Glycolytic interference blocks influenza A virus propagation by impairing viral polymerase-driven synthesis of genomic vRNA.

Influenza A virus (IAV), like any other virus, provokes considerable modifications of its host cell's metabolism. This includes a substantial increase in the uptake as well as the metabolization of glucose. Although it is known for quite some time that suppression of glucose metabolism restricts virus replication, the exact molecular impact on the viral life cycle remained enigmatic so far. Using 2-deoxy-d-glucose (2-DG) we examined how well inhibition of glycolysis is tolerated by host cells and which step of the IAV life cycle is affected. We observed that effects induced by 2-DG are reversible and that cells can cope with relatively high concentrations of the inhibitor by compensating the loss of glycolytic activity by upregulating other metabolic pathways. Moreover, mass spectrometry data provided information on various metabolic modifications induced by either the virus or agents interfering with glycolysis. In the presence of 2-DG viral titers were significantly reduced in a dose-dependent manner. The supplementation of direct or indirect glycolysis metabolites led to a partial or almost complete reversion of the inhibitory effect of 2-DG on viral growth and demonstrated that indeed the inhibition of glycolysis and not of N-linked glycosylation was responsible for the observed phenotype. Importantly, we could show via conventional and strand-specific qPCR that the treatment with 2-DG led to a prolonged phase of viral mRNA synthesis while the accumulation of genomic vRNA was strongly reduced. At the same time, minigenome assays showed no signs of a general reduction of replicative capacity of the viral polymerase. Therefore, our data suggest that the significant reduction in IAV replication by glycolytic interference occurs mainly due to an impairment of the dynamic regulation of the viral polymerase which conveys the transition of the enzyme's function from transcription to replication.

Conversion of dendritic cells into tolerogenic or inflammatory cells depends on the activation threshold and kinetics of the mTOR signaling pathway.

BACKGROUND: Restoring impaired peripheral immune tolerance is the primary challenge in treating autoimmune diseases. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs), a fraction of low molecular weight proteins, in inhibiting the progression of psoriatic arthritis, even in the presence of high levels of the proinflammatory cytokine TNFα in the bloodstream. When specifically targeting dendritic cells (DCs), SSPs transform them into tolerogenic cells, which efficiently induce the development of regulatory Foxp3+ Treg cells. In this study, we provide further insights into the mechanism of action of SSPs. RESULTS: We found that SSPs stimulate the activation of the mTOR signaling pathway in dendritic cells, albeit in a different manner than the classical immunogenic stimulus LPS. While LPS-induced activation is rapid, strong, and sustained, the activity induced by SSPs is delayed, less intense, yet still significant. These distinct patterns of activation, as measured by phosphorylation of key components of the pathway are also observed in response to other immunogenic and tolerogenic stimuli such as GM-CSF + IL-4 or IL-10 and TGFß. The disparity in mTOR activation between immunogenic and tolerogenic stimuli is quantitative rather than qualitative. In both cases, mTOR activation primarily occurs through the PI3K/Akt signaling axis and involves ERK and GSK3ß kinases, with minimal involvement of AMPK or NF-kB pathways. Furthermore, in the case of SSPs, mTOR activation seems to involve adenosine receptors. Additionally, we observed that DCs treated with SSPs exhibit an energy metabolism with high plasticity, which is typical of tolerogenic cells rather than immunogenic cells. CONCLUSION: Hence, the decision whether dendritic cells enter an inflammatory or tolerogenic state seems to rely on varying activation thresholds and kinetics of the mTOR signaling pathway.

Small spleen peptides prevent development of psoriatic arthritis via restoration of peripheral tolerance.

