Influenza is an acute respiratory infection causing high morbidity and mortality in annual outbreaks worldwide. Antiviral drugs are limited and pose the risk of resistance development, calling for new treatment options. IFN-α subtypes are immune-stimulatory cytokines with strong antiviral activities against IAV in vitro and in vivo. However, the clinical use of IFN-α2, the only licensed subtype of this multi-gene family, could not prevent or limit IAV infections in humans. However, the other subtypes were not investigated.Therefore, this study evaluated the induction and antiviral potential of all human IFN-α subtypes during H3N2 IAV infection in human lung explants. We found that subtypes with weak antiviral activities were preferentially induced during IAV infection in human lungs. Intriguingly, non-induced subtypes α16, α5 and α4 suppressed viral replication up to 230-fold more efficiently than α2. Furthermore, our results demonstrate that subtypes with stronger antiviral activities induce higher expression of IAV-specific restriction factors and that MxA expression is a determinant of the subtype-specific antiviral activity towards H3N2 IAV. These results corroborate that IFN-α subtypes exhibit differential antiviral activities and emphasize that subtypes α16, α5 and α4 should be further investigated for the prevention and treatment of severe infections with seasonal H3N2 IAV.

Severe influenza virus (IV) infections still represent a major challenge to public health. To combat IV infections, vaccines and antiviral compounds are available. However, vaccine efficacies vary with very limited to no protection against newly emerging zoonotic IV introductions. In addition, the development of resistant virus variants against currently available antivirals can be rapidly detected, in consequence demanding the design of novel antiviral strategies. Virus supportive cellular signaling cascades, such as the NF-κB pathway, have been identified to be promising antiviral targets against IV in in vitro and in vivo studies and clinical trials. While administration of NF-κB pathway inhibiting agents, such as LASAG results in decreased IV replication, it remained unclear whether blocking of NF-κB might sensitize cells to secondary bacterial infections, which often come along with viral infections. Thus, we examined IV and Staphylococcus aureus growth during LASAG treatment. Interestingly, our data reveal that the presence of LASAG during superinfection still leads to reduced IV titers. Furthermore, the inhibition of the NF-κB pathway resulted in decreased intracellular Staphylococcus aureus loads within epithelial cells, indicating a dependency on the pathway for bacterial uptake. Unfortunately, so far it is not entirely clear if this phenomenon might be a drawback in bacterial clearance during infection.

Human beings are exposed to microorganisms every day. Among those, diverse commensals and potential pathogens including Staphylococcus aureus (S. aureus) compose a significant part of the respiratory tract microbiota. Remarkably, bacterial colonization is supposed to affect the outcome of viral respiratory tract infections, including those caused by influenza viruses (IV). Since 30% of the world's population is already colonized with S. aureus that can develop metabolically inactive dormant phenotypes and seasonal IV circulate every year, super-infections are likely to occur. Although IV and S. aureus super-infections are widely described in the literature, the interactions of these pathogens with each other and the host cell are only scarcely understood. Especially, the effect of quasi-dormant bacterial subpopulations on IV infections is barely investigated. In the present study, we aimed to investigate the impact of S. aureus small colony variants on the cell intrinsic immune response during a subsequent IV infection in vitro. In fact, we observed a significant impact on the regulation of pro-inflammatory factors, contributing to a synergistic effect on cell intrinsic innate immune response and induction of harmful cell death. Interestingly, the cytopathic effect, which was observed in presence of both pathogens, was not due to an increased pathogen load.

