Blackwell Publishing LtdOxford, UKCMICellular Microbiology 1462-5814© 2005 The Authors; Journal compilation © 2005 Blackwell Publishing Ltd200583375386Review ArticleS. Ludwig, S. Pleschka, O. Planz and T. WolffInfluenza virus and
cell signalling
Cellular Microbiology (2006) 8(3), 375–386
doi:10.1111/j.1462-5822.2005.00678.x
First published online 27 January 2006
Microreview
Ringing the alarm bells: signalling and apoptosis
in influenza virus infected cells
Stephan Ludwig,1* Stephan Pleschka,2 Oliver Planz3
and Thorsten Wolff4
1
Institute of Molecular Virology (IMV) WestfaelischeWilhelms-University, Von-Esmarch Str. 56, D-48161
Muenster, Germany.
2
Institute of Medical Virology, Justus-Liebig-University,
Frankfurter Str. 107, D-35392 Giessen, Germany.
3
Institute of Immunology, Friedrich-Loeffler-Institute (FLI),
Paul-Ehrlich Str. 28, D-72076 Tübingen, Germany.
4
Robert-Koch Institute (RKI), Nordufer 20, 13353 D-Berlin,
Germany.
Summary
Small RNA viruses such as influenza viruses extensively manipulate host-cell functions to support their
replication. At the same time the infected cell induces
an array of defence mechanisms to fight the invader.
These processes are mediated by a variety of intracellular signalling cascades. Here we will review the
current knowledge of functional kinase signalling and
apoptotic events in influenza virus infected cells and
how these viruses have learned to misuse these cellular responses for efficient replication.
Cell fate decisions in response to extracellular agents,
including pathogenic invaders are commonly mediated by
intracellular signalling cascades that transduce signals
into stimulus specific actions, e.g. changes in gene
expression patterns, alterations in the metabolic state of
the cell or induction of programmed cell death (apoptosis).
Thus, these signalling molecules are at the bottleneck of
the control of cellular responses. Many DNA- and retroviruses are known to induce cellular signalling mainly to
drive cells into a proliferative state. The reason is quite
obvious because these pathogens partly employ the DNA
synthesis machinery for their replication. The consequences of signalling induced by RNA viruses, including
influenza viruses were less clear because this area was
not a focus of research for a long time. However, in the
last couple of years reports on functional signal transduction processes induced by RNA viruses rapidly accumulated. As detailed below, a majority of the more recent
reports address the signalling events concomitant with the
innate immune response. Nevertheless, there are also
findings suggesting that RNA viruses misuse cellular signals to support their replication. Here we will discuss
recent advances in the analysis of influenza virus-induced
signalling pathways and the insights these studies provide
for our understanding of the viral replication process.
Introduction
Influenza A viruses are highly contagious pathogens for
humans and several animal species. These viruses
belong to the family of Orthomyxoviridae and possess a
segmented negative-stranded RNA genome of roughly
13 kB, coding for at least 10 viral proteins (Lamb and
Krug, 2001). The viral genome is replicated and transcribed in the nucleus, a feature that requires bidirectional
transport through the nuclear membrane. Due to the limited coding capacity of the genome these viruses extensively employ functions of their host-cell for efficient
replication (Ludwig et al., 1999).
Received 2 November, 2005; accepted 30 November, 2005. *For
correspondence. E-mail [email protected]; Tel. (+49)
251 83 57791; Fax (+49) 251 83 57793.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd
Influenza viruses and MAP kinase cascades
Mitogen-activated protein kinase (MAPK) cascades are
important signalling pathways that convert a variety of
extracellular signals into a multitude of cellular responses
(reviewed in Pearson et al., 2001). These signalling cascades regulate cellular decision processes as diverse as
proliferation and differentiation, but also cell activation and
immune responses (Dong et al., 2002). Four different prototype members of the MAPK family that are organized in
separate cascades have been identified so far: ERK
(extracellular signal-regulated kinase), JNK (Jun-N-terminal kinase), p38 and BMK-1/ERK5 (Big MAP kinase)
(Pearson et al., 2001) (Fig. 1). For each MAP kinase different isoforms are known. All these enzymes have in
common that they are activated by a dual phosphorylation
event on threonine and tyrosine mediated by upstream
376 S. Ludwig, S. Pleschka, O. Planz and T. Wolff
Influenza Virus
IKK2
NA
?
RNA accumulation
NP
HA
Raf
ASK-1
?
P
M1
P
HA
IkBa
P
MKK4/7
P
P
P
MKK3/6
P
P
MEK5
P
PKCbII
p50/p65
P
P
TPY
JNK
P
p50/p65
?
P
MEK1/2
IkBa
P
P
T GY
p38
P
P
TEY
ERK5
P
P
TEY
ERK1/2
endocytosis
P
P
p50/p65
ATF-2
P
c-Jun
?