The major challenge in the treatment of autoimmune diseases is the restoration of the impaired peripheral immune tolerance that always accompanies the development of such diseases. Here, we show that small splenic peptides (SSPs) of whole spleen extract efficiently suppress the development of psoriatic arthritis in vivo, even in the presence of sustained levels of pro-inflammatory cytokines. SSPs target dendritic cells (DCs) and convert them into tolerogenic cells, which in turn differentiate naive CD4+ cells into Foxp3-expressing T regulatory cells (Tregs). The latter requires direct contact between SSP-activated DCs and naive CD4+ T cells via PD-1 and CTLA4 immune checkpoint receptors of T cells. Finally, depletion of Foxp3+ Tregs in vivo abrogated the protective effect of SSPs on psoriatic arthritis development. We hypothesize that SSPs represent an intrinsic component of the adaptive immune system responsible for the physiological maintenance of peripheral tolerance and that therapeutically administered SSPs are able to restore imbalanced peripheral tolerance in autoimmune diseases.

Phosphorylation of Influenza A Virus NS1 at Serine 205 Mediates Its Viral Polymerase-Enhancing Function.

Influenza A virus (IAV) nonstructural protein 1 (NS1) is a protein with multiple functions that are regulated by phosphorylation. Phosphoproteomic screening of H1N1 virus-infected cells revealed that NS1 was phosphorylated at serine 205 in intermediate stages of the viral life cycle. Interestingly, S205 is one of six amino acid changes in NS1 of post-pandemic H1N1 viruses currently circulating in humans compared to the original swine-origin 2009 pandemic (H1N1pdm09) virus, suggesting a role in host adaptation. To identify NS1 functions regulated by S205 phosphorylation, we generated recombinant PR8 H1N1 NS1 mutants with S205G (nonphosphorylatable) or S205N (H1N1pdm09 signature), as well as H1N1pdm09 viruses harboring the reverse mutation NS1 N205S or N205D (phosphomimetic). Replication of PR8 NS1 mutants was attenuated relative to wild-type (WT) virus replication in a porcine cell line. However, PR8 NS1 S205N showed remarkably higher attenuation than PR8 NS1 S205G in a human cell line, highlighting a potential host-independent advantage of phosphorylatable S205, while an asparagine at this position led to a potential host-specific attenuation. Interestingly, PR8 NS1 S205G did not show polymerase activity-enhancing functions, in contrast to the WT, which can be attributed to diminished interaction with cellular restriction factor DDX21. Analysis of the respective kinase mediating S205 phosphorylation indicated an involvement of casein kinase 2 (CK2). CK2 inhibition significantly reduced the replication of WT viruses and decreased NS1-DDX21 interaction, as observed for NS1 S205G. In summary, NS1 S205 is required for efficient NS1-DDX21 binding, resulting in enhanced viral polymerase activity, which is likely to be regulated by transient phosphorylation.IMPORTANCE Influenza A viruses (IAVs) still pose a major threat to human health worldwide. As a zoonotic virus, IAV can spontaneously overcome species barriers and even reside in new hosts after efficient adaptation. Investigation of the functions of specific adaptational mutations can lead to a deeper understanding of viral replication in specific hosts and can probably help to find new targets for antiviral intervention. In the present study, we analyzed the role of NS1 S205, a phosphorylation site that was reacquired during the circulation of pandemic H1N1pdm09 "swine flu" in the human host. We found that phosphorylation of human H1N1 virus NS1 S205 is mediated by the cellular kinase CK2 and is needed for efficient interaction with human host restriction factor DDX21, mediating NS1-induced enhancement of viral polymerase activity. Therefore, targeting CK2 activity might be an efficient strategy for limiting the replication of IAVs circulating in the human population.

Phosphorylation of JIP4 at S730 Presents Antiviral Properties against Influenza A Virus Infection.

