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1
Scientific RepoRts | 6:19922 | DOI: 10.1038/srep19922
www.nature.com/scientificreports
NF-κB promotes leaky expression
of adenovirus genes in a replication-
incompetent adenovirus vector
M. Machitani
1
, F. Sakurai
1,2
, K. Wakabayashi
1
, K. Nakatani
1
, K. Shimizu
1,3
, M. Tachibana
1
&
H. Mizuguchi
1,4,5,6,7
The replication-incompetent adenovirus (Ad) vector is one of the most promising vectors for gene
therapy; however, systemic administration of Ad vectors results in severe hepatotoxicities, partly
due to the leaky expression of Ad genes in the liver. Here we show that nuclear factor-kappa B (NF-
κB) mediates the leaky expression of Ad genes from the Ad vector genome, and that the inhibition
of NF-κB leads to the suppression of Ad gene expression and hepatotoxicities following transduction
with Ad vectors. Activation of NF-κB by recombinant tumor necrosis factor (TNF)-α signicantly
enhanced the leaky expression of Ad genes. More than 50% suppression of the Ad gene expression
was found by inhibitors of NF-κB signaling and siRNA-mediated knockdown of NF-κB. Similar results
were found when cells were infected with wild-type Ad. Compared with a conventional Ad vector, an
Ad vector expressing a dominant-negative IκBα (Adv-CADNIκBα), which is a negative regulator of
NF-κB, mediated approximately 70% suppression of the leaky expression of Ad genes in the liver. Adv-
CADNIκBα did not induce apparent hepatotoxicities. These results indicate that inhibition of NF-κB
leads to suppression of Ad vector-mediated tissue damages via not only suppression of inammatory
responses but also reduction in the leaky expression of Ad genes.
e replication-incompetent E1-deleted adenovirus (Ad) vector is the most promising vector for both gene ther-
apy and basic studies due to its advantages as a gene delivery vehicle, which include high titer production and high
transduction eciencies in both dividing and non-dividing cells
1,2
. In a conventional Ad vector, the E1 (E1A,E1B)
gene is replaced by a transgene expression cassette and the Ad vector is propagated in E1-expressing cell lines,
such as HEK293 cells. e E1A gene is the rst transcription unit to be expressed immediately aer wild-type Ad
infection, and transactivates the expression of other Ad genes. us, no Ad genes should be expressed from the
Ad vector genome due to the lack of the E1A and E1B genes in the Ad vector genome. However, expression of Ad
genes, including the E2, E4 and pIX genes, is indeed detected in the Ad vector-transduced cells, indicating that
the Ad genes are expressed from the Ad vector genome in an E1A gene-independent manner
3–6
. Further, the leaky
expression results in immune responses against Ad proteins and Ad protein-induced cellular toxicity, leading to
tissue damages such as hepatotoxicities
7–9
.
Various types of next-generation Ad vectors which can overcome these disadvantages have been developed
3,4,8
.
For example, an Ad vector lacking the E2A gene expression achieved a reduction in the immune responses
and the prolonged transgene expression proles
10
. A helper-dependent Ad (HD-Ad) vector, in which all viral
encoding genes were deleted, also showed a reduction in the production of inammatory cytokines and the
tissue damages
11–13
. In addition, we recently developed an Ad vector that suppresses the leaky expression of Ad
1
Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University,
1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
2
Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics,
Clinical Drug Development Unit, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka,
Suita, Osaka, 565-0871, Japan.
3
Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiorikita, Tondabayashi,
Osaka, 584-8540, Japan.
4
Laboratory of Hepatocyte Regulation, National Institutes of Biomedical Innovation, Health
and Nutrition, 7-6-8 Saito, Asagi, Ibaraki, Osaka 567-0085, Japan.
5
iPS Cell-Based Research Project on Hepatic
Toxicity and Metabolism, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita,
Osaka 565-0871, Japan.
6
Global Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2
Yamadaoka, Suita, Osaka 565-0871, Japan.
7
Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita,
Osaka 565-0871, Japan. Correspondence and requests for materials should be addressed to F.S. (email: sakurai@
phs.osaka-u.ac.jp) or H.M. (email: mizuguch@phs.osaka-u.ac.jp)
Received: 28 August 2015
Accepted: 21 December 2015
Published: 27 January 2016
OPEN
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Scientific RepoRts | 6:19922 | DOI: 10.1038/srep19922
genes by inserting microRNA-targeted sequences in the Ad vector genome
9
. Nonetheless, problems remain with
all of these novel Ad vectors, including the complexity of the Ad vector preparation and some remaining Ad
vector-induced hepatotoxicities. To overcome these drawbacks, the mechanisms of the E1A gene-independent
leaky expression of Ad genes should be claried.
Transduction with Ad vectors causes activation of a number of transcriptional factors. For example, injection
with recombinant Ad vectors immediately induces production of inammatory cytokines associated with innate
immune responses
14,15
, suggesting that several transcriptional factors would be activated in the innate immune
responses. In this study, among these transcriptional factors, we focused on nuclear factor-kappa B (NF-κ B)
because NF-κ B has been demonstrated to play a crucial role in the expression of numerous viral genes
16
. NF-κ B
is a ubiquitous transcriptional factor which promotes the expression of a large number of genes, particularly
those from gene families associated with host immune responses
16
. us, NF-κ B plays a crucial role in immune
responses. Although NF-κ B functions as a homodimer or heterodimer (p50/p50, p50/p65, and p52/RelB), canon-
ical NF-κ B is a heterodimer which is composed of p50 and p65. Under normal conditions, NF-κ B stays in the
cytoplasm via association with Iκ Bα , an NF-κ B-inhibitory factor. Upon the stimulation by cytokines and path-
ogens, Iκ Bα is phosphorylated and degraded, leading to localization of NF-κ B into the nucleus from the cyto-
plasm. In the nucleus, translocated NF-κ B promotes the expression of target genes by binding to the consensus
sequence (5′ GGGACT TTCC-3′ ) in their promoter region.