NF-kB
AP-1
IFNb expression RANTES expression vRNP
IFNb expression
export
Cytokine expression RANTES expression TNFa expression
IL-1 expression
TNFa expression
Apoptosis regulation Apoptosis regulation
Fig. 1. Schematic representation of intracellular signalling pathways that are activated upon influenza virus infection or treatment with viral
components and the proposed function of these cascades in the infected cell. For clarity, the major pathways of the antiviral IRF activation and
induction of the IFN response are shown in a separate figure (Fig. 2).
MAP kinase kinases (MEKs or MKKs) (Fig. 1). The MAP
kinase ERK1 and ERK2 are activated by the dual-specific
kinases MEK1 and 2 that are controlled by the serine
threonine kinase Raf. Raf, MEK and ERK form the prototype module of a MAP kinase pathway. This three-kinase
module is also known as the classical mitogenic cascade.
The MAP kinases p38 and JNK are activated by MKK3/6
and MKK4/7, respectively, and are predominantly activated by proinflammatory cytokines and certain environmental stress conditions. ERK5, also known as big-MAP
kinase (BMK-1) is activated by MEK5 (Fig. 1). This kinase
module is special because it is both activated by mitogens
and certain stress inducers.
Influenza virus infection of cultured cells results in the
activation of all four known MAPK family members (Kujime
et al., 2000; Ludwig et al., 2001; Pleschka et al., 2001)
(Fig. 1). Activation of p38 MAPK after influenza virus
infection has been linked to expression of RANTES and
IL-8, chemokines involved in the attraction of eosinophils
and neutrophils respectively (Kujime et al., 2000; Guillot
et al., 2005). Furthermore, inhibition of p38 and other
MAPKs results in decreased prostaglandin E2 release
(Mizumura et al., 2003), indicating that p38 MAPK activation controls the onset of an inflammatory response. This
is also supported by the recent finding that hyperinduction
of TNF-α upon infection with avian H5N1 influenza viruses
occurs in a p38-dependent manner (Lee et al., 2005).
The JNK subgroup of MAPKs came into focus in the
context of an influenza virus infection because a very early
activation of activator-protein 1 (AP-1) transcription factors
(Karin et al., 1997) was observed in productively infected
cells (Ludwig et al., 2001). AP-1 factors include c-Jun and
ATF-2 that are phosphorylated by JNKs to potentiate their
transcriptional activity (Karin et al., 1997). Accordingly,
activation of JNK was observed with different virus strains
in a variety of permissive cell lines (Kujime et al., 2000;
Ludwig et al., 2001). JNK activation required productive
replication and was induced by accumulating RNA produced by the viral polymerase.
Together with NF-κB and IRF-3 the JNK effectors c-Jun
and ATF-2 are critical to regulate the expression of IFNβ,
one of the most potent antiviral cytokines (Samuel, 2001)
(Figs 1 and 2). Accordingly, inhibition of the cascade by
dominant-negative mutants of c-Jun, JNK or the JNK acti-
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
Influenza virus and cell signalling 377
Virus
dsRNA
TLR-3
helicase
dsRNA
TRIF
RIG-I
dsRNA
ssRNA
helicase
TR
IF
?
TLR-7
TBK1
IPS-1
MyD88
NS1
TRAF6
TBK1 / IKKε
P
PP P
PP
PP
P
PP P
PP
PP
IRF-3/-7
P
PP P
?
PP
PP
P
PP P
PP
PP
IRF-3/-7
IRF-7
P
PP P
PP
PP
PP
PP
IFN-α/β
IFN-α/β
Fig. 2. Induction pathways of IFN-α/β genes during virus infection.
Left. Productive virus infection induces the appearance of dsRNA, that is recognized by the RNA helicase RIG-I. This leads, via the adapter
protein IPS-1, to the phosphorylation and activation of the key transcription factors IRF3 and/or IRF7 by the protein kinases TBK1 and/or IKK-ε.
Phosphorylated IRF-3/-7 dimerizes and translocates to the nucleus, where it becomes engaged in the activation of IFN-α/β genes. The influenza
virus NS1 protein is known to inhibit activation of IRF-3/7 indicating a blockade of dsRNA-dependent upstream signals at a currently unidentified
step.
Right. Uptake of influenza virus into plasmacytoid dendritic cells results in the exposure of virion single-stranded (ss) RNAs to Toll-like receptor
(TLR)-7 in an endosomal compartment. Agonist recognition of the TLR leads through the adapter MyD88 and TRAF6 to recruitment of an unknown
kinase that activates IRF-7. DsRNA released from infected cells can activate TLR3 either in the endosome or on the cell surface, which leads
through to the recruitment of the adapter protein TRIF and TBK1 to the activation of IRF-3.
vator MKK7 in the infected cell resulted in impaired transcription from the IFNβ promoter and an enhanced virus
production. Thus, the JNK pathway appears to be a crucial
mediator of the antiviral response to an influenza virus
infection by coregulating IFNβ expression (Ludwig et al.,
2001).