Influenza A virus (IAV) is the causative agent of flu disease that results in annual epidemics and occasional pandemics. IAV alters several signaling pathways of the cellular host response in order to promote its replication. Therefore, some of these pathways can serve as targets for novel antiviral agents. Here, we show that c-Jun NH2-terminal kinase (JNK)-interacting protein 4 (JIP4) is dynamically phosphorylated in IAV infection. The lack of JIP4 resulted in higher virus titers, with significant differences in viral protein and mRNA accumulation as early as within the first replication cycle. In accordance, decreased IAV titers and protein accumulation were observed during the overexpression of JIP4. Strikingly, the antiviral function of JIP4 does not originate from modulation of JNK or p38 mitogen-activated protein kinase (MAPK) pathways or from altered expression of interferons or interferon-stimulated genes but rather originates from a direct reduction of viral polymerase activity. Furthermore, the interference of JIP4 with IAV replication seems to be linked to the phosphorylation of the serine at position 730 that is sufficient to impede the viral polymerase. Collectively, we provide evidence that JIP4, a host protein modulated in IAV infection, exhibits antiviral properties that are dynamically controlled by its phosphorylation at S730. IMPORTANCE Influenza A virus (IAV) infection is a world health concern, and current treatment options encounter high rates of resistance. Our group investigates host pathways modified in IAV infection as promising new targets. The host protein JIP4 is dynamically phosphorylated in IAV infection. JIP4 absence resulted in higher virus titers and viral protein and mRNA accumulation within the first replication cycle. Accordingly, decreased IAV titers and protein accumulation were observed during JIP4 overexpression. Strikingly, the antiviral function of JIP4 does not originate from modulation of JNK or p38 MAPK pathways or from altered expression of interferons or interferon-stimulated genes but rather originates from a reduction in viral polymerase activity. The interference of JIP4 with IAV replication is linked to the phosphorylation of serine 730. We provide evidence that JIP4, a host protein modulated in IAV infection, exhibits antiviral properties that are dynamically controlled by its phosphorylation at S730.

Discrete spatio-temporal regulation of tyrosine phosphorylation directs influenza A virus M1 protein towards its function in virion assembly.

Small RNA viruses only have a very limited coding capacity, thus most viral proteins have evolved to fulfill multiple functions. The highly conserved matrix protein 1 (M1) of influenza A viruses is a prime example for such a multifunctional protein, as it acts as a master regulator of virus replication whose different functions have to be tightly regulated. The underlying mechanisms, however, are still incompletely understood. Increasing evidence points towards an involvement of posttranslational modifications in the spatio-temporal regulation of M1 functions. Here, we analyzed the role of M1 tyrosine phosphorylation in genuine infection by using recombinant viruses expressing M1 phosphomutants. Presence of M1 Y132A led to significantly decreased viral replication compared to wildtype and M1 Y10F. Characterization of phosphorylation dynamics by mass spectrometry revealed the presence of Y132 phosphorylation in M1 incorporated into virions that is most likely mediated by membrane-associated Janus kinases late upon infection. Molecular dynamics simulations unraveled a potential phosphorylation-induced exposure of the positively charged linker domain between helices 4 and 5, supposably acting as interaction platform during viral assembly. Consistently, M1 Y132A showed a defect in lipid raft localization due to reduced interaction with viral HA protein resulting in a diminished structural stability of viral progeny and the formation of filamentous particles. Importantly, reduced M1-RNA binding affinity resulted in an inefficient viral genome incorporation and the production of non-infectious virions that interferes with virus pathogenicity in mice. This study advances our understanding of the importance of dynamic phosphorylation as a so far underestimated level of regulation of multifunctional viral proteins and emphasizes the potential feasibility of targeting posttranslational modifications of M1 as a novel antiviral intervention.

Dynamic phospho-modification of viral proteins as a crucial regulatory layer of influenza A virus replication and innate immune responses.

Influenza viruses are small RNA viruses with a genome of about 13 kb. Because of this limited coding capacity, viral proteins have evolved to fulfil multiple functions in the infected cell. This implies that there must be mechanisms allowing to dynamically direct protein action to a distinct activity in a spatio-temporal manner. Furthermore, viruses exploit many cellular processes, which also have to be dynamically regulated during the viral replication cycle. Phosphorylation and dephosphorylation of proteins are fundamental for the control of many cellular responses. There is accumulating evidence that this mechanism represents a so far underestimated level of regulation in influenza virus replication. Here, we focus on the current knowledge of dynamics of phospho-modifications in influenza virus replication and show recent examples of findings underlining the crucial role of phosphorylation in viral transport processes as well as activation and counteraction of the innate immune response.