In the present study, we have demonstrated that NF-κ B promotes Ad gene expression following transduction
with an Ad vector and a wild-type Ad (WT-Ad). Further, an Ad vector expressing a dominant-negative Iκ Bα
(Adv-CADNIκ Bα ), which is a negative regulator of NF-κ B, mediated the signicant suppression of the leaky
expression of Ad genes, with the result that no apparent hepatotoxicities were induced.
Materials and Methods
Cell lines and animals. HEK293 (a transformed embryonic kidney cell line) and HeLa (a human epithe-
lial carcinoma cell line) cells were cultured in Dulbecco’s modied Eagle’s medium supplemented with 10%
fetal bovine serum (FBS), streptomycin (100 μ g/ml), and penicillin (100 U/ml). H1299 (a non-small cell lung
carcinoma cell line) cells were cultured in RPMI1640 supplemented with 10% FBS, streptomycin (100 μ g/ml),
and penicillin (100 U/ml). Female C57BL/6 mice aged 5–6 weeks were obtained from Nippon SLC (Shizuoka,
Japan). Rag2/Il2rγ double knockout mice of C57BL/6 background, aged 5–7 weeks, were obtained from Taconic
Farms (Hudson, NY)
17,18
. All mouse experimental procedures used in this study were approved by Animal
Experimentation Committee of Osaka University and performed in accordance with institutional guidelines for
animal experiments at Osaka University.
Reagents. Recombinant human and mouse tumor necrosis factor (TNF)-α were purchased from Invivogen
(San Diego, CA) and Peprotech (Rocky Hill, NJ), respectively. NF-κ B inhibitors, BAY11-7082 and MG-132, were
purchased from Invivogen. Recombinant human interferon-α (IFN-α ) was purchased from PBL interferon
source (Piscataway, NJ).
Plasmids. pNF-κ B-Luc, which contains the rey luciferase (FLuc) gene expression cassette driven by a pro-
moter containing a consensus sequence for the NF-κ B binding, was purchased from Agilent Technologies (Santa
Clara, CA). pHMCMV-RLuc, a reporter plasmid carrying a cytomegalovirus (CMV) promoter-driven renilla
luciferase (RLuc) expression cassette, was constructed previously
19
.
e reporter plasmids pGL4-E1A, -E1B, -E2, -E3, and -E4, which have the promoter sequences of the respec-
tive Ad genes upstream of the FLuc gene, were constructed as follows: the fragment containing the E2 promoter
sequence (bp 27061–27661) was amplified by PCR using the primers E2p-F and E2p-R, and ligated with a
HindIII/XhoI fragment of pGL4.10 (Promega, Madison, WI), resulting in pGL4-E2. pGL4-E1A, -E1B, -E3, and -E4,
which contain the E1A (bp 1–546), E1B (bp 1336–1702), E3 (bp 26987–27578), and E4 (bp 35530–35939) pro-
moter region, respectively, were similarly constructed using the corresponding primers. e mutated reporter
plasmids pGL4-E2-del1, -del2, and -del3, which have shortened E2 promoter sequences (bp 27061–27296, 27061–
27197, and 27061–27136, respectively) upstream of the FLuc gene, were constructed as follows. First, pGL4-E2
was digested with EcoRV/XhoI. e resulting fragments were then self-ligated aer the sticky end was converted
to a blunt end, resulting in pGL4-E2-del1. e fragment containing the shortened E2 promoter sequence (bp
27061–27197) was amplied by PCR using the primers E2-del2-F and E2-del2-R, and ligated with a HindIII/XhoI
fragment of pGL4.10, resulting in pGL4-E2-del2. An AII/XhoI fragment of pGL4-E2 was ligated with the oligo-
nucleotides, E2-del3-S and E2-del3-AS, which encode the shortened E2 promoter sequence (bp 27061–27136),
resulting in pGL4-E2-del3. A reporter plasmid, pGL4-del2.1, which has the shortened E2 promoter sequences
(bp 27061–27197) and lacks the predicted NF-κ B binding site in the E2 promoter sequence, was constructed as
follows: the predicted NF-κ B binding site was mutated by using a QuikChange Site-Directed Mutagenesis Kit
(Agilent Technologies) and the primers E2-del2.1-F and E2-del2.1-R, resulting in pGL4-E2-del2.1. e mutated
sequence of pGL4-E2-del2.1 is shown in Fig.1B. Sequences of the primers and oligonucleotides are shown in
Table S1.
pAdHM4-CMV-Luc, the Ad vector plasmid carrying a CMV promoter-driven luciferase expression cas-
sette, was constructed previously
9
. pAdHM4-CA-Luc and -DNIκ B α , the Ad vector plasmid carrying a CA
promoter (a β -actin promoter/CMV enhancer with a β -actin intron)-driven luciferase and dominant-negative
Iκ Bα gene expression cassette, respectively, were constructed as follows: pHMCA5
20
was digested with
NotI/XbaI, and ligated with the NotI/XbaI fragment of pCMVL1
9
, resulting in pHMCA5-Luc. e fragment
containing the dominant-negative Iκ Bα gene was amplied by PCR using pCMX-Iκ Bα M (Addgene plasmid
12329; Addgene, Cambridge, MA), which contains the dominant-negative Iκ Bα gene expression cassette, and
the primers DNIκ Bα -F and DNIκ Bα -R, and ligated with the NotI/XbaI fragment of pHMCA5, resulting in
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pHMCA5-DNIκ Bα . pHMCA5-Luc and -DNIκ Bα were digested with I-CeuI/PI-SceI, and then ligated with
I-CeuI/PI-SceI− digested pAdHM4
21
, resulting in pAdHM4-CA-Luc and pAdHM4-CA-DNIκ Bα , resp ectively.