Resolution failure of exacerbated inflammation triggered by Influenza A virus (IAV) prevents return of pulmonary homeostasis and survival, especially when associated with secondary pneumococcal infection. Therapeutic strategies based on pro-resolving molecules have great potential against acute inflammatory diseases. Angiotensin-(1-7) [Ang-(1-7)] is a pro-resolving mediator that acts on its Mas receptor (MasR) to promote resolution of inflammation. We investigated the effects of Ang-(1-7) and the role of MasR in the context of primary IAV infection and secondary pneumococcal infection and evaluated pulmonary inflammation, virus titers and bacteria counts, and pulmonary damage. Therapeutic treatment with Ang-(1-7) decreased neutrophil recruitment, lung injury, viral load and morbidity after a primary IAV infection. Ang-(1-7) induced apoptosis of neutrophils and efferocytosis of these cells by alveolar macrophages, but had no direct effect on IAV replication in vitro. MasR-deficient (MasR-/-) mice were highly susceptible to IAV infection, displaying uncontrolled inflammation, increased viral load and greater lethality rate, as compared to WT animals. Ang-(1-7) was not protective in MasR-/- mice. Interestingly, Ang-(1-7) given during a sublethal dose of IAV infection greatly reduced morbidity associated with a subsequent S. pneumoniae infection, as seen by decrease in the magnitude of neutrophil influx, number of bacteria in the blood leading to a lower lethality. Altogether, these results show that Ang-(1-7) is highly protective against severe primary IAV infection and protects against secondary bacterial infection of the lung. These effects are MasR-dependent. Mediators of resolution of inflammation, such as Ang-(1-7), should be considered for the treatment of pulmonary viral infections.

During influenza A virus (IAV) infections, viral proteins are targeted by cellular E3 ligases for modification with ubiquitin. Here, we decipher and functionally explore the ubiquitination landscape of the IAV polymerase proteins during infection of human alveolar epithelial cells by applying mass spectrometry analysis of immuno-purified K-ε-GG (di-glycyl)-remnant-bearing peptides. We have identified 59 modified lysines across the three subunits, PB2, PB1 and PA of the viral polymerase of which 17 distinctively affect mRNA transcription, vRNA replication and the generation of recombinant viruses via non-proteolytic mechanisms. Moreover, further functional and in silico analysis indicate that ubiquitination at K578 in the PB1 thumb domain is mechanistically linked to dynamic structural transitions of the viral polymerase that are required for vRNA replication. Mutations K578A and K578R differentially affect the generation of recombinant viruses by impeding cRNA and vRNA synthesis, NP binding as well as polymerase dimerization. Collectively, our results demonstrate that the ubiquitin-mediated charge neutralization at PB1-K578 disrupts the interaction to an unstructured loop in the PB2 N-terminus that is required to coordinate polymerase dimerization and facilitate vRNA replication. This provides evidence that IAV exploits the cellular ubiquitin system to modulate the activity of the viral polymerase for viral replication.

Influenza A viruses (IAV) can cause severe global pandemic outbreaks. The currently licensed antiviral drugs are not very effective and prone to viral resistance. Thus, novel effective and broadly active drugs are urgently needed. We have identified the cellular Raf/MEK/ERK signaling cascade as crucial for IAV replication and suitable target for an antiviral intervention. Since this signaling cascade is aberrantly activated in many human cancers, several clinically approved inhibitors of Raf and MEK are now available. Here we explored the anti-IAV action of the licensed B-RafV600E inhibitor Vemurafenib. Treatment of B-RafWT cells with Vemurafenib induced a hyperactivation of the Raf/MEK/ERK cascade rather than inhibiting its activation upon IAV infection. Despite this hyperactivation, which has also been confirmed by others, Vemurafenib still strongly limited IAV-induced activation of other signaling cascades especially of p38 and JNK mitogen-activated protein kinase (MAPK) pathways. Most interestingly, Vemurafenib inhibited virus-induced apoptosis via impaired expression of apoptosis-inducing cytokines and led to hampered viral protein expression most likely due to the decreased activation of p38 and JNK MAPK. These multiple actions resulted in a profound and broadly active inhibition of viral replication, up to a titer reduction of three orders of a magnitude. Thus, while Vemurafenib did not act similar to MEK inhibitors, it displays strong antiviral properties via a distinct and multi-target mode of action.

