46 Review TRENDS in Molecular Medicine
2 February 2003
Influenza-virus-induced signaling
cascades: targets for antiviral therapy?
Stephan Ludwig1, Oliver Planz2, Stephan Pleschka3 and Thorsten Wolff4
1
Institute of Molecular Medicine, Heinrich Heine University, Universitätsstr. 1, D-40225 Düsseldorf, Germany
2
Institute of Immunology, Bundesforschungsanstalt für Viruserkrankungen der Tiere, Paul Ehrlich Str. 28, D-72076 Tübingen,
Germany
3
Institute of Virology (FB10), Justus Liebig University, Frankfurter Str. 107, D-35392 Giessen, Germany
4
Robert Koch Institute, Nordufer 20, D-13353 Berlin, Germany
Influenza viruses continue to pose a severe threat
worldwide, causing thousands of deaths and an enor-
mous economic loss every year. The major problem in
fighting influenza is the high genetic variability of the
virus, resulting in the rapid formation of variants that
escape the acquired immunity against previous virus
strains, or have resistance to antiviral agents. Every
virus depends on its host cell and, hence, cellular func-
tions that are essential for viral replication might be
suitable targets for antiviral therapy. As a result, intra-
cellular signaling cascades induced by the virus, in par-
ticular mitogen-activated protein kinase pathways,
have recently come into focus.
Influenza is a highly contagious, acute respiratory disease
that affects all age groups and can occur repeatedly in any
particular individual. The etiological agent of the disease,
the influenza virus, is responsible for an average of
114 000 hospitalizations and 20 000 death each year in
the USA alone [1]. Epidemics occur almost every year,
owing to an antigenic drift in two viral surface glyco-
proteins, hemagglutinin (HA) and neuraminidase (NA)
(Fig. 1). In recent history, highly pathogenic strains of the
influenza A virus have emerged in an unpredictable
manner, causing pandemics such as the one in 1918 that
caused the death of 20 –40 million people worldwide [2].
Pandemic strains of the virus usually possess antigeni-
cally different, novel glycoproteins, resulting from anti-
genic shift, and hence the available vaccines are
ineffective.
Strains of avian influenza viruses can also appear in
humans, as happened in Hong Kong in 1997 [3]. These
viruses had an extremely high virulence in humans,
killing six out of 18 infected individuals. Fortunately, they
did not acquire the ability to spread in the human
population, although it is possible that a novel virus strain
emerging in the future could have such a capability.
Antiviral agents against influenza virus: the state of the
art
The most commonly employed and efficient way of
controlling influenza is by vaccination. However,
Corresponding author: Stephan Ludwig (stephan.ludwig@uni-duesseldorf.de).
http://tmm.trends.com 1471-4914/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. PII: S1471-4914(02)00010-2
Vol.9 No.
immunoprophylaxis against influenza has several limi-
tations in practice: (1) vaccination rates in the population
are too low to produce a herd immunity; (2) the efficacy of
vaccination is reduced in high-risk groups, such as the
elderly and in immunocompromised individuals; (3) the
constant antigenic drift demands ongoing development of
new vaccines for each season; and (4) there is a constant
risk of new viruses or shift variants emerging, against
which even the most recent vaccines are ineffective. Thus,
there is a clear need for potent antiviral therapies.
The clinically approved anti-influenza drugs available
today are directed against essential viral protein functions
(Table 1). Adamantane compounds, such as amantadine
and rimantadine, have a type-specific inhibitory effect on
(a) (b)
vRNA Segments Gene products
PB2 PB2
PB1 PB1
PA
PA
PB1-F2
HA HA
NP NP
NA NA
M M1
M2
NS NS1
NEP/NS2
TRENDS in Molecular Medicine
Fig. 1. The influenza A virus particle. (a) An electrophoretic separation of the influ-
enza virus genome, which consists of eight RNA segments of negative polarity
(reviewed in [49]). Each segment is named according to the viral protein(s) it
encodes. (b) The location of the different structural proteins within the virus par-
ticle. The nucleoprotein (NP) (green) associates with each of the eight viral RNA
(vRNA) segments to form ribonucleoprotein (RNP) complexes. The subunits of the
viral RNA-dependent RNA-polymerase complex PB1, PB2 and PA (red) are also
associated with the RNPs. The PB1 segment of many, but not all, influenza A virus
strains also contains a þ 1 reading frame encoding the recently discovered PB1-F2
protein [50]. The viral glycoproteins, hemagglutinin (HA) (blue) and neuraminidase
(NA) (yellow), are exposed on the virus surface as trimers and tetramers, respect-
ively. Matrix-protein-1 (M1) (purple) is derived from a colinear mRNA of segment 7
and forms the inner layer of the virion. A spliced mRNA of the same segment gives
rise to the transmembrane M2 protein (orange), which is a pH-dependent ion chan-
nel. Alternative splicing of segment 8 yields the non-structural protein 1 (NS1) and
the nuclear export protein (NEP; also known as NS2) (grey).
 Review TRENDS in Molecular Medicine Vol.9 No.2 February 2003 47