In contrast to the JNK pathway the Raf/MEK/ERK cascade appears to serve as a module that is beneficial for
the virus (Pleschka et al., 2001). Blockade of the pathway
by specific inhibitors of MEK, or dominant-negative
mutants of ERK or Raf resulted in a strongly impaired
growth of both influenza A and B viruses (Pleschka et al.,
2001; Ludwig et al., 2004). Conversely, virus titres were
enhanced in cells expressing active mutants of Raf or
MEK (Ludwig et al., 2004; Olschlager et al., 2004). This
has not only been demonstrated in cell culture but also in
vivo in infected mice expressing a constitutively active
form of the Raf kinase in the alveolar epithelial cells of the
lung (Olschlager et al., 2004). In the wild-type situation
influenza viruses primarily infected bronchiolar epithelial
cells, while in the alveolar layer replication occurs most
exclusively in cells carrying the transgene. As a consequence this resulted in an earlier death of the transgenic
animals (Olschlager et al., 2004).
Strikingly, inhibition of the pathway did not affect viral
RNA or protein synthesis (Pleschka et al., 2001). The
pathway rather appears to control the active nuclear
export of the viral RNP complexes. RNPs are readily
retained in the nucleus upon blockade of the signalling
pathway. This is most likely due to an impaired activity of
the viral nuclear export protein NEP (Pleschka et al.,
2001). Thus, active RNP export appears to be at least in
part an inducible event, a hypothesis supported by a late
activation of ERK in the viral life cycle. However, the
detailed mechanism of how ERK regulates export of the
RNPs remains to be elucidated.
The requirement of Raf/MEK/ERK activation for efficient
influenza virus replication suggests that this pathway may
be a cellular target for antiviral approaches. Besides the
antiviral action against both type A and B viruses (Ludwig
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
378 S. Ludwig, S. Pleschka, O. Planz and T. Wolff
et al., 2004), MEK inhibitors meet two further criteria
which are a prerequisite for a potential clinical use.
Although targeting an important signalling pathway in the
cell, the inhibitors showed surprisingly little toxicity (i) in
cell culture (Planz et al., 2001; Pleschka et al., 2001; Ludwig et al., 2004); (ii) in an in vivo mouse model (SeboltLeopold et al., 1999); and (iii) in clinical trials for the use
as anticancer agent (Cohen, 2002). In the light of the latter
finding it was hypothesized that the mitogenic pathway
may only be of major importance during early development of an organism and may be dispensable in adult
tissues (Cohen, 2002). Another very important feature of
MEK inhibitors is that they showed no tendency to induce
formation of resistant virus variants (Ludwig et al., 2004).
Although targeting of a cellular factor may still raise the
concern of potential side-effects of a drug, it appears likely
that local administration of an agent such as a MEK
inhibitor to the primary site of influenza virus infection, the
respiratory tract, is well tolerated. Here the drug primarily
affects differentiated epithelial cells, for which a proliferative signalling cascade such as the Raf/MEK/ERK cascade may be dispensable. Following this approach it was
recently demonstrated that the MEK inhibitor U0126 is
effective in reducing virus titres in the lung of infected mice
after local administration (Ludwig et al., 2003; Klumpp,
2004).
Protein Kinase C (PKC) as a regulator of viral entry
Activation of the kinase Raf within the Raf/MEK/ERK module is complex and involves phosphorylation by several
other kinases including members of the PKC family (Kolch
et al., 1993; Cai et al., 1997).
The PKC superfamily consists of at least 12 different
PKC isoforms that carry out diverse regulatory roles in
cellular processes by linking into several downstream signalling pathways (Toker, 1998). It has been noticed quite
some time ago that influenza virus infection or treatment
of cells with purified viral haemagglutinin results in rapid
activation of PKCs upon binding to host-cell surface
receptors (Rott et al., 1995; Arora and Gasse, 1998; Kunzelmann et al., 2000) (Fig. 1). However, the functional
consequences of this activation remained elusive. Given
the variety of downstream effectors of PKCs (Toker, 1998)
it appears likely that beside a regulation of the Raf/MEK/
ERK cascade and other downstream pathways, PKCs
may have additional functions during viral replication. This
assumption is supported by the finding that the viral M1
protein gets phosphorylated by PKC in vitro and binds to
the cellular receptor of activated C kinase RACK1 (Reinhardt and Wolff, 2000). A role of PKCs in the process of
entry of several enveloped viruses has been proposed
based on the action of protein kinase inhibitors H7 and
staurosporine (Constantinescu et al., 1991). In more
recent studies it was shown that the pan-PKC inhibitor
bisindolylmaleimide I prevented influenza virus entry and
subsequent infection in a dose-dependent and reversible
manner (Root et al., 2000). Using a dominant-negative
mutant approach this function was assigned to the PKCβII
isoform: Overexpression of a phosphorylation-deficient
mutant of PKCβII revealed that the kinase is a regulator
of late endosomal sorting. Accordingly, expression of the
PKCβII mutant resulted in a block of virus entry at the level
of late endosomes (Sieczkarski and Whittaker, 2002;
Sieczkarski et al., 2003). This identifies PKCβII as a specific regulator of influenza virus entry (Fig. 1).