Influenza is caused by influenza A virus (IAV), an enveloped, negative-stranded RNA virus that derives its envelope lipids from the host cell plasma membrane. Here, we examined the functional role of cellular cholesterol in the IAV infection cycle. We show that shifting of cellular cholesterol pools via the Ca(2+)-regulated membrane-binding protein annexin A6 (AnxA6) affects the infectivity of progeny virus particles. Elevated levels of cellular AnxA6, which decrease plasma membrane and increase late endosomal cholesterol levels, impaired IAV replication and propagation, whereas RNA interference-mediated AnxA6 ablation increased viral progeny titers. Pharmacological accumulation of late endosomal cholesterol also diminished IAV virus propagation. Decreased IAV replication caused by upregulated AnxA6 expression could be restored either by exogenous replenishment of host cell cholesterol or by ectopic expression of the late endosomal cholesterol transporter Niemann-Pick C1 (NPC1). Virus released from AnxA6-overexpressing cells displayed significantly reduced cholesterol levels. Our results show that IAV replication depends on maintenance of the cellular cholesterol balance and identify AnxA6 as a critical factor in linking IAV to cellular cholesterol homeostasis.

Infection with influenza A viruses (IAV) provokes activation of cellular defence mechanisms contributing to the innate immune and inflammatory response. In this process the cyclooxygenase-2 (COX-2) plays an important role in the induction of prostaglandin-dependent inflammation. While it has been reported that COX-2 is induced upon IAV infection, in the present study we observed a down-regulation at later stages of infection suggesting a tight regulation of COX-2 by IAV. Our data indicate the pattern-recognition receptor RIG-I as mediator of the initial IAV-induced COX-2 synthesis. Nonetheless, during on-going IAV replication substantial suppression of COX-2 mRNA and protein synthesis could be detected, accompanied by a decrease in mRNA half-life. Interestingly, COX-2 mRNA stability was not only imbalanced by IAV replication but also by stimulation of cells with viral RNA. Our results reveal tristetraprolin (TTP), which is known to bind COX-2 mRNA and promote its rapid degradation, as regulator of COX-2 expression in IAV infection. During IAV replication and viral RNA accumulation TTP mRNA synthesis was induced, resulting in reduced COX-2 levels. Accordingly, the down-regulation of TTP resulted in increased COX-2 protein expression after IAV infection. These findings indicate a novel IAV-regulated cellular mechanism, contributing to the repression of host defence and therefore facilitating viral replication.

Influenza A viruses (IAVs) are respiratory pathogens that are able to hijack multiple cellular mechanisms to drive their replication. Consequently, several viral and cellular proteins undergo posttranslational modifications such as dynamic phosphorylation/dephosphorylation. In eukaryotic cells, dephosphorylation is mainly catalyzed by protein phosphatase 2A (PP2A). While the function of kinases in IAV infection is quite well studied, only little is known about the role of PP2A in IAV replication. Here, we show, by using knockdown and inhibition approaches of the catalytic subunit PP2Ac, that this phosphatase is important for efficient replication of several IAV subtypes. This could neither be attributed to alterations in the antiviral immune response nor to changes in transcription or translation of viral genes. Interestingly, decreased PP2Ac levels resulted in a significantly reduced cell viability after IAV infection. Comprehensive kinase activity profiling identified an enrichment of process networks related to apoptosis and indicated a synergistic action of hyper-activated PI3K/Akt, MAPK/JAK-STAT and NF-kB signaling pathways, collectively resulting in increased cell death. Taken together, while IAV seems to effectively tap leftover PP2A activity to ensure efficient viral replication, reduced PP2Ac levels fail to orchestrate cell survival mechanisms to protect infected cells from early cell death.

Influenza A virus (IAV) is a common pathogen of respiratory disease. The IAV-induced seasonal epidemics and the sporadic pandemics are associated with high morbidity and mortality. Therefore, effective protection and therapy for IAV infections is an important challenge in countering this public health threat. Because vaccinations only protect against known circulating strains, and the currently available antivirals pose the risk of resistance formation, drugs targeting host cell factors needed for viral replication offer a promising therapeutic approach. In this study, we describe the use of the antifungal therapeutics posaconazole and itraconazole in the therapy of IAV. We show that both drugs efficiently inhibit the propagation of IAV in the cell culture model without being cytotoxic. The mode of action is probably based on several targets and includes both a priming of the interferon response and the induced imbalance of cellular cholesterol. The antiviral effect of itraconazole could be confirmed in the mouse model, where the administration of itraconazole led to a drastic reduction in mortality and a significant increase in the survival rate. Thus, our data indicate a promising therapeutic potential of at least itraconazole in influenza therapy.

