B) Accumulation of LC3
during HCMV infection is independent of the autophagic level. MRC5 cells
were infected with HCMV at an MOI of 1 during 6, 24, or 48 h and treated or
not with E64, followed by immunoblotting with anti-LC3 and anti-actin anti-
bodies. Note that the level of LC3-II in infected cells does not change in the
presence of lysosomal inhibitor.
HCMV Bidirectionally Modulates Autophagy
March 2012 Volume 86 Number 5jvi.asm.org 2575
strate, c-Jun, at later times (Fig. 5A and B). Treatment of infected
cells with SP600125, a selective JNK inhibitor, showed that Bcl-2
phosphorylation in infected cells was JNK specific (Fig. 5C). Ec-
topic expression of HCMV immediate-early protein 1 (IE1) was
previously shown to selectively induce RelB NF-?B transcription
and activity, via the JNK pathway (59–60). Similarly, we observed
in HCMV-infected cells a JNK-mediated upregulation of RelB
NF-?B subunit and its nuclear relocalization (Fig. 5D and E),
which might be responsible for the HCMV-mediated Bcl-2 up-
regulation. Finally, our results suggest that HCMV-induced Bcl-2
upregulation and phosphorylation of Bcl-2 are both mediated by
FIG 3 Inhibition of autophagic flux requires expression of viral genes. (A) Representative images of MRC5 cells transfected with mRFP-GFP-LC3 plasmid and
infected with HCMV or UV-inactivated HCMV. Red and yellow dots indicate GFP?RFP?and GFP?RFP?puncta, respectively. (B) Autolysosomes were
(t test). Amino acid-starved cells (EBSS) were used as a positive control (C) Immunoblot analysis of p62 levels in cells infected with HCMV or UV-inactivated
HCMV for the indicated times. Lysosomal inhibitors E64/pepstatin A were added to all conditions, to block the lysosomal degradation. Actin immunoblotting
was used as a loading control. (D) Effect of HCMV infection on long-lived protein degradation. [14C]valine-radiolabeled MRC5 cells were mock infected or
infected with HCMV or UV-inactivated HCMV for 8 or 24 h, and proteolysis was measured as described in Materials and Methods. Cells were treated with 200
nM wortmannin (Plus WN) or not (No WN). The results are the means of three independent experiments. *, P ? 0.05.
Chaumorcel et al.
jvi.asm.org Journal of Virology
these results could explain the mechanism by which the Beclin
1/Bcl-2 complex is disrupted during HCMV infection. While this
tion has the opposite effect. Therefore, we still needed to explore
the mechanisms for this HCMV-mediated inhibition of au-
The viral protein TRS1 inhibits starvation-induced au-
tophagy. As viral expression during HCMV infection is necessary
to inhibit autophagy, we sought to identify the viral protein(s)
responsible for autophagy inhibition. It has been previously
shown that several viral proteins prevent autophagy induction.
medium (EBSS), and in cells infected with HCMV at an MOI of 1 from 6 to 72 h or with UV-inactivated HCMV for 24 h. The results are the means of three
independent experiments. (B) Coimmunoprecipitation of Beclin 1 and Bcl-2 in MRC5 cells. MRC5 cells were mock infected, infected with HCMV (MOI ? 1)
for 2 days, or grown in rich medium (RM) for 4 h prior to coimmunoprecipitation. Rich medium is known to inhibit autophagy. Cell lysates were immuno-
precipitated with preimmune goat serum or a goat polyclonal anti-Beclin 1 antibody followed by immunoblotting with the indicated antibodies. (C and D)
Accumulation of hyperphosphorylated Bcl-2 during HCMV infection in MRC5 fibroblasts. (C) Immunoblot analysis of the phosphorylated Bcl-2 (Ser70).