Influenza Virus and the classical pathway
κB activation
of NF-κ
Another important signalling pathway which is commonly
activated upon virus infection is the IκB kinase (IKK)/NFκB signalling module (Hiscott et al., 2001). The NF-κB/IκB
family of transcription factors promote the expression of
well over 150 different genes, such as cytokine or
chemokine genes, or genes encoding for adhesion molecules or anti- and pro-apoptotic proteins (Pahl, 1999). The
classical mechanism of NF-κB activation includes activation of IKK that phosphorylates the inhibitor of NF-κB, IκB
and targets the protein for subsequent degradation (Karin
and Ben-Neriah, 2000) (Figs 1 and 3). This leads to the
release and migration of the transcriptionally active NFκB factors, such as p65 or p50 to the nucleus (Ghosh,
1999; Karin and Ben-Neriah, 2000). The IKK complex
consists of at least three components, namely the enzymatically active IKK1/IKKα and IKK2/IKKβ and the scaffold protein NEMO/IKKγ. The most important isozyme for
NF-κB activation via the degradation of IκB is IKK2 while
IKK1 seems to primarily phosphorylate other factors of the
NF-κB/IκB family namely p100/p52 (reviewed in Bonizzi
and Karin, 2004). The large IKK complex (Karin and Delhase, 2000) appears to contain still other kinases such as
MEKK1 (MAPK kinase kinase 1), TAK1 (TGFβ-activated
kinase), MLK-3 (Mixed-lineage kinase 3), NIK (NF-κB
inducing kinase), and the double-stranded (ds) RNA-activated protein kinase PKR (reviewed in Hiscott et al.,
2001).
Influenza virus infection results in an activation of NFκB (reviewed in Ludwig et al., 1999; 2003; Julkunen et al.,
2000) although the level of activation is kept in a certain
limit due to the action of the viral NS1 protein (Wang et al.,
2000), as will be further discussed below. Nevertheless,
the induced activity is sufficient to control expression of a
variety of genes (Wurzer et al., 2004; Bernasconi et al.,
2005). Viral induction of the transcription factor involves
activation of IKK2 (Flory et al., 2000; Wurzer et al., 2004;
Bernasconi et al., 2005) and is also achieved with isolated
influenza virus components. This includes single-stranded
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
Influenza virus and cell signalling 379
Influenza Virus
IKK2
P
P
p50/p65
PB1-F2
IκBα
κ α
P
p50/p65
p50/p65
TRAIL-R
Fas
IκBα
Fas, FasL
TRAIL
Caspases
vRNP export
NF-κB
Nucleus
Cytoplasm
Fig. 3. Model of the chain of events that links virus-induced NF-κB activation to caspase induction and enhanced RNP export. During productive
virus infection the proapoptotic factors TRAIL, Fas and FasL are expressed in an NF-κB-dependent manner. These factors induce caspase
activation in an auto- and paracrine fashion. Active caspases allow an enhanced release of RNP complexes from the nucleus, presumably due
to caspase-mediated disruption of the active nuclear pore complex (see text for further details). Alternatively, caspase activation can also be
achieved by expression of the viral PB1-F2 protein although this appears not to be linked to RNP export. It should be stressed that the mechanism
shown may not be the only event regulating the NF-κB- and caspase-mediated outcome on viral propagation.
(ss) RNA (Heil et al., 2004; Kawai et al., 2004) or doublestranded (ds) RNA (Chu et al., 1999) as well as overexpression of the viral HA, nucleoprotein (NP) or M1 proteins
(Pahl and Baeuerle, 1995; Flory et al., 2000) (Fig. 1). As
gene expression of many proinflammatory or antiviral
cytokines, such as IFNβ or TNF-α, is controlled by NF-κB
(Pahl, 1999) the concept emerged that IKK and NF-κB are
essential components in the innate immune response to
virus infections (Chu et al., 1999). Accordingly, influenza
virus-induced IFNβ promoter activity is strongly impaired
in cells expressing transdominant negative mutants of
IKK2 or IκBα (Wang et al., 2000; Wurzer et al., 2004).
Nevertheless, IKK and NF-κB might not only have antiviral functions. Two recent studies demonstrate that influenza viruses exhibit higher levels of replication in cells
where NF-κB is preactivated (Nimmerjahn et al., 2004;
Wurzer et al., 2004). Conversely, a dramatic reduction of
influenza virus titres could be observed in cells were NFκB signalling was impaired (Nimmerjahn et al., 2004;
Wurzer et al., 2004). This is different from the situation
with other RNA viruses, e.g. Borna disease virus (BDV)
where constitutive activation of NF-κB clearly leads to a
drop in virus titres (Bourteele et al., 2005). Thus, in the
context of an influenza virus infection NF-κB appears to
have a supportive function for viral replication that is dominant over the antiviral activity induced by the transcription
factor. On a molecular basis this was shown to be at least
in part due to the NF-κB-dependent expression of proapoptotic factors, such as TNF-related apoptosis inducing
ligand (TRAIL) or FasL (Wurzer et al., 2004). Inhibition of
virus induced expression of these factors results in
strongly impaired viral growth. These findings link the proinfluenza action of NF-κB to the induction of apoptosis, a
process that will be further discussed below (Fig. 3).
