RelAp43, a Member of the NF-κB Family Involved in Innate Immune Response against Lyssavirus Infection
NF-κB transcription factors are crucial for many cellular processes. NF-κB is activated by viral infections to induce expression of antiviral cytokines. Here, we identified a novel member of the human NF-κB family, denoted RelAp43, the nucleotide sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and tissues tested and exhibits all the properties of a NF-κB protein. Although its sequence does not include a transactivation domain, identifying it as a class I member of the NF-κB family, it is able to potentiate RelA-mediated transactivation and stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-β - three genes involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies, resulting in an inhibition of the NF-κB pathway. Taken together, our data provide the description of a novel functional member of the NF-κB family, which plays a key role in the induction of anti-viral innate immune response.
RelAp43, a Member of the NF-kB Family Involved in
Innate Immune Response against Lyssavirus Infection
, Olivier Delmas
*, Pierre-Olivier Vidalain
, Robert Weil
1Institut Pasteur, Unite
´Dynamique des Lyssavirus et Adaptation a
ˆte, Paris, France, 2Universite
´Paris Diderot, Sorbonne Paris Cite
´, Cellule Pasteur, Paris, France,
3Institut Pasteur, Unite
´nomique virale et vaccination, Paris, France, 4Institut Pasteur, Unite
´de Signalisation mole
´culaire et Activation cellulaire, Paris, France
NF-kB transcription factors are crucial for many cellular processes. NF-kB is activated by viral infections to induce expression
of antiviral cytokines. Here, we identified a novel member of the human NF-kB family, denoted RelAp43, the nucleotide
sequence of which contains several exons as well as an intron of the RelA gene. RelAp43 is expressed in all cell lines and
tissues tested and exhibits all the properties of a NF-kB protein. Although its sequence does not include a transactivation
domain, identifying it as a class I member of the NF-kB family, it is able to potentiate RelA-mediated transactivation and
stabilize dimers comprising p50. Furthermore, RelAp43 stimulates the expression of HIAP1, IRF1, and IFN-b- three genes
involved in cell immunity against viral infection. It is also targeted by the matrix protein of lyssaviruses, the agents of rabies,
resulting in an inhibition of the NF-kB pathway. Taken together, our data provide the description of a novel functional
member of the NF-kB family, which plays a key role in the induction of anti-viral innate immune response.
Citation: Luco S, Delmas O, Vidalain P-O, Tangy F, Weil R, et al. (2012) RelAp43, a Member of the NF-kB Family Involved in Innate Immune Response against
Lyssavirus Infection. PLoS Pathog 8(12): e1003060. doi:10.1371/journal.ppat.1003060
Editor: Matthias Johannes Schnell, Thomas Jefferson University, United States of America
Received April 3, 2012; Accepted October 14, 2012; Published December 13, 2012
Copyright: ß2012 Luco et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: S. L. was supported by an Allocation Specifique Normalien (ASN) from French Ministry of Higher Education and Research. O. D. was supported by a
grant Pasteur-Roux from the Institut Pasteur. This work was supported by European Union Project PREDEMICS (FP7-HEALTH-2011-278433). The funders had no
role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
.These authors have equally contributed to the work.
NF-kB proteins comprise a family of structurally-related
eukaryotic transcription factors involved in the control of many
physiological cellular processes . This family contains five
major Rel proteins in mammalian cells: p65/RelA, c-Rel, RelB,
p50 and p52. All Rel proteins share the N-terminal homology
domain (RHD) mediating homo- or hetero-dimerization, DNA
binding, nuclear localization and interaction with the IkB
proteins, the inhibitors of NF-kB. Only RelA, c-Rel and RelB
have a transactivation domain (TAD) in their C-terminal
region. In the vast majority of cell types, NF-kB is kept inactive
in the cytoplasm through association with an inhibitory protein
of the IkB family, which includes IkBa,IkBband IkBe,aswell
as p105 and p100, the cytoplasmic precursors of p50 and p52.
Most of the signals that lead to activation of NF-kB, such as
cytokines, various stress signals, and viral and bacterial
infections, activate a high molecular weight complex containing
a serine-specific IkBkinase(IKK).IKKislargelycomposedof
three distinct subunits: the two related catalytic kinases - IKKa
and IKKb, and NEMO. Activated IKK then phosphorylates
IkB on specific residues. Phosphorylated IkB are polyubiqui-
tinated, then degraded by the proteasome machinery. As a
consequence, free NF-kB dimers enter the nucleus and activate
transcription of their target genes by binding specific DNA
sequences named kB sites in the promoter region of numerous
The list of target genes controlled by NF-kB is extensive and
many are involved in key cellular processes such as cell survival,
proliferation and immunity. The duration, strength and specificity
of induction of these genes are tightly regulated . Accumulating
evidence suggests that alternative splicing events of NF-kB
signaling components could be involved in controlling NF-kB
signaling . A variety of post-translational modifications of NF-
kB constitute a second level of regulation . Furthermore, non-
Rel proteins that interact with NF-kB transcription factors within
the nucleus constitute a third level of modulation . These
mechanisms of regulation of NF-kB activity notably impact the
innate immune response. One of the major antiviral effectors
induced by NF-kB are type I interferons, like interferon-b(IFN-b).
RelA has been proposed to be crucial for early IFN-bexpression
that could prevent the replication of some RNA viruses . RelA
is also important for the maintenance of basal IFN-bexpression in
non-infected cells  that is important to prime a strong response
in case of viral infection . Viruses have evolved many strategies
to manipulate the NF-kB pathway to their own benefit, especially
to counteract the induction, signalling, or antiviral actions of the
IFN circuit and to modulate cell death and apoptosis [9,10].
Among RNA viruses, pathogenic strains of lyssavirus, the agent of
rabies, have been shown to evade host innate immune response
while non-pathogenic strains do not, indicating that response to
infection is strain dependent .
Here, we describe a new splicing variant of RelA, which we
named RelAp43, exhibiting all the properties of a NF-kB protein.
PLOS Pathogens | www.plospathogens.org 1 December 2012 | Volume 8 | Issue 12 | e1003060
Interestingly, RelAp43 is targeted by the matrix (M) protein of
field but not vaccine strains of lyssaviruses, therefore blocking
essential steps during activation of the innate immune response.
The lyssavirus M protein is a small protein (,20–25 kDa), forming
oligomers that bind to the outside of the nucleocapsid, giving
rigidity to the virion structure and providing a binding platform for
viral glycoprotein trimers and the envelope membrane [12,13]. It
is also involved in a number of essential steps during infection and
appears to issue multiple seemingly conflicting signals during the
replication cycle of the virus . Taken together, our data
describe a novel functional member of the NF-kB family, which
plays a key role in the induction of anti-viral innate immune
RelAp43, a variant of RelA, is expressed in human cells
Using the lyssavirus M protein as bait in a yeast two-hybrid
screen against a human spleen cDNA library (Invitrogen), we
found that it interacts with a protein initially assigned as RelA.
After full sequencing of the cDNA of this M protein cellular
partner, it appeared that it was a variant of RelA likely resulting
from an alternative splicing event. Indeed, the 59-end sequence of
the cDNA of this variant aligned perfectly with the sequence of
RelA mRNA (GeneBank refseq accession: NM_021975), while its
39-end sequence corresponded to a part of the intron between
exon 9 and 10 in the gene of RelA (Fig. 1A). The corresponding
transcript encodes a protein of 378 amino acids with a calculated
molecular weight of 43 kD which was accordingly named
RelAp43. It contains the full RHD present in RelA but lacks its
TAD, which is replaced by the short specific sequence of 33 amino
acids (from amino acid 345 to 378) (Fig. 1A).