Influenza viruses (IV) exploit a variety of signaling pathways. Previous studies showed that the rapidly accelerated fibrosarcoma/mitogen-activated protein kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) pathway is functionally linked to nuclear export of viral ribonucleoprotein (vRNP) complexes, suggesting that vRNP export is a signaling-induced event. However, the underlying mechanism remained completely enigmatic. Here we have dissected the unknown molecular steps of signaling-driven vRNP export. We identified kinases RSK1/2 as downstream targets of virus-activated ERK signaling. While RSK2 displays an antiviral role, we demonstrate a virus-supportive function of RSK1, migrating to the nucleus to phosphorylate nucleoprotein (NP), the major constituent of vRNPs. This drives association with viral matrix protein 1 (M1) at the chromatin, important for vRNP export. Inhibition or knockdown of MEK, ERK or RSK1 caused impaired vRNP export and reduced progeny virus titers. This work not only expedites the development of anti-influenza strategies, but in addition demonstrates converse actions of different RSK isoforms.

Recently, we showed that infection of primary lung tumor-bearing mice with oncolytic influenza A viruses (IAVs) led to strong virus-induced tumor cell lysis but also to restoration of immune competence of innate immune cells. Murine B16-F10 melanoma cells are known for their high lung tropism and progressive growth. As these cells are also highly permissive for IAVs, we analyzed their oncolytic and immunomodulatory efficiency against pulmonary B16-F10 lung metastases in vivo. IAV infection abrogated the melanoma-mediated immune suppression in the lung and induced a more than 50% cancer cell lysis. The oncolytic effect reached maximal efficacy 3 days post-infection, but it was not sustained over time. In order to maintain the virus-induced anti-tumor effect, mice with melanoma-derived lung cancers were treated in addition to influenza virus infection with an immune checkpoint inhibitor against programmed death-1 receptor (PD-1). The combined IAV and immune checkpoint inhibition (ICI) therapy resulted in a sustained anti-tumor efficacy, keeping the lung melanoma mass at day 12 of IAV infection still reduced by 50% over the control mice. In conclusion, ICI treatment strongly enhanced the oncolytic effect of influenza virus infection, suggesting that combined treatment is a promising approach against metastatic pulmonary melanoma.

The frequent emergence of new influenza viruses in the human population underlines the urgent need for antiviral therapeutics in addition to the preventative vaccination against the seasonal flu. To circumvent the development of resistance, recent antiviral approaches target cellular proteins needed by the virus for efficient replication. We investigated the contribution of the small GTPase Rac1 to the replication of influenza viruses. Inhibition of Rac1 by NSC23766 resulted in impaired replication of a wide variety of influenza viruses, including a human virus strain of the pandemic from 2009 as well as highly pathogenic avian virus strains. Furthermore, we identified a crucial role of Rac1 for the activity of the viral polymerase complex. The antiviral potential of NSC23766 was confirmed in mouse experiments, identifying Rac1 as a new cellular target for therapeutic treatment of influenza virus infections.

Influenza virus infections represent a major public health issue by causing annual epidemics and occasional pandemics that affect thousands of people worldwide. Vaccination is the main prophylaxis to prevent these epidemics/pandemics, although the effectiveness of licensed vaccines is rather limited due to the constant mutations of influenza virus antigenic characteristics. The available anti-influenza drugs are still restricted and there is an increasing viral resistance to these compounds, thus highlighting the need for research and development of new antiviral drugs. In this work, two semisynthetic derivatives of digitoxigenin, namely C10 (3β-((N-(2-hydroxyethyl)aminoacetyl)amino-3-deoxydigitoxigenin) and C11 (3β-(hydroxyacetyl)amino-3-deoxydigitoxigenin), showed anti-influenza A virus activity by affecting the expression of viral proteins at the early and late stages of replication cycle, and altering the transcription and synthesis of new viral proteins, thereby inhibiting the formation of new virions. Such antiviral action occurred due to the interference in the assembly of viral polymerase, resulting in an impaired polymerase activity and, therefore, reducing viral replication. Confirming the in vitro results, a clinically relevant ex vivo model of influenza virus infection of human tumor-free lung tissues corroborated the potential of these compounds, especially C10, to completely abrogate influenza A virus replication at the highest concentration tested (2.0 µM). Taken together, these promising results demonstrated that C10 and C11 can be considered as potential new anti-influenza drug candidates.

