Introduction: Influenza A viruses (IAV) are the causative agents of severe respiratory diseases, in some cases even leading to death. The majority of these fatal outcomes are linked to secondary bacterial pneumonia, caused by pathogens such as Staphylococcus aureus (S. aureus). While this problem is known for a long time, there is only scarce knowledge about the interplay of viruses with bacteria during infection on a molecular level. Thus, we assessed how S. aureus may interfere with IAV-induced signalling in vitro.

Schwere und akute Infektionen des Respirationstrakts gehören mit weltweit jährlich 3,9 Mio. fataler Fälle zu den häufigsten Todesursachen durch Infektionserreger. Insbesondere bei Kindern unter 5 Jahren kommt es jährlich zu 120 Mio. Fällen von Pneumonien mit zumeist viraler Ätiologie mit bis zu 1,4 Mio. Todesfällen. Darüber hinaus beobachtet man immer wieder das Auftreten neuer und hochgefährlicher respiratorischer Erreger, wie z.B. dem SARS-Coronavirus oder den hochpathogenen Influenza-Viren. Die Weltgesundheitsorganisation (WHO) hat aufgrund dieser Bedrohung in einer Initiative „Battle against Respiratory Viruses (BRaVe)“ ein „Call to Action“-Papier verfasst, das den dringenden Bedarf an sicheren und effektiven Therapeutika mit möglichst breitem antiviralen Spektrum hervorhebt. Hier rücken auch Pflanzenprodukte aus der traditionellen Medizin zur Abwehr von respiratorischen Viren mehr und mehr in den Fokus wissenschaftlicher Untersuchungen.

Influenza- und Rhinoviren sind als hauptsächliche Verursacher für akute Infektionen des Atmungstraktes verantwortlich. Influenzaviren sind im oberen und unteren Respirationstrakt zu finden und können die Ursache für schwere Infektionen sein. Im Gegensatz dazu werden Erkältungskrankheiten vorwiegend durch Rhinoviren ausgelöst, die hauptsächlich im oberen Atmungstrakt zu finden sind. Gerade die Schweinegrippepandemie hat gezeigt, dass neue Wirkstoffe benötigt werden, die Schutz gegen eine Influenzavirusinfektion bieten. Solche neuen Wirkstoffe sind auch für die Kontrolle von Rhinovirusinfektionen notwendig. Wir zeigen hier, dass Ladania067, ein Extrakt aus Blättern der Schwarzen Johannisbeere (Ribes nigrum L.) antivirale Aktivität gegen Influenza- und Rhinoviren in Zellkultur und im Maus-Modell besitzt.

Previous observations suggested that the entry process of influenza A viruses (IAV) is at least in part a signaling-regulated event. Although it is well known that sialic acids on the cell surface serve as direct receptors for IAV by binding to the viral HA, the requirement of early signaling events for viral entry suggests the involvement of signal transmitting receptors. However, the nature of these receptors that could transmit entry relevant signals across the membrane, are so far unknown. Our recent observation that the phosphatidylinositol-3 kinase (PI3K), that is an effector enzyme of growth factor receptors, is involved in IAV entry [1] lead to the hypothesis that receptor tyrosine kinases may play a role as cellular signaling receptors upon virus binding to cells. In this study we introduce the EGFR, a prominent member of the receptor tyrosine kinase family as a novel player to be involved in IAV entry processes. Inhibition of tyrosine kinases in general by small molecule inhibitors as well as specific inhibition of the EGFR by inhibitors, siRNA mediated knock-down or treatment of cells with EGFR blocking antibodies results in reduced viral uptake and subsequently to reduced progeny virus titers. In contrary, overexpression of the EGFR or treatment with EGF during infection leads to enhanced uptake and increased virus titers. Furthermore, infection results in a redistribution of the EGFR similar to that observed upon stimulation with the ligand EGF. IAV at least in part co-localizes with the EGFR and both, viral particles on the surface and the receptor are localized in lipid rafts. According to our data we propose, that influenza virus is a multivalent agent that induces a clustering of EGFR and other signaling receptors into lipid rafts, by binding to sialic acid coupled proteins. This may lead to a low level induction of the receptor-induced signaling cascades, such as PI3K/Akt that facilitates viral entry. Thus, we could identify for the first time the EGFR as an indirect viral receptor to form a lipid raft-based signaling platform required for efficient IAV uptake.