Cellular Protein Phosphatase 2A Regulates Cell Survival Mechanisms in Influenza A Virus Infection.

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.

Altered Signal Transduction in the Immune Response to Influenza Virus and S. pneumoniae or S. aureus Co-Infections.

Influenza virus is a well-known respiratory pathogen, which still leads to many severe pulmonary infections in the human population every year. Morbidity and mortality rates are further increased if virus infection coincides with co-infections or superinfections caused by bacteria such as Streptococcus pneumoniae (S. pneumoniae) and Staphylococcus aureus (S. aureus). This enhanced pathogenicity is due to complex interactions between the different pathogens and the host and its immune system and is mainly governed by altered intracellular signaling processes. In this review, we summarize the recent findings regarding the innate and adaptive immune responses during co-infection with influenza virus and S. pneumoniae or S. aureus, describing the signaling pathways involved and how these interactions influence disease outcomes.

Advances in Transgenic Mouse Models to Study Infections by Human Pathogenic Viruses.

Medical research is changing into direction of precision therapy, thus, sophisticated preclinical models are urgently needed. In human pathogenic virus research, the major technical hurdle is not only to translate discoveries from animals to treatments of humans, but also to overcome the problem of interspecies differences with regard to productive infections and comparable disease development. Transgenic mice provide a basis for research of disease pathogenesis after infection with human-specific viruses. Today, humanized mice can be found at the very heart of this forefront of medical research allowing for recapitulation of disease pathogenesis and drug mechanisms in humans. This review discusses progress in the development and use of transgenic mice for the study of virus-induced human diseases towards identification of new drug innovations to treat and control human pathogenic infectious diseases.

Employing RNA viruses to fight cancer: novel insights into oncolytic virotherapy.

Within recent decades, viruses that specifically target tumor cells have emerged as novel therapeutic agents against cancer. These viruses do not only act via their cell-lytic properties, but also harbor immunostimulatory features to re-direct the tumor microenvironment and stimulate tumor-directed immune responses. Furthermore, oncolytic viruses are considered to be superior to classical cancer therapies due to higher selectivity towards tumor cell destruction and, consequently, less collateral damage of non-transformed healthy tissue. In particular, the field of oncolytic RNA viruses is rapidly developing since these agents possess alternative tumor-targeting strategies compared to established oncolytic DNA viruses. Thus, oncolytic RNA viruses have broadened the field of virotherapy facilitating new strategies to fight cancer. In addition to several naturally occurring oncolytic viruses, genetically modified RNA viruses that are armed to express foreign factors such as immunostimulatory molecules have been successfully tested in early clinical trials showing promising efficacy. This review aims to provide an overview of the most promising RNA viruses in clinical development, to summarize the current knowledge of clinical trials using these viral agents, and to discuss the main issues as well as future perspectives of clinical approaches using oncolytic RNA viruses.

Evidence for a Novel Mechanism of Influenza Virus-Induced Type I Interferon Expression by a Defective RNA-Encoded Protein.

Influenza A virus (IAV) defective RNAs are generated as byproducts of error-prone viral RNA replication. They are commonly derived from the larger segments of the viral genome and harbor deletions of various sizes resulting in the generation of replication incompatible viral particles. Furthermore, small subgenomic RNAs are known to be strong inducers of pattern recognition receptor RIG-I-dependent type I interferon (IFN) responses. The present study identifies a novel IAV-induced defective RNA derived from the PB2 segment of A/Thailand/1(KAN-1)/2004 (H5N1). It encodes a 10 kDa protein (PB2∆) sharing the N-terminal amino acid sequence of the parental PB2 protein followed by frame shift after internal deletion. PB2∆ induces the expression of IFNß and IFN-stimulated genes by direct interaction with the cellular adapter protein MAVS, thereby reducing viral replication of IFN-sensitive viruses such as IAV or vesicular stomatitis virus. This induction of IFN is completely independent of the defective RNA itself that usually serves as pathogen-associated pattern and thus does not require the cytoplasmic sensor RIG-I. These data suggest that not only defective RNAs, but also some defective RNA-encoded proteins can act immunostimulatory. In this particular case, the KAN-1-induced defective RNA-encoded protein PB2∆ enhances the overwhelming immune response characteristic for highly pathogenic H5N1 viruses, leading to a more severe phenotype in vivo.