MRC5 cells were mock infected or infected with HCMV at an MOI of 1 from 12 to 72 h. Actin immunoblotting was used as a loading control. (D) MRC5 cells
were mock infected or infected with HCMV at an MOI of 1 from 18 to 72 h and at an MOI of 3 from 18 to 48 h. An increased staining of phosphorylated Bcl-2
(juxtanuclear green staining) was observed in the HCMV-infected cells compared to mock-infected cells (nuclear red staining shows HCMV IE antigens).
HCMV Bidirectionally Modulates Autophagy
March 2012 Volume 86 Number 5 jvi.asm.org 2577
autophagy by interaction with Beclin 1, whereas the viral homo-
(35, 44, 48). Despite the fact that HCMV belongs to the Herpes-
viridae family, the HCMV genome does not contain any ortholog
of the HSV-1 ?134.5 gene or viral Bcl-2 and FLIP homologs, like
those known to be present in gammaherpesviruses. However,
ICP34.5 was previously described to preclude the host shutoff of
protein synthesis via the PKR/eIF2? signaling pathway and
HCMV possesses a functional homolog of ICP34.5, called TRS1
(11, 26). TRS1 binds double-stranded RNA and interferon-
induced kinase PKR and inhibits the PKR/eIF2? signaling path-
way (10, 26). To determine whether TRS1 interacts with Beclin 1
and antagonizes its autophagy function, like ICP34.5, we per-
formed coimmunoprecipitation studies. In MCR5 cells infected
with HCMV at an MOI of 1 for 24 h, we found that endogenous
impact of TRS1 on the autophagic pathway, different assays to
FIG 5 Activation of the JNK pathway during HCMV infection. (A) HCMV activates JNK phosphorylation. MRC5 cells were mock infected or infected with
(P-JNK). (B) Immunoblot analysis of the phosphorylated c-Jun during HCMV infection. MRC5 cells were mock infected or infected with HCMV at MOIs of 1
and 3 from 24 to 96 h. (C) Cells were infected with HCMV during 48 h and treated with SP600125, a selective JNK inhibitor, during different time frames as
indicated in the diagram (conditions 1 to 4). SP600125 blocks Bcl-2 overexpression when added during the first 24 h of infection (lane 2) and phosphorylation
of Bcl-2 when added from 24 until 48 h p.i. (lane 3). SP600125 blocks phosphorylation of c-Jun when added during 24 or 48 h (lanes 3 and 4) (D) RelB mRNA
analysis by real-time PCR. MRC5 cells were mock infected or infected with HCMV at an MOI of 1 for 6 to 72 h, infected with UV-inactivated HCMV for 24 h,
or grown in starvation medium (EBSS) for 4 h. The results are the means of three independent experiments. (E) Confocal analysis of the expression of RelB in
it has a diffuse (nuclear and cytoplasmic) staining in mock-infected cells. The cells were observed under a confocal laser scanning microscope (Zeiss LSM 510)
and analyzed with Imaris software for three-dimensional reconstruction.
Chaumorcel et al.
jvi.asm.org Journal of Virology
since they are much more transfectable than MRC5 cells. HeLa
sion vector or an empty vector, and autophagy was induced by
starvation. A decrease of LC3-II was observed by immunoblot
analysis in TRS1-transfected cells (Fig. 6B). Comparable results
were obtained in HEK cells (data not shown). To determine
the autophagic flux, we used the tandem mRFP-GFP-LC3 probe
(Fig. 6C). HeLa cells were transiently cotransfected with TRS1 or
empty vector and mRFP-GFP-LC3 plasmids. After starvation for
immunofluorescence and GFP?RFP?(yellow) or GFP?RFP?
(red) puncta were quantified in those cells (Fig. 6C and D). HeLa
cells transfected with TRS1 displayed decreased total LC3 puncta
Similar results were obtained with the GFP-LC3 probe (data not
puncta, corresponding to autophagosome and a strong decrease
indicate that TRS1 inhibits starvation-induced autophagic flux in
HeLa cells. To complement these results, we examined cellular
expression levels of p62 in TRS1-transfected cells that were cul-
tured in starvation medium. In TRS1-transfected cells, p62 pro-
tein levels were approximately increased by 40% compared to
control cells, confirming inhibition of the autophagic flux by
the diminution of autolysosomes per cell detected with mRFP-
of p62 in cells transfected with TRS1 demonstrate that this viral
protein can inhibit the formation of autophagosome.