The non-structural protein 1 (A/NS1) of influenza A viruses harbors several src homology domain (SH)-binding motifs that are required for interaction with cellular proteins, such as the p85 beta subunit of PI3-kinase. Besides the A/NS1 interaction with p85 beta it could be shown, that the SH3-binding motif 2 (aa 212–217 [PPLPPK]) within A/NS1 is essential for binding to the cellular adaptor proteins Crk and CrkL. Both regulate diverse pathways in the cell including activation of the MAP kinase JNK, that was previously shown by us to mediate antiviral responses [1,2]. To elucidate Crk/CrkL functions in the infected cell we knocked-down expression of the adaptor proteins by a siRNA approach. We could demonstrate that only those influenza A viruses that encode a A/NS1-protein harboring the Crk/CrkL SH3-binding motif 2 PPLPPK are attenuated upon downregulation of Crk/CrkL. It could also be observed that the PPLPPK site-harboring candidate strains exhibit a stronger viral activation of the JNK/ATF-2 signaling module compared to other strains and that knock-down of the adaptor proteins resulted in an even stronger activation of this virus-induced antiviral acting pathway. Consistent with this observation, overexpression of Crk or CrkL resulted in a reduced virus-induced JNK activation. Further analysis revealed that the localization of the A/NS1 is altered in Crk overexpressing cells and that the CrkL-phosphorylation pattern is changed upon binding to A/NS1. The data so far suggest that A/NS1 binding to Crk or CrkL contributes to the suppression of the antiviral acting JNK/-ATF-2 pathway. The Crk/CrkL binding capability may have only evolved in virus strains that over-induce this antiviral signaling module to suppress its detrimental action.

We have previously shown that the cellular PI3K/Akt pathway is strongly activated upon influenza A virus infection in later stages of the infection cycle and that this activation was dependent on the expression of the viral non-structural protein 1 (A/NS1) [1]. Later it was demonstrated by us and others that activation occurs upon direct interaction of the A/NS1 to the regulatory subunits of PI3K, p85 alpha and beta [2,3]. Several reports proposed that two src homology (SH)-binding motifs within A/NS1 (aa 89 [YXXXM] (SH2-binding motif) and aa 164–167 [PXXP] (SH3-binding motif 1)) may mediate binding to p85 beta. Our work confirms that tyrosine 89 within the A/NS1 is required for the interaction of A/NS1 with p85 beta, subsequent PI3K-activation. However, mutant viruses that carry a phenylalanine instead of the tyrosine at position 89 of the NS1 only showed marginal differences to wt viruses with regard to their replication fitness. More detailed analysis revealed that both, wt type and mutant viruses induced similar PI3K activation levels late in infection, suggesting that besides expression of the NS1 there are alternative virus-induced mechanisms to activate the kinase. Here we demonstrate that this additional inducer is viral 5'triphosphate RNA that accumulates late in the infection cycle. Thus, PI3K activity is regulated by a NS1 protein-dependent as well as a vRNA-dependent mechanism, presumably via the RIG-I sensory pathway. Since NS1 is also a negative regulator of RIG-I, we suggest that influenza viruses have developed multiple mechanisms to achieve a well-balanced PI3K activation at later phases of the infection cycle.

Infections with influenza A viruses still pose a major threat to humans and several animal species. The occurrence of highly pathogenic influenza viruses of H5N1 subtype capable to infect and kill humans highlights the urgent need for new efficient anti-viral drugs. Here we demonstrate that a polyphenol rich plant extract, CYSTUS052 from the Mediterranean plant Cistus incanus exerts a potent anti-influenza virus activity in cells infected with various influenza viruses including those of the H5N1 type [1]. CYSTUS052 did not exhibit apparent harming effects on cell viability, metabolism or proliferation [2]. Furthermore, viruses did not develop resistance to CYSTUS052 upon consecutive passaging. Mechanistically, the protective effect appears to be due to a binding of the CYSTUS052-ingredients to the virus surface, preventing virus-binding to cellular receptors. Thus, local application of CYSTUS052 to the respiratory tract may be a promising approach to prevent influenza virus infection.