Table 1. Inhibitors of influenza virus propagation

Inhibitor Action
Inhibitors that target a viral protein or function
Sialic-acid analogs Block the receptor-binding pocket of
hemagglutinin (HA)
Quinone- and hydroquinone-derivatives Inhibit the low-pH-dependent
conformational change of HA
Anionic polymers Prevent the hydrophobic membrane
attachment
M2 inhibitorsa (amantadine and Inhibit early Hþ influx into the virion and late
rimantadine) Hþ efflux from transport vesicles
Inhibitors of the viral RNA-polymerase Inhibit the endonucleolytic (‘cap-snatching’)
subunit, PB2 activity of PB2
Neuraminidase inhibitorsa (e.g. zanamivir, Inhibit the cleavage of the glycosidic bond of
oseltamivir) terminal sialic-acid residues on receptor
determinants
Synthetic capped RNA and Inhibit viral polymerase activities
phosphorothioate oligonucleotides
Affected step in viral replication
Attachment of the virion to cell surface
receptors
Acquirement of the fusion-active
conformation of HA
Insertion of the fusion peptide into the cell
membrane
Early release of the viral genome into the
cytoplasm and late induction of HA
fusion conformation
Transcription of viral mRNAs
Detachment of the virion from the cell
Transcription and replication of the viral
genome

Inhibitors of host-cell functions

Protein kinase C (PKC) inhibitorsb (e.g. Block the PKC activity required for a late Entry of the virion by endocytosis
bisindolylmaleimide) endosomal process (in particular the
PKCbII isoform)
Protease inhibitors (exogenous and Inhibit proteolytic cleavage of HA Processing of the HA0 precursor into HA1
endogenous) and HA2
Hþ-ATPase inhibitors Inhibit the acidification of endocytotic pH-dependent activation of the HA fusion
vesicles activity
Ribavarin Inhibits cellular synthesis of the 7mGPPP- Transcription of viral mRNAs
cap of mRNAs
MAPK/ERK kinase (MEK) inhibitorsb Inhibit the Raf –MEK –ERK signaling pathway Nuclear-export-protein-mediated export of
(e.g. U0126) that is required for nuclear export. ribonucleoproteins
Abbreviations: ERK, extracellular-signal-regulated kinase; MAPK, mitogen-activated protein kinase.
a
Inhibitors approved for clinical use.
b
Novel signaling inhibitors effective in preclinical studies.

type-A but not on type-B influenza viruses (reviewed in
[4]). These agent block the ion-channel activity of the viral
M2 protein (Fig. 1), which is mainly required during virus
entry in the early phase of the replication cycle (Fig. 2).
However, amantadine-sensitive influenza A virus strains
rapidly develop resistance in vitro and in vivo [5].
Zanamivir and oseltamivir, two products of modern
drug design, block the receptor-destroying activity of the
viral NA protein, thereby preventing viral release from the
cell membrane [6]. Both drugs efficiently inhibited influenza
virus A and B strains in clinical studies [7,8], and are
approved and commercially available in many countries.
However, escape from the selective pressure of NA inhibitors
has been observed in cell culture and in patients, as a result
of various mutations, although on a much smaller scale than
with adamantane compounds [9,10].
Ongoing studies are attempting to find new anti-
influenza drugs that target the virus replication cycle
at many different steps (Table 1) [4], but none has yet
been approved for clinical use. All these approaches
have the major disadvantage that they attack a viral
function and/or structure. Hence, even though strongly
conserved elements will not change easily, the virus will
eventually adapt to and escapes from the selective pressure
exerted by the drug.
As a result, specific cellular functions essential for virus
replication have attracted increasing interest, because a
missing cellular function is more difficult for the virus to
http://tmm.trends.com
adapt to, and should affect replication independent of the
type, strain and antigenic properties of the invading
influenza virus.
Intracellular signaling cascades and influenza virus
infection
Cell-fate decisions in response to extracellular agents,
including pathogenic invaders, are commonly mediated by
phosphorylation-regulated signaling cascades that trans-
duce signals into stimulus-specific actions, such as changes
in gene expression patterns.
Influenza viruses induce expression of a variety of
cytokine and chemokine genes, such as interferons IFN-a
and IFN-b, tumor-necrosis-factor-a (TNF-a), interleukins
IL-1b, IL-6 and IL-8, monocyte chemo-attractant protein 1
(MCP-1) and RANTES (reviewed in [11,12]). In a recent
transcriptional-profiling study, 84 out of an array of 13 000
genes were shown to be deregulated in response to infec-
tion in a lung epithelial cell line [13], suggesting that the
activity of many intracellular signaling mediators might
be altered. Signaling processes can be initiated by the cell
as a defense against the invader, but can also be used by
the virus to support replication.
Protein kinase C: a regulator of influenza virus entry
The protein kinase C (PKC) superfamily consists of at
least 12 different isoforms, which carry out diverse
regulatory roles in cellular processes by linking into
48 Review TRENDS in Molecular Medicine Vol.9 No.2 February 2003

