Respiratory syncytial virus decreases p53 protein to prolong survival of airway epithelial cells.
ABSTRACT Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immunosuppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53 to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1-6 h after infection resulting in a decrease in p53 6-24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity. Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association. Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53 enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway epithelial cells.
- SourceAvailable from: Cliff Taggart[Show abstract] [Hide abstract]
ABSTRACT: Respiratory syncytial viral (RSV) infections are a frequent cause of chronic obstructive pulmonary disease (COPD) exacerbations, which are a major factor in disease progression and mortality. RSV is able to evade antiviral defenses to persist in the lungs of COPD patients. Though RSV infection has been identified in COPD, its contribution to cigarette smoke-induced airway inflammation and lung tissue destruction has not been established. Here we examine the long-term effects of cigarette smoke exposure, in combination with monthly RSV infections, on pulmonary inflammation, protease production and remodeling in mice. RSV exposures enhanced the influx of macrophages, neutrophils and lymphocytes to the airways of cigarette smoke exposed C57BL/6J mice. This infiltration of cells was most pronounced around the vasculature and bronchial airways. By itself, RSV caused significant airspace enlargement and fibrosis in mice and these effects were accentuated with concomitant smoke exposure. Combined stimulation with both smoke and RSV synergistically induced cytokine (IL-1α, IL-17, IFN-γ, KC, IL-13, CXCL9, RANTES, MIF and GM-CSF) and protease (MMP-2, -8, -12, -13, -16 and cathepsins E, S, W and Z) expression. In addition, RSV exposure caused marked apoptosis within the airways of infected mice, which was augmented by cigarette smoke exposure. RSV and smoke exposure also reduced protein phosphatase 2A (PP2A) and protein tyrosine phosphates (PTP1B) expression and activity. This is significant as these phosphatases counter smoke-induced inflammation and protease expression. Together, these findings show for the first time that recurrent RSV infection markedly enhances inflammation, apoptosis and tissue destruction in smoke-exposed mice. Indeed, these results indicate that preventing RSV transmission and infection has the potential to significantly impact on COPD severity and progression.PLoS ONE 02/2014; 9(2):e90567. · 3.53 Impact Factor
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ABSTRACT: Respiratory syncytial virus (RSV) is a major worldwide pathogen for which there is still no effective vaccine or antiviral treatment available, and immunoprophylaxis with RSV-specific antibodies (e.g., palivizumab) is used in limited clinical settings. In this review, we discuss virus–host interactions relevant to RSV pathobiology and how advances in cell and systems biology have accelerated knowledge in this area. We also highlight recent advances in understanding the relationship between RSV bronchiolitis and sequelae of recurrent wheezing and asthma, new findings into an intriguing interaction between RSV and air pollution, and exciting developments toward the goal of realizing a safe and effective RSV vaccine.Current Pediatrics Reports. 09/2013; 1(3).
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ABSTRACT: Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how Respiratory Syncytial Virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here we studied the effect of RSV infection on airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell® inserts and infected with RSV strain A2. We analyzed: (i) epithelial apical junctional complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to FITC-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pre-treated with and without protein kinase D (PKD) inhibitors. UV irradiated RSV served as negative control. RSV infection led to a significant reduction in TEER, and increase in permeability. Additionally it caused disruption of the AJC, and remodeling of the apical actin cytoskeleton. Pre-treatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin-binding protein cortactin in a PKD-dependent manner. UV inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potentially new therapeutic target for RSV mediated lung diseases.Journal of Virology 08/2013; · 4.65 Impact Factor
Respiratory Syncytial Virus Decreases p53 Protein to Prolong
Survival of Airway Epithelial Cells1
Dayna J. Groskreutz,2* Martha M. Monick,* Timur O. Yarovinsky,* Linda S. Powers,*
Dawn E. Quelle,†Steven M. Varga,‡Dwight C. Look,* and Gary W. Hunninghake*
Respiratory syncytial virus (RSV) is a clinically important pathogen. It preferentially infects airway epithelial cells causing
bronchiolitis in infants, exacerbations in patients with obstructive lung disease, and life-threatening pneumonia in the immuno-
suppressed. The p53 protein is a tumor suppressor protein that promotes apoptosis and is tightly regulated for optimal cell growth
and survival. A critical negative regulator of p53 is murine double minute 2 (Mdm2), an E3 ubiquitin ligase that targets p53 for
proteasome degradation. Mdm2 is activated by phospho-Akt, and we previously showed that RSV activates Akt and delays
apoptosis in primary human airway epithelial cells. In this study, we explore further the mechanism by which RSV regulates p53
to delay apoptosis but paradoxically enhance inflammation. We found that RSV activates Mdm2 1–6 h after infection resulting
in a decrease in p53 6–24 h after infection. The p53 down-regulation correlates with increased airway epithelial cell longevity.