Platelet activation and aggregation promote lung inflammation and influenza virus pathogenesis.

RATIONALE: The hallmark of severe influenza virus infection is excessive inflammation of the lungs. Platelets are activated during influenza, but their role in influenza virus pathogenesis and inflammatory responses is unknown. OBJECTIVES: To determine the role of platelets during influenza A virus infections and propose new therapeutics against influenza. METHODS: We used targeted gene deletion approaches and pharmacologic interventions to investigate the role of platelets during influenza virus infection in mice. MEASUREMENTS AND MAIN RESULTS: Lungs of infected mice were massively infiltrated by aggregates of activated platelets. Platelet activation promoted influenza A virus pathogenesis. Activating protease-activated receptor 4, a platelet receptor for thrombin that is crucial for platelet activation, exacerbated influenza-induced acute lung injury and death. In contrast, deficiency in the major platelet receptor glycoprotein IIIa protected mice from death caused by influenza viruses, and treating the mice with a specific glycoprotein IIb/IIIa antagonist, eptifibatide, had the same effect. Interestingly, mice treated with other antiplatelet compounds (antagonists of protease-activated receptor 4, MRS 2179, and clopidogrel) were also protected from severe lung injury and lethal infections induced by several influenza strains. CONCLUSIONS: The intricate relationship between hemostasis and inflammation has major consequences in influenza virus pathogenesis, and antiplatelet drugs might be explored to develop new antiinflammatory treatment against influenza virus infections.

ß-catenin promotes the type I IFN synthesis and the IFN-dependent signaling response but is suppressed by influenza A virus-induced RIG-I/NF-κB signaling.

BACKGROUND: The replication cycle of most pathogens, including influenza viruses, is perfectly adapted to the metabolism and signal transduction pathways of host cells. After infection, influenza viruses activate several cellular signaling cascades that support their propagation but suppress those that interfere with viral replication. Accumulation of viral RNA plays thereby a central role. Its sensing by the pattern recognition receptors of the host cells leads to the activation of several signal transduction waves that result in induction of genes, responsible for the cellular innate immune response. Type I interferon (IFN) genes and interferon-stimulated genes (ISG) coding for antiviral-acting proteins, such as MxA, OAS-1 or PKR, are primary targets of these signaling cascades. ß- and γ-catenin are closely related armadillo repeat-containing proteins with dual roles. At the cell membrane they serve as adapter molecules linking cell-cell contacts to microfilaments. In the cytosol and nucleus, the proteins form a transcriptional complex with the lymphoid enhancer factor/T-cell factor (LEF/TCF), regulating the transcription of many genes, thereby controlling different cellular functions such as cell cycle progression and differentiation. RESULTS: In this study, we demonstrate that ß- and γ-catenin are important regulators of the innate cellular immune response to influenza A virus (IAV) infections. They inhibit viral replication in lung epithelial cells by enhancing the virus-dependent induction of the IFNB1 gene and interferon-stimulated genes. Simultaneously, the prolonged infection counteracts the antiviral effect of ß- and γ-catenin. Influenza viruses suppress ß-catenin-dependent transcription by misusing the RIG-I/NF-κB signaling cascade that is induced in the course of infection by viral RNA. CONCLUSION: We identified ß- and γ-catenin as novel antiviral-acting proteins. While these factors support the induction of common target genes of the cellular innate immune response, their functional activity is suppressed by pathogen evasion.

