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RESEARCH PAPER
Induction of DNA double-strand breaks and cellular senescence by human
respiratory syncytial virus
Isidoro Mart
ınez
a
,
y
, Ver
onica Garc
ıa-Carpizo
b
,
y
, Trinidad Guijarro
a
, Ana Garc
ıa-Gomez
a
, Diego Navarro
b
,
Ana Aranda
b
, and Alberto Zambrano
a
a
Department of Molecular Pathology, Spanish National Center for Microbiology, Institute of Health Carlos III, Madrid, Spain;
b
Department of
Endocrine Physiopathology and Nervous System, Institute for Biomedical Research (IIBM), CSIC-UAM, Madrid, Spain
ARTICLE HISTORY
Received 12 August 2015
Revised 11 January 2016
Accepted 14 January 2016
ABSTRACT
Human respiratory syncytial virus (HRSV) accounts for the majority of lower respiratory tract
infections during infancy and childhood and is associated with significant morbidity and mortality.
HRSV provokes a proliferation arrest and characteristic syncytia in cellular systems such as
immortalized epithelial cells. We show here that HRSV induces the expression of DNA damage
markers and proliferation arrest such as P-TP53, P-ATM, CDKN1A and gH2AFX in cultured cells
secondary to the production of mitochondrial reactive oxygen species (ROS). The DNA damage foci
contained gH2AFX and TP53BP1, indicative of double-strand breaks (DSBs) and could be reversed
by antioxidant treatments such as N-Acetylcysteine (NAC) or reduced glutathione ethyl ester
(GSHee). The damage observed is associated with the accumulation of senescent cells, displaying a
canonical senescent phenotype in both mononuclear cells and syncytia. In addition, we show signs
of DNA damage and aging such as gH2AFX and CDKN2A expression in the respiratory epithelia of
infected mice long after viral clearance. Altogether, these results show that HRSV triggers a DNA
damage-mediated cellular senescence program probably mediated by oxidative stress. The results
also suggest that this program might contribute to the physiopathology of the infection, tissue
remodeling and aging, and might be associated to long-term consequences of HRSV infections.
KEYWORDS
cellular senescence; DNA
damage; human respiratory;
ROS; syncytial virus
Introduction
HRSV is an enveloped virus that belongs to the Pneumo-
virus genus of the Paramixoviridae family.
5,14
HRSV rep-
licates in airway epithelial cells activating a variety of
mediators involved in lung immune/inflammatory
responses such as proinflammatory cytokines, chemo-
kines, interferons and adhesion molecules.
24,26
The gene
expression of these mediators is regulated by key tran-
scription factors including nuclear factor-kB (NF-kB),
the signal transducers and activators of transcription
(STATs) and interferon regulatory factors (IRFs).
Through its action on the signaling pathways implicated
in the activation of these transcription factors, reactive
oxygen species (ROS) formation can regulate the expres-
sion of the immune/inflammatory responses.
1,24
Exces-
sive ROS production, however, can lead to oxidative
stress and cause severe damage to cells. Thus, oxidative
stress has been increasingly recognized as a contributing
factor in aging and in multiple pathologies including
lung inflammatory diseases.
38,51
HRSV infection is
accompanied by the induction of ROS whose production
by the pulmonary epithelial and endothelial cells is
involved in the activation of transcription factors, oxida-
tive stress and lung damage in infected cells in both ani-
mals and children.
9,10,24,30,31,32,37
Compelling evidence
indicate that antioxidants treatments inhibit both viral
HRSV replication and the activation of NF-kB, STATs
and IRFs.
9,31,37,43
Moreover, antioxidants reduce HRSV-
induced oxidative stress and clinical disease in a mouse
model of infection suggesting a causal relationship
between increased ROS production and lung disease.
10
DNA double-strand breaks (DSBs) may arise from a
variety of exogenous and endogenous sources and in
general by cell stress coming from any deviation from
normal physiological conditions.
4,47,55
One of the most
CONTACT Alberto Zambrano azambra@isciii.es; Department of Molecular Pathology, Spanish National Center for Microbiology, Institute of Health
Carlos III, Ctra. Majadahonda-Pozuelo Km 2, Majadahonda, Madrid 28220, Spain; Alberto Zambrano aaranda@iib.uam.es Instituto de Investigaciones
Biomédicas “Alberto Sols”Arturo Duperier, 4, Madrid-28029, Spain
Color versions of one or more of the figures in this article can be found online at www.tandfonline.com/kvir.
y
These authors contributed equally to this work.
Supplemental data for this article can be accessed on the publisher’s website.
© 2016 Taylor & Francis
VIRULENCE
2016, VOL. 7, NO. 4, 427–442
http://dx.doi.org/10.1080/21505594.2016.1144001
important sources of DSBs is the generation of ROS in
cells. ROS are produced primarily by the mitochondria
and can induce base oxidation, abasic sites, and even
both single and double-DNA strand breaks.
7,16,20,66
A
significant number of the DNA single-stranded lesions
induced by ROS are converted in DSBs, either by a direct
mechanism or by the repair process itself.
63,72
A compel-
ling amount of evidence has strengthened the notion
that oxidative stress can trigger cellular senescence, a cel-
lular state characterized primarily by an irreversible
proliferative arrest. Besides the absence of proliferation,
senescent cells display a number of morphological
changes and markers that in aggregate define the senes-
cent phenotype. These include the expression of a senes-
cence-associated b-galactosidase activity (SA-bgal),
expression of tumor suppressors and cell cycle inhibitors,
secretion of diverse cytokines and often also the presence
of DNA damage lesions phenotype.
8,15,18,61
There are a
number of stimuli that induce senescence that ultimately
converge in the activation of TP53 and of key cyclin-
dependent kinase (CDK) inhibitors such as CDKN2A
(p16), CDKN2B (p15), CDKN1A (p21) and CDKN1B
(p27). These mediators trigger a proliferation arrest and
the hypo-phosphorylation of the tumor suppressor RB1
that implements the cellular senescence program.
53
Senescence as well as apoptosis has emerged as physio-
logical mechanisms to prevent proliferation of damaged
cells. However, recent evidence has expanded its role
beyond DNA damage or stress. For instance, senescence
may occur during development
52,53,70
(developmental
senescence) and as a physiological program in adult cells,
such as normal megakaryocytes and placenta syncytio-
trophoblasts,
73
in association with DNA damage
markers, TP53, CDKN2A and CDKN1A.