Viruses. WT-Ad (human Ad serotype 5) was obtained from the American Type Culture Collection (ATCC).
Recombinant Ad vectors were prepared by an in vitro improved ligation method as follows
22,23
; all Ad vector
plasmids used in this study have PacI-anked Ad vector genome. Ad vector plasmids, pAdHM4-CMV-Luc,
pAdHM4-CA-Luc, and pAdHM4-CA-DNIκ Bα , were digested with PacI to release the recombinant Ad vector
genome, and were each transfected into HEK293 cells using Lipofectamine 2000 (Life Technologies, Carlsbad,
CA), resulting in Adv-CMVLuc, Adv-CALuc, and Adv-CADNIκ Bα , respectively. Adv-CALacZ, an Ad vector
expressing LacZ, was constructed previously
24
. ese Ads were amplied and puried by two rounds of cesium
chloride-gradient ultracentrifugation, dialyzed, and stored at − 80 °C
22
. e virus particles (VP) were deter-
mined by a spectrophotometrical method
22
. Determination of infectious units (IFU) was accomplished using an
Adeno-X Rapid Titer Kit (Clontech, Mountain View, CA).
Prediction of NF-κB binding sites in the Ad genome. Transcriptional factors binding to the E2 pro-
moter region and the putative binding sites in the promoter region were predicted using TFSEARCH (http://
www.cbrc.jp/research/db/TFSEARCH.html).
Analysis of the promoter activities using reporter plasmids. Cells were seeded in a 24-well plate at a
density of 5 × 10
4
cells/well. On the following day, cells were co-transfected with FLuc-expressing reporter plas-
mids (500 ng/ml) (constructed as shown above) and pHMCMV-RLuc (160 ng/ml) using Lipofectamine 2000 (Life
Technologies). Following a 6-h incubation, the medium was changed. Luciferase activity in cells was determined
using the Dual Luciferase Reporter Assay System (Promega) 24 h aer transfection.
Figure 1. Reduction in the E2 promoter activity by deletion of an NF-κB binding site. (A,B) HeLa cells were
co-transfected with pHMCMV6-RLuc and reporter plasmids carrying the E2-promoter-driven FLuc expression
cassette (pGL4, pGL4-E2, pGL4-E2-del1, pGL4-E2-del2, pGL4-E2-del2.1, or pGL4-E2-del3). Aer a 24-h
incubation, luciferase activity was determined. e data show FLuc activity normalized by RLuc activity in the
cells. Schematic diagrams of each promoter are shown at the le of the graph. (B) Nucleotide sequence around
the NF-κ B binding site (italic type) in the E2 promoter region. e NF-κ B binding sequence is deleted in pGL4-
E2-del2.1. ese data are expressed as the means ± S.D. (n = 4). ***p < 0.001.
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Scientific RepoRts | 6:19922 | DOI: 10.1038/srep19922
For evaluation of the eects of TNF-α on the promoter activities, cells were treated with TNF-α (100 ng/
ml) 24 h aer transfection with plasmids as described above. Luciferase assay was performed 24 h aer TNF-α
treatment.
Quantitative RT-PCR analysis of Ad gene expression. Cells were seeded in a 24-well plate at a den-
sity of 5 × 10
4
cells/well. On the following day, WT-Ad and Ad vectors were added to the cells at the indicated
titers. Following the indicated incubation periods, total RNA was isolated from cells using ISOGEN (Nippon
Gene, Tokyo, Japan). cDNA was synthesized using 500 ng of total RNA with a Superscript VILO cDNA synthesis
kit (Life Technologies). Real-time RT-PCR analysis was performed using Fast SYBR Green Master Mix (Life
Technologies) and StepOnePlus real-time PCR systems (Life Technologies). Sequences of the primers used in this
study are described in Table S1.
For evaluation of the eects of TNF-α on Ad gene expression, cells were pre-treated with TNF-α at a concen-
tration of 100 ng/ml for 5 h, followed by incubation with WT-Ad and Ad vectors. Total RNA was recovered for
quantitative RT-PCR analysis 12 h aer addition of WT-Ad and Ad vectors.
For inhibition of NF-κ B, cells were pre-treated with BAY11-7082 and MG-132 at 10 μ M and 2.5 μ M, respec-
tively, for 1 h, followed by incubation with WT-Ad and Ad vectors. Total RNA was recovered 12 h aer the addi-
tion of WT-Ad and Ad vectors.
For knockdown of p50, cells were transfected with an siRNA against p50 (sip50) (Gene Design, Osaka, Japan)
using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s instructions. e target sequence
of sip50 was 5′ -aaggggctataatcctggact-3′ . Control siRNA was purchased from Qiagen (Allstars Negative Control
siRNA; Qiagen, Hilden, Germany). Cells were treated with WT-Ad and Ad vectors 48 h aer siRNA transfection.
Total RNA was recovered 12 h aer addition of WT-Ad and Ad vectors.