Importantly, inhibition of the PI3K/Akt pathway blocks the activation of Mdm2 by RSV and preserves the p53 response. The
effects of RSV infection are antagonized by Nutlin-3, a specific chemical inhibitor that prevents the Mdm2/p53 association.
Nutlin-3 treatment increases endogenous p53 expression in RSV infected cells, causing earlier cell death. This same increase in p53
enhances viral replication and limits the inflammatory response as measured by IL-6 protein. These findings reveal that RSV
decreases p53 by enhancing Akt/Mdm2-mediated p53 degradation, thereby delaying apoptosis and prolonging survival of airway
epithelial cells. The Journal of Immunology, 2007, 179: 2741–2747.
exacerbations in patients with obstructive lung disease, and life-
threatening pneumonia in immunosuppressed patients. In addition,
RSV infection early in life has been associated with the subsequent
development of asthma (1–8). RSV is a member of the Paramyxo-
viridae family and consists of a negative strand RNA genome in
a nucleocapsid surrounded by an envelope (9). Entry into the
host respiratory epithelium is by cell surface fusion, and infec-
tion leads to viral replication and subsequent host inflammatory
The tumor suppressor protein, p53, is a potent inhibitor of cell
proliferation (16, 17) and the most frequently inactivated gene in
human cancers (18, 19). The p53 protein is a transcription factor
that is usually short-lived and expressed at very low levels in nor-
espiratory syncytial virus (RSV)3is a ubiquitous patho-
gen causing upper respiratory infections in healthy
adults, bronchiolitis and pneumonia in young children,
mal cells. When activated by cellular stresses such as DNA dam-
age, p53 induces the expression of gene products that promote
apoptotic cell death or permanent cell cycle withdrawal (19–22).
In this way, p53 eliminates damaged and potentially transformed
cells from an organism, thereby protecting against the develop-
ment of cancer.
The regulation of p53 is complex and controlled by many fac-
tors. One protein that is essential for restricting p53 function is the
murine double minute protein 2 (Mdm2) (23). Mdm2 is a nuclear
phosphoprotein and an E3 ubiquitin ligase that binds to p53, ubiq-
uitinates it, and targets it for proteosome degradation (24–27).
Notably, Mdm2 is a transcriptional target of p53; thus, the inter-
dependent activities of p53 and Mdm2 comprise a negative auto-
regulatory feedback loop (28, 29).
The regulation and activation of Mdm2 has been extensively
studied. Mayo and Donner (30) demonstrated that activation of the
PI3K/Akt pathway leads to phosphorylation of Mdm2 at Ser166
and Ser186and a decrease in p53 protein while inhibition of the
PI3K/Akt pathway increases the levels of p53 and augments tran-
scription. These findings were confirmed by another study that
showed that phosphorylation of Mdm2 at Ser166by Akt leads to
inhibition of self-ubiquitination, stabilization of Mdm2, and a con-
sequent down-regulation of p53 (31).
Recent studies have investigated the effect of viral infection on
p53. Inflammatory cells in influenza pneumonia activate p53 di-
rectly, leading to apoptosis (32). A subsequent paper examined the
effect of influenza infection on p53 in respiratory epithelial cells
and found that the mechanism of increased p53 in influenza infec-
tion is increased transcription (33). One study investigated the ef-
fects of multiple viruses on HT1080 and HepG2 cells and found
that the amount of p53 was decreased in encephalomyocarditis
virus and human parainfluenza virus type 3 infection due to protein
kinase R-mediated inhibition of translation (34). Another study
concluded that p53 actually enhances the ability of human CMV to
*Division of Pulmonary, Critical Care, and Occupational Medicine,†Department of
Pharmacology, and‡Department of Microbiology and Interdisciplinary Graduate Pro-
gram in Immunology, University of Iowa Roy J. and Lucille A. Carver College of
Medicine and Veterans Administration Medical Center, Iowa City, IA 52242
Received for publication June 18, 2007. Accepted for publication June 19, 2007.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by a Veterans Affairs Merit Review grant, by Grants
HL-60316, HL-077431, and HL079901-01A1 from the National Institutes of Health,
and by Grant RR00059 from the General Clinical Research Centers Program, Na-
tional Center for Research Resources, National Institutes of Health.
2Address correspondence and reprint requests Dr. Dayna Groskreutz, Division of
Pulmonary, Critical Care, and Occupational Medicine, 100 Eckstein Medical Re-
search Building, University of Iowa Roy J. and Lucille A. Carver College of Medi-
cine, Iowa City, IA 52242. E-mail address: Dayna-Groskreutz@uiowa.edu
3Abbreviations used in this paper: RSV, respiratory syncytial virus; MOI, multiplic-
ity of infection; HTBE, human tracheobronchial epithelial; Mdm2, murine double
Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00
The Journal of Immunology
replicate in fibroblasts (35). Finally, a recent study suggested that
poliovirus induces p53 degradation in a proteasome, promyelo-
cytic leukemia-dependent and in an Mdm2-dependent manner in
human glioblastoma astrocytoma cells (36). These conflicting re-
sults suggest that the effect of viral infection on the amount of p53
may be virus and/or cell type-specific.