Staphylococcus aureus necrotizing pneumonia is a life-threatening disease that is frequently preceded by influenza infection. The S. aureus toxin Panton-Valentine leukocidin (PVL) is most likely causative for necrotizing diseases, but the precise pathogenic mechanisms of PVL and a possible contribution of influenza virus remain to be elucidated. In this study, we present a model that explains how influenza virus and PVL act together to cause necrotizing pneumonia: an influenza infection activates the lung epithelium to produce chemoattractants for neutrophils. Upon superinfection with PVL-expressing S. aureus, the recruited neutrophils are rapidly killed by PVL, resulting in uncontrolled release of neutrophil proteases that damage the airway epithelium. The host counteracts this pathogen strategy by generating PVL-neutralizing antibodies and by neutralizing the released proteases via protease inhibitors present in the serum. These findings explain why necrotizing infections mainly develop in serum-free spaces (eg, pulmonary alveoli) and open options for new therapeutic approaches.

The type I interferon (IFN) response represents the first line of defence to invading pathogens. Internalized viral ribonucleoproteins (vRNPs) of negative-strand RNA viruses induce an early IFN response by interacting with retinoic acid inducible gene I (RIG-I) and its recruitment to mitochondria. Here we employ three-dimensional stochastic optical reconstruction microscopy (STORM) to visualize incoming influenza A virus (IAV) vRNPs as helical-like structures associated with mitochondria. Unexpectedly, an early IFN induction in response to vRNPs is not detected. A distinct amino-acid motif in the viral polymerases, PB1/PA, suppresses early IFN induction. Mutation of this motif leads to reduced pathogenicity in vivo, whereas restoration increases it. Evolutionary dynamics in these sequences suggest that completion of the motif, combined with viral reassortment can contribute to pandemic risks. In summary, inhibition of the immediate anti-viral response is 'pre-packaged' in IAV in the sequences of vRNP-associated polymerase proteins.

Phosphorylation and dephosphorylation acts as a fundamental molecular switch that alters protein function and thereby regulates many cellular processes. The non-structural protein 1 (NS1) of influenza A virus is an important factor regulating virulence by counteracting cellular immune responses against viral infection. NS1 was shown to be phosphorylated at several sites; however, so far, no function has been conclusively assigned to these post-translational events yet. Here, we show that the newly identified phospho-site threonine 49 of NS1 is differentially phosphorylated in the viral replication cycle. Phosphorylation impairs binding of NS1 to double-stranded RNA and TRIM25 as well as complex formation with RIG-I, thereby switching off its interferon antagonistic activity. Because phosphorylation was shown to occur at later stages of infection, we hypothesize that at this stage other functions of the multifunctional NS1 beyond its interferon-antagonistic activity are needed.