To check for the transcription of RelAp43 gene, its specific
sequence corresponding to amino acids 345 to 378 was blasted to
search for human mRNA or expressed sequence tags (EST). We
found one mRNA (accession number: BC116830) isolated from a
human pancreas epithelioid carcinoma that codes for RelAp43,
and three human EST isolated from lung (DA58560), brain
(AW054862) and prostate (DA873834) which have a sequence
similar to the specific part of RelAp43, indicative of a potential
expression of RelAp43 in various human organs. To confirm this
hypothesis, we set up a real-time RT-PCR assay that amplified the
respective specific parts of either RelAp43 or RelA mRNA. The
quantification of the number of mRNA copies of RelA and
RelAp43 was performed in several cell lines of human origin:
HeLa cells, a cervical carcinoma cell line; 293T cells derived from
embryonic kidney; SK-N-SH and IMR5, neuroblastoma cell lines.
Even in low amount, RelAp43 gene transcription was detected in
all the cell lines tested from about 1,100 copies in 293T cells to
15,000 in IMR5 cells per 100 ng of total RNA (Fig. 1B). As a
control, we checked that no detection was noticed in the absence
of reverse transcription (data not shown). The RelA/RelAp43
mRNA ratio is highly contingent upon the investigated cell type
(from 2 in IMR5 cells, 10 in HeLa cells to 22 in 293T cells) further
suggesting that the regulation of RelAp43 transcription is
independent of that of RelA. As NFkB signaling is involved in
many cellular pathways such as the balance between cell death
and survival, inflammation, or the control in cell proliferation
[15,16], the expression level of RelAp43 could then be modified in
immortalized cells compared to primary tissues. Therefore, we
extracted total RNA from several normal human tissues and
subjected them to quantification. We collected RNA samples from
spinal cord, cerebral trunk, frontal cortex, cerebellum, peripheral
blood mononucleated cells (PBMC) and skin biopsy. For each
sample, we tested 100 ng of total RNA. As already shown,
variations in the number of RelAp43 RNA copies (3,000 in skin
biopsy to 32,000 in PBMC) (Fig. 1C) as well as in the RelA/
RelAp43 mRNA ratio (2 in skin, 3 in PBMC, 9 in frontal cortex)
were observed. To assess the expression of RelAp43, we
performed a western blot analysis on 50 mg of total proteins from
direct HeLa cell lysates after transfection with specific and control
siRNAs, or FLAG-tagged RelAp43. We observed a 43 kD band,
which intensity was reduced when specific siRNA was transfected
(Fig. 1D, and Fig. S1). This specific siRNA did not affect the
transcription of RelA mRNA (Fig. S1). The expression of RelAp43
was also observed in total cell lysates from HeLa and SK-N-SH
after immunoprecipitation (IP) using an antibody targeting
RelAp43 N-terminal part and western blot analysis using a
specific antibody raised against the C-terminal extremity of
RelAp43 (Fig. 1E).
Together, this demonstrates the transcription and the expres-
sion of a novel variant of RelA named RelAp43 in every cell line
and tissue tested, such that it could be ubiquitous.
RelAp43 can interact with members of the NF-kB family
To determine whether RelAp43 retains the ability to interact
with the other proteins of the NF-kB family, FLAG-tagged
RelAp43 was immunoprecipitated from transfected cells (HEK-
293T and HeLa) and co-immunoprecipitation (co-IP) of endog-
enous members of the NF-kB pathway was analyzed by Western
Blot (Fig. 2A). As expected, RelAp43 interacts with p105/p50,
p100/p52, RelA, RelB and c-Rel. In addition, RelAp43 was found
to interact with the NF-kB inhibitor IkBa. None of these partners
were immunoprecipitated from non-transfected cells nor from
FLAG-tagged CAT expressing cells, confirming the specificity of
the co-IP (Fig. 2A). The same results were obtained with FLAG-
tagged RelA, which indicates that RelAp43 has the same capacity
of interaction with the other NF-kB family members as RelA
(Fig. 2A). It is interesting to mention that the interaction of
RelAp43 with RelA and IkBawas also demonstrated by yeast two-
hybrid (data not shown).
RelAp43 is retained in the cytoplasm by IkBa
To test whether RelAp43 could similarly to RelA enter the
nucleus and whether its translocation could be inhibited by IkBa,
we performed immunofluorescence experiments in transfected
The homeostasis of living cells is tightly regulated by
signaling pathways, most of them being pleiotropic, which
makes their understanding crucial in biology. One of them,
the NF-kB pathway, includes a family of transcription
factors involved in cell survival, proliferation, differentia-
tion, and cell immunity. In this study, we identified a novel
human member of the NF-kB family that we named
RelAp43. It shares all the main characteristics of the already
known NF-kB family members. Moreover, we demonstrat-
ed that RelAp43 induced specifically the expression of
genes involved in the innate immune response against
viruses. Interestingly, we showed that RelAp43 is specifi-
cally targeted by the matrix protein of rabies virus,
which contributes to the pathogenesis of the virus and
its escape from innate immune response. Taken together,
our data provide the description of a novel functional
member of the NF-kB family, which is involved in the
induction of innate immune response against virus
RelAp43: Role in Anti-Viral Innate Immunity
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Figure 1. RelAp43 is a variant of RelA expressed in cell lines and tissues. (A) Schematic representations of RelA, RelAp43 and of the exons
and introns present in their corresponding mRNA. Exons (EX on the Figure) are represented by boxes numbered with arabic numbers, and introns are
indicated by red lines with roman numbers. While RelA protein is encoded by a mRNA composed of all 11 exons, a part of the intron IX (figured as a
red box in the picture), followed by a stop codon, is found in the mRNA transcript encoding RelAp43. Rel Homology domains (RHD) of RelA and
RelAp43: Role in Anti-Viral Innate Immunity
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HeLa cells expressing V5-tagged RelAp43 alone or in association
with 3xFLAG-tagged IkBa(Fig. 2B). Apotome analysis of the
transfected cells indicates that in absence of IkBa, RelAp43
exhibits extensive colocalization with the DAPI staining of the
nucleus, whereas in presence of IkBa, it is mostly cytoplasmic and
co-localizes with IkBa(Fig. 2B). This was confirmed by nuclear
cytosol fractionation experiments and analysis by Western Blot,
which showed that RelAp43 was no more detectable in the nuclear
RelAp43 are depicted here, such as the transactivation domain (TAD) of RelA. (B) Absolute quantification of RelA- (black bars) and RelAp43- (grey bars)
encoding mRNA in the indicated cell line. (C) Absolute quantification of RelA (black bars) and RelAp43 (grey bars) transcription in human primary
tissues. SC: spinal cord; CT: cerebral trunk; FC: frontal cortex; Ce: cerebellum; MC: peripheral blood mononucleated cells; SB: skin biopsy. Results are
the mean mRNA level obtained after 3 independent experiments. (D) Expression of RelAp43 in HeLa cells transfected either with a control siRNA, a
specific siRNA directed against RelAp43 (RelAp43 siRNA) or overexpressing FLAG-tagged RelAp43 (RelAp43 F). Western blot analysis was performed
with an antibody targeting RelAp43 C-terminal part on 50 mg of total cell lysates of each condition. See also Fig. S2. (E) Immunoprecipitation of
RelAp43 in HeLa and SK-N-SH (SK) cells transfected either with a control siRNA, a specific siRNA directed against RelAp43 (RelAp43 siRNA) or
overexpressing FLAG-tagged RelAp43 (RelAp43 F) or non-transfected. Immunoprecipitations were performed with a pre-immune serum or with
antibody directed against RelAp43 N-terminal part then analyzed by western blot revealed either with an antibody targeting RelAp43 C-terminal part
or with an anti 3xFLAG antibody. Only 30% of the sample corresponding to HeLa cells overexpressing RelAp43F was loaded on the gel in comparison
with the other samples that were loaded in totality after immunoprecipitation.