Finally, viral need for NF-κB activity suggests that this
pathway may be suitable as a target for antiviral intervention. To this end it has been shown recently that pharmacological inhibitors of NF-κB act antiviral in vivo without
toxic side-effects or the tendency to induce resistant virus
variants (Ludwig et al., 2003).
Mounting the antiviral state – signalling events
inducing the type I interferon response
A large proportion of the signalling events in infected
epithelial cells at the primary viral replication sites is
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
380 S. Ludwig, S. Pleschka, O. Planz and T. Wolff
devoted to the induction of cellular responses that aim to
prevent the spread of the invading virus in the tissue and
the establishment of a persistent infection. A major part
of the antiviral response is the expression and secretion
of the interferons (IFN)-α and -β. These antiviral cytokines
bind to the IFN-α/β receptor, which by signalling through
the JAK-STAT pathway leads to the formation of the trimeric transcription factor ISGF3 that in turn activates a
multitude of latent gene products, many of which have
strong antiviral activities such as the Mx, p56 and 2′-5′oligoadenylate synthetases (Samuel, 2001). The potency
of the IFN-α/β system is illustrated by the exquisite sensitivity of STAT1–/– mice to viral infections (Durbin et al.,
1996). However, during coevolution with their hosts probably most viruses have evolved gene products that interfere with the IFN-α/β system at the induction or effector
level as a prerequisite for efficient replication (GarciaSastre, 2004). Thus, depending on the particular virus and
the target(s) of its antagonistic gene product one may
observe high or low contributions of distinct cellular components to the establishment of an antiviral state (Leib
et al., 2000).
It has been known for decades that dsRNA is a molecular pattern commonly associated with viral infections and
that the intracellular appearance of dsRNA mediates a
strong induction of IFN-α/β genes in many cell types
(summarized in Majde, 2000). However, although it was
established a while ago that IFN induction is controlled by
the transcription factors IRF3/-7, NF-κB and ATF-2/c-Jun
(Wathelet et al., 1998), the molecular events facilitating
this antiviral reaction had remained elusive for a long time.
Lately, several breakthrough studies have elegantly identified molecular sensors and at least some of the mediators that ultimately lead to the activation of IRF-3/-7 and
hence, IFN-α/β genes. Yoneyama and colleagues recently
showed that intracellular dsRNA, a frequent by-product of
viral replication, is detected by the dsRNA helicases RIGI (Yoneyama et al., 2004; Kato et al., 2005) and its homologue mda-5 (Andrejeva et al., 2004), that both contain
two caspase recruitment domains (CARD) (Fig. 2). RIG-I
mediates the activation of the transcription factors IRF3
and NF-κB and expression of IFN-α/β after virus infection
by interaction with another CARD protein, termed interferon-β promoter stimulator 1 (IPS-1; also identified as
MAVS, VISA, Cardif) (Kawai et al., 2005; Meylan et al.,
2005; Seth et al., 2005; Xu et al., 2005). This protein was
shown to interact with overexpressed TBK-1 and IKK-ε
(Meylan et al., 2005; Xu et al., 2005) that are two members of the IKK family known to activate the latent IRF-3
and IRF-7 transcription factors by phosphorylation
(Fitzgerald et al., 2003; Sharma et al., 2003) (Fig. 2). Furthermore, IPS-1 facilitated the recruitment of IRF-3 to
RIG-I (Xu et al., 2005) and cells with abolished or
‘knocked-down’ levels of RIG-I or IPS-1, respectively, were
severely impaired in IFN secretion and produced highly
increased virus titres (Kato et al., 2005; Kawai et al., 2005;
Seth et al., 2005). Collectively, these findings span the
framework for a model, in which RIG-I and IPS play central
roles in initiating the antiviral response (Fig. 2).
In our consideration of how RNA viruses are detected
in body cells we like to distinguish between the processes
described above for cells that support productive virus
replication from the uptake of viruses in professional antigen presenting cells such as dendritic cells (DCs), which
mainly serves to initiate an adaptive immune response
(Fig. 2). Immature DCs express a variety of pattern recognition receptors belonging to the Toll-like receptor (TLR)
family, that recognize various microbial components such
as lipopolysaccharide (LPS), peptidoglycan or CpG motifcontaining DNA (Akira and Takeda, 2004). Agonist binding
to TLRs triggers via the adapter molecules MyD88 and
TRIF the induction of pro-inflammatory and antiviral cytokine genes including IFN-α and/or IFN-β, which results in
DC maturation (Takeda and Akira, 2005) (Fig. 2). As the
TLR family members 3 (dsRNA sensor) (Alexopoulou
et al., 2001), 7 and 8 (ssRNA sensors) (Heil et al., 2004;
Kawai et al., 2004) are known to be activated by genetic
materials also found in RNA viruses, it appeared possible
that TLRs might mediate an RIG-I-independent detection
of influenza virus infections also in non-haematopoetic
cells (Guillot et al., 2005). However, the unaltered course
and pathogenesis of influenza- and other negative strand
RNA virus infections in TLR3–/– and MyD88–/– mice (Edelmann et al., 2004; Lopez et al., 2004; Barchet et al., 2005)
argues against such a generalized role for TLRs as antiviral effectors in the early phase of infection.