Influenza still represents a major threat to humans. The appearance of highly pathogenic avian influenza viruses of the H5N1 subtype being able to infect humans reveals the urgent need for new and efficient countermeasures against this disease. Several antiviral compounds have been developed against influenza virus, their long-term efficacy is often limited, because of their toxicity or the emergence of drug-resistant virus mutants. Moreover, neuraminidase inhibitors the most common anti-influenza agents are less effective against new H5N1 isolates. In this regard, we were able to show that a polyphenol rich plant extract from a special variety of Cistus incanus named CYSTUS052 exhibits antiviral activity against influenza viruses in vitro, in a mouse model and in clinical patents. The protective effect of CYSTUS052 appears to be mainly due to binding of the polymeric polyphenol components of the extract to the virus surface, thereby inhibiting binding of the hemagglutinin to cellular receptors. In addition, we investigated the antiviral potential of CYSTUS052 in comparison to oseltamivir against various H5N1 influenza viruses. We tested the antiviral efficacy of a single treatment with CYSTUS052 or oseltamivir, against six H5N1 viruses with avian or human origin. Using an in vitro infectivity inhibition assay we found that during the first 24 hours after infection a single treatment of CYSTUS052 is up to 100 fold more effective against these H5N1 viruses compared to oseltamivir. We conclude that CYSTUS052 given prior to infection might be an effective antiviral with prophylactic potential against influenza viruses including A/H5N1.

Expression of the antiviral cytokines IFN-α/β is among the most potent innate defenses of higher vertebrates to virus infections, which is controlled by the inducible transcription factor IFN regulatory factor (IRF)3. Borna disease virus (BDV) establishes persistent noncytolytic infections in animals and tissue culture cells, indicating that it can circumvent this antiviral reaction by an unexplained activity. In this study, we identify the BDV P protein as microbial gene product that associates with and inhibits the principal regulatory kinase of IRF3, Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK-1). We demonstrate that the P protein counteracts TBK-1-dependent IFN-β expression in cells and, hence, the establishment of an antiviral state. Furthermore, our data show that the BDV P protein itself is phosphorylated by TBK-1, suggesting that P functions as a viral decoy substrate that prevents activation of cellular target proteins of TBK-1. Thus, our findings provide evidence for a previously undescribed mechanism by which a viral protein interferes with the induction of the antiviral IFN cascade.

ABSTRACT The inducible transcription factor NF-κB is commonly activated upon RNA virus infection and is a key player in the induction and regulation of the innate immune response. Borna disease virus (BDV) is a neurotropic negative-strand RNA virus, which replicates in the nucleus of the infected cell and causes a persistent infection that can lead to severe neurological disorders. To investigate the activation and function of NF-κB in BDV-infected cells, we stably transfected the highly susceptible neuronal guinea pig cell line CRL with a constitutively active (IKK EE) or dominant-negative (IKK KD) regulator of the IKK/NF-κB signaling pathway. While BDV titers were not affected in cells with impaired NF-κB signaling, the expression of an activated mutant of IκB kinase (IKK) resulted in a strong reduction in the intracellular viral titer in CRL cells. Electrophoretic mobility shift assays and luciferase reporter gene assays revealed that neither NF-κB nor interferon regulatory factors (IRFs) were activated upon acute BDV infection of wild-type or vector-transfected CRL cells. However, when IKK EE-transfected cells were used as target cells for BDV infection, DNA binding to an IRF3/7-responsive DNA element was detectable. Since IRF3/7 is a key player in the antiviral interferon response, our data indicate that enhanced NF-κB activity in the presence of BDV leads to the induction of antiviral pathways resulting in reduced virus titers. Consistent with this observation, the anti-BDV activity of NF-κB preferentially spread to areas of the brains of infected rats where activated NF-κB was not detectable.

Influenza A and B viruses are still a major worldwide threat. We demonstrate that influenza B virus infection induces signaling via the Raf/MEK/ERK cascade, a process required for efficient virus production. Expression of dominant-negative Raf and ERK mutants or treatment with a MEK inhibitor (U0126) strongly impaired viral propagation, while selective activation of the pathway resulted in increased virus titers. MEK inhibition appears to interfere with a distinct viral nuclear export process. Most importantly, no resistant virus variants emerged in the presence of U0126 demonstrating that influenza viruses cannot easily adapt to the missing cellular function.