Figure 2. RelAp43 interacts with all human members of the NF-kB family and is supported by IkBa.All experiments were performed
three times independently. (A) IP using anti-FLAG antibody of FLAG-tagged RelAp43 (RelAp43 F) or RelA (RelA F) or CAT (CAT F) expressing cells or
non-transfected cells (NT). The presence of FLAG-tagged protein or endogenous transcription factors of the NF-kB family was analyzed by western
blot using specific antibodies in cell lysates either before (left panel) or after IP (right panel). (B) Cellular localization of transfected RelAp43 (green)
analyzed by immunofluorescence and apotome imaging in absence (left panel), or in presence of transfected IkBa(red, right panel). Nuclei were
visualized using DAPI staining. The scale bar corresponds to 2 mm. (C) Western Blot analysis of nuclear (N) and cytosolic fractions (C) of RelAp43 V5
and IkBaF co-transfected cells after nuclear-cytosolic fractionation. Nuclear and cytosolic fractions were controlled using anti histone H3 and anti
actin bantibodies, respectively. See also Figure S2.
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fraction of cells co-transfected with IkBa(Fig. 2C). As a control,
we checked that transfection of RelAp43 did not induce the
production of IkBa(Fig. S2). Overexpression of RelAp43 could
then overcome its retention by endogenous IkBaand be targeted
in the nucleus. Thus, when IkBawas co-transfected with
RelAp43, this induced the cytoplasmic retention of RelAp43.
This suggests that RelAp43 can shuttle in different cell compart-
ments notably via its interaction with IkBa(Fig. 2C).
RelAp43 is a class I member of the NF-kB family
To assess that RelAp43 could associate with DNA and interfere
with DNA-bound NF-kB complexes, we performed electrophore-
sis mobility assays (EMSA) experiments and supershift experiments
using an anti-FLAG antibody on nuclear fractions of unstimulated
or TNF-a-induced HeLa cells expressing either FLAG-tagged
CAT or FLAG-tagged RelAp43. While we could not detect
nuclear NF-kB DNA-binding activity in unstimulated control
HeLa cells (Fig. 3A, lane 1), this was strongly induced after the
addition of TNF-afor 15 min (Fig. 3A, band A, lane 2). When the
cytokine was removed, NF-kB activity started decreasing following
a chase period of 2 hours (Fig. 3A lane 5). Interestingly, a higher
basal NF-kB activation was observed following transient expres-
sion of RelAp43 (Fig. 3A lane 8) compared to CAT (Fig. 3A lane
1). NF-kB activation was potentiated by treatment with TNF-a
(Fig. 3A lane 9). During the chase period, RelAp43 sustained NF-
kB DNA-binding activity (Fig. 3A lanes 10–14) to a higher level
and for a longer period than CAT (Fig. 3A lanes 3–7). This result
probably indicates that RelAp43 sustained the interaction between
endogenous NF-kB in HeLa cells and the kB probe. Incubation of
nuclear fractions of RelAp43-transfected cells (Fig. 3B), with anti-
RelA antibody which recognizes the unique C-terminus part of
RelA, supershifted the complex to a higher molecular weight
(Fig. 3B lane 5) similar to that observed in CAT- expressing cells
(Fig. 3B lane 2), whereas the migration of this complex was not
modified by pre-immune serum (Fig. 3B lanes 1 and 4). Moreover,
an additional band that migrates faster appeared in FLAG-tagged
RelAp43 compared to FLAG-tagged CAT expressing cells (Fig. 3A
band B, lanes 8–14), suggesting some interaction between FLAG-
tagged RelAp43 and the kB probe. In agreement with this
hypothesis, this band was supershifted when the nuclear extract of
RelAp43-transfected cells was incubated with anti-FLAG antibody
(Fig. 3B lane 6) but not following incubation with anti RelA
antibody (Fig. 3B lane 5). Although we could not conclude about
the partners of RelAp43 from the supershift experiments, they did
clearly indicate that a complex containing RelAp43 was associated
with kB probe. This was not a tag artifact since FLAG-tagged
CAT was not able to associate with the same probe than FLAG-
To test whether RelAp43 presents a transactivation activity
while its sequence differs from that of RelA on its C-terminal
extremity, we cloned it in fusion with the DNA binding domain of
Gal4 (DB), a yeast modular transcription factor, and measured the
expression of firefly luciferase gene under control of a promoter
sequence containing binding sites for Gal4-DB. The same
experiment was performed with RelA as a positive control. As
expected, DB-RelA induced a strong expression of the luciferase
gene under control of Gal4 promoter. In contrast, DB-RelAp43
did not induce expression of luciferase gene under control of Gal4
promoter since the luminescence measured was not significantly
different from that of untransfected cells (Fig. 3C). Thus and as
expected considering its sequence, RelAp43, like p50 or p52,
contains a full RHD but lacks TAD even in its C-terminal specific
extremity of 33 amino acids. Since RelAp43 interacts with RelA
(as shown by co-IP, see Fig. 2A), we co-transfected DB-RelA with
RelAp43 or CAT as a control to test the effect of RelAp43 on DB-
RelA dependant induction of luciferase gene and checked the level
of expression of RelAp43 and CAT (Fig. 3D). Increasing doses of
RelAp43 co-expressed with constant amounts of DB-RelA, clearly
indicated a stimulating role of RelAp43 (Fig. S3A and S3B). We
then used 160 ng of CAT- and RelAp43-encoding plasmids for
transfection as this dose exhibited the best trade-off between the
intensity and the variability of the luminescence signal (Fig. S3A).
In that case, the co-expression of CAT had no effect on DB-RelA-
dependant luciferase expression, whereas the co-expression of
RelAp43 increased the luciferase expression more than 3 fold
(Fig. 3D). Overall, these results indicate that RelAp43 is a class I
member of the NF-kB family which can potentiate the
transactivation potential of RelA.
RelAp43 modifies the equilibrium of p50-comprising
dimers and potentiates the transcription of several NF-kB
Dimerization is required for NF-kB binding to DNA and 15
homo- and heterodimers have so far been described. We
hypothetized that RelAp43, as a class I NF-kB protein, could
modify the equilibrium between the different combinations of NF-
kB dimers. As active NF-kB complexes are mainly composed of
RelA-p50 dimers, we analyzed the stability of these dimers in
response to cycloheximide, a protein synthesis inhibitor, in
RelAp43- or CAT-transfected cells (used as control cells). RelA
decay was identical in CAT- and RelAp43-transfected cells. Most
interestingly, the expression of RelAp43 (Fig. 4A) but not that of
CAT (used as a control) (Fig. 4B) prevented p50 degradation. We
then investigated the propensity of RelAp43 to modulate the
formation of p50-RelA dimers by co-IP experiments using an anti-
p50 monoclonal antibody. In cells transfected with p50 and RelA,
RelAp43 but not CAT overexpression affected the formation of
RelA-p50 dimers (Fig. 4C). To confirm if this modulation exerted
by RelAp43 in transfected cells could also be seen in more
physiological conditions, we performed the same type of exper-
iment in cells partially depleted for RelAp43 by specific siRNA
(52%; see Fig. S1 and Fig. 4D). Following this depletion, we
observed that the amount of endogenous RelA involved in dimer
formation with p50 was higher than in normal conditions (Fig. 4D).