Western blotting analysis. Western blotting assay was performed as previously described
13
. Briefly,
whole-cell extracts were prepared and electrophoresed on 10% sodium dodecyl sulfate (SDS)-polyacrylamide
gels under reducing conditions, followed by electrotransfer to PVDF membranes (Millipore, Bedford, MA). Aer
blocking with 5% skim milk prepared in TBS-T (tween-20, 0.1%), the membrane was incubated with a mouse
anti-E1A antibody (clone name) (Abcam, Cambridge, UK), a rabbit anti-p50 antibody (H-119) (Santa Cruz
Biotechnology, Santa Cruz, CA), or mouse anti-β -actin (Sigma-Aldrich, St. Louis, MO), followed by incubation
in the presence of horseradish peroxidase (HRP)-labeled anti-rabbit or anti-mouse IgG antibody (Cell Signaling
Technology, Danvers, MA). e intensity of protein bands was quantied by Image J soware.
Determinaton of Ad genome copy numbers. Cells were treated with WT-Ad and Ad vectors in a man-
ner similar to that described above. Following the indicated incubation periods, total DNA, including Ad genomic
DNA, was isolated from the cells infected with Ads using a DNeasy Blood & Tissue Kit (Qiagen). Aer isolation,
Ad genome copy numbers were quantied using a StepOnePlus real-time PCR system (Life Technologies) as pre-
viously described
25
. Sequences of the primers and probes used in this study are described in Table S1.
Chromatin immunoprecipitation (ChIP) assay. HeLa cells were infected with WT-Ad at 100 VP/cell.
Following a 24-h incubation, cells were treated with formaldehyde at a nal concentration of 1% for crosslinking,
and then genomic DNA was fragmented by sonication. e DNA fragment-protein complexes were immuno-
precipitated using a rabbit anti-p65 antibody (ab7970) (Abcam) or normal rabbit IgG (Cell Signaling). e ChIP
assay kit was purchased from Merck Millipore (Darmstadt, Germany). e precipitated DNA copy numbers were
determined by quantitative PCR using the primers shown in Table S1.
In vivo Ad gene expression analysis. Ad vectors were intravenously administered to mice at a dose of 10
9
IFU/mouse via a tail vein. Total RNA was extracted from the livers 48 h aer administration, and the Ad gene
mRNA levels were determined by quantitative RT-PCR analysis.
Analysis of Ad vector-mediated hepatotoxicities. Ad vectors were intravenously administered to mice
at a dose of 10
9
IFU/mouse via a tail vein. e blood samples were collected via retro-orbital bleeding at the
indicated days, and the serum samples were obtained aer centrifugation. e serum alanine aminotransferase
(ALT) and aspartate aminotransferase (AST) levels were determined using a transaminase-CII-test kit (Wako,
Osaka, Japan).
Statistical analysis. Statistical signicance was determined using Student’s t-test. Data are presented as the
means ± S.D or S.E.
Results
The NF-κB binding site is essential for the E2 promoter activity. In order to identify the transcrip-
tional factors crucial for leaky expression of the Ad genes, functional analysis of a promoter of the E2 gene, which
shows leaky expression from the Ad vector genome
5
, was performed using reporter plasmids (Fig.1A). e lucif-
erase expression levels from pGL4-E2-del3, which has the shortened promoter sequence (bp 27061–27136) of
the E2 gene upstream of the FLuc gene, was signicantly lower than that from pGL4-E2, which has the promoter
sequence (bp 27061–27661) of the E2 gene upstream of the FLuc gene (Fig.1A), suggesting that the sequence
between bp 27136–27197 in the E2 promoter was signicantly involved in the E2 promoter activity. Next, we
performed in silico analysis using TFSEARCH, a program that searches for transcriptional factor binding sites,
to identify transcriptional factor binding sites in the sequence between bp 27136–27197. is analysis revealed
an NF-κ B binding site (5′ -gggaatttcc-3′ ) in this sequence (Fig.1B). To examine whether the NF-κ B binding site
is essential for the E2 promoter activity, the NF-κ B binding site in the E2 promoter sequence of pGL4-E2-del2
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Scientific RepoRts | 6:19922 | DOI: 10.1038/srep19922
was deleted. e deletion of the NF-κ B binding site resulted in a signicant reduction in the luciferase activity of
pGL4-E2-del2 (Fig.1B). ese results suggested that the NF-κ B binding site was important for the E2 promoter
activity.
Activation of NF-κB promotes Ad gene expression and Ad replication. In order to examine
whether NF-κ B is involved in the leaky expression of Ad genes, including the E2 gene, HeLa cells were pre-treated
with recombinant human TNF-α (hTNF-α ), followed by infection with WT-Ad. The reporter assay using
pNF-κ B-Luc, which carries the luciferase gene expression cassette driven by a promoter containing a consensus
sequence for NF-κ B binding, showed that hTNF-α treatment signicantly activated NF-κ B signaling (Fig.2A).
Activation of the NF-κ B signaling by hTNF-α signicantly (1.4-2-fold) enhanced the mRNA levels of the Ad
early genes, including the E1A, E2, E3, and E4 genes, in HeLa cells following infection with WT-Ad (Fig.2B). e
E1A protein levels were also elevated by TNF-α treatment in WT-Ad-infected HeLa cells (Fig.2C). WT-Ad repli-
cated more eciently in HeLa cells pre-treated with hTNF-α in a dose-dependent manner (Fig.2D). In addition,
hTNF-α treatment signicantly enhanced the leaky expression of the E2 and E4 genes in HeLa cells following
transduction with Adv-CMVLuc, a replication-incompetent Ad vector expressing FLuc (Fig.2E). ese results
suggested that the activation of NF-κ B by TNF-α promoted Ad gene expression and Ad replication.