The influenza virus NS1 protein interferes with the
antiviral response
Although IFN-α/β were originally discovered during the
study of influenza virus (Lindenmann et al., 1957), it is
ironic to note that reverse genetic studies revealed that
these viruses actually possess the capacity to suppress
the expression of these cytokines. In comparison to wild
type, influenza viruses with deleted NS1 genes proofed to
be much stronger inducers of IFN-α/β genes and their
growths were highly attenuated in hosts with an intact IFN
system (Garcia-Sastre et al., 1998; Dauber et al., 2004).
A second known activity of the 26 kDa NS1 protein is to
inhibit activation of the antiviral dsRNA-dependent protein
kinase R (PKR), which is probably due to NS1’s capacity
to bind to dsRNA produced during virus replication (Lu
et al., 1994; Bergmann et al., 2000). PKR can phosphorylate the translation factor eIF2α, which leads to a sustained arrest in cellular translation and, hence, to an
inhibition of viral gene expression (Samuel, 2001). The
strong IFN induction during influenza virus infection in the
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
Influenza virus and cell signalling 381
absence of NS1 expression correlated directly with a
prominent activation of the transcription factors IRF-3/-7,
NF-κB and ATF-2/c-jun (Ludwig et al., 2003). Thus, the
NS1 protein is capable of inhibiting the virus-induced
dsRNA signals leading to activation of antiviral cytokine
genes (Fig. 2). Whether NS1 functions merely by intracellular sequestration of dsRNA molecules or by direct targeting of any of the recently described factors such as
RIG-I or IPS-1 that transduce these signals is currently
under investigation. Preliminary data showed that recombinant influenza viruses expressing NS1 proteins with
inactivated RNA binding can still effectively block viral IFN
induction suggesting that dsRNA sequestration plays only
a minor role in IFN antagonization (B. Dauber and T. W.,
unpubl. obs.). The NS1 protein has also been described
to inhibit the maturation of cellular pre-mRNAs raising the
possibility that this activity additionally reduces secretion
of IFN-α/β from infected cells (reviewed in Krug et al.,
2003). The strong induction of IFN-α in plasmacytoid DCs
by wild-type influenza viruses (Diebold et al., 2004) suggests that the viral NS1 protein does not interfere significantly with TLR-dependent signals, but rather specifically
targets the RIG-I-dependent pathway of IFN induction.
Influenza virus-induced caspase activation and
apoptosis – submerging a suicide program
An important cellular signalling response commonly
observed upon virus infections is the induction of the
apoptotic cascade. Apoptosis is a morphologically and
biochemically defined form of cell death (Kerr et al., 1972)
and has been demonstrated to play a role in a variety of
diseases including infections by viruses (Razvi and Welsh,
1995; Fischer and Schulze-Osthoff, 2005). Apoptosis is
mainly regarded to be a host cell defence because many
viruses express antiapoptotic proteins to prevent this cellular response. The central component of the apoptotic
machinery is a proteolytic system consisting of a family of
cysteinyl proteases, termed caspases (for review see
Cohen, 1997; Thornberry and Lazebnik, 1998). Two
groups of caspases can be distinguished: upstream initiator caspases such as caspase 8 or caspase 9 which
cleave and activate other caspases and downstream
effector caspases, including caspases 3, 6 and 7, cleaving
a variety of other cellular substrates, thereby disassembling cellular structures or inactivating enzymes (Thornberry and Lazebnik, 1998). Caspase 3 is the most
intensively studied effector caspase. Work on MCF-7
breast carcinoma cells which are deficient in caspase 3
due to a deletion in the casp3 gene has revealed the
existence of a crucial caspase 3 driven feedback loop
which mediates the apoptotic process (Janicke et al.,
1998; Slee et al., 1999). Thus, caspase 3 is a central
player in apoptosis regulation and the level of procaspase
3 in the cell determines the impact of a given apoptotic
stimulus.