TRS1 inhibits autophagy independently of its interaction
with PKR. With the finding that TRS1 blocks autophagy, we in-
vestigated which domain of TRS1 is responsible for inhibition of
6?His-tagged TRS1 constructs to inhibit starvation-induced au-
tophagy in HeLa cells (Fig. 7). TRS1 contains a domain required
for PKR binding located at its carboxy-terminal region and a
minus (Fig. 7A). The percentage of GFP-LC3-positive cells with
GFP-LC3 dots was measured in starved cells. We observed that
result suggests that PKR binding is not required for autophagy
inhibition. This was confirmed by the observation that TRS1 was
able to block starvation-induced autophagy in murine fibroblasts
lacking PKR (Fig. 8). PKR?/?and PKR?/?MEFs were cotrans-
fected with plasmids expressing GFP-LC3 and either HSV-1
starvation-induced autophagy was compared in wild-type versus
PKR-deficient cells. We observed that both TRS1 and ICP34.5
failed to inhibit starvation-induced autophagy (Fig. 7B and C).
The number of GFP-LC3 positive cells was clearly increased after
transfection with TRS1 (45-795) and autophagy induction. Thus,
of TRS1. Next, we performed a series of coimmunoprecipitation
experiments with different His-tagged forms of TRS1 transfected
in HeLa cells. We observed that TRS1 (1-738) and (1-679) bound
to Beclin 1 whereas TRS1 (45-795) and (93-795) did not bind
FIG 6 The viral protein TRS1 interacts with Beclin 1 and inhibits autophagic
flux. (A) Coimmunoprecipitation of Beclin 1 and TRS1 in HCMV-infected
MRC5 cells. MRC5 cells were mock infected or infected with HCMV at an
serum or a goat polyclonal anti-Beclin 1 antibody followed by immunoblot-
ting with an anti-TRS1 antibody. (B) Immunoblot analysis of LC3 in HeLa
cells transfected with TRS1 and cultured in starvation medium. Actin immu-
noblotting was used as a loading control. (C) Representative images of HeLa
cells cotransfected with mRFP-GFP-LC3 and TRS1 plasmids for 24 h and
dots indicate GFP?RFP?and GFP?RFP?puncta, respectively. Bar, 10 ?m.
cell, total puncta per cell (GFP?RFP?and GFP?RFP?puncta), GFP?RFP?
puncta per cell, and GFP?RFP?puncta per cell. Only TRS1-expressing cells
were quantified as described in Materials and Methods. The results are the
means of three independent experiments. Twenty cells were analyzed per as-
say. *, P ? 0.05; **, P ? 0.01 (t test). (E) Immunoblot analysis of p62 protein
in HeLa cells transfected with TRS1 plasmids and cultured in starvation me-
dium. Actin immunoblotting was used as a loading control.
HCMV Bidirectionally Modulates Autophagy
March 2012 Volume 86 Number 5 jvi.asm.org 2579
efficiently to Beclin 1 (Fig. 7D). These results demonstrated that
the deletion of the PKR-interacting domain of TRS1 did not im-
pair the interaction of the viral protein with endogenous Beclin 1
but that the N-terminal domain is required for the interaction
with Beclin 1. These findings, together with the observation that
TRS1 blocks starvation-induced autophagy in PKR?/?cells, sup-
port the hypothesis that TRS1 inhibits autophagy as a result of its
interaction with Beclin 1.
bition in fibroblasts. The observations that TRS1 interacts with
Beclin 1 and inhibits starvation-induced autophagy to the same
extent as ICP34.5 support the role of TRS1 in the inhibition of
autophagy in HCMV-infected cells. To test whether the virally
expressed TRS1 is necessary for the inhibition of autophagy in
HCMV-infected cells, we analyzed autophagy in cells infected
indistinguishable from those of mock-infected fibroblasts (Fig.