Inhibition of NF-κB negatively regulates Ad gene expression and Ad replication. Next, in order
to examine the involvement of NF-κ B in Ad gene expression and Ad replication, HeLa cells were pre-treated
with the NF-κ B inhibitors BAY11-7082 and MG-132, followed by infection with WT-Ad. e cell viabilities
were not signicantly reduced following treatment with BAY11-7082 and MG-132 (Figure S1). e reporter
assay using pNF-κ B-Luc showed that the treatment with BAY11-7082 and MG-132 largely inhibited NF-κ B sig-
naling (Fig.3A). Inhibition of NF-κ B signaling by BAY11-7082 and MG-132 resulted in more than 80% reduc-
tion in the E1A, E2, E3, and E4 gene expressions in WT-Ad-infected HeLa cells (Fig.3B). Copy numbers of the
WT-Ad genome were also suppressed by more than 80% in HeLa cells following treatment with BAY11-7082
and MG-132 (Fig.3C). In addition, treatment with BAY11-7082 and MG-132 reduced the leaky expression of
the E2 and E4 genes by approximately 50% in HeLa cells following transduction with Adv-CMVLuc (Fig.3D).
Next, in order to further examine the eects of NF-κ B on Ad gene expression and Ad replication, p50, which
is a component of NF-κ B, was knocked down in HeLa cells. Transfection with sip50 resulted in a signicant
knockdown of p50 at both the mRNA and protein levels (Fig.4A,B). e E1A, E2, E3, and E4 mRNA levels
following infection with WT-Ad were suppressed by more than 50% in p50-knockdown cells, compared with
those in control cells (Fig.4C). An approximately 90% reduction in copy numbers of the WT-Ad genome was
found in p50-knockdown HeLa cells (Fig.4D). Knockdown of p50 also led to approximately 70% decreases in
the leaky expression of the E2 and E4 genes following transduction with Adv-CMVLuc (Fig.4E). Similar results
were found in H1299 cells (Figure S2). ese results indicated that NF-κ B was essential for Ad gene expression
and replication of WT-Ad, and that NF-κ B induced the leaky expression of the Ad genes following transduction
with Ad vectors.
NF-κB binds to the E2 and E3 promoter. In order to examine whether NF-κ B enhances Ad gene pro-
moter activities, HeLa cells were transfected with reporter plasmids carrying the Ad gene promoter-driven lucif-
erase expression cassette, followed by treatment with hTNF-α . e E2 and E3 promoter activities, but not the
activities of the E1A, E1B, or E4 promoter, were more than 3-fold enhanced by hTNF-α treatment (Fig.5A). Next,
we performed a ChIP assay using an antibody against p65, which is another component of NF-κ B, to examine
whether NF-κ B directly binds to the E2 and E3 promoter region. e E2 and E3 promoter is a bidirectional pro-
moter, which drives transcription of both the E2 and E3 genes. e ChIP assay demonstrated that NF-κ B directly
bound to the E2 and E3 promoter region (Fig.5B), indicating that NF-κ B activates the E2 and E3 gene expression
via direct binding to the promoter region.
IFN-α treatment suppresses Ad gene expression and Ad replication. Next, we investigated the
involvement of other transcriptional factors in Ad gene expression. In order to activate transcriptional factors
involved in innate immune responses, HeLa cells were pre-treated with recombinant human IFN-α , which acti-
vates STAT1, STAT2, and IRF9, followed by infection with WT-Ad. e reporter assay using pNF-κ B-Luc showed
that IFN-α treatment signicantly inhibited NF-κ B signaling (Figure S3A). Contrary to TNF-α treatment, IFN-α
treatment signicantly suppressed the mRNA levels of the Ad early genes, including the E1A, E2, E3, and E4
genes, in HeLa cells following infection with WT-Ad (Figure S3B). WT-Ad replicated less eciently in HeLa cells
pre-treated with IFN-α (Figure S3C). ese results suggested that transcriptional factors activated by IFN-α did
not promote Ad gene expression and Ad replication.
NF-κB is involved in Ad gene expression in the mouse liver. Next, in order to examine the eects of
NF-κ B on the Ad gene expression in vivo, mice were intravenously administered WT-Ad and Ad vectors, followed
by intravenous injection of recombinant mouse TNF-α (mTNF-α ) 24 h aer Ad injection. Ad gene expression
levels in the liver were determined by quantitative RT-PCR analysis. e liver is a main organ which an Ad vector
are distributed to following intravenous administration. Ad gene expression from the WT-Ad genome in the liver
increased by more than 9-fold by mTNF-α administration (Fig.6A). mTNF-α treatment also mediated a signif-
icant increase in the leaky expression of the E2 and E4 genes from the Ad vector genome in the liver (Fig.6B).
Next, to inhibit NF-κ B signaling in the liver, we constructed an Ad vector expressing a dominant-negative Iκ Bα
(Adv-CADNIκ Bα ), which is a negative regulator of NF-κ B
26
. Adv-CADNIκ Bα induced signicant inhibition of
NF-κ B signaling, compared with a conventional Ad vector expressing LacZ (Adv-CALacZ) (Fig.7A). Compared
with the leaky expression levels of the Ad genes from Adv-CALuc, Adv-CADNIκ Bα mediated an approximately
70% suppression of the leaky expression of the Ad genes in HeLa cells (Fig.7B) and in the liver (Fig.7C). ese
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Scientific RepoRts | 6:19922 | DOI: 10.1038/srep19922
results indicated that NF-κ B mediated the leaky expression of the Ad genes following administration with Ad
vectors in not only cultured cells but also the organs.