In contrast, recent studies have investigated the effect of p53 on
viral replication. HT1080 cells were treated with p53 small inter-
fering RNA, and vesicular stomatitis virus replication increased
(34). Similarly, poliovirus replication increased when U2OS cells
were treated with p53 small interfering RNA (36). In contrast,
other studies suggested that p53 enhances viral replication. When
fibroblast p53 null cells were treated with human CMV, viral titers
were attenuated (35), and cells expressing functional p53 allowed
for more adenoviral replication than cell lines deficient in func-
tional p53 (37).
The link between p53 and inflammation is less clear. A study of
non-small cell lung carcinoma surgical specimens showed a cor-
relation between IL-8 mRNA expression and p53 mutations (38).
Another study of psoriasis also showed an inverse relationship
between IL-8 and p53: a decrease in p53 expression and an in-
crease in IL-8 expression was demonstrated in psoriatic skin le-
sions (39). One study demonstrated that p53 has suppressive ac-
tivity on the inflammatory pathways leading to activation of AP-1
and NF-?B, and this activity is mediated by phosphatase and tensin
homolog (40). Suppression of inflammatory pathways might be
one other mechanism by which p53 further potentiates cell death.
We have previously shown that RSV activates both proapoptotic
and antiapoptotic pathways in airway epithelial cells, and the an-
tiapoptotic effects of RSV during the first few hours of infection
are mediated through PI3K and the downstream mediator, Akt (41,
42). Recent work in our lab also indicated that epidermal growth
factor receptor and subsequent ERK activation lead to an alteration
in the Bcl-2 protein balance, favoring survival and delayed apo-
ptosis (42). One key regulator of cell death is p53, which may be
regulated by Akt. We hypothesized that RSV delays cellular apo-
ptosis and prolongs cell survival by activating Akt, which phos-
phorylates Mdm2 and leads to p53 proteosome degradation. This
decrease in p53 and delay in apoptosis paradoxically does not aug-
ment viral replication and allows for an enhanced host cell inflam-
Materials and Methods
Chemicals were obtained from Sigma-Aldrich and Calbiochem. Protease
inhibitors were obtained from Roche Applied Science. Nutlin-3 (catalog
no. 444143) and LY294002 (catalog no. 440202) were purchased from
Calbiochem. Mouse monoclonal IgG2a (DO-1) Ab to p53 was obtained
from Santa Cruz Biotechnology (sc-126), and rabbit polyclonal IgG (FL-
393) Ab to p53 was obtained from Santa Cruz Biotechnology (sc-6243).
Rabbit polyclonal Ab to phospho-p53 (Ser15) was purchased from Cell
Signaling Technology (no. 9284). Rabbit polyclonal Ab to phospho-Mdm2
(Ser166) was obtained from Cell Signaling Technology (no. 3521). Rabbit
polyclonal IgG Ab to Mdm2 (N-20) was obtained from Santa Cruz Bio-
technology (sc-813). Rabbit polyclonal Ab to phospho-Akt (Ser473) (no.
9271), cleaved caspase 7 (Asp198) (no. 9491), and cleaved poly(ADP-
ribose) polymerase (Asp214) (no. 9541) were obtained from Cell Signaling
Biotechnology. Rabbit mAb to cleaved caspase 3 (Asp175) (no. 9664) was
obtained from Cell Signaling Biotechnology. Mouse mAb to ?-actin was
obtained from Sigma-Aldrich.
Human tracheobronchial epithelial (HTBE) cells
Human TBE cells were obtained under a protocol approved by the University
of Iowa Institutional Review Board. Epithelial cells were isolated from
tracheal and bronchial mucosa by enzymatic dissociation and cultured in
Laboratory of Human Carcinogenesis LHC-8e medium on plates coated
with collagen/albumin for study up to passage 10 as previously described
(43). For infection, cells at 80% confluency were treated with human RSV
strain A-2 at a multiplicity of infection (MOI) of 2. Viral stocks were
obtained from Advanced Biotechnologies. The initial stock (1 ? 109
TCID50) was aliquoted and kept frozen at ?135°F, and a fresh aliquot was
thawed for each experiment. The virus was never refrozen. UV light-in-
activated RSV was prepared by exposure of a 1/30 dilution of the live virus
with PBS to 18 J of UV light at 4°C.