HRSV induces a proliferation arrest by activating
transforming growth factor band increasing the abun-
dance of cell-cycle regulatory molecules involved in
G
0
/G
1
phase control.
3,25,74
In addition, the infection
courses with delayed low levels of apoptosis due to differ-
ent antiapoptotic (resistance) mechanisms.
12,19,42,49,50
This evidence together with the fact that HRSV is able to
activate the DNA damage response (DDR)
21
led us to
investigate the role of HRSV in DNA damage and cellu-
lar senescence.
We show here the occurrence of cellular senes-
cence during the infection of HRSV. HRSV induces
the expression of DNA damage markers and prolifer-
ation arrest such as P-TP53, P-ATM, CDKN1A and
gH2AFX (H2A histone family member X, phosphory-
lated on Ser 139) in cultured cells secondary to the
production of mitochondrial ROS. The DNA damage
foci contained gH2AFX and TP53BP1, indicative of
DSBs and could be reversed by antioxidants
treatments such as N-Acetylcysteine (NAC) or
reduced glutathione ethyl ester (GSHee). The damage
observed is associated with the accumulation of senes-
cent cells, displaying all the hallmarks of the senes-
cence phenotype in both mononuclear cells and
syncytia. In addition, we show signs of DNA damage
and aging such as gH2AFX and CDKN2A expression
in the lungs of infected mice at different times post-
infection. Altogether, these results show that oxidative
stress induced by HRSV triggers a DNA damage-
mediated cellular senescence program. The results
also suggest that the DNA damage inflicted and the
senescence program might contribute to the physio-
pathology of the infection, tissue remodeling and
aging, and might be associated to long-term conse-
quences of HRSV infections.
Results
HRSV induces cellular senescence in cultured cells
We observed that the infection of A549 and HEp-2 cells
by HRSV induces the accumulation of senescent cells
positive for SA-bgal activity. This activity could also be
detected in spontaneous syncytia of both immortalized
cell lines (»20–40%), but was greatly magnified in both
mononuclear cells and syncytia upon HRSV infection
(Fig. 1a-d and Fig. S1a-d). The absence of significant
levels of SA-bgal in the majority of the spontaneous
syncytia suggests that besides the fusion process there is
aspecific mechanism responsible for the SA-bgal activ-
ity associated to the infection of these immortalized
cells. We performed the infections 24 h after seeding
cells at two densities, 5200 cells/cm
2
(Fig. 1a-d)and
31200 cells/cm
2
(Fig. S1a-b) These cellular densities
correspond approximately to 18% and 33% of the typi-
cal density used in conventional infections (93750 cells/
cm
2
) and prevent the typical SA-bgal background due
to cell over confluence. Under these conditions, expo-
sure of A549 cells to HRSV produces a drastic increase
in the levels of senescence, being higher at cellular den-
sities that favor virus dissemination (31200 cells/cm
2
).
This effect was magnified when HEp-2 cells were used
(Fig. 1c,d and Fig. S1b,c). This cell line of laryngeal ori-
gin, the cell line of choice for virus production, shows
high levels of chromosomal instability and is very per-
missive for HRSV replication. Due to the high levels of
senescence found upon HRSV infection, we used an
assay consisting of a combination of SA-bgal activity
and immunocytochemical staining. This technique
termed here Immuno-SA-bgal allows the detection of
senescent cells expressing high levels of the antigen of
choice, in this case, HRSV antigens. As shown in
428 I. MART
INEZ ET AL.
Figure 1. Occurrence of cellular senescence in cells infected with HRSV. (A) Occurrence of cellular senescence during HRSV lytic infection
of A549 cells seeded at 5200 cells/cm
2
. The cells were processed for SA-bgal assay at 48 h.p.i. (MOI D3). Magnification: 200X, scale bar:
50 mm. (B) Quantification of SA-bgal expression in A549 cells (in total population and syncytia). (C) and (D) Similar assays in HEp-2 cells.
(E) Immuno-SA-bgal, an assay that allows the detection of SA-bgal expression in massively infected HEp-2 cells stained with a-HRSV
antibodies (red staining). Magnification: 200X; scale bar: 50 mm. (F) Occurrence and quantification of cellular senescence in a culture of
HEp-2 cells persistently infected by HRSV. Magnification: 200X; scale bar: 50 mm. (G) Quantification of IL-6 and TNF-ain supernatants of
cultured cells. Bars in the graphs represent the mean §SD of 2–3 experiments. n D3 replicates. (H) Analysis of apoptosis/necrosis. Eight
thousand cells/well were plated in 6-well plates and were infected with HRSV the following day at MOI D3. At 60 h.p.i., the cells were
processed for flow cytometry. After treatment with the fluorescent probes (AnnexinV-AlexaFluor488TM and SytoxTM) apoptotic cells
show green fluorescence, dead cells show brighter green fluorescence, and live cells show little. These populations were distinguished
in the FL1 channel of a FACSCalibur flow cytometer. Treatment of cells with 20 mM camptothecin (CPT) for 12 h was used as positive
control for apoptosis. (I) SA-bgal assay using the fluorogenic bgalactosidase substrate C12FDG and flow cytometry (upper panel) or con-
ventional SA-bgal (bottom panel). Eight thousand cells/well were plated in 6-well plates and were infected following day with HRSV at
MOI D3. At 60 h.p.i., the cells were incubated with Bafilomycin A1 and C12FDG and processed for flow cytometry or conventional SA-
bgal assay (bottom panel) as described in materials and methods. Magnification: 200X; scale bar: 50 mm.
VIRULENCE 429
Fig. 1e, nearly 100% of the cells were infected (red
staining) and a significant proportion of them (approxi-
mately 70%) were also positive for SA-bgal. Although
to a lesser extent than in lytic infections of HEp-2 cells,
significant levels of senescence could be detected in
HEp-2 cells persistently infected with HRSV, i.e., cells
that survived a lytic infection and were grown to obtain
a culture that can be propagated indefinitely (Fig. 1f).
These cells exhibited a constant virus production for
more than twenty passages and a high heterogeneity
regarding the expression of viral antigens, ranging from
high to undetectable levels.
42
Thus, the coexistence of
uninfected and infected cells in these complex cultures
may account for the high levels of senescence observed
compared to uninfected HEp-2 cells.