Inhibition of NF-κB leads to suppression of the leaky expression of Ad genes and reduction
in the hepatotoxicities. Several groups, including ours, previously reported that the leaky expression of
Ad genes from the Ad vector genome induced tissue damages such as hepatotoxicities
7–9
. erefore, in order to
Figure 2. Promotion of Ad early gene expression and replication by TNF-α stimulation. (A) HeLa cells
were transfected with pNF-κ B-Luc, followed by treatment with recombinant hTNF-α at 100 ng/ml. Aer 24-h
incubation, luciferase activity was determined. The data show FLuc activity normalized by RLuc activity.
(B,C) HeLa cells were pre-treated with hTNF-α at 100 ng/ml for 5 h, followed by infection with WT-Ad at
100 VP/cell. Aer 12-h incubation, the E1A, E2, E3, and E4 mRNA levels in the cells were determined by
quantitative RT-PCR (B). Aer 24-h incubation, the E1A protein levels in the cells were determined by western
blotting analysis (C). e intensity of E1A expression was quantied using Image J soware. (D) HeLa cells
were pre-treated with hTNF-α at the indicated concentration for 5 h, followed by infection with WT-Ad at
100 VP/cell. After 24-h incubation, Ad genome copy numbers in the cells were determined by quantitative
PCR. (E) HeLa cells were pre-treated with hTNF-α at 100 ng/ml for 5 h, followed by transduction
with Adv-CMVLuc at 100 VP/cell. Aer 12-h incubation, Ad gene mRNA levels in the cells were similarly
determined. ese data are expressed as the means ± S.D. (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 3. Suppression of Ad early gene expression and replication by NF-κB inhibitors. (A) HeLa cells were
pre-treated with BAY11-7082 and MG-132 at 10 mM and 2.5 mM, respectively, for 1 h, followed by transfection
with pNF-κ B-Luc. Aer 24-h incubation, luciferase activity was determined. e data show FLuc activity
normalized by RLuc activity. (B,C) HeLa cells were pre-treated with BAY11-7082 and MG-132 at 10 mM and
2.5 mM, respectively, for 1 h, followed by infection with WT-Ad at 100 VP/cell. Aer 12-h incubation, the E1A,
E2, E3, and E4 mRNA levels in the cells were determined by quantitative RT-PCR (B). Aer 24-h incubation,
Ad genome copy numbers in the cells were determined by quantitative PCR (C). (D) HeLa cells were pre-treated
with BAY11-7082 and MG-132 at 10 mM and 2.5 mM, respectively, for 1 h, followed by infection with Adv-
CMVLuc at 100 VP/cell. Aer 12-h incubation, Ad gene mRNA levels in the cells were similarly determined.
ese data are expressed as the means ± S.D. (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001.
Figure 4. Suppression of Ad early gene expression and replication by NF-κB knockdown. (A,B) HeLa and
H1299 cells were transfected with siControl or sip50. Aer 48-h incubation, mRNA and protein levels of p50
in the cells were determined by quantitative RT-PCR (A) and western blotting analysis (B), respectively. p50
mRNA levels in the cells transfected with siControl were normalized to 1. (C,D) HeLa cells were transfected
with sip50 at 50 nM, followed by infection with WT-Ad at 100 VP/cell. Aer 12-h incubation, the E1A, E2,
E3, and E4 mRNA levels in the cells were determined by quantitative RT-PCR (C). Aer 24-h incubation, Ad
genome copy numbers in the cells were determined by quantitative PCR (D). (E) HeLa cells were transfected
with sip50 at 50 nM, followed by transduction with Adv-CMVLuc at 100 VP/cell. Aer 12-h incubation,
Ad gene mRNA levels in the cells were similarly determined. ese data are expressed as the means ± S.D.
(n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001.
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Figure 5. NF-κB-mediated enhancement of the E2 and E3 promoter activity. (A) HeLa cells were
transfected with reporter plasmids carrying the expression cassette of Ad early gene promoter-driven FLuc
(pGL4-E1A, -E1B, -E2, -E3, and -E4) for 24 h, followed by treatment with hTNF-α at 100 ng/ml. Aer 24-h
incubation, luciferase activity was determined. (B) HeLa cells were infected with WT-Ad at 100 VP/cell. Aer
24-h incubation, NF-κ B binding DNA levels were determined by ChIP assay. ese data are expressed as the
means ± S.D. (n = 3–4). **p < 0.01, ***p < 0.001.
Figure 6. Promotion of Ad early gene expression by TNF-α stimulation in mouse liver. (A,B) C57BL/6
mice were intravenously administered 10
9
IFU of WT-Ad (A) or Adv-CALuc (B), followed by intravenous
administration of recombinant mTNF-α at 0.5 mg/mouse. Twenty-four hours aer the administration, the
E1A, E2, E3, and E4 mRNA levels in the mouse liver were determined by quantitative RT-PCR. ese data are
expressed as the means ± S.E. (n = 5–6). *p < 0.05, **p < 0.01, ***p < 0.001.
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examine whether inhibition of NF-κ B leads to the suppression of Ad vector-mediated hepatotoxicities via reduc-
tion in the leaky expression of Ad genes, serum ALT and AST levels, which are representative biomarkers of hepa-
totoxicities, were measured at the indicated days aer administration with Adv-CALuc and Adv-CADNIκ Bα
(Fig.8A). ALT and AST levels were not apparently elevated following administration with Adv-CADNIκ Bα ,
while Adv-CALuc showed signicant elevation in serum ALT and AST levels.