Vero cells were cultured in MEM (Invitrogen Life Technologies) supple-
mented with 10% FBS (JRH Biosciences), penicillin-streptomycin, L-glu-
tamine, non-essential amino acids, and sodium pyruvate (all from Invitro-
gen Life Technologies).
Cell protein isolation
Whole cell protein was prepared by lysing the cells on ice for 20 min in 300
?l of lysis buffer (0.05 M Tris (pH 7.4), 0.15 M NaCl, 1% Nonidet P-40,
with added protease and phosphatase inhibitors: 1 protease minitab/10 ml
(Roche Applied Science) and 100 ?l of 100? phosphatase inhibitor mix-
ture/10 ml (no. 524625; Calbiochem). Lysates were sonicated on ice con-
tinuously for 20 s at a 70% duty cycle with a microtip limit of 2, using a
Tekmar Sonic Disruptor. Lysates were kept at 4°C for 30 min and spun at
15,000 ? g for 10 min to remove insoluble debris, and the supernatant was
saved. Protein concentrations were determined using a commercial Protein
Assay kit from Bio-Rad. Cell lysates were stored at ?70°C until use.
Western blot analysis
Protein (40 ?g) was mixed 1:1 with 2? sample buffer (20% glycerol, 4%
SDS, 10% 2-ME, 0.05% bromphenol blue, and 1.25 M Tris (pH 6.8)) and
separated using SDS-PAGE. Cell proteins were transferred to polyvinyli-
dene difluoride membranes (Bio-Rad). Equal loading of proteins was eval-
uated using Ponceau S dye staining (Sigma-Aldrich). The polyvinylidene
difluoride membrane was saturated with methanol, washed, and then in-
cubated with primary Ab. Blots were washed four times and incubated with
HRP-conjugated anti-IgG Ab (1/5000 to 1/40000). Immunoreactive bands
were developed using a chemiluminescent substrate, ECL and ECL Plus
(Amersham Biosciences) and detected by autoradiography. Protein levels
were quantified using densitometry via a FluorS scanner and Quantity One
software for analysis (Bio-Rad). Densitometry is expressed as the fold
increase of experimental value per control value.
Total RNA was isolated using the Absolutely RNA RT-PCR Miniprep kit
(Stratagene) following the manufacturer’s instructions. RNA was quanti-
tated using RiboGreen kit (Invitrogen Life Technologies). Total RNA (1
?g) was reverse-transcribed to cDNA using iScript cDNA Synthesis kit
(Bio-Rad) according to the manufacturer’s instructions. PCR was per-
formed using 2 ?l of cDNA and 48 ?l of master mix containing iQ SYBR
Green Supermix (Bio-Rad), 15 pmol of forward primer and 15 pmol of
reverse primer, in a MyiQ Single-Color Real-Time PCR Detection System
as follows: 3 min at 95°C, followed by 45 cycles of 20 s at 95°C, 20 s 60°C
and 20 s at 72°C. The fluorescence signal generated with SYBR Green I
DNA dye was measured during annealing steps. Specificity of the ampli-
fication was confirmed using melting curve analysis. Data were collected
and recorded by MyiQ Optical System Software version 2.0 (Bio-Rad) and
expressed as a function of threshold cycle (Ct). Relative gene expression
was normalized to HPRT or GAPDH mRNA using ?Ctmethod as previ-
ously described (44). Specific primer sets used are as the following: HPRT
(forward) 5?-TTGGAAAGGGTGTTTATTCCTC-3? (reverse) 5?-TCC
GTGAG-3? (reverse) 5?-CCAGTGTGATGGTGAGG-3?; and RSV N-gene
(forward) 5?-GCTCTTAGCAAAGTCAAGTTGAATGA-3? (reverse) 5?-
TGCTCCGTTGGATGGTGTATT-3?. Gene-specific primers were custom-
synthesized and purchased from Integrated DNA Technologies based on
design using gene-specific nucleotide sequences from the National Center
for Biotechnology Information sequence databases and PrimerQuest Web
interface (Integrated DNA Technologies) or Primer3 Web interface (45).
Cell death assay
Human TBE cells were plated at 50% confluence. The cells were pretreated
with either 10 ?M Nutlin-3 or control medium followed by RSV (MOI of
2) for 0, 24, 48, or 72 h. Nonadherent cells were collected to 1.5-ml tubes,
and adherent cells were detached by incubation with trypsin followed by
neutralization with complete medium (MEM, 10% FBS, and gentamicin).
The detached cells were pooled with the corresponding nonadherent cells
and stained with Guava Technologies ViaCount reagent, which contains
2742 RSV AND p53
propidium iodide and a cell-permeable dye for nucleated cells LDS-751,
following the manufacturer’s protocol. Personal Cell Analysis flow cytom-
eter (Guava Technologies) was used to identify nucleated cells (total cells)
and propidium iodide stained nucleated cells (dead cells) to determine the
percentage of cell death.