Therefore, the selective stabilization of different members of the
NF-kB family mediated by RelAp43 could influence their relative
amounts in the nucleus and their interaction with DNA. To
explore the impact of RelAp43 on genes expression, we focused on
eight genes belonging to the apoptosis pathway and innate
immune response, in which NF-kB signaling is known to be
involved. To this aim, we designed RT-PCR sets of primers to
quantify the transcription of Bcl2,XIAP,MCP1,cFLIP,HIAP1,
HIAP2,IRF1 and IFN-bgenes in HeLa cells overexpressing
RelAp43 or RelA for 48 h, compared to HeLa cells overexpressing
CAT for 48 h. For five of these genes, transcription levels were not
significantly different when RelAp43 was overexpressed compared
to those observed with CAT (Fig. 4E). In contrast, the
transcription of HIAP1,IRF1 and IFN-bgenes was significantly
enhanced in the presence of RelAp43 (Fig. 4E, left panel). As
expected, transfection of RelA, which activates by itself NF-kB
pathway, significantly induced the transcription of all eight genes
since they are all known to respond to NF-kB (Fig. 4E, right
panel). HIAP1, IRF1 and IFN-binduction mediated by RelAp43
represented respectively 68.9%, 51.7% and 3.05% of the
induction mediated by RelA, further suggesting that RelAp43
induces a gene modulation, probably through its association with
class II NF-kB family members. Furthermore, the observed
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Figure 3. RelAp43 binds on DNA kB sites and potentiates RelA-mediated transactivation of gene expression. All experiments were
performed three times independently. Error bars indicate standard deviations. *p,0.05. (A) EMSA analysis of nuclear extracts from FLAG tagged-CAT
and FLAG tagged-RelAp43 transfected HeLa cells, using a radioactive kB probe. Cells were either left untreated (2, lanes 1 and 8), treated for 15 min
with 10 ng/mL TNF-a(+, lanes 2 and 9) or treated for 15 min with 10 ng/mL TNF-afollowed by chase periods of 30 min to 6 hours (lanes 3–7 and 10–
14). Band A: RelA bound to kB-probe; band B: RelAp43 bound to kB-probe, as demonstrated in Figure 3B. (B) Supershift analysis of the complexes
bound to the kB-probe. 100 ng of a-RelA antibody, a-FLAG antibody or pre-immune serum was incubated with the EMSA reaction mixture before gel
electrophoresis. (C) Measurement of the luminescence of cells expressing luciferase under control of the Gal4 promoter and RelA or RelAp43 fused to
the Gal4 DNA Binding domain (named as DB-RelA or DB-RelAp43 on the figure, respectively) or non transfected (NT). DB-RelA was arbitrary set to 1.
(D) Luciferase assay of RelA transactivation properties in presence of CAT or RelAp43. RelAp43- or CAT-encoding plasmids were added to the
transfection mix in the same conditions as in (C) (see also Figure S3). Their expression level was controlled by the western blot analysis showed on the
right of the Figure. The level of luminescence obtained in the presence of DB-RelA and CAT encoding plasmids was arbitrary set to 1.
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Figure 4. RelAp43 modifies the equilibrium of p50-comprising dimers and specifically potentiates the transcription of several NF-
kB dependent genes. Analysis of the stability of RelA and p50 using cycloheximide blockage of protein translation in HeLa cells overexpressing
RelAp43 (A) or CAT (B). Quantification of the RelA and p50 expression, corrected by the actin controls, are given under each RelA and p50 bands.
Expression levels in cells not treated with cycloheximide (time 0) were used as controls and set up to 100. (C) Modulation of transfected RelA-p50
dimer formation according to the amount of RelAp43. Dimer formation involving transfected RelA and p50 was analyzed by co-IP using anti-p50
antibody on protein extracts of HeLa cells cotransfected by FLAG-tagged RelA, V5-tagged p50 and FLAG-tagged RelAp43 or CAT as a control. The
amount of FLAG-tagged RelA interacting with V5-tagged p50 was assessed using anti-FLAG antibodies. The quantification levels presented at the
bottom of the lanes are the means and standard deviations (SD) of the intensity of the band corresponding to RelA obtained by substracting the
intensity of the band observed with the pre-immune serum to the one obtained after p50 immunoprecipitation. These experiments (A, B and C) were
repeated 3 times independently and results presented are representative of the three repetitions. (D) Modulation of endogenous RelA-p50 dimer
formation according to the amount of endogenous RelAp43. Quantification of RelA obtained after co-IP using anti-p50 antibody (IP: p50) was
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pattern is different of that mediated by RelA in terms of target
genes and intensity of their transcription.
Collectively these data indicate that RelAp43 is preferentially
involved in dimer formation with p50 thereby limiting the number
of RelA-p50 dimers. Furthermore, RelAp43 increases the expres-
sion of NF-kB target genes like HIAP1,IRF1 and IFN-bin a
specific manner compared to RelA.
Lyssavirus M proteins from field isolates interact with
RelAp43 and inhibit NF-kB signaling
Next we investigated the role of RelAp43 during viral infection.
We first determine the interaction of various M proteins from
different lyssaviruses with RelAp43. In the yeast two-hybrid
system, we found that M proteins of field isolates of four species of
lyssavirus (M-Tha, M-Mok, M-Lag, M-EBLV-1) could interact
with RelAp43, whereas M proteins from laboratory strains or
vaccine strains (M-PV and M-SAD) did not (Fig. 5A). To validate
these results, we performed co-IP experiments (Fig. 5B). In
agreement with two-hybrid screen, we observed that IP of FLAG-
tagged M-Tha but not of M-SAD led to the recovery of V5-tagged
RelAp43. The specificity of the interaction of M-Tha with
RelAp43 was further confirmed since neither FLAG-tagged
CAT expressing cells nor control cells that did not express any
FLAG-tagged protein (NT) allowed the precipitation of RelAp43
with a FLAG antibody. Moreover, this interaction was specific to
RelAp43 since neither RelA nor the common part of RelA and
RelAp43 encompassing the RHD (RHDb in Fig. 5B) could
interact with M-Tha in the same conditions. This result suggests
that the unique region of RelAp43 is necessary for the interaction
with M-Tha. However, since we could not express efficiently the
unique C-terminal 33 amino acids of RelAp43, we could not
formally demonstrate that this region is involved in the interaction
with M-Tha. Since RelAp43 was targeted by lyssavirus M we
tested whether infection by lyssavirus could modulate RelAp43
transcription. The transcription of RelAp43 was increased from 2
to 3.5 fold at 48 h p.i. compared to cells infected for only 1 h (Fig.
To test whether M proteins from field isolates but not from
vaccine strains could modulate NF-kB pathway, we used a
luciferase reporter gene under control of kB sites. Interestingly,
cells expressing M-Tha, M-Mok, M-Lag and M-EBLV-1 which all
interact with RelAp43 showed a reduced NF-kB-dependent
luciferase activity in response to TNF-a(5 h) compared to M-
SAD or M-PV expressing cells which do not target RelAp43
(Fig. 5C). To further investigate the modulation of NF-kB pathway
later in infection, cells were transfected with increasing amounts of
59-triphosphate RNA and cultured during 24 hours. Indeed, 59-
triphosphate RNA is a virus-associated molecular pattern that
triggers antiviral responses, including NF-kB activation. In this
latter condition, a clear inhibitory effect of M-Tha compared to
M-SAD and CAT (used as a control) was observed (Fig. 5D).