In addition to being flat and bigger than their normal
counterparts, senescent cells display a senescence-associ-
ated secretory phenotype (SASP), characterized by
expression of proinflammatory cytokines, growth factors
and proteases. HRSV induces a rapid activation of sev-
eral mediators involved in the immune/inflammatory
responses. Strikingly, the broad array of proinflamma-
tory cytokines induced by HRSV resembles the expressed
SASP by canonical senescent cells.
5,14,60
As reported by
others, we detected significantly higher levels of IL-6 and
TNF-ain supernatants of infected cells when compared
with mock-infected cells (Fig. 1g). In order to know
whether cellular senescence can be associated to cell sur-
vival, we assessed the levels of apoptosis and senescence
after 60 h.p.i. The levels of apoptosis were determined
with the Annexin V/Sytox assay. This assay allows the
detection of live cells (low green fluorescence), apoptosis
(moderate green fluorescence due to Annexin V binding)
and necrosis (intense green fluorescence due to the Sytox
green dye which is impermeable to live cells). The levels
of apoptosis were similar for mock-infected and infected
cells (4% approximately) and relatively low compared to
the levels found when cells were treated with camptothe-
cin, a known inducer of apoptosis (Fig. 1h). On the other
hand, the majority of cells that survived a lytic infection
showed higher intensities of C12FDG fluorescence
(senescence) as determined by flow cytometry analysis
using this fluorogenic b-galactosidase substrate (Fig. 1i,
upper panel). Cells showing FL1-H intensities over value
10 represented less than 4% of the control cultures, how-
ever, this proportion increased to 30 in the case of
infected cultures. Finally, approximately 60% of cells that
survived a lytic infection were senescent as determined
by the conventional SA-bgal assay (Fig. 1i, bottom
panel). Together, these findings suggest that cellular
senescence is a biological program associated to HRSV
infections and might be associated to cell survival.
HRSV-induced cellular senescence is associated
to DNA damage
As senescence is primarily a mechanism to prevent pro-
liferation of damaged cells, we reasoned that the senes-
cence signs observed in virus-infected cells could be
associated to DNA damage. Of the different types of
DNA lesions, DSBs are the most deleterious, being
potent inducers of chromosomal rearrangements such as
deletions, translocations or amplifications.
67,75
The phos-
phorylated histone H2AFX (gH2AFX) and TP53BP1 are
accurate markers of DNA damage.
2,56,62,64,65
When DSBs
are detected, a signaling cascade is initiated by the phos-
phorylation of H2AFX by Ataxia-Telangiectasia Mutated
kinase (ATM) near the break site, followed by the rapid
recruitment of TP53BP1 on the chromatin surrounding
the DNA lesion. TP53BP1, among other functions, acts
as a molecular scaffold in damaged chromatin during
non-homologous end-joining (NHEJ) repair mecha-
nism.
6,56
We analyzed the expression of DNA damage
(DD) foci containing gH2AFX and TP53BP1 (Fig. 2a,b).
First, we examined their confocal colocalization in A549
cells. As extensively reported before in other cellular sys-
tems, we corroborated their significant colocalization
and observed an increase of DD foci in infected-A549
cells, in both mononuclear cells and syncytia (Fig. 2a).
Next, we performed conventional microscopy to analyze
and quantify the levels of DD in the same cells and in
HEp-2 cells (Fig. 2b, Fig. S1e, f). We observed a signifi-
cant increase of cells containing DD foci and cells con-
taining numerous foci (>5/nucleus) after virus infection.
This effect was exacerbated in the HEp-2 cell line
(Fig. S1e, f). We further examined various markers of the
DNA damage response (DDR), proliferation arrest and
cellular senescence in A549 cells. As shown in Figure. 2c,
the infection of A549 cells induces the phosphorylation
and activation of ATM, TP53 and H2AFX, an elevation
in CDKN1A and CDKN2A expression and RB1 hypo-
phosphorylation. Altogether these findings indicate that
cellular senescence is a common phenomenon during
HRSV infection and is associated with the presence of
DNA damage.
Reactive oxygen species (ROS) generated during
HRSV infection induce DNA damage
Induction of ROS by HRSV has been extensively docu-
mented.
9,10,24,30,31,32,37
As previously reported, we
observed an elevation in the total ROS levels during the
infection of A549 cells by using the widely used probe
DCFH-DA
9,34
(Fig. 3a). As expected, we also observed a
significant increase in the intracellular amount of
430 I. MART
INEZ ET AL.
oxidized glutahione (GSSG) concomitantly with a
decrease in reduced glutathione (GSH), upon HRSV
infection as previously reported
32,48
(Fig. 3b). As ROS
are produced primarily by the mitochondria we further
examined whether HRSV could trigger the formation of
superoxide (O
2
.
¡
), the main mitochondrial ROS. HRSV
infection induced a drastic increase in superoxide
generation, detected with the specific mitochondrial
probe MitoSOX by immunofluorescence and by flow
cytometry (Fig. 3c,d, Fig. S2 a). Therefore, ROS triggered
by HRSV have at least in part a mitochondrial origin. As
ROS are able to induce DSBs and HRSV behaves as a
potent stressor inducing mitochondrial superoxide, we
reasoned that the DD observed could be alleviated by
Figure 2. HRSV induces DNA damage. (A) Analysis of confocal colocalization of the DNA damage markers gH2AFX and TP53BP1 in
HRSV-infected A549 cells (48 h.p.i. MOI D3). Magnification: 600X; scale bar: 10 mm (B) Conventional indirect immunofluorescence show-
ing the presence of gH2AFX and TP53BP1 in mock and infected A549 cells (48 h.p.i. MOI D3). Magnification: 600X; scale bar: 10 mm.
Quantification of the DNA damage foci containing gH2AFX and TP53BP1 is shown at the right panel. (C) Detection by western-blotting
of various DNA damage and proliferation arrest markers in mock and infected A549 cells (30 h.p.i. MOI D3: panels P-TP53, CDKN2A, P-
RB1, P-ATM); 48 h.p.i. MOI D3: panels CDKN1A and gH2FAX. KDa: kilodaltons. Bars in the graphs represent the mean §SD of 2 experi-
ments, n D3 replicates. DNA damage foci were counted from >150 cells for each experimental condition. Data from panel c are of a
representative experiment.