Small Spleen Peptides (SSPs) Shape Dendritic Cell Differentiation through Modulation of Extracellular ATP Synthesis Profile.

Restoring peripheral immune tolerance is crucial for addressing autoimmune diseases. An ancient mechanism in maintaining the balance between inflammation and tolerance is the ratio of extracellular ATP (exATP) and adenosine. Our previous research demonstrated the effectiveness of small spleen peptides (SSPs) in inhibiting psoriatic arthritis progression, even in the presence of the pro-inflammatory cytokine TNFα, by transforming dendritic cells (DCs) into tolerogenic cells and fostering regulatory Foxp3+ Treg cells. Here, we identified thymosins as the primary constituents of SSPs, but recombinant thymosin peptides were less efficient in inhibiting arthritis than SSPs. Since Tß4 is an ecto-ATPase-binding protein, we hypothesized that SSPs regulate exATP profiles. Real-time investigation of exATP levels in DCs revealed that tolerogenic stimulation led to robust de novo exATP synthesis followed by significant degradation, while immunogenic stimulation resulted in a less pronounced increase in exATP and less effective degradation. These contrasting exATP profiles were crucial in determining whether DCs entered an inflammatory or tolerogenic state, highlighting the significance of SSPs as natural regulators of peripheral immunological tolerance, with potential therapeutic benefits for autoimmune diseases. Finally, we demonstrated that the tolerogenic phenotype of SSPs is mainly influenced by adenosine receptors, and in vivo administration of SSPs inhibits psoriatic skin inflammation.

A new splice variant of the human guanylate-binding protein 3 mediates anti-influenza activity through inhibition of viral transcription and replication.

Guanylate-binding proteins (GBPs) belong to the family of large GTPases that are induced in response to interferons. GBPs contain an N-terminal globular GTPase domain and a C-terminal α-helical regulatory domain that are connected by a short middle domain. Antiviral activity against vesicular stomatitis virus and encephalomyocarditis virus has been shown for hGBP-1; however, no anti-influenza virus properties for GBPs have been described to date. Here we show that hGBP-1 and hGBP-3 possess anti-influenza viral activity. Furthermore, we have identified a novel splice variant of hGBP-3, named hGBP-3ΔC, with a largely modified C-terminal α-helical domain. While all three GBP isoforms were up-regulated on influenza virus infection, hGBP-3ΔC showed the most prominent antiviral activity in epithelial cells. Mutational analysis of hGBPs revealed that the globular domain is the principal antiviral effector domain, and GTP-binding, but not hydrolysis, is necessary for antiviral action. Furthermore, we showed that hGBP-3ΔC strongly represses the activity of the viral polymerase complex, which results in decreased synthesis of viral vRNA, cRNA, mRNA, and viral proteins, as well.

H5N1 virus activates signaling pathways in human endothelial cells resulting in a specific imbalanced inflammatory response.

H5N1 influenza virus infections in humans cause a characteristic systemic inflammatory response syndrome; however, the molecular mechanisms are largely unknown. Endothelial cells (ECs) play a pivotal role in hyperdynamic septic diseases. To unravel specific signaling networks activated by H5N1 we used a genome-wide comparative systems biology approach analyzing gene expression in human ECs infected with three different human and avian influenza strains of high and low pathogenicity. Blocking of specific signaling pathways revealed that H5N1 induces an exceptionally NF-κB-dependent gene response in human endothelia. Additionally, the IFN-driven antiviral program in ECs is shown to be dependent on IFN regulatory factor 3 but significantly impaired upon H5N1 infection compared with low pathogenic influenza virus. As additional modulators of this H5N1-specific imbalanced gene response pattern, we identified HMGA1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not antiviral response, whereas NFATC4 was found to regulate transcription of specifically H5N1-induced genes. We describe for the first time, to our knowledge, defined signaling patterns specifically activated by H5N1, which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of an overwhelming proinflammatory and impaired antiviral gene program.

Replication-incompetent influenza A viruses armed with IFN-γ effectively mediate immune modulation and tumor destruction in mice harboring lung cancer.