Over-expression of DNIκ B α in immune cells might inhibit the activation of immune cells via innate
immune responses, leading to a reduction in the hepatotoxicities. Ad vector-induced hepatotoxicities
are mediated by cytotoxic T cells as well as Ad proteins themselves
7–9
. An Ad vector transduces not only
hepatocytes but also dendritic cells, which are professional antigen presenting cells, in the spleen following
intravenous administration
27
. Next, in order to examine whether the suppression of Ad vector-mediated
hepatotoxicities by DNIκ B α expression is induced even in immune-deficient mice, Adv-CALuc and
Adv-CADNIκ Bα were intravenously administered to Rag2/Il2rγ double knockout mice, which have global
defects in both cellular and humoral immune responses due to the lack of T, B, and natural killer (NK)
cells
17,18
. Compared with the leaky expression levels of the E2 genes in the liver of Rag2/Il2rg double knock-
out mice following intravenous administration with Adv-CALuc, Adv-CADNIκ Bα exhibited significantly
lower expression levels of the E2 genes in the liver (Fig.8B). Rag2/Il2rγ double knockout mice showed sig-
nificant elevation in serum ALT and AST levels following intravenous administration with Adv-CALuc, but
not with Adv-CADNIκ B α (Fig.8C). These results indicated that NF-κ B-mediated leaky expression of Ad
genes induced hepatotoxicities, and that Ad-CADNIκ Bα did not induce any apparent hepatotoxicity due to
significant suppression of the leaky expression of Ad genes.
Figure 7. Suppression of Ad early gene expression by expression of a dominant-negative IκBα gene.
(A) HeLa cells were transfected with pNF-κ B-Luc, followed by transduction with Adv-CALuc or
Adv-CADNIκ Bα at an MOI of 5. Aer 48-h incubation, luciferase activity was determined. e data show FLuc
activity normalized by RLuc activity. (B) HeLa cells were transduced with Adv-CALuc or Adv-CADNIκ Bα at
an MOI of 5. Aer 48-h incubation, the E2 and E4 mRNA levels in the cells were determined by quantitative
RT-PCR. ese data (A,B) are expressed as the means ± S.D. (n = 4). (C) C57BL/6 mice were intravenously
administered 10
9
IFU of Adv-CALuc or Adv-CADNIκ Bα . Forty-eight hours aer the administration, the E2,
E4, and pIX mRNA levels in the mouse liver were determined by quantitative RT-PCR. e data are expressed
as the means ± S.E. (n = 5–6). **p < 0.01, ***p < 0.001.
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Discussion
Several studies have demonstrated that the leaky expression of Ad genes from a replication-incompetent
Ad vector genome leads to tissue damages such as hepatotoxicities
7–9
; however, the mechanism of the E1A
gene-independent expression of Ad genes following transduction has remained unclear. e aim of this study
was to identify cellular factors involved in the E1A gene-independent expression of Ad genes and to provide
insights into the development of a safer Ad vector. e results showed that NF-κ B promoted Ad gene expression
following treatment with WT-Ad and an Ad vector (Figs2–4 and 6). Adv-CADNIκ Bα , which eciently inhibited
NF-κ B sig naling via overexpression of DNIκ Bα , also induced a signicant suppression of the leaky expression
of Ad genes (Fig.7). In addition, Adv-CADNIκ Bα did not lead to any apparent hepatotoxicity following admin-
istration (Fig.8).
is study demonstrated that NF-κ B is largely responsible for Ad gene expression. Several types of viruses,
including CMV and herpes simplex virus type-1 (HSV-1), utilize NF-κ B signaling to facilitate their replication
16
.
ese viruses possess NF-κ B binding sites in their viral gene promoters, and thus the NF-κ B signaling triggers the
enhancement of their viral gene expressions, leading to promotion of the viral replication. For example, infection
with CMV results in up-regulation of Sp1 activity, which induces NF-κ B activation. e activated NF-κ B triggers
the expression of CMV genes, leading to productive CMV infection
28
. HSV-1 also exploits NF-κ B signaling for
their ecient replication. HSV-1 infection-induced activation of NF-κ B signicantly promotes the HSV-1 rep-
lication via up-regulation of expression of the viral late proteins VP16 and gC
29,30
. On the other hand, there has
been no study reporting the relationship between NF-κ B and Ad replication, although Deryckere et al. reported
that NF-κ B up-regulated E3 gene expression. Rather, previous reports demonstrated that the adenoviral E1A
31
and E3/19K gene products
32
activated NF-κ B in Ad infection. Our present study showed that NF-κ B activation
enhanced Ad gene expression and Ad replication. ese ndings suggest that, like other viruses, Ad utilizes
NF-κ B, which is activated by Ad-mediated innate immunity, for its ecient replication.
Gribaudo et al. has previously demonstrated that IFN-α treatment inhibited enhancer activities of murine
CMV early genes via down-regulation of NF-κ B activities
33
. On the other hand, as shown in Figure S3, IFN-α
treatment signicantly inhibited NF-κ B signaling, Ad gene expression, and Ad replication. ese results suggest
that similarly to murine CMV, Ad infection would be also inhibited by IFN-α signaling via down-regulation of
NF-κ B activities. Further studies will be needed to clarify the mechanism underlying IFN-α -mediated suppres-
sion of Ad gene expression.
This study provided evidence that NF-κ B is largely involved in not only the gene expression of WT-Ad
but also leaky expression of the Ad genes from Ad vectors. Leaky expression of Ad genes is responsible for
Ad vector-mediated tissue damages, including hepatotoxicities. Several groups previously reported that Ad
vector-mediated hepatotoxicities were signicantly reduced by treatment with immunosuppressive agents such
as cyclosporine
34
and FK506
35
, permitting long-term expression of the transgene. In these studies, the diminished
Figure 8. Suppression of Ad vector-mediated hepatotoxicities by expression of a dominant-negative
IκBα gene. C57BL/6 mice were intravenously administered 10
9
IFU of Adv-CALuc or Adv-CADNIκ Bα . At
the indicated time points, the serum ALT and AST levels were determined. ese data are expressed as the
means ± S.E. (n = 5–6). (B,C) Rag2/IL2rgc double knockout mice were intravenously administered 10
9
IFU of
Adv-CALuc or Adv-CADNIκ Bα . (B) Forty-eight hours aer the administration, the E2 and E4 mRNA levels
in the mouse liver were determined by quantitative RT-PCR. ese data are expressed as the means ± S.E.