VIRULENCE 431
treatments with antioxidants as previously reported.
39,77
We treated cells with N-Acetylcysteine (NAC) and found
a drastic reduction in the number of damaged nuclei and
a concomitant moderate reduction on virus titers
(Fig. 4A-C). This is consistent with previous reports indi-
cating that antioxidant treatment inhibits HRSV infec-
tion
9,31,37,43
and ameliorates clinical disease and
pulmonary inflammation in mice.
10,31
Similar results
were obtained when cells were treated with reduced glu-
tathione ethyl ester (GSHee), a permeable source of GSH
(Fig. 4D-F). Treatment of cells with GSHee increased the
intracellular pool of GSH and induced a reduction in
DD foci and virus titers. When GSHee or NAC were also
included during virus adsorption, virus titers were very
strongly reduced (data not shown), indicating that these
antioxidants might have also an effect on virus integrity,
attachment or entry into the cells. Remarkably, neither
of the two treatments altered substantially the low levels
of constitutive DD foci, suggesting that the antioxidants
are only effective against the de novo DD foci induced by
HRSV. Antioxidants treatment also reduced the occur-
rence of senescence in infected cultures (Fig. S2 b), and
the expression or activation of DD markers such as P-
TP53, gH2AFX and CDKN1A (Fig. S2 c). In addition,
the partial depletion of TP53BP1 mediated by small-
interfering RNAs (siRNAs) increased significatively the
levels of senescence in infected cultures (Fig. S2 d,e)
probably due to an additional increase of unrepaired
DNA damage. In order to determine whether the oxida-
tive stress itself was responsible of the DD observed, we
transfected a plasmid encoding human catalase to HRSV
infected cells and evaluated the presence of DD foci.
Figure 3. HRSV induces mitochondrial reactive oxygen species (ROS). (A) Measurement of total ROS levels of mock and infected A549
cells (48 h.p.i. MOI D3) using the DCFH-DA probe and fluorometry. (B) Measurement of reduced (GSH) and oxidized (GSSG) glutathione
of mock and infected A549 cells (24 h.p.i. MOI D3). (C) Evaluation of mitochondrial ROS in mock and infected A549 cells (24 h.p.i. MOI
D3) with MitoSOX and fluorescence microscopy. Positive control: cells treated with 100 mM Paraquat. Images of cells treated with Para-
quat, acquired at the same exposure to that of the rest of the samples (equivalent exposure) and at a reduced exposure, are shown.
Magnification: 400X; scale bar: 20 mm. (D) Assessment of mitochondrial ROS levels of mock and infected A549 cells (24 h.p.i. MOI D3)
with MitoSOX and flow cytometry. The left panel shows representative histograms. The mean intensity of MitoSOX fluorescence and the
percentage of positive cells are shown at the right panel. Bars represent the mean §SD of two experiments, ANOVA of data D:
P<0,0001; n D3 replicates.
432 I. MART
INEZ ET AL.
Figure 4. HRSV-induced DNA damage is reduced with antioxidant treatments (A) Quantification of DNA damage foci containing gH2AFX
and TP53BP1 in mock and infected A549 cells (48 h.p.i. MOI D3), treated or not with N-Acetylcysteine (NAC). Cells were treated with
5 mM NAC 90 min before the infection and then after virus adsorption until the end of the experiment. (B) Virus titers in supernatants
of A549 infected cells (48 h.p.i. MOI D3) treated or not with 5 mM NAC. (C) Representative images of DNA damage foci of mock and
infected A549 cells (48 h.p.i. MOI D3), treated or not with N-Acetylcysteine (NAC). Magnification: 600X; scale bar: 10 mm. Arrowheads:
DD foci. (D) Increase of intracellular reduced glutathione (GSH) in mock and infected A549 cells (24 h.p.i. MOI D3) upon treatment with
2,5 mM GSH ethyl ester (GSHee). (E) Quantification of DNA damage foci containing gH2AFX and TP53BP1 in mock and infected A549
cells (48 h.p.i. MOI D3) treated or not with GSHee. Cells were treated with GSHee 90 min before the infection and then after virus
adsorption until the end of the experiment. Virus titers in supernatants of A549 infected cells (48 h.p.i. MOI D3) treated or not with
2,5 mM GSHee (right panel). (F) Representative images of DNA damage foci in mock and infected A549 cells (48 h.p.i. MOI D3), treated
or not with GSHee. Magnification: 600X; scale bar: 10 mm. (G) Left panel: Expression of transfected catalase (flagged-catalase, in green)
and DD foci (TP53BP1 in red) in A549 cells infected with HRSV (48 h.p.i. MOI D3) and transfected with the plasmid pCMV3-CAT-N-FLAG.
Cells labeled as “b”and “c”show catalase overexpression and the absence of DD foci; syncytium labeled as “a”shows the absence of sig-
nificant catalase overexpression and the presence of nuclei harboring numerous DD foci. Right panel: expression of DD foci (TP53BP1)
and HRSV-F glycoprotein (green) in A549 infected cells (48 h.p.i. MOI D3) Magnification 600X, Scale bar: 10 mM. (h) Left: panel: Quanti-
fication of DD foci in A549 cells infected with HRSV (48 h.p.i. MOI D3) and transfected with the plasmid pCMV3-CT-N-FLAG. CAT
¡
: cells
without significant catalase overexpression, CAT
C
: cells overexpressing catalase. Right panel: virus titers of culture supernatants of A549
cells infected with HRSV (48 h.p.i. MOI D3) and transfected with either empty vector as a control or catalase (CAT) plasmid. Bars repre-
sent the mean §SD of two experimenst; n D3 replicates. DNA damage foci were counted from >150 cells for each experimental
condition.
VIRULENCE 433
Catalase has a preeminent role in protecting cells from
oxidative damage caused by ROS.