(a) Adsorption
(i) Budding

Bisindolyl- (h) Packaging ?

maleimide I
Post-translational
Raf
processing
MEK U0126
(f) Translation
PKCβII (b) Endocytosis
ERK

(c) Fusion and

(c) release
(e) (g) RNP
mRNA export

vRNA (–)
(d) Import
cRNA (+) Nucleus

TRENDS in Molecular Medicine

Fig. 2. The lifecycle of the influenza A virus. The virus first binds to sialic-acid-containing receptors (dark blue) on the cell surface via the hemagglutinin protein (a). It then
enters the cell by endocytosis (b) and the viral RNA (vRNA) is released by fusion of the viral and endosomal membranes (c). The viral genome is transported into the
nucleus (d) where transcription and genome amplification take place (e). Viral transcripts are translated in the cytoplasm (f). Late in the infection cycle the viral genome is
transported out of the nucleus in the form of ribonucleoprotein (RNP) complexes (g). RNPs are packaged (h) and progeny virions are then released from the cell surface (i).
The replication cycle is completed within 6–16 h, depending on the particular virus strain and the cell type. Endocytosis of viral particles appears to be controlled by the
protein-kinase-C isoform, PKCbII, and RNP export is dependent upon the Raf –MEK –ERK cascade. Thus, specific inhibition of PKC or of the Raf– MEK– ERK pathway by
bisindolylmaleimides or by the MEK inhibitor U0126, respectively, results in impaired virus propagation.

several downstream signaling pathways [14]. Based on
the action of two protein-kinase inhibitors, H7 and
staurosporine, it has been suggested that PKCs have a
role in the entry process of several enveloped viruses
[15]. The influenza virus and the viral HA can rapidly
activate PKCs upon binding to host-cell surface
receptors (Fig. 3) [16 – 18]. Furthermore, the PKC
inhibitor, bisindolylmaleimide I, has been shown to
prevent influenza virus entry and subsequent infection
in a dose-dependent and reversible manner [19]. It is
likely that the PKCbII isoform performs this function;
overexpression of a phosphorylation-deficient dominant-
negative mutant form of PKCbII showed that it is a
regulator of late endosomal sorting, and hence blocks
virus entry at the level of late endosomes (G. Whittaker,
pers. commun.) (Fig. 3). Therefore, a specific pharma-
cological interference with PKCbII might help to
prevent influenza virus infections at the most effective
level: the initial entry of virions into their target
cells (Fig. 2).
Mitogen-activated protein kinase cascades and influenza
virus infection
Mitogen-activated protein kinase (MAPK) cascades are
important signal transducers in the conversion of a
variety of extracellular signals into changes in gene
http://tmm.trends.com
expression [20]. In this way, MAPK cascades regulate
numerous cellular responses, such as proliferation and
differentiation, and also cell activation and immune
response [21]. Four different members of the MAPK
family, which are organized in separate cascades,
have been identified to date (Fig. 3) [20]. Three of
these, extracellular-signal-regulated kinase (ERK), JUN
N-terminal kinase (JNK), and p38 have been reported to
be activated upon infection by an influenza virus [22 – 24]
and there is also evidence of viral activation of ERK5 (also
known as big mitogen-activated protein kinase) (S. Ludwig
et al., unpublished).
Recent work has helped to clarify the functions of the
p38, JNK and ERK signaling pathways. Using specific
kinase inhibitors, p38 and JNK but not ERK have been
linked with virus-induced expression of RANTES, a
chemokine involved in the attraction of eosinophils during
an inflammatory response (Fig. 3) [22]. The importance of
the JNK subgroup of MAPKs in influenza virus infection
was further suggested by the observation that activator-
protein-1 (AP-1) transcription factors are activated early
in productively infected cells [23]; AP-1 factors include
c-Jun and activating-transcription-factor-2 (ATF-2), whose
transcriptional activity is potentiated by phosphorylation
by JNK [25]. Both c-Jun and ATF-2 are phosphorylated
upon influenza virus infection [23,26]. Furthermore,
 Review TRENDS in Molecular Medicine Vol.9 No.2 February 2003 49