It is long known that infection with A and B type influenza viruses results in the induction of apoptosis both in
permissive and non-permissive cultured cells as well as
in vivo (Takizawa et al., 1993; Fesq et al., 1994; Hinshaw
et al., 1994; Mori et al., 1995; Ito et al., 2002). Interestingly, viral activation of MAPKs or upstream kinases has
been linked to the onset of apoptosis. In a mouse model
for a neurovirulent influenza infection, JNK but not p38
activity correlated with apoptosis induction in the infected
brain (Mori et al., 2003). In embryonic fibroblasts deficient
for the MAPK kinase kinase ASK-1 (Fig. 1) virus-induced
p38 and JNK activation was blunted concomitant with an
inhibition of caspase 3 activation and virus-induced apoptosis (Maruoka et al., 2003). As an extrinsic mechanism
of viral apoptosis induction it has been noted quite early
on that the Fas/FasL apoptosis inducing receptor/ligand
system (Takizawa et al., 1993; 1995; Wada et al., 1995;
Fujimoto et al., 1998) is expressed in a PKR-dependent
manner in infected cells (Takizawa et al., 1996). This most
likely contributes to virus-induced cell death via the receptor mediated FADD/caspase 8-dependent pathway (Balachandran et al., 2000). Another mode of viral apoptosis
induction might occur via activation of TGF-β, a known
apoptosis inducer that is converted from its latent form by
the viral neuraminidase (Schultz-Cherry and Hinshaw,
1996). Within the infected cell the apoptotic program is
mediated by activation of caspases (Takizawa et al., 1999;
Zhirnov et al., 1999; Lin et al., 2002) with a most crucial
role of caspase 3 (Wurzer et al., 2003).
Although it is now well established that influenza virus
infection induces caspases and subsequent apoptosis,
the consequence of this activation for virus replication or
host cell defence is still under a heavy debate (reviewed
in Schultz-Cherry et al., 1998; Ludwig et al., 1999; Lowy,
2003). Early studies demonstrated that overexpression of
the antiapoptotic protein Bcl-2 results in impaired virus
production correlating with a misglycosylation of the viral
surface protein haemagglutinin (Hinshaw et al., 1994;
Olsen et al., 1996). Furthermore, it has been shown that
the viral NS1 protein has pro-apoptotic features and
induces apoptosis when ectopically expressed (SchultzCherry et al., 2001). These data have been challenged by
the finding that a recombinant influenza virus lacking the
NS is a stronger apoptosis inducer than the wild type
suggesting an antiapoptotic function of NS1 (Zhirnov
et al., 2002a). The findings link viral apoptosis induction
to the antiviral type I interferon (IFNα/β) response,
because the NS1 protein was shown to be an efficient
IFN-α/β antagonist (Garcia-Sastre, 2004) and type I interferons are believed, besides the Fas/FasL system, to be
main inducers of influenza virus induced apoptosis (Balachandran et al., 2000). Another finding in favour of an
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
382 S. Ludwig, S. Pleschka, O. Planz and T. Wolff
antiviral role of apoptosis is caspase-mediated cleavage
of the NP of human influenza virus strains (Zhirnov et al.,
1999). The truncated form of the NP is not packaged into
viral particles, an observation that has led to the suggestion that caspases may act to limit amounts of virus protein
for proper assembly. Furthermore it has been demonstrated in an in vitro binding assay that the viral M1 protein
specifically binds to caspase 8 and weakly to caspase 7,
suggesting interference of M1 with a caspase 8 mediated
apoptosis pathway (Zhirnov et al., 2002b).
The PB1-F2 protein has recently been characterized as
a new pro-apoptotic influenza A virus protein that is
expressed from a + 1 reading frame of the PB1 polymerase gene segment (Chen et al., 2001). PB1-F2
induces apoptosis via the mitochondrial pathway when
added to cells and infection with recombinant viruses lacking this protein results in reduced apoptotic rates of lymphocytes (Chen et al., 2001) (Fig. 3). The protein contains
a mitochondrial target sequence (Gibbs et al., 2003;
Yamada et al., 2004) and interacts with the inner mitochondrial membrane adenine nucleotide translocator 3
(ANT3) and the outer mitochondrial membrane voltagedependent anion channel 1 (VDAC), both of which are
implicated in the mitochondrial permeability transition during apoptosis (Zamarin et al., 2005).
These results have let to the assumption that apoptosis
induction by PB1-F2 may be required for the specific
depletion of lymphocytes during an influenza virus infection, a process which is observed in infected animals (Van
Campen et al., 1989; Tumpey et al., 2000). Others have
suggested that apoptosis may serve also to boost the
induction of cytotoxic T cell responses, because apoptotic
cells or materials are efficiently taken up by macrophages
or DCs by phagocytosis (Albert et al., 1998; Watanabe
et al., 2002). Furthermore, it was reported that viral induction of the apoptotic process limits the release of proinflammatory cytokines and thereby may reduce the
severity of the inflammatory response to infection (Brydon
et al., 2003). However, no direct proof for each of the
suggested functions has been given so far.
A recent study adds a new aspect to the open discussion by the surprising observation that influenza virus
propagation was strongly impaired in the presence of
caspase inhibitors (Wurzer et al., 2003). This dependence
on caspase activity was most obvious in cells where
caspase 3 was partially knocked-down by siRNA (Wurzer
et al., 2003). Consistent with these findings, poor replication efficiencies of influenza A viruses in cells deficient for
caspase 3 could be boosted 30-fold by ectopic expression
of the protein. Mechanistically, the block in virus propagation appeared to be due to the retention of viral RNP
complexes in the nucleus preventing formation of progeny
virus particles (Wurzer et al., 2003) (Fig. 3). As influenza
viral apoptosis induction has been linked to the pro-viral
activity of NF-κB due to a virus-supportive effect of NFκB-dependent proapoptotic factor (see above Wurzer
et al., 2004) a chain of events from NF-κB induction to
caspase-mediated regulation of RNP export unravels
(Fig. 3).