36,76
Catalase calalyzes
the decomposition of H
2
O
2
to H
2
O and O
2
.H
2
O
2
is a
harmful byproduct of many normal metabolic processes
including the reaction of superoxide dismutase (SOD)
enzyme that converts superoxide radical into O
2
and
H
2
O. As shown in Figure 4g (cells labeled as “b”and “c”
in the picture on the left), the overexpression of catalase
reduced the occurrence of DD foci while cells with
apparent signs of infection (syncytium labeled as “a”in
Fig. 4g) showed numerous DD foci similar to those typi-
cal syncytia stained with HRSV-F glycoprotein (Fig. 4g,
picture on the right). The analysis of the DD foci and the
expression of catalase in the infected/transfected popula-
tion indicated that the expression of DD foci was signifi-
catively reduced in cells overexpressing catalase (Fig. 4h,
CAT
C
cells, left panel). In contrast to antioxidant treat-
ments, transfection of the plasmid encoding catalase did
not alter significantly virus titers compared to cells
infected and transfected with the empty vector (control
culture) (Fig. 4h, right panel). These results suggest that
oxidative stress is directly responsible of the damage
observed and link the occurrence of DD damage with
cellular senescence. The physical presence of DNA
breaks in infected cells was also directly detected by
using the terminal deoxynucleotidyl transferase (TdT)
enzyme,
77
(Fig. S2f). We also assessed the repair capabil-
ity of infected cells after irradiation with g-rays (Fig. S3
a-d). Due to the progression of the infection and the sub-
sequent accumulation of de novo foci, the DD load was
still very high compared to control cells. Analysis of the
DDR at a single time suggested that HRSV does not
affect significantly the early DDR signaling (Fig. S3e, f).
Thus, the DD inflicted by HRSV is most likely due to
oxidative stress and does not appear to be caused by a
gross defect in cellular DSBs repair capacity.
Signs of DNA damage and aging in lungs of young
mice infected with HRSV
Mice were either mock infected (n D5) or infected with
HRSV (strains Long and A2, n D6) and the lungs were
analyzed by a highly sensitive multiple immunofluores-
cence assay to detect both gH2AFX, a marker of DD and
aging,
44
and HRSV antigens. We found no significant
differences in DD expression and viral antigens between
the two strains used. As shown in Figure 5b and Fig. S3g,
we observed signs of severely damaged DNA in 23%§2
of HRSV-infected cells, in »30% of the columnar epithe-
lia of bronchioles (CSCE: ciliated simple columnar epi-
thelium). Moderate DNA damage, 1 or 2 foci/nucleus
approximately, could be also detected in »30% of the
alveolar epithelium of infected mice (26%§5 of alveolar
cells with significant reactivity to HRSV antigens showed
DD foci)(data not shown). In contrast, no significant
gH2AFX reactivity was detected in the lungs of mock-
infected cells (Fig. 5A,C). We further evaluated the
expression of both gH2AFX and CDKN2A by conven-
tional immunohistochemistry at different times post
infection (4, 11 and 30 days). HRSV-infected mice nor-
mally show a viral replication that peaks at days 4–5 and
becomes undetectable by day 7.
33
As expected, we did
not observe reactivity to HRSV antigens at days 11 and
30 post infection in the lungs of infected mice (data not
shown), however, we could still detect signs of DNA
damage and senescence in the respiratory epitelium of
some bronchioles (»18%) at those times post infection
(Fig. 5E, F). These results suggest the occurrence of DNA
damage and senescence long time after a primary
infection.
Discussion
We show for the first time the occurrence of cellular
senescence in cultured immortalized epithelial cells
infected with HRSV. These infected cells show the
canonical cellular senescence phenotype, including the
SA-bgal activity, components of the SASP phenotype
and the presence of accurate markers of DNA damage
and proliferation arrest such as the phosphorylation and
activation of ATM, TP53 and H2AFX, an elevation in
CDKN1A and CDKN2A expression and RB1 hypo-
phosphorylation. SA-bgal activity is evident at 24 h.p.i.
(data not shown) but increased progressively with time
and under conditions that favor virus dissemination
(31200 cells/cm
2
), although it could be also detected in
severely damaged mononuclear infected cells. Besides
HRSV, fusion of cells may be also triggered by patho-
genic viruses such as HIV, ERVWE1 virus, Measles, and
Influenza.
11,28,35,46,69
This fusogenic property seems to be
a mechanism to support virus dissemination and repro-
duction and has been associated to senescence in some
cases.
11
In the case of HRSV and the immortalized con-
text of the cellular systems used, it seems that cellular
senescence is driven by the DNA damage induced by
HRSV rather than the cell fusion process itself as signs of
cellular senescence can be detected in spontaneous syncy-
tia and in mononuclear infected cells.
As recently reported by others,
21
HRSV is able to acti-
vate the DDR. Here we show evidence indicating that
the DNA damage inflicted by HRSV are DSBs. DSBs
observed are primarily of oxidative nature, probably due
to ROS generation. Several reports have previously
reported that HRSV induces ROS expression by different
mechanisms including nicotinamide adenine dinucleo-
tide phosphate (NADPH)-oxidase dependent
434 I. MART
INEZ ET AL.
mechanisms and inhibition of antioxidant enzymes.
22,24
According to the results obtained from the use of the
mitochondrial superoxide indicator MitoSOX, ROS for-
mation by HRSV has at least in part a mitochondrial ori-
gin. ROS are involved in transcription factor activation
and chemokine gene expression, oxidative stress and
lung damage in infected cells in both animals and chil-
dren.
9,10,30,32,37
Antioxidants treatments inhibit viral
HRSV replication and block the activation of
transcription factors implicated in the regulation of
immune/inflammatory responses.
9,31,37,43
In addition,
antioxidants reduce HRSV-induced oxidative stress and
clinical symptoms in mice suggesting a link between
increased ROS production and lung disease.
10
We show
that damage inflicted by HRSV can be alleviated to a
great extent by antioxidants that also inhibited moder-
ately virus production and reversed the reduced glutathi-
one (GSH)/oxidized GSSG ratio as reported before.
32,48
Antioxidant treatments concomitantly reduced the levels
of cellular senescence induced by HRSV and the activa-
tion of DD markers in cell cultures suggesting that the
cellular senescence observed is mainly triggered by DNA
damage. According to this, the ectopic expression of the
enzyme catalase, that protect cells from oxidative damage
from ROS, reduced the expression of DD foci in infected
cells without altering virus production, in contrast to
antioxidant treatments, suggesting a direct role of the
oxidative stress in the damage observed. The DD
inflicted does not seems to be a consequence of a gross
deficiency in the cellular DD repair capabilities and the
downregulation of one of the major components of DSB
repair, TP53BP1, slightly increased the levels of senes-
cence induced by HRSV probably due to increased unre-
paired damage. These results suggested a direct link
between DNA damage and the occurrence of senescence.