NA HA Influenza virus infection

dsRNA
Extracellular
TLR-3 Intracellular
Intracellular ?
NP
RNA accumulation
M1 HA (dsRNA)
PKR Raf
?
PKCβII
MKK4 MKK7 MKK6 MEK5 MEK1/2
IKK

Endocytosis P P P P P P P P
P T PY
ΙκBα ΙκBα T GY T EY T EY
p50–p65 JNK p38 ERK5 ERK

P P
p50–p65 ATF-2 c-Jun
? RNP export
NF-κB AP-1
TNF-α expression IFN-β expression IFN-β expression RANTES Nucleus
IL-1 expression IL-8 expression RANTES expression expression
TRENDS in Molecular Medicine

Fig. 3. Influenza-virus-induced signaling processes and their functions in the infected host cell. Induction of signaling by influenza viruses can be divided in early events,
such as binding of the virus, hemagglutanin (HA), neuraminidase (NA) or free double-strand RNA (dsRNA) to cellular receptors, and late events, which commonly require
productive virus replication. Entry of the virus into the cell requires the activation of the protein kinase C bII (PKCbII) isoform. Intracellular expression of the viral proteins
HA, nucleoprotein (NP) and matrix-protein-1 (M1), or accumulation of viral RNA species, leads to activation of nuclear factor kB (NF-kB) (made up of subunits p50 and p65)
and upregulated expression of several antiviral cytokines, such as interferon-b (IFN-b) and interleukin-8 (IL-8). In addition, four members of the mitogen-activated protein
kinase (MAPK) family – JUN N-terminal kinase (JNK), p38, extracellular-signal-regulated kinase (ERK) and ERK5 – are also induced in response to infection by the influenza
virus, by phosphorylation at specific threonine and tyrosine residues. JNK and p38 mediate antiviral signaling via the transcription factor AP-1, whereas ERK signaling is
proviral, inducing export of viral ribonucleoprotein (RNP) from the nucleus. Abbreviations: IkB, inhibitor of NF-kB; IKK, IkB inhibitor; MEK, MAPK/ERK kinase; MKK, MAPK
kinase; P, phosphate; PKR, protein kinase R; TLR-3, Toll-like receptor 3; TNF-a, tumor-necrosis-factor-a.