Viral titers of HTBE RSV-infected cells were measured by standard plaque
assay using 90% confluent Vero cells. Briefly, HTBE cells were exposed to
RSV (MOI of 2) or Nutlin-3 and RSV (MOI of 2) for 72 h. The supernatant
and adherent cells were removed, sonicated for 20 s on ice as described,
and frozen at ?70°C to be assayed later by plaque assay. Vero cells were
treated with serial 10-fold dilutions of the HTBE supernatant/lysed cell
mixture. The cell cultures were incubated at 37°C, 5% CO2for 90 min,
with gentle rocking of the plates every 15 min. Overlay, consisting of
EMEM (Cambrex), 10% FBS (JRH Biosciences), L-glutamine (Invitrogen
Life Technologies), penicillin-streptomycin (Invitrogen Life Technolo-
gies), and 1% SeaKem ME Agarose (Cambrex) was prepared, and 4 ml of
cooled overlay was added to each sample. Samples were gently swirled to
mix and then allowed to cool in a laminar flow hood for 15 min. When the
agar solidified, plates were incubated for 5 days at 37°C, 5% CO2. Cells
were stained by adding 2 ml of neutral red (Fisher Scientific) overlay and
incubated for 24 h. The next day, plaques were counted in each well over
a light box and the concentration of virus calculated.
Primary HTBE cells were plated at ?80% confluence and exposed to con-
trol medium of 10 ?M Nutlin-3, RSV (MOI of 2), or RSV and Nutlin-3.
Supernatants were collected and frozen at ?70°C. Human IL-6 and IL-8
concentrations in cell culture supernatants were determined using DuoSet
ELISA kits from R&D Systems.
Statistical analysis was performed on densitometry, the cell death assay,
real-time PCR, and ELISA data. Significant differences between two
groups were determined by Student’s t test with (GraphPad statistical anal-
ysis software). Significant differences for over two groups were confirmed
by one-way ANOVA with a Bonferroni’s test for multiple comparisons
(Graphpad statistical analysis software).
RSV decreases p53 protein
In previous studies, we showed that RSV triggers proapoptotic
events late in the course of infection (41, 42). One key regulator of
cell death is p53. Recent studies investigated the effect of viral
infection on p53 levels, and the findings varied depending on the
virus and cell-type studied. We began by looking at how RSV
infection affects the level of p53 protein. As shown in Fig. 1A,
RSV reduces the amount of p53 and activated phospho-p53 protein
in primary HTBE cells, first apparent 6 h after RSV infection but
more significantly after 16 and 24 h of RSV infection. The amount
of p53 mRNA does not change with RSV infection as measured by
quantitative real-time PCR (Fig. 1B), suggesting that regulation is
at a posttranscriptional level.
Inhibition of the proteosome preserves p53 protein in RSV
Because the change in p53 did not appear to be due to a change in
transcription, we investigated whether the mechanism for reduc-
tion in p53 protein was increased degradation. We exposed HTBE
cells to RSV infection for 24 h with and without the proteosome
inhibitor, MG132. As shown in Fig. 2A, the amount of p53 protein
was again reduced after 24 h of RSV infection, but this reduction
RSV (MOI of 2) for the indicated periods of time. Whole cell lysates were
obtained, and Western analysis performed for p53 protein. Western blot
shown is representative of three experiments. Densitometry was performed
on Western blots from three separate experiments and averaged. One-way
ANOVA with a Bonferroni’s test for multiple comparisons shows a sta-
tistically significant difference between cells exposed to RSV for 16 and
24 h as compared with control cells. ?, p ? 0.01. B, RSV does not change
p53 mRNA. HTBE cells were exposed to RSV (MOI of 2) for the indicated
periods of time. Cells were harvested, RNA isolated, cDNA made, and
quantitative real-time PCR performed. Data reflect mean and SE of three
RSV decreases p53 protein. A, HTBE cells were exposed to
infection. A, HTBE cells were exposed to MG132 10 ?M and/or RSV
(MOI of 2) for 24 h. Whole cell lysates were obtained and Western analysis
performed for p53 protein. In both control cells and cells exposed to RSV,
inhibition of the proteosome prevents degradation of p53. B, RSV increases
active phospho-Mdm2 but not total Mdm2. HTBE cells were exposed to
RSV (MOI of 2) for the indicated periods of time. Cells were then har-
vested for cellular protein, and Western blot analysis was performed. There
is no change in total Mdm2, but phospho-Mdm2 (Ser166) increases by 1–6
h of RSV exposure. The phospho-Mdm2 Western blot shown is represen-
tative of three experiments.
Inhibition of the proteosome preserves p53 protein in RSV
2743 The Journal of Immunology
was prevented with the use of the proteosome inhibitor. In both
RSV-infected cells and control cells, p53 protein increased when
the proteosome was inhibited, confirming that the mechanism for
reduction of p53 both in RSV infection and under normal condi-
tions is proteosome degradation.