decreased in HCMV-infected cells. Moreover, the number of
?TRS1 mutant virus was not able to block starvation-induced
autophagy. We found that Bcl-2 is overexpressed and hyperphos-
phorylated in ?TRS1 mutant virus-infected cells similarly to re-
sults in HCMV-infected cells (Fig. 9C and D). These results con-
firmed that increased expression of Bcl-2 plays no role in
inhibition of autophagy by HCMV.
In this report, we report that HCMV stimulates autophagy in hu-
of viral protein synthesis and is able to inhibit autophagosome
formation and maturation by 24 h postinfection, as a result of
expression of one or more viral proteins. We identify the viral
protein TRS1 of HCMV as a negative modulator of autophagy,
independently of its previously described function on the kinase
PKR but likely via its interaction with the autophagy protein Be-
clin 1 (26).
The observation that autophagy is stimulated at early time
points after infection independently of the viral replication is in a
good agreement with a recent study by McFarlane et al. (40). The
significance of the early induction of autophagy by HCMV re-
infection and may aid in an adaptive immune response by pro-
moting viral antigen presentation by major histocompatibility
complex (MHC) class II molecules. It might also contribute to an
early innate immune response by degradation of viruses into au-
tophagolysosomes, referred to as xenophagy (45, 56). In fact,
by interacting with cell surface receptors and/or soon after their
entry in the cell cytoplasm (22, 28). For example, the binding of
measles virus (Edmonston strain) to CD46 at the surface of hu-
man cells induces de novo formation of autophagosome (28). The
mechanism by which HCMV triggers autophagy remains to be
better defined. McFarlane et al. proposed that autophagy is stim-
FIG7 The N-terminal region of TRS1 is required for inhibition of autophagy
of the region required for PKR binding (amino acids 679 to 738) and the
dsRNA-binding domain (amino acids 74 to 248). Different TRS1 constructs
fragment [TRS1 (1-679)], and N-terminal deleted fragments [TRS1 (45-795)
and (93-795)]. (B) Representative images of GFP-LC3 staining in HeLa cells
cotransfected for 48 h with GFP-LC3 and indicated TRS1 constructs. Four
hours before fixation, cells were grown in starvation medium to induce au-
tophagy. TRS1-transfected cells were visualized by an anti-His antibody, and
GFP-LC3-positive cells with GFP-LC3 dots were quantified (C). The results
are the means of three independent experiments. Between 50 and 100 cells
were analyzed per assay. *, P ? 0.05 (t test). (D) Coimmunoprecipitation of
endogenous Beclin 1 and indicated His-tagged mutant TRS1 in HeLa cells
transfected with the indicated plasmids.
Chaumorcel et al.
jvi.asm.org Journal of Virology
may be that contact of the viral particle with the cell surface or
interaction between a viral structural component and a cellular
sensor triggers autophagy. In line with this possibility, we ob-
served that the binding of HCMV to cell surface heparan sulfate
HCMV interacts with Toll-like receptor 2 (TLR2) via two viral
tion independently of viral entry (29). Since TLR activation by
different ligands induces autophagy, it will be interesting to test
whether activation of TLR2 by HCMV triggers autophagy (15).