activation of JNK was observed using different virus
strains in a variety of permissive cell lines [22,23]; it
required productive replication and was induced by the
accumulating RNA made by the viral polymerase. Two
MAPK kinases, MKK4 and MKK7, have been identified as
upstream activators in the viral context. The AP-1 factors,
c-Jun and ATF-2, are crucial for the expression of
interferon-b (IFN-b), a potent antiviral cytokine [27],
and hence inhibition of the JNK signaling cascade by
dominant-negative mutant forms of MKK7, JNK or c-Jun
during a virus infection resulted in impaired transcription
from the IFN-b promoter and an enhancement of virus
production. Thus, the JNK pathway appears to be a crucial
mediator of the antiviral response to an influenza virus
infection [23].
Interestingly, suppression of ERK using U0126 [a
specific inhibitor of the upstream MAPK/ERK kinase
(MEK)], or using a dominant-negative mutant form of
ERK or the MEK activator Raf, results in a strong
impairment of virus propagation [24]. At a molecular
level, it is the nuclear export of viral ribonucleoprotein
complexes that is affected by inhibition of the ERK
signaling pathway (Fig. 2), probably as a result of
impaired activity of the viral nuclear export protein,
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NEP [24]. Thus, in contrast to the JNK cascade,
activation of the Raf – MEK – ERK pathway is required
for efficient virus growth. The replication of other
viruses, such as Borna-disease virus [28], Visna virus
[29] or Coxsackie B3 virus [30], is also impaired by
MEK inhibition. Interestingly, MEK inhibitors meet
two further criteria of a potential anti-influenza agent:
(1) the inhibitors were not toxic in a variety of cell
types tested [24,28] and (2) there is no tendency
towards induction of the formation of resistant virus
variants. Hence, the Raf – MEK – ERK pathway appears
to be a suitable target for antiviral intervention.
IKK –NF-kB pathway
Activation of the transcription factor, nuclear factor kB
(NF-kB), is a hallmark of most infections by viral
pathogens [31], including influenza viruses (reviewed
in [11,12]). Induction of NF-kB by a virus involves
activation of IKK [inhibitor of NF-kB (IkB) kinase].
Isolated components of the influenza virus, such as
double-strand RNA [32] or overexpressed viral HA,
nucleoprotein (NP) or matrix-protein-1 (M1) [33], can
also activate IKK (Fig. 3). Gene expression of many
pro-inflammatory or antiviral cytokines, such as IFN-b
50 Review TRENDS in Molecular Medicine
and tumor-necrosis-factor-a (TNF-a), is controlled by
NF-kB and, therefore, it was suggested that IKK and
NF-kB are essential components in the innate immune
response to virus infections [32]. In accordance with
this idea, influenza-virus-induced activity of the IFN-b
promoter is strongly impaired in cells expressing
transdominant-negative mutants of IKK2 or IkBa
[34]. However, recent experiments have shown that
titers of influenza viruses grown in cells with impaired
NF-kB signaling were dramatically lower than those
from wild-type cells (S. Ludwig et al., unpublished).
This suggests that NF-kB activity is also required for
efficient virus replication, probably by regulating
expression of an as yet unidentified proviral cellular
protein.
Influenza viral inducers or inhibitors of signaling
A specific pharmacological interference with virus-induced
signaling could be achieved by blocking the interaction of
the inducing viral component with its cellular receptor.
Unfortunately, little is known about the molecular inter-
actions of influenza viral inducers with cellular sensors,
although several influenza virus proteins have been
reported to exhibit signal-inducing capacity. It was noted
more than 20 years ago that influenza strains differ in
their ability to induce IFN-a and IFN-b, and that this
correlates with the NA activity of these strains [35]. It was
subsequently shown that treatment of cells with recombin-
ant NA, but not HA, induces the production of IL-1 and
TNF-a in murine macrophages (Fig. 3) [36]. In another
study, NA was shown to convert transforming-growth-
factor-b (TGF-b) from the latent to the active form to an
extent sufficient to induce TGF-b-dependent apoptosis
[37]. Hence, although the exact molecular mechanisms
and the pathways involved in NA-induced signaling
remain unknown, both direct and indirect events might
be involved.
HA can stimulate signaling from both inside the cell
and at the cell surface (Fig. 3): binding of recombinant
HA to cell-surface receptors leads to a rapid induction
of PKC signaling [17], whereas overexpression of HA
inside the cell leads to the activation of NF-kB [38],
probably via an endoplasmic-reticulum (ER)-overload
mechanism. Interestingly, NF-kB can also be activated
by expression of M1 and NP, which are not processed
in the ER [33]. This suggests that there might be
different, and as yet unidentified, cellular sensors for
all three proteins. Induction of IKK and NF-kB by HA,
NP and M1 appears to be specific because MAPK
signaling pathways, and transcription factors from the
AP-1 and Ets families, are not activated [33].
The most potent viral signaling component, however, is
probably dsRNA. It is generally believed that most RNA