Interestingly, the findings are consistent with a much
earlier report showing that upon infection of cells overexpressing the antiapoptotic protein Bcl-2 the viral RNP
complexes were retained in the nucleus (Hinshaw et al.,
1994) resulting in repressed virus titres (Olsen et al.,
1996). The observation of a caspase requirement for
RNP nuclear export was quite puzzling because this
export process was shown before to be mediated by the
active cellular export machinery involving the viral nuclear
export protein (NS2/NEP) (O’Neill et al., 1998; Neumann
et al., 2000) and the antiapoptotic Raf/MEK/ERK cascade
(Pleschka et al., 2001). Caspase activation does not support, but rather inhibit the active nuclear export machinery by cleavage of transport proteins. This suggests the
existence of an alternative strategy by which caspases
may regulate RNP export, e.g. by directly or indirectly
increasing the diffusion limit of nuclear pores (Faleiro and
Lazebnik, 2000) to allow passive diffusion of larger proteins. Such a scenario is supported by the finding that
isolated NPs or RNP complexes, which are nuclear if
ectopically expressed, can partially translocate to the
cytoplasm upon stimulation with an apoptosis inducer in
a caspase 3-dependent manner (Wurzer et al., 2003).
These findings can be merged into a model in which the
RNPs are transported via an active export mechanism in
intermediate steps of the virus life cycle. Once caspase
activity increases in the cells, proteins of the transport
machinery get destroyed, however, widening of nuclear
pores may allow the viral RNPs to use a second mode of
exit from the nucleus (Faleiro and Lazebnik, 2000)
(Fig. 3). Such mechanism could further enhance RNP
migration to the cytoplasm in late phase of the viral life
cycle and thereby support virus replication. The model of
a complementary use of both, active Raf/MEK/ERKdependent and passive caspase-dependent transport
mechanisms is supported by the observation that concentrations of MEK and caspase inhibitors, which can not
block influenza virus replication completely, impaired
virus propagation much more efficiently when used in
combination (Wurzer et al., 2003). Thus, while both pathways do not interfere with each other (Wurzer et al.,
2003), they appear to synergize to mediate RNP export
via different routes.
Taken together, one may conclude that influenza virus
has acquired the capability to take advantage of supposedly antiviral host cell responses to support its propagation. This includes early induction of caspase activity, but
not necessarily execution of the full apoptotic process that
most likely is an antiviral response.
© 2006 The Authors
Journal compilation © 2006 Blackwell Publishing Ltd, Cellular Microbiology, 8, 375–386
Influenza virus and cell signalling 383
Conclusions
A variety of signalling pathways induced by influenza
viruses have been described in the last few years and
evidences and suggestions for the activating components
and functions in the cell have been provided. Still other
pathways are beginning to unravel, such as the Phosphatidylinositol 3 kinase (PI3K)-Akt pathway or the Rac1/
p21-activated kinase (PAK) module, that are both activated upon influenza virus infection (Ehrhardt et al., 2004;
Guillot et al., 2005) and appear to be required for TBK-1independent (Sarkar et al., 2004) or TBK-1-dependent
IRF-3 activation respectively (Ehrhardt et al., 2004).
The picture emerging implicates, that most of the signalling events are initiated by the infected cell as an alert
signal to fight the invader. Thus, virus induced cellular
signalling can be considered as an antiviral response.
Nevertheless, viruses have not only acquired the capability to suppress these responses but also to misuse the
remaining activities to support their replication. A prominent example is the NF-κB pathway. Viral NF-κB activation
is partially suppressed by the NS1 protein, presumably to
prevent an overshooting expression of IFNβ, but at the
same time the virus appears to take advantage of the
remaining NF-κB activity for apoptosis related and virus
supporting processes. This is an economic way for the
virus to control efficient replication without the need for
specific viral inducers of cellular activities. Thus, there
appears to be no black or white situation. Cellular antiviral
responses may not only be partially misused to support
viral replication at some point during the life cycle, but
even may be turned into a proviral activity. Such a potential bivalent function should be considered when evaluating the impact a given signalling pathway has on virus
growth. If a virus-supportive activity is dominant over an
antiviral action one may even consider to use these cellular signalling components as targets for an antiviral intervention (Ludwig et al., 2003; Nimmerjahn et al., 2004).
Acknowledgements
This work is dedicated to the 75th birthday of Christoph Scholtissek, a pioneer in influenza virus research. We are thankful for
support by different grants from the Deutsche Forschungsgemeinschaft (DFG) and by the Fonds der Chemischen Industrie
(FdChI). Furthermore, this work is part of the activities of the
VIRGIL European Network of Excellence on Antiviral Drug Resistance supported by a grant (LSHMCT-2004-503359) from the
Priority 1 ‘Life Sciences, Genomics and Biotechnology for Health’
programme in the 6th Framework Programme of the EU.
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