(n = 4). (C) At the indicated time points, the serum ALT and AST levels were determined. ese data (B,C)
are expressed as the means ± S.E. (n = 6–8). *p < 0.05, **p < 0.01, ***p < 0.001 (Adv-CALuc versus Adv-
CADNIκ Bα ).
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hepatotoxicities by the immunosuppressive agents were considered to be mainly attributable to the suppression
of Ad vector-induced immune responses; however, immunosuppressive agents also inhibit NF-κ B activation,
leading to the suppression of leaky expression of Ad genes and the subsequent hepatotoxicities. is study showed
that the inhibition of NF-κ B via overexpression of DNIκ Bα resulted in a reduction of the Ad vector-mediated
hepatotoxicities (Fig.8).
As shown in Fig.5A, stimulation by recombinant TNF-α did not activate the E1A, E1B, and E4 promoters,
although the E2 and E3 promoter activities were signicantly enhanced by TNF-α treatment. e E2 and E3
promoter regions possess the NF-κ B binding site (Figs1B and 5B). On the other hand, in silico analysis predicted
no NF-κ B binding site in the E1A, E1B, and E4 promoter regions, suggesting that NF-κ B indirectly induced
the enhancement of the E1A, E1B, and E4 gene expressions. Lusky et al. reported that the DNA binding protein
encoded by the E2A gene is involved in the leaky expression of other Ad genes in Ad vector-transduced cells
4
. It
would be interesting to examine whether NF-κ B-induced E2 gene products are involved in other Ad gene expres-
sion. Alternatively, cellular factors which are activated by NF-κ B signaling would be involved in up-regulation
of Ad gene expression. Further studies will be needed to clarify the mechanism underlying NF-κ B-mediated
enhancement of Ad gene expression.
Although we demonstrated that Adv-CADNIκ Bα achieved a reduction in the expression levels of Ad genes,
and thereby induced no evident hepatotoxicity (Fig.8), previous studies have reported that an Ad vector-mediated
expression of Iκ Bα eciently inhibited NF-κ B activities, resulting in signicant apoptosis of hepatocytes fol-
lowed by severe hepatotoxicities
26,36
. Because NF-κ B functions as an inhibitor of apoptosis
37
, ecient inhibition
of NF-κ B by overexpression of DNIκ Bα induced the apoptosis of hepatocytes in these previous reports. In the
present study, 10
9
IFU of Adv-CADNIκ Bα was intravenously administered to mice, while the previous studies
evaluated the hepatotoxicities following administration with Ad vectors expressing Iκ Bα at doses of more than
4 × 10
9
IFU
26,36
. ese dierences in the injection doses were likely responsible for the dierences in hepatotox-
icity levels.
In order to suppress or eliminate the leaky expression of Ad genes, various types of replication-incompetent Ad
vectors have been developed
3,4,8
. Following transduction with E2 and/or E4-deleted Ad vectors, the leaky expres-
sion of other Ad proteins was signicantly suppressed, leading to decreases of cytotoxic T lymphocyte (CTL)
responses and hepatotoxicities
3,4,8
. In addition, an HD-Ad vector, in which almost all viral coding sequences were
deleted, has been developed
11–13
. is HD-Ad vector achieved a reduction in inammatory cytokine production
and tissue damages, leading to persistent transgene expression in vivo. However, in vivo application of HD-Ad
vectors has been limited due to the low titers of HD-AD vectors and complicated production method, which
requires special packaging cells and a helper virus for the propagation of HD-Ad vectors. As shown in Fig.8
Adv-CADNIκ Bα , which was propagated to a high titer in a conventional method, mediated the reduced leaky
expression of Ad genes and hepatotoxicities. In addition, Ad vector-induced inammatory immune responses are
suppressed by the overexpression of DNIκ Bα . An Ad vector carrying an expression cassette of DNIκ Bα would be
a promising framework for safer and more ecient Ad vectors, although we should pay attention to the injection
dose, as described above.
In summary, we have demonstrated that NF-κ B induces the leaky expression of Ad genes, and that inhibition
of NF-κ B leads to suppression of Ad vector-mediated hepatotoxicities by not only Ad vector-induced immune
responses but also the Ad proteins themselves. is study provides important clues toward the development of
promising Ad vectors.
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Acknowledgements
e authors declare no conict of interest. We thank Sayuri Okamoto and Eri Hosoyamada (Graduate School of
Pharmaceutical Sciences, Osaka University, Osaka, Japan) for their help. is work was supported by grants-in-
aid for Scientic Research (A) and (B) from the Ministry of Education, Culture, Sports, Science, and Technology
(MEXT) of Japan, and by a grant-in-aid from the Ministry of Health, Labor, and Welfare (MHLW) of Japan and
Japan Agency for Medical Research and Development (AMED).
Author Contributions
M.M. designed and performed the experiments, analyzed data, and wrote the manuscript; F.S. designed and
supervised the projects, analyzed data, and wrote the manuscript; K.W., K.N. and K.S. supported the experiments;
M.T. analyzed data. H.M. supervised the projects, interpreted data, and wrote the manuscript.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Machitani, M. et al. NF-κB promotes leaky expression of adenovirus genes in a
replication-incompetent adenovirus vector. Sci. Rep. 6, 19922; doi: 10.1038/srep19922 (2016).
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