viruses produce dsRNA-like intermediates and that these
represent a shared molecular pattern that triggers
antiviral signaling within the cell [39]. Indeed, dsRNA
induces activation of pathways that regulate the type-I-IFN
response and expression of other antiviral cytokines.
Several signaling pathways are involved, including the
IKK –NF-kB pathway, and both the JNK and p38 MAPK
cascades [32].
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Vol.9 No.2 February 2003
dsRNA is the best characterized of the viral signaling
inducers in terms of its cellular receptors (Fig. 3). At least
two cellular gene products bind to dsRNA and induce
activation of NF-kB: the IFN-inducible RNA-dependent
protein kinase R (PKR) [27] and the Toll-like receptor 3
(TLR-3) [40]. PKR acts as an intracellular sensor, whereas
TLR-3 is thought to detect extracellular dsRNA species
released from dying cells [40]. Extracellular RNAs have
been detected as relatively stable structures in the super-
natants of influenza-virus-infected cells [41]. Thus, the cell
has developed a two-step method of dsRNA sensing: TLR-3
confers immediate protective signaling initiated from the
outside, which does not require virus entry, whereas PKR
senses the dsRNA produced at later stages within the
infected cell (Fig. 3).
The influenza non-structural-protein-1: a universal
antagonist of dsRNA-induced signaling processes
Many of the signaling functions in a virus-infected cell,
especially those induced by dsRNA, are thought to be
part of an antiviral program and, hence, it is not
surprising that influenza viruses have evolved strate-
gies to counteract this response. For example, recent
analyses have shown that a major in vivo function of
the RNA-binding influenza non-structural-protein-1
(NS1) is to antagonize the antiviral type-I-IFN system
[42]. Strikingly, deletion of the coding region of NS1 in
a recombinant virus resulted in a complete loss of
virulence in wild-type mice [43]. However, this delNS1
mutant virus could still kill mice with genetic knock-
outs of either the STAT1 (signal-transducer-and-
activator-of-transcription-1) protein (which transduces
IFN-mediated signals to the transcriptional machinery)
[43], or PKR (whose expression is upregulated by IFN)
[44]. Moreover, multicyclic replication of the mutant
virus was strongly attenuated in IFN-competent but
not in IFN-deficient tissue-culture cells. Further
comparisons of wild-type and delNS1 viruses revealed
that NS1 inhibits activation of the gene encoding
IFN-b, which is controlled by the transcriptional
activators NF-kB, interferon-regulatory-factor-3, and
ATF-2/c-Jun [26,34,45]. Significantly, induction of the
IFN-b gene in the absence of NS1 correlates with a
strong upregulation of the three dsRNA-responsive
transcription factors, demonstrating that NS1 inter-
feres with upstream signaling events. The inhibitory
activity of NS1 was shown to be dependent on its
RNA-binding domain, and it is thought that NS1
sequesters intracellular dsRNA produced during viral
replication, thereby denying access of the antiviral
enzyme PKR and of other cellular dsRNA-sensor
proteins. Inhibition of the processing of residual IFN
mRNAs or IFN-induced mRNAs by the NS1 protein
might further contribute to its antagonistic activity
[46]. However, influenza patients actually secrete some
IFN-a [47], and some IFN-dependent genes are still
activated upon infection by the NS1-expressing wild-
type virus [13]. Therefore, the IFN-antagonistic activ-
ity of NS1 might not be considered an absolute but
rather a relative, exhaustible function that boosts viral
replication in the early phase of infection.
 Review TRENDS in Molecular Medicine
In conclusion, the NS1 protein is a well-characterized
example of an IFN-antagonistic protein, and has a
crucial role in viral virulence. Further support for this
role of the NS1 protein came from the finding that the
high virulence of the avian H5N1 influenza viruses
that were transmitted to humans in Hong Kong in
1997 was caused largely by a specific amino-acid
position in the NS1 protein [48]. These viruses were
particularly resistant to the antiviral action of IFNs
and TNF-a, and this resistance could be transmitted to
other strains of the virus by transfer of the H5N1-virus
NS gene segment [48].
Hence, strengthening the innate antiviral response by
using pharmacological measures to interfere with the
NS1-mediated block of antiviral signaling might be a
legitimate strategy for restraining the replication and
spread of influenza viruses.
Concluding remarks
Influenza virus infections impose a considerable socio-
economic burden upon society, despite annual vaccin-
ation campaigns. Therefore, efficient antiviral therapy is
required for control of the virus. Current anti-influenza
drugs target viral components (Table 1) and are there-
fore prone to compensatory viral escape mechanisms.
However, blocking specific host-cell functions that are
required for viral replication, but not for short-term cell
survival, might reduce the frequency at which resistant
variants arise.
A variety of cellular signaling cascades are activated
by influenza virus infection. Some of these pathways
are purely antiviral, whereas others appear to support
virus replication. Hence, it should be possible to inter-