To further understand the functional interplay between M
proteins and RelAp43, M-Tha and M-SAD were coexpressed with
RelAp43 and tested on the previously described RelA-mediated
transcription luciferase assay. Our results indicate that DB-RelA
alone (RelA fused to Gal4 DNA binding domain) induced
expression of luciferase under control of Gal4 promoter and that
CAT, M-SAD and M-Tha had no effect on this basal level
(Fig. 5E). Interestingly, when RelAp43 is added to DB-RelA,
which was previously shown to induce RelA-mediated transcrip-
tion of luciferase (see Fig. 3D), the level of luciferase recovered was
significantly decreased in the presence of M-Tha only (Fig. 5E).
This further confirms that RelA-mediated transcription is specif-
ically modulated by interaction between M-Tha and RelAp43 and
is probably involved in M-mediated inhibition of NF-kB pathway.
RelAp43 is involved in IFN-btranscription during
Since RelAp43 appears to be involved in IFN-bpathway
(Fig. 4E), we wanted to correlate the induction of IFN-bexpression
by different strains of lyssavirus to the ability of their M protein to
interact with RelAp43. To this aim, we first used an in vitro assay
system to measure luciferase activity under control of IFN-b
promoter in cells expressing M-Tha, M-SAD or CAT used as a
control (Fig. 6A). IFN-bpathway was stimulated in these cells
using different amounts of 59-triphosphate RNA molecules. A
significant difference in the level of luciferase activity following the
expression of M-Tha compared to M-SAD or CAT was observed.
The activation of IFN-bpromotor was strongly reduced in cells
expressing M-Tha, compared to other transfected cells, especially
at the highest dose of 59-triphosphate RNA (Fig. 6A). Thus, the
expression of M-Tha alone was sufficient to inhibit IFN-b
To better understand the importance of RelAp43 in the
induction of IFN-btranscription after viral infection, the
expression of RelAp43 was silenced using specific RNAi (Fig. 6B
and 6C). Knock-down of RelAp43 was similar (nearly 40 to 50%
of depletion) in infected cells at 48 h post infection (p.i.) and in
non-infected cells analyzed at the same time (Fig. 6C). After
infection at an MOI of 1, infectious supernatant virus production
of the vaccine strain SAD (highly adapted to cell culture) reached
fluorescent forming units (FFU)/ml
at day 1 and 2 p.i., respectively (Fig. 6B). As expected, the field
isolate Tha virus, showed a marked delay in virus production and
yielded slightly less than 10
FFU/ml at day 2 p.i. The
transfection of a specific anti RelAp43 siRNA or a control siRNA
did not modified the production of any of the two viruses (Fig. 6B).
The transcription of IFN-busing real-time quantitative PCR was
studied at 48 h p.i.. As expected, in infected cells transfected with
the control RNAi, the level of transcription of IFN-bwas increased
when compared with non-infected cells (Fig. 6D). When RelAp43
expression was turned down, IFN-bmRNA level after Tha- or
SAD- infection was half-reduced whereas it was not modified in
In Tha infected cells, IFN-bmRNA level even reached the same
level than in non-infected cells in the presence of anti-RelAp43
corrected by that observed using a pre-immune serum (IP: pre-immune). Expression levels in cells transfected with control siRNA were used as
controls and set up to 100. The experiments were repeated twice independently and the quantification levels presented at the bottom of the lanes
are the means and standard deviations (SD) obtained. (E) Relative level of transcription of a set of apoptosis genes in HeLa cells transfected with
RelAp43- or CAT-encoding plasmids (on the left) and with RelA- or CAT- encoding plasmids (on the right). The levels of RelAp43 and RelA mRNAs
were normalized to the level of GAPDH mRNA and reported relatively to the level measured in CAT-expressing cells used as control (set to 1, not
figured). Results presented here are the mean of three independent experiments. For one given repetition, all eight genes were studied on the same
cDNAs using specific set of primers. The percentage of induction of HIAP1, IRF1 and IFN-bmediated by RelAp43 and relatively to that mediated by
RelA is indicated on the corresponding bars (on the left). *p,0.05.
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RelAp43: Role in Anti-Viral Innate Immunity
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siRNA, thus further demonstrating a role for RelAp43 in IFN-b
transcription cascade during virus infection. The remaining
difference in the induction of IFN-bbetween Tha and SAD when
cells are transfected with anti-RelAp43 siRNA may be due to the
fact that the expression of RelAp43 was not completely knocked
down (Fig. 6C), and that the interaction between M-Tha and the
remaining RelAp43 further inhibited the residual RelAp43-
mediated IFN-bgene transcription compared to SAD. However,
other explanations may also be considered. In particular, it could
not be ruled out that the effect could be partly due to the
differences in virus titers. Further, other components than the M
protein in SAD virus may play a role in an RelAp43-independant
IFN-bgene transcription or that higher SAD replication allowed
higher IFN-bmRNA level.
Altogether these results demonstrate that IFN-bresponse is
modulated by the ability of M proteins to bind RelAp43 and that
RelAp43 is involved in the IFN-bresponse to lyssavirus infection.
The activity of NF-kB transcription factors is tightly regulated
by the cell dimer repertoire . Homeostasis of the cell is thus
clearly related to the association and dissociation of each of the 15
monomer pairs, and this process could be even more complex if
the NF-kB included additional transcription factors. In this
context, the characterization of alternative splicing events leading
to the translation of additional members of NF-kB family  is
paramount to understand the regulation of this signaling pathway.
The current study has identified a new RelA variant with a
molecular weight of 43 kD - RelAp43. It shares with RelA a full
and active RHD, but lacks the TAD that is replaced by a specific
domain encoded by an intron sequence located between exons 9
and 10 of the human relA gene . This sequence of 33 amino
acids does not show any transcription activation activity; thus
RelAp43 seems to be a new class I member of the Rel protein
family. Therefore, the structure of the RelAp43 gene differs from
that of 3 previously described splicing variants of RelA (p65D,
p65D2, and p65D3) which strictly result from deletion in RelA
sequence coding for the RHD [19,20,21,22]. The most studied of
these splice variants, p65D, looses its association with p50, presents
a weak association with p65, and a reduced ability to bind DNA.
The identification of RelAp43 further illustrates the potential role
of alternative splicing or truncated forms in the NF-kB signaling
pathways as already demonstrated in a mice model after injury
 and during Leishmania infection .
RelAp43 mRNA is present in all tissues and cell lines tested,
although its amount is about 10 times less than that of RelA. This
indicates that NF-kB dimers including RelAp43 should belong to
the common dimer repertoire of human cells. However, the
different mRNA ratios of RelA and RelAp43 observed in different
cell lines and tissues indicate some variations in these repertoires as
reported previously for RelA  and other RHD polypeptide
synthesis . How this new member of the Rel family interferes
with NF-kB dimer generation and with transient immunological
stimuli remains a crucial question. RelAp43 retains all activities
related to dimer formation and DNA binding through its RHD
domain. As suggested by the co-IP experiments, RelAp43 acts as a
binding partner for cRel, RelB, RelA, p50, p52, p100 and p105 as
previously demonstrated for p50 and p52, two other RHD
polypeptides that also lack TAD. Therefore, the formation and the
stability of the different NF-kB dimers including those with
RelAp43 may depend on the level of expression of RelAp43 and
on the capacity of the different types of dimers to associate and
dissociate . The action of RelAp43 on the dimer equilibrium
could result from several processes (Fig. 7). First and as for RelA,
RelAp43 function could be regulated by the IkBs as indicated by
the ability of RelAp43 to interact with IkBaand by the fact that
overexpression of IkBare-localized RelAp43 to the cytoplasm.