We have also shown the presence of two accurate bio-
markers of DNA damage and aging in HRSV-infected
lungs of young mice, gH2AFX and CDKN2A. HRSV
infection was evident in some distal and terminal airways
as well as in the respiratory epithelium. A significant
number of gH2AFX foci were found in some infected
cells lining the terminal airways. However only 1–2foci/
Figure 5. Expression of markers of DNA damage and aging in
lungs of mice infected by HRSV. (A) Epithelial lung tissue from
mock-infected mice stained with antibodies: aHRSV (pool of anti-
bodies against HRSV) and gH2AFX. (B) Representative image of
multiple immunofluorescence labeling with aHRSV and gH2AFX
antibodies of lung tissue from HRSV-infected mice stained. (C),
(D) Representative images of negative controls (no primary) of
the multiple immunofluorescence labeling of epithelial lung tis-
sue. Magnifications: 600X; scale bar: 10 mm. Arrowheads signal
cells showing reactivity to gH2AFX. AE: alveolar epithelium, Br:
bronchiole, CSCE: ciliated simple columnar epithelium. (E) Repre-
sentative images of conventional immunohistochemistry of
g-H2AFX and CDKN2A on mice lung tissue (mock-infected or
infected with HRSV) at 4 days post-infection. (F) Representative
images of conventional immunohistochemistry of gH2AFX and
CDKN2A on mice lung tissue at 11 and 30 days post-infection.
Arrowheads signal cells showing reactivity to gH2AFX and
CDKN2A. Two experiments, n D5 (mock-infected) or 6 (infected)
replicates (mice) per condition.
VIRULENCE 435
nucleus were observed in infected cells of the alveolar
fields (data not shown). Distal airways of the mouse are
lined with a simple columnar epithelium containing cili-
ated and secretory cells and are an important site for
HRSV replication. The staining of HRSV antigens found
was similar to that observed by others in the human and
mouse airway columnar epithelium.
13,24
gH2AFX and
CDKN2A expression was still evident at 11 and 30 days
post infection indicating a persistence of the primary
damage induced by HRSV during the acute infection.
We found that CDKN2A expression pattern was diffuse
nuclear and cytoplasmic. Although nuclear expression of
CDKN2A is usually associated to cell-cycle control, cyto-
plasmic accumulation has also been associated to senes-
cent phenotype induced by stress.
68
Histological studies suggest that non-ciliated secre-
tory cells give rise to ciliated cells and function as a
long-term self-renewing stem cell population in the
intrapulmonary airways.
59
In addition, it has been
reported that the adult mouse lung contains a minor
population of multipotent epithelial stem cells with the
capacity of self-renewal and to give rise to all epithelial
progenitors that reside in discrete microenviromental
niches along the proximal-distal axis.
45
It is tempting
to speculate that such stem cells could be targets of
HRSV. Indeed, it has been recently reported that HRSV
can infect proliferating airway basal cells and alter cel-
lular composition of the epithelium contributing to air-
way epithelial remodeling.
58
Thus, the DNA damage
inflicted and the occurrence of senescence might influ-
ence events long time after the virus has cleared from
thelungbymechanismsbasedonakindof“hit-and-
run”phenomenon that has been proposed to explain
how transient infections can cause long term airway
disease.
29
Our findings might have substantial implications for
the pathophysiology of HRSV, the leading cause of bron-
chiolitis and pneumonia in infants and young chil-
dren.
27,54
During the acute infection, the SASP might
contribute to alert the immune system about the damage
inflicted. The SASP components would mediate this pro-
cess, facilitating the attraction of immune cells to the
infection sites and the further clearance of infected cells.
Finally, the SASP and cellular senescence may serve as
therapeutic targets.
53,57,71
Although it is too premature to
determine whether cellular senescence would be positive
or detrimental for the pathological consequences of
HRSV infection, current evidence indicates that both
prosenescent and antisenescent therapeutic approaches
can be useful depending on the context. To confirm this
hypothesis further investigations in human primary cells
and in more permissive animal models supporting
HRSV infections are needed.
Methods
Cell culture, viruses, mice and transfections
HEp-2 and A549 cells (ATCC) were maintained in DMEM
medium supplemented with 10% FBS (Biowest), 2 mM
glutamine and 100 U/ml of penicillin and streptomycin
(Lonza). The Long and A2 strains of HRSV were propa-
gated in HEp-2 cells in medium supplemented with 2%
FBS, glutamine and antibiotics. Viruses were purified and
titrated in HEp-2 cells as previously described.
41
All experi-
ments were performed with the Long strain except for the
in vivo experiments in which the A2 strain was also used.
Two-month-old female mice (BALB/c strain) were intra-
nasally infected with 3,3£10
7
pfus of purified virus. At 4,
11 and 30 days post-infection, mice were sacrified by cervi-
cal dislocation and the lungs were processed for immuno-
histochemistry. All experiments were performed following
the regulations of the Instituto Carlos III for animal care
and handling. HEp-2 persistently infected by HRSV (L39)
were described elsewhere.
42
N-Acetylcysteine (NAC,
Sigma) and reduced glutathione reduced ethyl ester
(GSHee, Sigma) were prepared as indicated by the manu-
facturers and used at 5 mM and 2,5 mM, respectively.
Tansfections of siRNAs were performed with Lipofect-
amine RNAimax (cat.#13778, Invitrogen) following manu-
facturers instructions. Briefly, cells were transfected with
siRNAs (control or TP53BP1 duplexes, sc-37007 and sc-
37455, respectively, Santa Cruz Biotechnology) and the fol-
lowing day infected at MOI 3 with HRSV. After 48 h.p.i.,
the cells were processed for western-blotting and SA-bgal
assays. Transitory transfections of the plasmid pCMV3-
CT-N-FLAG (HG12084-NF, Sino Biological Inc.) express-
ing human catalase or empty vector (pCMV) were per-
formed with Lipofectamine 2000 (Invitrogen, cat. 11668)
in 8-well chamber slides following manufacturers instruc-
tions. Cells were transfected and the following day infected
at MOI 3 with HRSV. After 48 h.p.i., culture supernatants
were collected for virus titration and the cells were proc-
essed for immunofluorescence.