RSV activates Mdm2
A negative regulator of p53 is the E3 ubiquitin ligase, Mdm2,
which is activated by phosphorylation at Ser166to ubiquitinate p53
(26). Ubiquitination targets p53 for proteasome degradation. West-
ern blot shows that although RSV does not alter the amount of
total Mdm2, it does increase the amount of active phospho-
Mdm2 (Ser166) (Fig. 2B). This increase at 1–6 h after RSV
infection temporally precedes the reduction in p53 at 16 h.
These data demonstrate that RSV infection decreases the level
of p53 protein, perhaps by Mdm2-mediated degradation.
Inhibition of the PI3K/Akt pathway decreases phospho-Mdm2
and protects p53 from degradation
We have previously shown that RSV phosphorylates Akt (Ser473)
and that this activation of Akt is inhibited by the PI3K chemical
inhibitor, LY294002, in both control and RSV-infected cells (41).
We further investigated this mechanism by examining how the
PI3K/Akt activity affects Mdm2 and p53 in RSV infection. We
exposed HTBE cells to RSV in the presence or absence of the
PI3K/Akt pathway inhibition, LY294002. As shown in Fig. 3,
the amount of phospho-Mdm2 (Ser166) increases after 6 h of RSV
infection, but in the presence of the PI3K inhibitor, LY294002,
phospho-Mdm2 is significantly decreased in cells exposed to RSV.
We selected this time point of 6 h because a significant activation
of Mdm2 occurs in RSV infection from 1 to 6 h (Fig. 2), and our
previous studies have shown that the PI3K/Akt pathway is an im-
portant prosurvival pathway early in RSV infection (41). We also
investigated the effect of PI3K/Akt inhibition on the amount of
p53. As also shown in Fig. 3, RSV infection results in a decrease
increases p53 protein. HTBE cells were exposed to medium alone, 20 ?M
LY294002, RSV (MOI of 2), or 20 ?M LY294002 followed by RSV for
6 h. Cells were harvested for cellular protein after 6 h and Western blot
analysis was performed for phospho-Mdm2 (Ser166) and p53. Inhibition of
the PI3K/Akt pathway with a chemical inhibitor results in less activation of
Mdm2 and prevents the RSV-induced reduction of p53. Western blots
shown are representative of three experiments.
Inhibition of AKT decreases phospho-Mdm2 (Ser166) and
airway epithelial cells and leads to earlier cell death.
A, HTBE cells were exposed to control buffer, 10
?M Nutlin-3, RSV (MOI of 2), or 10 ?M Nutlin-3
followed by RSV. Cells were then harvested for cel-
lular protein after 24, 48, and 72 h, and Western blot
analysis was performed for p53. Nutlin-3 leads to
stabilization of p53 protein, both in control cells and
cells exposed to RSV. Western blots shown are rep-
resentative of three experiments. Next, hTBE cells
exposed to control medium alone, 10 ?M Nutlin-3,
RSV (MOI of 2), or Nutlin-3 followed by RSV for
24, 48, and 72 h and were mobilized and stained
with propidium iodide. Flow cytometry was per-
formed to detect the percentage of cells with ?2n
DNA content, indicating cell death. One-way
ANOVA with a Bonferroni’s test for multiple com-
parisons shows a statistically significant difference
(?, p ? 0.05) between cells exposed to RSV and
Nutlin-3 as compared with cells only exposed to
RSV. Data are representative of three experiments.
B, UV light-inactivated RSV does not decrease p53
protein. HTBE cells were exposed to RSV (MOI of
2) or UV light-inactivated RSV for 16 h, with and
without Nutlin-3 pretreatment. Whole cell lysates
were obtained and Western blot analysis was per-
formed for p53 protein.
Nutlin-3 maintains p53 protein in
2744 RSV AND p53
in p53 protein first apparent 6 h after infection, but this decrease is
prevented by the addition of LY294002. These results suggest that
the PI3K/Akt pathway activates Mdm2 and leads to the degrada-
tion of p53 in RSV infection.
Nutlin-3 maintains p53 protein in airway epithelial cells and
leads to earlier RSV-mediated cell death
Nutlin-3 (Calbiochem) is a chemical inhibitor of Mdm2 that pre-
vents the Mdm2/p53 association, thus inhibiting Mdm2-mediated
degradation of p53. This inhibition increases the level of endoge-
nous p53 protein. As shown in Fig. 4A, adding 10 ?M Nutlin-3 to
primary HTBE cells increases the amount of p53 protein both in
cells only treated with Nutlin-3 and those subsequently exposed to
RSV for 24, 48, and 72 h. The RSV-induced reduction in p53 is
reversed by Nutlin-3 at all time points. To confirm that this reduc-
tion in p53 is the result of viral mechanisms, we compared the
effect of live RSV and UV-inactivated RSV on p53 protein. Fig.