Whatever the mechanism responsible for the induction of au-
tophagy by HCMV, we observed that the virus is able to inhibit
The inhibition of autophagy has been shown by the use of differ-
ent readouts for autophagy: accumulation of GFP-LC3 and GFP-
RFP-LC3 puncta, degradation of the autophagy cargo p62, and
of wortmannin. On the basis of these criteria, we conclude that
HCMV blocks autophagy at the stage of autophagosome forma-
is still able to stimulate autophagy after 24 h of infection. This
Western blotting, a usually reliable method to detect the form of
the protein associated with autophagosomes. However, we previ-
ously noticed that LC3-I and -II accumulated in HCMV-infected
cells independently of the accumulation of autophagosomes (9)
coronaviruses to provide the membranous support for viral rep-
lication complex, in an autophagy-independent way (49). More-
over, the final envelopment of HCMV in the viral assembly com-
plex requires a remodeling of membranes from different
organelles, such as the trans-Golgi network (TGN) and endo-
as Atg9 (37, 62). Therefore, it would be interesting to investigate
whether LC3 and/or some Atg proteins could have a role during
HCMV infection, independently of their function in autophagy.
To understand the mechanisms by which HCMV inhibits au-
tophagy, we decided to focus on the HCMV protein TRS1, which
ing the PKR/eiF2? pathway. Activation of this signaling pathway
recombinant virus, and the viral protein ICP34.5 is capable of
time TRS1 as a HCMV protein able to block autophagy and to
interact with Beclin 1. Consistent with the timing of HCMV-
mediated autophagy inhibition, we observed that TRS1 accumu-
h p.i. (data not shown). Two functional domains of TRS1 have
been identified: the carboxy-terminal part, which is required for
interaction with PKR, and an unconventional dsRNA-binding
domain located in a 175-amino-acid stretch close to the N termi-
nus (between amino acids 74 and 248) (25). We thus explored
which domain of TRS1 is involved in the inhibition of the au-
venting phosphorylation of eIF2?, we were surprised to discover
FIG 8 TRS1 inhibits autophagy independently of PKR. Murine PKR?/?and
PKR?/?cells were cotransfected for 48 h with GFP-LC3 and His-TRS1 or
FLAG-ICP34.5 constructs. Four hours before fixation, cells were grown in
starvation medium to induce autophagy. TRS1-transfected cells were visual-
ized by an anti-His antibody, and ICP34.5-transfected cells were visualized by
an anti-FLAG antibody. (A) Representative images of GFP-LC3 in PKR?/?
and PKR?/?cells. Inserts show the viral protein staining. (B) GFP-LC3 posi-
independent experiments. Twenty cells were analyzed per assay. *, P ? 0.05;
**, P ? 0.01 (t test).
HCMV Bidirectionally Modulates Autophagy
March 2012 Volume 86 Number 5 jvi.asm.org 2581
that this domain is not necessary for inhibiting autophagy in our
is no longer able to block autophagy. This observation, together
with the fact that the N-terminal part of TRS1 is required for
strong binding to Beclin 1, suggests that TRS1 blocks autophagy
TRS1 inhibit autophagy not via their ability to interfere with the
activation of PKR but via their interaction with Beclin 1 (44).
Since numerous viral proteins block the PKR-eIF2? signaling
pathway to preclude the shutoff of protein synthesis, one impor-
tant question that arises is whether autophagy can be inhibited by
the unique blockade of PKR-dependent signaling by a viral pro-
We observed that the form of virally expressed TRS1 is suffi-
9). It is interesting to note that even though ?TRS1 mutant virus
did not inhibit autophagy, we did not observe a stimulation of
autophagy such as the one induced by UV-HCMV. HCMV ex-
also antagonize autophagy. It is likely that IRS1, which has an
amino terminus identical to that of TRS1, duplicates this TRS1
function. Indeed, the N-terminal 549-amino-acid regions of the
two proteins are identical and although their C-terminal regions
diverge, they nevertheless remain 55% identical in the divergent
region (3). Construction of ?TRS1/?IRS1 recombinant mutant
virus revealed that IRS1 can compensate for TRS1 regarding PKR
inactivation (38). Unfortunately, the mutant virus lacking both
genes is totally unable to replicate in human fibroblasts (38). An
alternative approach could be to use a chimeric ?34.5-HSV-1 re-
combinant virus which expresses the HCMV gene TRS1. Indeed,
Cassady showed that TRS1 can complement ?34.5-HSV-1 re-
combinant virus regarding wild-type viral protein synthesis in in-
fected cells (6). Whereas infection with a ?34.5-HSV-1 recombi-
nant virus stimulates autophagy, it is not known whether TRS1
can also restore the inhibitory effect of ICP34.5 on autophagy in
infected cells. Monitoring the autophagy level after infection with
the HCMV/HSV-1 chimeric virus would address this possibility.