Immunofluorescence
For immunofluorescence, cells were seeded in 8-well
chambers (Thermo Fisher Scientific) at a density of
50000 cells/well. The following day the cells were
infected or treated as indicated in the corresponding
experiments. Immunofluorescence was performed as
previously described.
77
Basically, cells were fixed in
2% PFA in PBS for 10 min at RT and permeabilized
with 0.1% Triton X-100 and 0.1% sodium citrate
(5 min at RT). Preparations were then washed with
PBS and washing solution (PBS/0.25% BSA/0.1%
Tween 20), blocked for 30 min with blocking
436 I. MART
INEZ ET AL.
solution (washing solution, 2.5% BSA, and 5% nor-
mal serum), and incubated overnight with antibodies
against gH2AFX (1:1000, cat.# 05–636 Millipore),
TP53BP1 (1:1000, cat.# NB-100–304, Novus Biologi-
cals), antibody 47F (1:50) directed against the F gly-
coprotein of HRSV and anti-Flag (1:500, cat.#
F1804, Sigma-Aldrich). Preparations were then
washed with washing solution and incubated with
secondary antibodies (AF488, AF546 or AF647, Life
Technologies) for 1 h at RT. Nuclei were counter-
stainedwithDAPI,andsamplesweremountedwith
ProLong Diamond (Life Technologies). Cell images
were captured with a fluorescence microscopy (Zeiss
Axio) equipped with a camera (Axiocam MRm) and
AxioVision software. DNA damage foci were counted
from >150 cells for each experimental condition.
Analysis of colocalization was performed with a con-
focal microscope (LEICA DMI 600; 63X/1.4 len),
equipped with the following laser lines: Argon (five
spectral lines 488 to 514 nm), HeNe (633 nm), diode
violet(405nm)anddiodesolidstate(561nm).
Images were obtained with software LAS AF (Leica).
Protein analysis
Cell monolayers were washed with ice-cold PBS and
lysed in triple-detergent lysis buffer [50 mM Tris-HCl,
pH 8.0, 150 mM NaCl, 0.02% sodium azide, 0.1% SDS,
1% NP-40, 0.5% sodium deoxycholate, 100 mg/ml
PMSF, 2 mg/ml pepstatin, 2 mg/ml aprotinin, 2 mg/ml
leupeptin, and phosphatase inhibitors cocktail 2 and 3
(Sigma-Aldrich)]. SDS-PAGE and immunoblotting were
performed under standard conditions. Basically, samples
in Laemmli buffer (30 mg/lane) were separated through
8% or 12% gels and transferred to nitrocellulose mem-
branes for 90 min at RT in the presence of 20% methanol
and 0,1% SDS. Membranes were blocked with 3% BSA in
PBS-Tween 0,05% (PBST-BSA) and incubated ON at
4C with specific antibodies diluted 1:1000 in PBST-
BSA. Antibodies used were: gH2AFX (05–636, Milli-
pore), P-CHEK2 (AF1626, R&D Systems), P-RB1 (sc-
16670-R, Santa Cruz Biotech.), P-TP53 (9286S, Cell Sig-
naling), P-ATM (05–740, Millipore), CDKN1A (ab7960,
Abcam), CDKN2A (04–239, Millipore), TP53BP1 (cat.#
NB-100–304, Novus Biologicals) and TUBA1A (used at
1:2000, clone DM1A, Sigma-Aldrich).
Senescence-associated b-galactosidase assays
(SA-bgal) and Immuno-SA-bgal
Conventional SA-bgal assays were performed as previ-
ously described by Dimri et al.
18
SA-bgal detection by
flow cytometry using Bafilomycin A1 (cat.#B1793,
Sigma-Aldrich) and the fluorogenic bgalactosidase sub-
strate C12FDG (cat.# 11590276, Fisher Scientific) was
performed as described by Debacq-Chainiaux et al.
17
Immuno-SA-bgal is an assay consisting of a combina-
tion of immunocytochemistry and SA-bgal assays. Cells
growing in 6-well plates (MW6) were washed twice
with PBS and fixed for 3 minutes with 2% formalde-
hyde and 0,2% glutaraldehyde. Cells were permeabi-
lized for 5 min at RT with 0.1% Triton X-100 and 0.1%
sodium citrate, washed with PBS and blocked with BSA
1% in PBS (30 min at RT). Cells were then incubated
for 45 min at RT with a pool of antibodies directed
against HRSV [021/1G, 021/2G, 47F, 67P].
23,40
The
plates were washed twice in a bath of tap water and
incubated for 45 min at RT with a-mouse-HRP second-
ary (ab97040, Abcam) 1:1000 in PBS-BSA 1%. The
plates were washed with tap water before adding the
peroxidase substrate in ELISA buffer [Citric acid
25 mM/Na
2
HPO
4
50 mM pH 5, 0,033 mg/ml AEC sub-
strate (A6926-100TAB, Sigma), 2 ml/ml (30% H
2
O
2
)].
The plates were maintained in the dark until the
appearance of signal (5–10min).Afterthat,thesub-
strate was removed, the plates were washed twice with
PBS and the SA-b-gal reaction mixture was added. The
plates were incubated in the dark at 37for 24 hours.
Micrographs were taken in a microscope TS100F
(Nikon) equipped with a digital camera DS-L1 (Nikon).
ROS analyses and quantification of GSSG (oxidized
glutathione) and GSH (reduced glutathione)
Cellular ROS levels were assessed with 20 mM
H
2
DCFDA (20,70dichlorodihydrofluorescein diacetate,
Sigma-Aldrich) and a microplate fluorometer Synergy
(Biotek) as previously described.
77
For the determination
of mitochondrial ROS (superoxide) we used MitoSOX
Red (Life Technologies), fluorescence microscopy and
flow citometry. Treatments of cells were carried-out in
triplicates in 8-well chambers (for microscopy) and in
MW6 plates (for flow cytometry). Positive controls con-
sisted of cells treated with 5 mM MitoSOX for 15 min,
then with 100 mM Paraquat for 30 min followed by an
additional 30 min treatment with 5 mM MitoSOX. The
rest of the samples were treated with 5 mM MitoSOX for
30 min. Blank cells consisted of cells treated with vehicle.
The cells growing in 8-well chambers were fixed with
PFA 2% in PBS for 10 min, washed and incubated with
DAPI for 15 minutes. Then, the cells were washed with
PBS, mounted with ProLong Diamond (Life Technolo-
gies) and examined under fluorescence microscopy. For
flow cytometry, the cells were trypsinized and counted.