Low pathogenic influenza A viruses (IAVs) have shown promising oncolytic potential in lung cancer-bearing mice. However, as replication-competent pathogens, they may cause side effects in immunocompromised cancer patients. To circumvent this problem, we genetically engineered nonreplicating IAVs lacking the hemagglutinin (HA) gene (ΔHA IAVs), but reconstituted the viral envelope with recombinant HA proteins to allow a single infection cycle. To optimize the therapeutic potential and improve immunomodulatory properties, these replication-incompetent IAVs were complemented with a murine interferon-gamma (mIFN-γ) gene. After intratracheal administration to transgenic mice that develop non-small cell lung cancer (NSCLC), the ΔHA IAVs induced potent tumor destruction. However, ΔHA IAVs armed with mIFN-γ exhibited an even stronger and more sustained effect, achieving 85% tumor reduction at day 12 postinfection. In addition, ΔHA-mIFN-γ viruses were proven to be efficient in recruiting and activating natural killer cells and macrophages from the periphery and in inducing cytotoxic T lymphocytes. Most important, both viruses, and particularly IFN-γ-encoding viruses, activated tumor-associated alveolar macrophages toward a proinflammatory M1-like phenotype. Therefore, replication-incompetent ΔHA-mIFN-γ-IAVs are safe and efficient oncolytic viruses that additionally exhibit immune cell activating properties and thus represent a promising innovative therapeutic option in the fight against NSCLC.

Cell-intrinsic genomic reassortment of pandemic H1N1 2009 and Eurasian avian-like swine influenza viruses results in potentially zoonotic variants.

Influenza A viruses (IAV) cause annual epidemics and occasional pandemics in humans. The most recent pandemic outbreak occurred in 2009 with H1N1pdm09. This virus, which most likely reassorted in swine before its transmission to humans, was reintroduced into the swine population and continues circulating ever since. In order to assess its potential to cause reassortants on a cellular level, human origin H1N1pdm09 and a recent Eurasian avian-like H1N1 swine IAV were (co-)passaged in the newly generated swine lung cell line C22. Co-infection with both viruses gave rise to numerous reassortants that additionally carry different mutations which can partially be found in nature as well. Reassortment most frequently affected the PB1, PA and NA segments with the swine IAV as recipient. These reassortants reached higher titers in swine lung cells and were able to replicate in genuine human lung tissue explants ex vivo, suggesting a possible zoonotic potential. Interestingly, reassortment and mutations in the viral ribonucleoprotein complex influence the viral polymerase activity in a cell type and species-specific manner. In summary, we demonstrate reassortment promiscuity of these viruses in a novel swine lung cell model and indicate a possible zoonotic potential of the reassortants.

Inhibition of p38 signaling curtails the SARS-CoV-2 induced inflammatory response but retains the IFN-dependent antiviral defense of the lung epithelial barrier.

SARS-CoV-2 is the causative agent of the immune response-driven disease COVID-19 for which new antiviral and anti-inflammatory treatments are urgently needed to reduce recovery time, risk of death and long COVID development. Here, we demonstrate that the immunoregulatory kinase p38 MAPK is activated during viral entry, mediated by the viral spike protein, and drives the harmful virus-induced inflammatory responses. Using primary human lung explants and lung epithelial organoids, we demonstrate that targeting p38 signal transduction with the selective and clinically pre-evaluated inhibitors PH-797804 and VX-702 markedly reduced the expression of the pro-inflammatory cytokines IL6, CXCL8, CXCL10 and TNF-α during infection, while viral replication and the interferon-mediated antiviral response of the lung epithelial barrier were largely maintained. Furthermore, our results reveal a high level of drug synergism of both p38 inhibitors in co-treatments with the nucleoside analogs Remdesivir and Molnupiravir to suppress viral replication of the SARS-CoV-2 variants of concern, revealing an exciting and novel mode of synergistic action of p38 inhibition. These results open new avenues for the improvement of the current treatment strategies for COVID-19.