It is possible that HCMV possesses more than one mechanism
for inhibiting the autophagic pathway. Cellular Bcl-2 (cBcl-2) is
with Beclin 1 (48). Despite the fact that other herpesviruses, such
as HHV8 and MHV-68, express a viral homolog of Bcl-2 able to
inhibit autophagy, HCMV does not possess one. Although
HCMV stimulates the expression of cBcl-2, it does not use this
strategy to block autophagy. Indeed, Bcl-2 is overexpressed in
?TRS1 mutant virus-infected cells whereas autophagy is not in-
ylation, as well as the induction of the expression of Bcl-2, is de-
pendent on the activity of JNK. Previous studies have shown that
the JNK-dependent phosphorylation of Bcl-2 triggers the dissoci-
ation of its complex with Beclin 1 (47, 61). It is interesting to note
that the gammaherpesvirus Bcl-2 homologue constitutively
blocks autophagy via its interaction with Beclin 1, because vBcl-2
does not have the N-terminal loop that contains amino acid sub-
strates for JNK and thus cannot be phosphorylated (61). Another
possibility concerns the autophagy regulatory complex mTORC1
sion of TRS1 and IEA in MRC5 cells infected with HCMV wild-type and
?TRS1 mutant virus. (B) MRC5 cells were infected with HCMV wild-type or
with GFP-LC3. When indicated, cells were grown in starvation medium
(EBSS) 4 h before fixation to induce autophagy. Autophagy was quantified in
IEA-positive cells by counting the number of GFP-LC3 puncta per cell. The
results are the mean of three independent experiments. Twenty cells were
analyzed per assay. *, P ? 0.05; ns, not significant (t test). (C) Immunoblot
analysis of Bcl-2 protein in cells infected with wild-type HCMV or ?TRS1
mutant virus at an MOI of 1, from 24 to 72 h. (D) Accumulation of hyper-
phosphorylated Bcl-2 during ?TRS1 mutant virus infection in MRC5 fibro-
blasts at 24, 48, and 72 h p.i. Nuclear red staining corresponds to viral IE
Chaumorcel et al.
jvi.asm.orgJournal of Virology
turns on the protein synthesis involved in cell growth but also
suppresses autophagy (reviewed in reference 20). HCMV influ-
ences multiple cellular pathways that communicate with
mTORC1, such as inhibition of the tuberous sclerosis complex
TSC1/2 by the HCMV UL38 protein (43). Moreover, the activa-
tion and the perinuclear localization of mTORC1 are maintained
in HCMV-infected cells, even during nutrient starvation, which
normally inhibits mTOR and activates autophagy (12).
HSV-1 to block autophagy by interacting with Beclin 1. Herpes-
viruses have developed redundant strategies to block autophagy
by interfering with the function of Beclin 1 or other Atg proteins
involved in the biogenesis of autophagosomes. Our results dem-
to block autophagy independently of their ability to produce a
viral form of Bcl-2.
We thank T. Yoshimori for providing us with the GFP-LC3 and mRFP-
precipitation and proteolysis experiments. We thank Claudine Delomé-
nie (IFR141 innovation thérapeutique, Châtenay-Malabry) for providing
assistance with quantitative reverse transcription-PCR studies and Caro-
line Tran Van (UPS, Châtenay-Malabry).
This work was supported by institutional funding from The Institut
National de la Santé et de la Recherche Médicale (INSERM) and from
(ANR MIME 2007) to A.E., from the National Institutes of Health
(AI26672) to A.P.G., and the Deutsche Forschungsgemeinschaft
(BR1730/3-1) to W.B.
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