RelAp43 could not only modulate the formation of active NF-kB
dimers, but be also part of NF-kB dimers that bind to DNA
through kB sites and induce transcription [26,27] as indicated by
the EMSA experiments showing a stabilization of the RelA
complex by RelAp43 in response to TNF-astimulation. Further-
more, it remains possible that RelAp43 may also act in
conjunction with enhanceosome at distant sites for a complete
activation of certain NF-kB responsive genes. This has been shown
for the IFN-bgene for which a cluster of kB sites 39downstream to
the gene is also needed for a maximal gene expression after LPS
treatment . Finally, RelAp43 could form new transcription
factor(s) in association with other members of the Rel protein
family, as suggested by the co-IP experiments that show
interactions with the other members of the NF-kB family. Some
of the NF-kB dimers, and especially those including RelAp43 are
more stable than others as indicated by the EMSA experiments.
RelAp43 could therefore regulate NF-kB dimer formation by
potential competition in dimerization between the different pools
of NF-kB monomers. RelAp43, like RelA, binds efficiently to p50
and diminishes its processing while it does not affect the processing
of the other TAD-containing members of the NF-kB family as
shown by cycloheximide and co-IP experiments in cells transfected
with anti-RelAp43 siRNA. Although these results do not provide a
direct demonstration of the possibility of some exchange of NF-kB
dimers, they highly suggest a potential replacement of RelA by
Figure 5. Specific interaction of RelAp43 with the M protein of rabies virus and inhibition of NF-kB pathway. The results presented
here are representative of three independent experiments. Error bars indicate standard deviations. *p,0.05. (A) Determination of RelAp43 interaction
with various lyssavirus M proteins in the yeast two-hybrid system. Yeast cells expressing Gal4-DB fused to M proteins from tested lyssaviruses were
co-transformed to express Gal4-AD fused to RelAp43. Cells were plated on synthetic medium lacking histidine so that yeast growth is determined by
M interaction with RelAp43 and activation of HIS3 reporter gene. (B) Western blot analysis of co-IP involving M proteins and RelA or RelAp43. Co-IP
was performed using anti-FLAG antibody on cells co-transfected with on the one hand plasmid encoding RelA V5, RelAp43 V5 or the common part
between them (RHDb) and on the other hand plasmid encoding FLAG-tagged M-Tha, M-SAD, CAT or non-transfected cells (NT). IP of FLAG-tagged
proteins (not figured) and co-purification of V5-tagged protein were assessed (WB:V5 on the figure). Initial cell lysates were checked for V5- (cell lysate
blot V5) and FLAG-tagged (cell lysate blot 3xFLAG) proteins expression. (C) Modulation of NF-kB activation in the presence of M protein from
different lyssavirus strains after short-term stimulation. The NF-kB pathway was exogenously activated using 10 ng/mL TNF-aduring 5 h (grey bars)
or left untreated (black bars). The M protein of vaccinal strain SAD-B19 was arbitrary considered as a reference. The expression levels of M proteins in
each condition were assessed by western blot (bottom of the figure). (D) Modulation of NF-kB activation in the presence of M-Tha (black bars), M-SAD
(dark grey bars) or CAT (light grey bars) after extended stimulation. Increasing amounts of tri-phosphate RNA mimicking viral infection during 24 h
were used. The expression levels of M-Tha, M-SAD and CAT in each condition were assessed by western blot (bottom of the figure). (E) Luciferase
assay of RelA transactivation properties either without any other plasmid (light grey bars) or with a plasmid encoding RelAp43 (dark grey bars) plus a
plasmid coding for the indicated M protein (SAD, Tha) or CAT or no additional plasmid (Mock).
RelAp43: Role in Anti-Viral Innate Immunity
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RelAp43 in p50-containing dimers, resulting in a higher stability
of these complexes.
RelAp43-containing dimers may exhibit a specific transcrip-
tional activity as shown for classical NF-kB dimers [25,29]. In
contrast to p65D, p65D2, and p65D3, RelAp43 does not act as a
negative regulator of NF-kB signaling pathway but rather as a co-
activator of the pathway. This effect was here particularly
exemplified on three different genes involved in innate immune
response: HIAP1,IRF1 and IFN-b(Fig. 4E). HIAP1, which
possesses a RING domain, functions as an E3 ubiquitin ligase
[30,31] and promotes the degradation of caspase-3 and -7 by the
proteasome , thereby protecting neurons from cell death .
RelAp43 could then regulate the balance between cell death and
survival. Furthermore, we show that RelAp43 activates the
transcription of IRF1, a member of the interferon regulatory
transcription factor (IRF) family. Upon induction, IRF1 binds to
interferon signalling regulatory elements (ISRE) located in
promoters of target genes  and enhances transcription of
IFN-aand -band of several secondary response genes. IRF1 has
also been shown to play roles in regulating apoptosis [35,36] and
is, at least for some viruses, necessary for the antiviral action of
IFNs . Interestingly, RelAp43 also exerts a specific induction
of the transcription of IFN-bwhich is dependent on the NF-kB
pathway, therefore completing the potential actions of RelAp43 in
the innate immune response against viruses.
Viruses possessing negative-single-strand RNA genomes (Order
Mononegavirales) induce a strong innate immune response and in
particular NF-kB-mediated type I interferon response [9,38].
Among them, lyssavirus are recognized by RIG-I cellular sensor
 which signals through its interaction with the mitochondrial
IPS-1 protein (also referred to as MAVS, Cardif or VISA) .
Finally, AP1, IRF3, and NF-kB transcription factors are activated,
leading to IFN-bproduction [11,41,42] and inhibition of lyssavirus
infection both in vitro and in vivo [43,44]. Lyssaviruses have
developed several mechanisms to counteract innate immune
response [44,45]: the nucleoprotein has been shown to prevent
RIG-I recognition , the phosphoprotein P prevents IRF3
phosphorylation, dimerization and nuclear import, it also blocks
STATs thus inhibiting interferon expression [47,48] and the
cytoplasmic portion of the G protein trigger survival pathways
promoting neuronal survival .
The fact that the M protein inhibits NF-kB response probably
through direct interaction with RelAp43 further demonstrates the
complex role played by this structural protein in viral virulence. M
proteins of field lyssavirus isolates have conserved the ability to
interact with RelAp43, in contrast to laboratory adapted (PV) or
vaccine strains (SAD B-19) which may have lost this feature during
virus adaptation to cell culture or during selection while preparing
a vaccine. This suggests that the inhibition of the NF-kB response
and of the transcription of several RelAp43 dependant genes
(HIAP1,IRF1 and IFN-b) is critical for the success of the infection
and the hence pathogenicity of lyssaviruses. This mechanism of
action is independent of those induced by the P which are
conserved by the SAD B-19 vaccine strain and which do not
modify the NF-kB response [50,51]. The M protein, by targeting
RelAp43 and inhibiting the transcription of HIAP1, a factor
protecting neurons from cell death , could also regulate the
balance between cell death and survival. This indirect action of the
Figure 6. RelAp43 is involved in IFN-btranscription during
lyssavirus infection. The results presented are the means of five (A)
or three (B, C, D) independent experiments. Error bars indicate standard
deviations. *p,0.05. (A) Luciferase assay of IFN-bpromoter activation in
presence of M protein. M-Tha (black bars), M-SAD (dark grey bars), or
CAT (light grey bars) as a control were used. The measure obtained with
M-SAD was arbitrary set to 1. The expression levels of M-Tha, M-SAD
and CAT in each condition were assessed by western blot (bottom of
the figure). (B) Infectious titres of Tha and SAD viruses from supernatant
of Hela cells infected at an MOI of 1 at 24 and 48 hours p. i. in the
presence of siRNA control (siRNAc) or anti-RelAp43 siRNA (siRelAp43).