Activation of the transcription factor NF-kappaB is a hallmark of infections by viral pathogens including influenza viruses. Because gene expression of many proinflammatory and antiviral cytokines is controlled by this factor, the concept emerged that NF-kappaB and its upstream regulator IkappaB kinase are essential components of the innate antiviral immune response to infectious pathogens. In contrast to this common view we report here that NF-kappaB activity promotes efficient influenza virus production. On a molecular level this is due to NF-kappaB-dependent viral induction of the proapoptotic factors tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and FasL, which enhance virus propagation in an autocrine and paracrine fashion. Thus, NF-kappaB acts both proapoptotically and provirally in the context of an influenza virus infection.

The anti-viral type I interferon (IFN) response is initiated by the immediate induction of IFN beta, which is mainly controlled by the IFN-regulatory factor-3 (IRF-3). The signaling pathways mediating viral IRF-3 activation are only poorly defined. We show that the Rho GTPase Rac1 is activated upon virus infection and controls IRF-3 phosphorylation and activity. Inhibition of Rac1 leads to reduced IFN beta promoter activity and to enhanced virus production. As a downstream mediator of Rac signaling towards IRF-3, we have identified the kinase p21-activated kinase (PAK1). Furthermore, both Rac1 and PAK1 regulate the recently described IRF-3 activators, I kappa B kinase- and TANK-binding kinase-1, establishing a first canonical virus-induced IRF-3 activating pathway.

ABSTRACT Transition from G 2 to M phase, a cell cycle checkpoint, is regulated by the Cdc2-cyclin B1 complex. Here, we report that persistent infection with Borna disease virus (BDV), a noncytolytic RNA virus infecting the central nervous system, results in decelerated proliferation of infected host cells due to a delayed G 2 -to-M transition. Persistent BDV-infected rat fibroblast cells showed reduced proliferation compared to uninfected cells. In pull-down assays we observed an interaction of the viral nucleoprotein with the Cdc2-cyclin B1 complex. Transfection of the viral nucleoprotein but not of the phosphoprotein also results in decelerated proliferation. This phenomenon was found in BDV-susceptible primary rat fibroblast cells and also in primary mouse cells, which are not susceptible to BDV infection. This is the first evidence that the noncytolytic Borna disease virus can manipulate host cell functions via interaction of the viral nucleoprotein with mitotic entry regulators. BDV preferentially infects and persists in nondividing neurons. The present report could give an explanation for this selective choice of host cell by BDV.

Apoptosis is a hallmark event observed upon infection with many viral pathogens, including influenza A virus. The apoptotic process is executed by a proteolytic system consisting of a family of cysteinyl proteases, termed caspases. Since the consequences of apoptosis induction and caspase activation for the outcome of an influenza virus infection are not clear, we have addressed this issue by interfering with expression or function of a major virus-induced apoptosis effector, caspase 3. Surprisingly, influenza virus propagation was strongly impaired in the presence of an inhibitor that blocks caspase 3 and in cells where caspase 3 was partially knocked down by small interfering RNAs. 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 retention of the viral RNP complexes in the nucleus, preventing formation of progeny virus particles. Our findings indicate that caspase 3 activation during the onset of apoptosis is a crucial event for efficient influenza virus propagation.

CD95 is a major apoptosis receptor that induces caspase activation and programmed cell death in susceptible cells. CD95-induced apoptosis can be blocked by peptidic caspase inhibitors such as benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone or Ile-Glu-Thr-Asp-fluoromethyl ketone. Here we show that stimulation of CD95 in the presence of these inhibitors induces necrosis and expression of various proinflammatory cytokines in primary T lymphocytes, such as TNF-alpha, IFN-gamma and granulocyte/macrophage colony-stimulating factor. In the absence of caspase inhibition CD95 stimulation did not result in cytokine expression, indicating that this proinflammatory signaling pathway is suppressed by active caspases. Further analysis with A3.01 T cells revealed that the proinflammatory signaling activity of CD95 was mediated by MEK/ERK, p38 and NF-kappaB signaling pathways. These findings point to a pivotal role of caspases not only as mediators of apoptosis but also as enzymes that prevent proinflammatory signaling during CD95-induced apoptosis. Moreover, our findings may be useful for the development of novel pharmacological strategies.

The influenza A virus nonstructural NS1 protein is known to modulate host cell gene expression and to inhibit double-stranded RNA (dsRNA)-mediated antiviral responses. Here we identify NS1 as the first viral protein that antagonizes virus- and dsRNA-induced activation of the stress response-signaling pathway mediated through Jun N-terminal kinase.