fere with virus replication either by enhancing anti-
viral signaling or by inhibiting proviral signaling,
especially if this intervention is well tolerated in the
normal cell.
The suitability of this concept has so far been best
proven for the Raf– MEK– ERK signaling cascade. Block-
ing this cascade using dominant-negative mutants or a
specific inhibitor resulted in a strong impairment of virus
propagation, and no severe cytotoxicity or tendency to
induce resistant variants was detected. Although target-
ing of a cellular factor will always raise concerns about the
side effects of a drug, it appears that local administration
of an agent, such as a MEK inhibitor, to the primary site of
influenza virus infection, the lung, is well tolerated. Here,
the drug primarily affects differentiated lung epithelial
cells, in which a proliferative signaling cascade like
Raf – MEK–ERK might be dispensable. Using this
approach, we have recently demonstrated in a mouse
infection model that local administration of the MEK
inhibitor U0126 reduces virus titers in the lung (S. Ludwig
et al., unpublished).
In addition to the Raf – MEK – ERK cascade, other
signaling pathways and mediators could be possible
targets, including PKCs, other MAPK pathways and the
IKK – NF-kB module, or the apoptotic cascades, which
are suspected to have some proviral activity in infected
cells.
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Vol.9 No.2 February 2003 51
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (grant
Lu477/4 – 3 to S.L.) and by the Fonds der Chemischen Industrie.
References
1 Simonsen, L. et al. (1997) The impact of influenza epidemics on
mortality: introducing a severity index. Am. J. Public Health 87,
1944– 1950
2 Taubenberger, J.K. et al. (2000) The 1918 influenza virus: a killer
comes into view. Virology 274, 241 – 245
3 Hatta, M. and Kawaoka, Y. (2002) The continued pandemic threat
posed by avian influenza viruses in Hong Kong. Trends Microbiol. 10,
340 – 344
4 Luscher-Mattli, M. (2000) Influenza chemotherapy: a review of the
present state of art and of new drugs in development. Arch. Virol. 145,
2233– 2248
5 Fleming, D.M. (2001) Managing influenza: amantadine, rimantadine
and beyond. Int. J. Clin. Pract. 55, 189 – 195
6 Gubareva, L.V. et al. (2000) Influenza virus neuraminidase inhibitors.
Lancet 355, 827 – 835
7 Dreitlein, W.B. et al. (2001) Zanamivir and oseltamivir: two new
options for the treatment and prevention of influenza. Clin. Ther. 23,
327 – 355
8 McClellan, K. and Perry, C.M. (2001) Oseltamivir: a review of its use in
influenza. Drugs 61, 263– 283
9 Gubareva, L.V. et al. (1998) Evidence for zanamivir resistance in an
immunocompromised child infected with influenza B virus. J. Infect.
Dis. 178, 1257– 1262
10 Gubareva, L.V. et al. (2002) Detection of influenza virus resistance to
neuraminidase inhibitors by an enzyme inhibition assay. Antiviral
Res. 53, 47 – 61
11 Ludwig, S. et al. (1999) A fatal relationship – influenza virus
interactions with the host cell. Viral Immunol. 12, 175 – 196
12 Julkunen, I. et al. (2000) Inflammatory responses in influenza A virus
infection. Vaccine 19, S32– S37
13 Geiss, G.K. et al. (2002) Cellular transcriptional profiling in influenza
A virus-infected lung epithelial cells: the role of the nonstructural NS1
protein in the evasion of the host innate defense and its potential
contribution to pandemic influenza. Proc. Natl. Acad. Sci. U. S. A. 99,
10736 – 10741
14 Toker, A. (1998) Signaling through protein kinase C. Front. Biosci. 3,
D1134– D1147
15 Constantinescu, S.N. et al. (1991) Effects of protein kinase C inhibitors
on viral entry and infectivity. FEBS Lett. 292, 31 – 33
16 Rott, O. et al. (1995) B cell superstimulatory influenza virus
(H2-subtype) induces B cell proliferation by a PKC-activating,
Ca2þ-independent mechanism. J. Immunol. 154, 2092 – 2103
17 Arora, D.J. and Gasse, N. (1998) Influenza virus hemagglutinin
stimulates the protein kinase C activity of human polymorphonuclear
leucocytes. Arch. Virol. 143, 2029 – 2037
18 Kunzelmann, K. et al. (2000) Influenza virus inhibits amiloride-
sensitive Naþ channels in respiratory epithelia. Proc. Natl. Acad. Sci.
U. S. A. 97, 10282 – 10287
19 Root, C.N. et al. (2000) Entry of influenza viruses into cells is inhibited
by a highly specific protein kinase C inhibitor. J. Gen. Virol. 81,
2697– 2705
20 Widmann, C. et al. (1999) Mitogen-activated protein kinase: conserva-
tion of a three-kinase module from yeast to human. Physiol. Rev. 79,
143– 180
21 Dong, C. et al. (2002) MAP kinases in the immune response. Annu. Rev.
Immunol. 20, 55 – 72
22 Kujime, K. et al. (2000) p38 mitogen-activated protein kinase and
c-jun-NH2-terminal kinase regulate RANTES production by influenza
virus-infected human bronchial epithelial cells. J. Immunol. 164,
3222– 3228
23 Ludwig, S. et al. (2001) Influenza virus-induced AP-1-dependent gene
expression requires activation of the JNK signaling pathway. J. Biol.
Chem. 276, 10990 – 10998
24 Pleschka, S. et al. (2001) Influenza virus propagation is impaired by
inhibition of the Raf/MEK/ERK signalling cascade. Nat. Cell Biol. 3,
301– 305
25 Karin, M. et al. (1997) AP-1 function and regulation. Curr. Opin. Cell
Biol. 9, 240– 246
52 Review TRENDS in Molecular Medicine Vol.9 No.2 February 2003