Titers are given in Fluorescent focus units per ml (FFU/ml). (C) Decrease
of the number of RelAp43 mRNA copies in Hela cells infected with Tha
or SAD virus and in non-infected cells (NI) in the presence of anti-
RelAp43 siRNA (siRNA RelAp43) compared to siRNA control (siRNAc)
and measured by quantitative RT-PCR. The level of transcription of
RelAp43 in Hela cells measured 48 hours after infection with SAD and in
the presence of control siRNA was arbitrary set to 1. (D) Modulation of
IFN-bmRNA levels detected by quantitative RT-PCR in HeLa cells
transfected by either a control siRNA or an anti RelAp43 siRNA, and
infected with Tha or SAD virus compared to non-infected cells (NI).
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M protein on HIAP1 together with those related to the induction
of apoptosis [52,53,54] further demonstrate the crucial role of the
M protein in cell death, although the precise mechanisms which
may link RelAp43, HIAP1 and M-mediated apoptosis remain
unknown. Therefore, it is tempting to speculate that RelAp43
targeting could be involved in a global reprogramming of the NF-
kB pathway to the benefit of lyssavirus infection. A recent study
demonstrated the blockade of the NF-kB pathway in rabies-
infected neurons, and suggested that one or more viral proteins
may directly interact with this pathway . According to our
results, we postulate that the M protein could play a role in this
blockade, and that its action is mediated by the targeting of
Figure 7. Proposed model for the role of RelAp43 and of the M protein of lyssavirus in the regulation of the NF-kB dimer repertoire
and downstream action on the transcription of different genes involved in innate immune response. RelAp43 is designed as p43, M
protein of lyssavirus wild isolates in orange; laboratory adapted and vaccine strains in green.
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RelAp43. Viral manipulations of the NF-kB signaling are a
common feature during infections, thus rabies virus would not
make exception. Positive strand RNA viruses like the picornavi-
ruses are known to degrade RelA and therefore suppress the
innate immune response [56,57]. DNA viruses of the Herpesviridae
family also prevent the transcriptional activity of NF-kB . In
measles virus, a paramyxovirus, the V protein produced from the
P gene has been shown to specifically bind to the RHD of RelA
and therefore to suppress NF-kB activity . More generally,
other regulatory effects of RelAp43 on cellular processes such as
inflammation and oncogenesis may also be expected, as observed
in humans with other alternative splicing events of cRel and which
serves as a marker of tumorigenesis .
Materials and Methods
Cells and viruses
Human carcinoma epithelial cells (HeLa), human epithelial
kidney cells (HEK-293T), and human neuroblastoma cells (IMR5
and SK-S-SH) were cultured as previously described . If
indicated, cells were treated with recombinant human TNF-a
(R&D systems) at a final concentration of 10 ng/ml and incubated
for indicated time at 37uC. When indicated, cycloheximide
(Sigma) was added to culture medium at 100 mg/mL for indicated
time. Virus infection was performed in 6-well plates during
indicated times at 37uC and using different viruses at a multiplicity
of infection (MOI) of 1. Thailand virus, referred as Tha (isolate
8743THA), is a field strain of the species rabies virus (RABV)
isolated in Thailand from a human bitten by a dog. SAD-B19
virus (SAD) and Pasteur Virus (PV) are vaccine strains of RABV.
Field isolates 8720MOK (Mok), 8619NGA (Lag) and 8918FRA
(EBLV-1) were used as representative strains of species Mokola
virus, Lagos Bat virus and European Bat Lyssavirus-1 (EBLV-1),
The following antibodies were used: mouse a-V5 antibody
(Invitrogen); mouse a-FLAG M2 antibody and rabbit a-FLAG
antibody (Sigma); rabbit a-p105/p50, a-p100/p52, a-c-Rel, a-
RelB, a-RelA and a-IkBaprovided by R Weil; a-RelAp43
polyclonal antibody generated on rabbit immunized with a
peptide coding for the specific part of RelAp43 (NH
COOH) (Eurogentec); conjugated a-rabbit Alexa 488 (Molecular
probes); a-mouse Alexa 555 (Molecular probes); HRP-linked a-
mouse antibody and HRP-linked a-rabbit antibody (GE Health-
RNA isolation, reverse transcription and quantitative real-
Total RNA was isolated using Nucleospin RNA II kit
(Macherey Nagel). Reverse transcription was performed on 2 mg
of RNA using Superscript II (Invitrogen) with 2 pmol of oligodT
primers (Fermentas) in a final volume of 20 ml. Transcription
analysis was performed on 100 ng of total RNA using Taqman
Power SYBR Green (Applied Biosystems) in a 7500 instrument
(Applied Biosystems) and specific primers (Table S1), following
manufacturer instruction. Relative quantification was performed
using GAPDH gene as endogenous control gene. Absolute
quantification of RelAp43 and RelA was performed using serial
dilutions of known quantity of cloned RelA and RelAp43. Results
were analyzed using 7500 SDS software version 2 (Applied
Open reading frame (ORF) coding for M proteins were
amplified by reverse transcription-PCR using total RNAs from
infected samples . PCRs were carried out with 2 ml of cDNA
using primers containing a Gateway cloning site and a matrix
specific sequence (Table S1). ORF coding for RelAp43 and RelA
were amplified from the cDNA bank that was used for two-hybrid
screen using primers indicated in the Supplementary Table S1.
Each PCR product was cloned in the pDONR221 vector using
Gateway recombinase (Invitrogen). Several final destination
vectors were used: yeast two-hybrid vectors pDEST32 and
pPC86 in which the Gateway cassette was inserted (Invitrogen),
pCDNA3.1N-V5-dest (Invitrogen), pCI-neo-3xFLAG that is a
pCI-neo vector (Promega) modified with a 3xFLAG tag (Sigma) in
N-terminus of the Gateway cassette (Invitrogen) , pM vector
(Clontech) in which the Gateway cassette was inserted in order to
make the ORF be expressed as a fusion protein with the Gal4
DNA binding domain (Gal4-DB) in N-terminus.
Western blot analysis was performed using NuPAGE gels
(Invitrogen). Protein transfer on nitrocellulose membrane was
performed using iBlot transfer system (Invitrogen), as indicating by
provider. Membranes were saturated for 1 h in PBS-Tween 0.1%
with 5% non-fat dried milk. Immunoblotting procedure consisted
in incubation for 1 h with indicated primary antibody diluted in
5% dried milk PBS-Tween, then washed three times for 20 min in
PBS-Tween, then incubated 1 h with indicated HRP conjugated
secondary antibody. Blots were revealed by chemiluminescence
(Pierce) and exposure to X-ray films (Amersham) for different time
to avoid saturation. Films were digitized and blot relative
quantification was performed using Scion Image software.
Immunofluorescence experiments were performed using as
primary antibodies a rabbit anti-FLAG antibody (Sigma) and a
mouse anti-V5 (Invitrogen) antibody and as secondary antibodies
a conjugated anti-rabbit Alexa 488 and anti- mouse Alexa 555
(Molecular probes) and analysed using Zeiss ApoTome system as
described previously . HeLa cells were cultured and transfect-
ed with Lipofectamine 2000 in glass Labteck multi-chambers
slides. Twenty-four hours post transfection, cells were fixed with
cold methanol and permeabilized with acetone.
EMSA and supershift experiments
Nuclear extracts of HeLa cells were prepared and EMSA were
performed as previously described using the kB site derived from
the enhancer of the immunoglobulin light chain gene as a probe
(Table S1) . For supershift analysis, 100 ng of indicated
antibody was incubated to the EMSA reaction mixture. Gel
electrophoresis and data collection were performed as described
for the EMSA.