4B again shows that following RSV infection, the amount of p53
protein is diminished, but this decrease is not seen when cells are
treated with UV-RSV. When control cells, RSV-treated cells, and
UV-inactivated RSV-treated cells are pretreated with Nutlin-3, the
p53 protein is increased. Next we evaluated the biological effect of
increasing p53 protein on cell viability. Primary (HTBE) cells
were treated with 10 ?M Nutlin-3 with and without RSV exposure.
Control and Nutlin-3-treated cells showed no increase in cell death
in the absence of RSV infection as measured by propidium iodide
staining of whole cells and quantification of cell death with flow
cytometry after 0, 24, 48, and 72 h of RSV exposure (Fig. 4A). The
fact that Nutlin-3 treatment alone showed no increase in cell death
despite marked up-regulation of p53 protein reflects published ob-
servations that stressed cells are killed by exposure to Nutlin-3
while unstressed control cells are largely unaffected (46). By com-
parison, RSV-treated cells show a gradual increase in cell death
over time that is markedly enhanced by Nutlin-3 at all time points
(Fig. 4A). These data demonstrate that increased endogenous p53
protein accelerates the onset and increases the magnitude of cell
death in RSV-infected airway epithelial cells. Preventing RSV-
mediated reduction in p53 shortens survival of RSV-infected ep-
Preventing RSV-mediated degradation of p53 leads to increased
We wanted to determine whether preserving endogenous p53 in
RSV infection leads to enhanced apoptosis. As shown in Fig. 5,
adding 10 ?M Nutlin-3 to primary HTBE cells treated with RSV
for 24 h increases the amount of multiple markers of apoptosis,
including cleaved caspase 3, cleaved caspase 7, and cleaved poly-
(ADP-ribose) polymerase protein. These results indicate that RSV
decreases p53 to delay apoptosis. Preservation of endogenous p53
in RSV infection leads to increased apoptosis as measured by
cleaved caspase products.
Increasing p53 enhances viral replication
We next examined the effect of p53 protein on RSV replication.
Previous studies investigating the effect of p53 protein on viral
replication have yielded disparate results. While p53 promotes ad-
enoviral and CMV replication, it limits poliovirus and vesicular
stomatitis virus replication (34–37). Primary HTBE cells were ex-
posed to RSV (MOI of 2) or 10 ?M Nutlin-3 followed by RSV and
incubated for 72 h. To quantify viral replication, cells were har-
vested, RNA was isolated, and quantitative real-time PCR was
optosis. HTBE cells were exposed to control buffer, 10 ?M Nutlin-3, RSV
(MOI of 2), or 10 ?M Nutlin-3 followed by RSV. Cells were then har-
vested for cellular protein after 24 h, and Western blot analysis was per-
formed for cleaved caspase 3, cleaved caspase 7, and cleaved poly(ADP-
ribose) polymerase. Results indicate that all these markers of apoptosis are
increased in RSV-infected cells treated with Nutlin-3.
Nutlin-3 maintains p53 protein and leads to increased ap-
exposed to RSV (MOI of 2) or 10 ?M Nutlin-3 followed by RSV for 72 h.
Cells were harvested, RNA isolated, cDNA made, and quantitative real-
time PCR performed. Primers targeted the RSV N-gene. Unpaired Stu-
dent’s t test indicates a statistically significant difference (?, p ? 0.05)
between cells exposed to RSV alone and RSV pretreated with Nutlin-3
(n ? 3 experiments).
p53 protein increases RSV replication. HTBE cells were
exposed to RSV (MOI of 2) or 10 ?M Nutlin-3 followed by RSV. Both
cells in the supernatant and adherence cells were collected and sonicated,
and 10-fold dilutions were performed. Dilutions were added to 90% con-
fluent Vero cells, overlayed, and incubated for 5 days. Neutral red was
added and viral plaques counted. The experiment was performed twice, and
photos show representative plaque assays.
p53 protein increases RSV replication. HTBE cells were
2745The Journal of Immunology
performed with primers specific for the well-conserved RSV N-
gene. Fig. 6 shows that the amount of viral RNA is increased
?5-fold when the cells are pretreated with 10 ?M Nutlin-3. To
confirm these results, a plaque assay was performed. Again, HTBE
cells were exposed to RSV (MOI of 2) or 10 ?M Nutlin-3 fol-
lowed by RSV and incubated for 72 h. The supernatant and ad-
herent cells were removed, combined, and sonicated, and a plaque
assay was performed. Fig. 7 demonstrates visually and in graphical
form that pretreatment with Nutlin-3 increases the amount of RSV
?3-fold as measured by plaque assay. Increased p53 protein en-
hances RSV replication.
p53 decreases IL-6 protein
Finally, we wanted to determine the effect of p53 on inflammation
in RSV infection, so we investigated whether p53 alters IL-6 pro-
tein levels. HTBE cells were exposed to control medium, 10 ?M
Nutlin-3, RSV (MOI of 2), or 10 ?M Nutlin-3 followed by RSV
infection for 24, 48, or 72 h. Supernatants were collected, and
ELISA was performed to detect IL-6 protein. Fig. 8 shows that
increasing the level of p53 attenuates the amount of IL-6 protein in
RSV-infected cells at all time points. Similar results were seen
when ELISA was performed for IL-8 protein (data not shown).