26 Ludwig, S. et al. (2002) The influenza A virus NS1 protein inhibits
activation of Jun N-terminal kinase (JNK) and AP-1 transcription
factors. J. Virol. 76, 11166 – 11171
27 Stark, G.R. et al. (1998) How cells respond to interferons. Annu. Rev.
Biochem. 67, 227– 264
28 Planz, O. et al. (2001) MEK-specific inhibitor U0126 blocks spread of
Borna disease virus in cultured cells. J. Virol. 75, 4871– 4877
29 Barber, S.A. et al. (2002) Visna virus-induced activation of MAPK is
required for virus replication and correlates with virus-induced
neuropathology. J. Virol. 76, 817– 828
30 Luo, H. et al. (2002) Coxsackievirus B3 replication is reduced by
inhibition of the extracellular signal-regulated kinase (ERK) signaling
pathway. J. Virol. 76, 3365– 3373
31 Hiscott, J. et al. (2001) Hostile takeovers: viral appropriation of the
NF-kB pathway. J. Clin. Invest. 107, 143 – 151
32 Chu, W.M. et al. (1999) JNK2 and IKKb are required for activating the
innate response to viral infection. Immunity 11, 721– 731
33 Flory, E. et al. (2000) Influenza virus-induced NF-kB-dependent gene
expression is mediated by overexpression of viral proteins and involves
oxidative radicals and activation of IkB kinase. J. Biol. Chem. 275,
8307 – 8314
34 Wang, X. et al. (2000) Influenza A virus NS1 protein prevents
activation of NF-kB and induction of a/b interferon. J. Virol. 74,
11566– 11573
35 Chomik, M. (1981) Interferon induction by influenza virus: signifi-
cance of neuraminidase. Arch. Immunol. Ther. Exp. 29, 109– 114
36 Houde, M. and Arora, D.J. (1990) Stimulation of tumor necrosis factor
secretion by purified influenza virus neuraminidase. Cell. Immunol.
129, 104 – 111
37 Schultz-Cherry, S. and Hinshaw, V.S. (1996) Influenza virus neur-
aminidase activates latent transforming growth factor b. J. Virol. 70,
8624 – 8629
38 Pahl, H.L. and Baeuerle, P.A. (1995) Expression of influenza virus
hemagglutinin activates transcription factor NF-kB. J. Virol. 69,
1480 – 1484
39 Majde, J.A. (2000) Viral double-stranded RNA, cytokines, and the flu.
J. Interferon Cytokine Res. 20, 259 – 272
40 Alexopoulou, L. et al. (2001) Recognition of double-stranded RNA
and activation of NF-kB by Toll-like receptor 3. Nature 413,
732 – 738
41 Majde, J.A. et al. (1998) Spontaneous release of stable viral double-
stranded RNA into the extracellular medium by influenza virus-
infected MDCK epithelial cells: implications for the viral acute phase
response. Arch. Virol. 143, 2371– 2380
42 Garcia-Sastre, A. (2001) Inhibition of interferon-mediated antiviral
responses by influenza A viruses and other negative-strand RNA
viruses. Virology 279, 375– 384
43 Garcia-Sastre, A. et al. (1998) Influenza A virus lacking the NS1
gene replicates in interferon-deficient systems. Virology 252,
324 – 330
44 Bergmann, M. et al. (2000) Influenza virus NS1 protein counter-
acts PKR-mediated inhibition of replication. J. Virol. 74,
6203 – 6206
45 Talon, J. et al. (2000) Activation of interferon regulatory factor 3 is
inhibited by the influenza A virus NS1 protein. J. Virol. 74,
7989 – 7996
46 Kim, M.J. et al. (2002) Human influenza viruses activate an interferon-
independent transcription of cellular antiviral genes: Outcome with
influenza A virus is unique. Proc. Natl. Acad. Sci. U. S. A. 99,
10096 – 10101
47 Hayden, F.G. et al. (1998) Local and systemic cytokine responses
during experimental human influenza A virus infection. Relation
to symptom formation and host defense. J. Clin. Invest. 101,
643 – 649
48 Heui-Seo, S. et al. (2002) Lethal H5N1 influenza viruses escape host
anti-viral cytokine responses. Nat. Med. 8, 950– 954
49 Lamb, R.A. and Krug, R.M. (2001) Orthomyxoviridae: the viruses and
their replication. In Virology (Fields, B.N., ed.), pp. 1487 – 1531,
Lippincott – Raven
50 Chen, W. et al. (2001) A novel influenza A virus mitochondrial protein
that induces cell death. Nat. Med. 7, 1306 – 1312

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