Luciferase reporter gene assays
HEK-293T cells were plated in 96-well plates with 15,000 cells
per well under 100 ml of culture medium. After 24 h, cells were
transfected with Lipofectamine 2000 (Invitrogen), as recommended
by the provider. To measure the NF-kB response, we transfected
cells with a mix containing (per well) 160 ng V5-tagged M protein or
CAT encoding plasmid, 10 ng of pNF-kB-Luc (Agilent Technolo-
gies) coding for firefly luciferase under control of kBsites,and2 ngof
EF1-b-gal (gift from S. Memet, Institut Pasteur) encoding b-
galactosidase under control of EF1 promoter insensitive to NF-kB
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activation. Finally, cells were stimulated or not with recombinant
TNF-aat 10 ng/mL (R&D systems). To measure RelA transactiva-
tion, we transfected cells with a mix of plasmids composed of 10 ng
of pM vector encoding for Gal4-DB fused to RelA, 20 ng of
luciferase under control of a Gal4-UAS promoter sequence, and
2ngofEF1-b-gal. If indicated, we further added 160 ng of plasmid
encoding V5-tagged RelAp43, with or without 80 ng of plasmid
encoding either V5-tagged M protein or CAT. After 24 h, luciferase
and galactosidase activities were measured using Steady-glo and
Beta-glo assays (Promega) respectively, as indicated by the provider.
Results are the mean of three measures of each activity per
transfection and are expressed as the luciferase/beta-galactosidase
activity ratio. To measure the late NF-kB response, HEK-293T cells
were plated and transfected simultaneously using JetPrime reagent
(Polyplus transfection). Transfection mix was composed of 30 ng of
pNF-kB-Luc (Agilent Technologies), 3 ng of TK-Renilla luciferase
encoding plasmid, 30 ng of plasmid encoding FLAG-tagged M
protein or CAT and the indicated amount of 59-triphosphate RNA.
After 24 h, luciferase activities in the lysate were determined using
Bright-Glo Luciferase assay system (Promega). The same protocol
was used to measure the IFN-binduction, using pIFN-b-Luc, a
plasmid encoding firefly luciferase under control of IFN-bpromoter.
Yeast two-hybrid screening
Yeast two-hybrid screen was performed using a human spleen
cDNA library cloned in the Gal4-AD pPC86 vector (Invitrogen)
and previously established in Y187 yeast strain (Clontech) .
We subsequently determined the interaction of various M proteins
from different lyssaviruses with RelAp43. ORFs coding for
lyssavirus M proteins and RelAp43 were recombined from
pDONR221 into pDEST32 and pPC86 yeast two-hybrid vectors,
respectively. Yeast cells (AH109 strain, Clontech) were co-
transformed with these constructs before platting on a selective
medium lacking tryptophan, leucine, histidine and supplemented
with 1 mM of 3-aminotriazole (3-AT) (Sigma-Aldrich) to test the
interaction-dependent transactivation of the HIS3 reporter gene.
Twenty-four hours post transfection, cells were lysed in Ripa
buffer (Santa Cruz). For IP of FLAG-tagged proteins, 100 mgof
total proteins were incubated with anti-FLAG M2 gel (Sigma) in
TNE Triton 1% (w/v) buffer (Tris 25 mM, NaCl 150 mM,
antiprotease cocktail (complete Roche) overnight at 4uCona
wheel. Gel was washed with TNE Triton 1% (w/v) buffer.
Proteins were eluted directly using loading sample buffer
(Invitrogen) and heated 95uC for 10 min. After centrifugation,
eluted proteins were analyzed by western blot with the indicated
antibody. For IP of non FLAG-tagged proteins, extracts contain-
ing 200 mg of total proteins were immunoprecipitated with
indicated antibody using washed protein A Sepharose (Sigma).
The rest of the protocol is similar as above.
RNA silencing of RelAp43
HeLa cells were plated out in 6-well plates to reach around 75%
confluence the following day. Two small interfering RNAs were
designed, one specifically targeting RelAp43 and the other used as
a control (Table S1). 20 pmol of siRNA targeting RelAp43 per
well were transfected using RNAiMAX Lipofectamine reagent
(Invitrogen) according to the manufacturer’s instructions. RelAp43
silencing was confirmed by quantitative real-time PCR (Fig. S1).
To turn down endogenous RelAp43 expression on infected HeLa
cells, control- or antiRelAp43-siRNA were transfected as de-
scribed above, 3 h post infection.
Single comparisons of data were performed by Student’s ttests
using the GraphPad Prism software.
Figure S1 Decrease of the number of RelAp43 but not
RelA mRNA copies in the presence of specific siRNA
directed against anti RelAp43 expression. RelAp43 and
RelA mRNA transcription were measured by quantitative RT-
PCR in HeLa cells transfected by either a control siRNA (light
gray bars) or an anti-RelAp43 siRNA (dark grey bars). For each
mRNA, the level of transcription measured in the presence of
control siRNA was arbitrary set to 1. The number of RelA mRNA
is not modified by anti-RelAp43 siRNA. Results presented here
are the mean mRNA level obtained after 3 independent
experiments. Significant effects (p,0,05) are indicated by asterisk
and error bars indicate standard deviations.
Figure S2 Transfection of RelAp43 did not induce the
production of IkBa.HeLa cells were transfected with either
FLAG-tagged CAT, RelA or RelAp43. After 24 hours, the NF-kB
pathway was exogenously activated using 10 ng/mL TNF-a
during indicated times. The levels of expression of IkBa,
phosphorylated IkBa(IkB-P) and actin were determined by
western blot using specific antibodies.
Figure S3 Luciferase assay of RelA transactivation
properties in presence of CAT or RelAp43. Using luciferase
under control of the Gal4 promoter, and RelA fused to the Gal4
DNA Binding domain (named as DB-RelA on the figure).
Increasing quantities of RelAp43- or CAT-encoding plasmids
were added to the transfection mix in the same conditions as in
Figure 3C. (A) Results presented here are the mean luminescence
signal obtained after 3 independent experiments. Significant
effects (p,0,05) are indicated by asterisk and error bars indicate
standard deviations. (B) Transformation of the luminescence units
in arbitrary units corresponding to signal with RelAp43/signal
Figure S4 Quantification of RelAp43 mRNA on cells
infected with Tha or SAD-B19 virus. Total RNA were
extracted from cells at indicated time post infection. Results are
the mean mRNA level obtained after 3 independent experiments.
The level of RelAp43 mRNA from cells infected with SAD 1 h p.i.
was arbitrary set to 1. Significant effects compared to SAD 1 h p.i.
(p,0,05) are indicated by asterisk and error bars indicate standard
Table S1 Forward primers (For) and reverse primers
(Rev) used in our study. For Gateway cloning primers,
gateway recombination sites are indicated in lower font and ORF
specific part in upper font; initiation codon on the forward primer
and stop codon on reverse primer are in bold.
We are grateful to E. Perret and S. Shorte at the Plate-forme d’Imagerie
Dynamique (PFID) at the Institut Pasteur for invaluable experimental help,
discussion and advice on data processing. We thank C. Pre´haud for
providing SK-N-SH cells.
RelAp43: Role in Anti-Viral Innate Immunity
PLOS Pathogens | www.plospathogens.org 14 December 2012 | Volume 8 | Issue 12 | e1003060
Conceived and designed the experiments: SL OD POV RW FT HB.
Performed the experiments: SL OD POV RW. Analyzed the data: SL OD
POV RW HB. Contributed reagents/materials/analysis tools: SL OD
POV FT RW HB. Wrote the paper: SL OD POV FT RW HB.
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