In this study, we demonstrate that RSV decreases the amount of
p53 in airway epithelial cells. The biological effect of this de-
creased p53 is prolonged cell survival (Fig. 9). This study is the
first to show that RSV alters the amount of p53 in airway ep-
ithelial cells, that the mechanism for this is alteration of p53 via
Akt and Mdm2, that RSV alteration of p53 prolongs cell sur-
vival, that p53 enhances RSV replication, and that p53 attenu-
ates inflammation in RSV infection.
Other studies have investigated the effect of viral infection on
p53. There have been no studies to date that have suggested an
alteration in p53 protein in RSV infection. In fact, Marques et al.
(34) saw no effect of RSV on p53 protein levels after 16 h of
exposure in HepG2 cells. However, the study did find that en-
cephalomyocarditis virus, human parainfluenza virus type 3,
Sendai virus, and VVE3L (a varicella virus mutant) induced
down-regulation of p53 via inhibition of the protein kinase R
and the RNase L pathways. Again, this difference may reflect
cell-specific responses to RSV infection or differences in the
strain and infectivity of the virus.
Conversely, studies have investigated the effect of p53 on viral
infection. p53 protein promotes adenoviral and CMV replication
and limits poliovirus and vesicular stomatitis virus replication
(34–37). We found that similar to the studies of adenovirus and
CMV, p53 protein increases RSV replication. The reason for this
finding is unclear and is the subject of current investigation. Mul-
tiple studies have demonstrated that RSV induces NF-?B (12, 13,
47), yet our data suggest that when p53 is maintained with Nutlin-3
in RSV infection, IL-6, which is an NF-?B-dependent protein, is
reduced. Perhaps increased p53 limits the RSV-induced NF-?B
host cell inflammatory response and allows for more viral
The regulation of p53 is complex and controlled by many dif-
ferent factors (19). We have previously shown that RSV increases
Akt activity and delays apoptosis (41). Other studies have shown
that Akt phosphorylates Mdm2 at Ser166, and this phosphorylated
Mdm2 tags p53 for ubiquitination and proteasome degradation
(24–27, 30, 31). Other pathways may be involved, but our study
indicates that this pathway is key because inhibition of the PI3K/
Akt signaling significantly affects activation of Mdm2 and, subse-
quently, p53 protein levels. Its inhibition virtually eliminates phos-
pho-Mdm2 (Ser166) and preserves p53 in RSV infection.
RSV is a clinically important pathogen, particularly for infants,
patients with obstructive lung disease, and the immunosuppressed.
Our study demonstrates that RSV prolongs infected cell survival
by delaying cell death via posttranslational degradation of p53.
The effect of this alteration in p53 protein on viral replication and
host cell inflammatory responses will continue to be the subject of
future studies. These observations suggest that p53, the PI3K/Akt
pathway, and Mdm2 may be important targets for therapy in RSV
We thank Lori Manzel and for assistance with primary airway epithelial
cells. We also thank Sara Hinde and Chris Barrett for technical assistance.
posed to control medium, 10 ?M Nutlin-3, RSV (MOI of 2), or Nutlin-3
followed by RSV for the indicated periods of time. Supernatants were
collected and analyzed for IL-6 protein by ELISA. One-way ANOVA with
a Bonferroni’s test for multiple comparisons indicates a statistically sig-
nificant difference (?, p ? 0.01) between IL-6 protein in cells exposed to
RSV and Nutlin-3 as compared with cells only exposed to RSV at all time
points. Data are representative of three experiments.
p53 protein decreases IL-6 protein. HTBE cells were ex-
tivates Mdm2. Activated Mdm2 ubiquitinates p53, targeting it for proteo-
some degradation. The Mdm2/p53 association can be prevented by Nut-
lin-3. A decrease in p53 allows for increased cell survival, decreased viral
replication, and increased inflammation. If p53 is not ubiquitinated, it can
be phosphorylated and activated, decreasing cell survival, increasing viral
replication, and limiting inflammation.
Overview. RSV activates Akt that phosphorylates and ac-
2746RSV AND p53
The authors have no financial conflict of interest.
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