The same number of cells for each condition was fixed
with PFA, washed and transferred to FACS tubes. The
VIRULENCE 437
measurements were carried out using FASCanto (BD
Bioscience) cytometer. MitoSOX was excited by a laser at
488 nm and the data collected at the FSC, SSC, 585/
42 nm bandpass filter. Cell debris, represented by distinct
low forward and side scatter, were gated out for analysis.
50000 gated events for each condition were
analyzed with the FACSCanto and Flow Jo software. The
mean intensity of MitoSOX fluorescence and the per-
centage of positive cells were calculated. For the determi-
nation of GSSG and GSH we used the EnzyChromTM
GSH/GSSG Assay kit (Bioassay Systems) and followed
manufacturer’s instructions.
Apoptosis/necrosis by flow cytometry
This assay was performed by using the single channel
dead cells apoptosis kit with Annexin
V-Alexa-fluor488TM and SytoxTM green dyes from Fisher
Scientific (cat.# V13240). Eight thousand cells per well
were plated in MW6 and were infected with HRSV at
MOI 3 the following day. Sixty h.p.i., attached cells were
processed for flow cytometry as indicated by the manu-
facturers. Treatment of cells with 20 mM camptothecin
(CPT) for 12 h (cat.#C9911, Sigma-Aldrich) was used as
positive control for apoptosis. After treatment with both
probes, apoptotic cells show green fluorescence, dead
cells show brighter green fluorescence, and live cells
show little. These populations were distinguished in the
FL1 channel of a FACSCalibur flow cytometer (Becton
Dickinson). Twenty thousand events for each condition
were analyzed with the FACSCalibur and Flow Jo
software.
Immunohistochemistry
Mice were sacrificed at 4, 11 and 30 days post-infection
by cervical dislocation and lung pieces (5 £5 mm,
approximately) were dissected, fixed in 4% buffered for-
malin and embedded in paraffin wax. Multiple antigen
labeling was performed following the double-immuno-
fluorescent labeling protocol and reagents provided by
Vector Laboratories (cat. # A-2011, A-2016, SP-2001,
PK-6102). Sequential incubations of the antibodies were
made overnight for the first antibody (a-HRSV: pool of
Abs 021/1G, 021/2G, 47F, 67P, 1:100) and 3 h at RT for
the second antibody (gH2AFX, 1:400). Antigen retrieval
was performed with citrate buffer (pH 6) using a Micro-
wave Tender Cooker (Nordic Ware) and a 700 W micro-
wave (15 min 700W/15 min 350W). Endogenous
peroxidase activity was inhibited with 0.3% H
2
O
2
in
methanol (25 min). Nuclei staining, mounting, and
microscopy acquisition were performed as for the indi-
rect immunofluorescence. Images of Figure 5A-D were
processed by deconvolution in the green channel with
Huygens Essential (Scientific Volume Imaging, Hilver-
sum, the Netherlands) based on the classicmaximum
likelihood estimation method. Conventional immuno-
histochemistry was performed following the labeling
protocol and reagents provided by Vector Laboratories
(PK-6101, PK-6102, SP-2001). Antibodies used were
gH2AFX (1:200 cat.# 05–636 Millipore) and CDKN2A
(1:200, cat.# 04–239, Millipore). Counterstaining with
nuclear fast, mounting and images acquisition were per-
formed as previously described.
77
Immunoassay
The determination of the concentration of human IL-6
and TNF-afrom cell culture supernatatants was assessed
by the ProcartaPlexTM multiplex Immunoassay kit -Mag-
netic beads (eBioscence) based on the Luminex
R
technol-
ogy. Procarta PlexTM Analyst 1.0 software was used for
analysis.
Irradiation of cells with g-ray
Cells growing in 8-well chambers and MW6 plates were
irradiated with 3 Gy (1.2 Gy/min) in a shelf-shielded J. L.
Shepherd Mark I irradiator with a Cesium-137 source.
After irradiation cells were processed for immunofluo-
rescence or immunoblotting at the corresponding times.
DNA breaks detection
To detect DNA breaks, we performed an assay based in
the incorporation of dUTP by Terminal deoxynucleoi-
tidyl Transferase enzyme (TdT) and posterior detection
by immunocytochemistry as previously described.
77
Statistical analysis
Statistical significance of data was determined by apply-
ing a two-tailed Student’s t test or analysis of variance
followed by the Newman–Keuls or Bonferroni post-tests
for experiments with more than two experimental
groups. P <0.05 is considered significant. Significance of
analysis of variance post-test or the Student’s t test is
indicated in the figures as
(P <0.05),
(P <0.01) and
(P <0.001). Statistics were calculated with the Prism
5 software (GraphPad Software). The results presented
in the figures are means §SD. Experiments were
repeated at least two times.
438 I. MART
INEZ ET AL.
Ethics statement
All experiments with animals were approved by the
Commitee of Bioethics and Animal Welfare of the Insti-
tuto de Salud Carlos III (file reference: PA 31). Protocols
used followed the guidelines for animal protection
reported by the Spanish national law RD 53/2013.
Abbreviations
CPT Camptothecin
DD DNA damage
DDR DNA damage response
DSB double-strand breaks
GSH reduced glutathione
GSHee reduced glutathione ethyl ester
GSSG oxidized glutathione
h.p.i hours post-infection
MOI multiplicity of infection
MW6 6-well plates
NAC N-Acetylcysteine
ROS reactive oxygen species
siRNA small interfering RNAs
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank the core facilities of the Instituto de Investigaciones
Biom
edicas de Madrid and of the Centro Nacional de Micro-
biolog
ıa for technical help.
Funding
This work was supported by Grants MPY-1038/14 to Alberto
Zambrano, PI 11/00590 to Isidoro Mart
ınez and RD12/0036/
0030 to Ana Aranda from FIS (Instituto de Salud Carlos III)
and Grant BFU2011-28958, from Ministerio de Econom
ıay
Competitividad to Ana Aranda. The authors have no conflict-
ing financial interests.
Author contributions
IM performed and designed experiments. V G-C performed
and designed experiments. TG, A G-G and DG performed
experiments, AA designed experiments and wrote paper. AZ
performed experiments, designed experiments, wrote paper
and conceived the project.
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