Infections by influenza A viruses (IAV) are a major health burden to mankind. The current antiviral arsenal against IAV is limited and novel drugs are urgently required. Medicinal plants are known as an abundant source for bioactive compounds, including antiviral agents. The aim of the present study was to characterize the anti-IAV potential of a proanthocyanidin-enriched extract derived from the aerial parts of Rumex acetosa (RA), and to identify active compounds of RA, their mode of action, and structural features conferring anti-IAV activity. In a modified MTT (MTTIAV) assay, RA was shown to inhibit growth of the IAV strain PR8 (H1N1) and a clinical isolate of IAV(H1N1)pdm09 with a half-maximal inhibitory concentration (IC50) of 2.5 µg/mL and 2.2 µg/mL, and a selectivity index (SI) (half-maximal cytotoxic concentration (CC50)/IC50)) of 32 and 36, respectively. At RA concentrations>1 µg/mL plaque formation of IAV(H1N1)pdm09 was abrogated. RA was also active against an oseltamivir-resistant isolate of IAV(H1N1)pdm09. TNF-α and EGF-induced signal transduction in A549 cells was not affected by RA. The dimeric proanthocyanidin epicatechin-3-O-gallate-(4β→8)-epicatechin-3'-O-gallate (procyanidin B2-di-gallate) was identified as the main active principle of RA (IC50 approx. 15 µM, SI≥13). RA and procyanidin B2-di-gallate blocked attachment of IAV and interfered with viral penetration at higher concentrations. Galloylation of the procyanidin core structure was shown to be a prerequisite for anti-IAV activity; o-trihydroxylation in the B-ring increased the anti-IAV activity. In silico docking studies indicated that procyanidin B2-di-gallate is able to interact with the receptor binding site of IAV(H1N1)pdm09 hemagglutinin (HA). In conclusion, the proanthocyanidin-enriched extract RA and its main active constituent procyanidin B2-di-gallate protect cells from IAV infection by inhibiting viral entry into the host cell. RA and procyanidin B2-di-gallate appear to be a promising expansion of the currently available anti-influenza agents.

Influenza virus infections and their associated morbidity and mortality are a major threat to global health. Vaccination is an effective influenza prevention measure; however, the effectiveness is challenged by the rapid changes in the influenza virus genome leading to viral adaptation. Emerging viral resistance to the neuraminidase inhibitor oseltamivir limits the treatment of acute influenza infections. Targeting influenza virus‑host interactions is a new and emerging field, and therapies based on the combination of virus‑ and host‑directed drugs might significantly improve treatment success. We therefore assessed the combined treatment with oseltamivir and the repurposed antifungal drug itraconazole on infection of polarized broncho‑epithelial Calu-3 cells with pdm09 or Panama influenza A virus strains. We detected significantly stronger antiviral activities in the combined treatment compared to monotherapy with oseltamivir, permitting lower concentrations of the drug than required for the single treatments. Bliss independence drug interaction analysis indicated that both drugs acted independently of each other. The additional antiviral effect of itraconazole might safeguard patients infected with influenza virus strains with heightened oseltamivir resistance.

The type I interferon (IFN) system is a first line of defense against viral infections. Viruses have developed various mechanisms to counteract this response. So far, the interferon antagonistic activity of influenza A viruses was mainly observed on the level of IFNbeta gene induction via action of the viral non-structural protein 1 (NS1). Here we present data indicating that influenza A viruses not only suppress IFNbeta gene induction but also inhibit type I IFN signaling through a mechanism involving induction of the suppressor of cytokine signaling-3 (SOCS-3) protein. Our study was based on the observation that in cells that were infected with influenza A virus and subsequently stimulated with IFNalpha/beta, phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was strongly reduced. This impaired STAT1 activation was not due to the action of viral proteins but rather appeared to be induced by accumulation of viral 5' triphosphate RNA in the cell. SOCS proteins are potent endogenous inhibitors of Janus kinase (JAK)/STAT signaling. Closer examination revealed that SOCS-3 but not SOCS-1 mRNA levels increase in an RNA- and nuclear factor kappa B (NF-kappaB)-dependent but type I IFN-independent manner early in the viral replication cycle. This direct viral induction of SOCS-3 mRNA and protein expression appears to be relevant for suppression of the antiviral response since in SOCS-3 deficient cells a sustained phosphorylation of STAT1 correlated with elevated expression of type I IFN-dependent genes. As a consequence, progeny virus titers were reduced in SOCS-3 deficient cells or in cells were SOCS-3 expression was knocked-down by siRNA. These data provide the first evidence that influenza A viruses suppress type I IFN signaling on the level of JAK/STAT activation. The inhibitory effect is at least in part due to the induction of SOCS-3 gene expression, which results in an impaired antiviral response.