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.

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.

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.

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.

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.

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.

Metabolic Modifications by Common Respiratory Viruses and Their Potential as New Antiviral Targets.

Respiratory viruses are known to be the most frequent causative mediators of lung infections in humans, bearing significant impact on the host cell signaling machinery due to their host-dependency for efficient replication. Certain cellular functions are actively induced by respiratory viruses for their own benefit. This includes metabolic pathways such as glycolysis, fatty acid synthesis (FAS) and the tricarboxylic acid (TCA) cycle, among others, which are modified during viral infections. Here, we summarize the current knowledge of metabolic pathway modifications mediated by the acute respiratory viruses respiratory syncytial virus (RSV), rhinovirus (RV), influenza virus (IV), parainfluenza virus (PIV), coronavirus (CoV) and adenovirus (AdV), and highlight potential targets and compounds for therapeutic approaches.

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.

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.

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.

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.

The Two Sides of the Same Coin-Influenza Virus and Intracellular Signal Transduction.

Cells respond to extracellular agents by activation of intracellular signaling pathways. Viruses can be regarded as such agents, leading to a firework of signaling inside the cell, primarily induced by pathogen-associated molecular patterns (PAMPs) that provoke safeguard mechanisms to defend from the invader. In the constant arms race between pathogen and cellular defense, viruses not only have evolved mechanisms to suppress or misuse supposedly antiviral signaling processes for their own benefit but also actively induce signaling to promote replication. This creates viral dependencies that may be exploited for novel strategies of antiviral intervention. Here, we will summarize the current knowledge of activation and function of influenza virus-induced signaling pathways with a focus on nuclear factor (NF)-κB signaling, mitogen-activated protein kinase cascades, and the phosphatidylinositol-3-kinase pathway. We will discuss the opportunities and drawbacks of targeting these signaling pathways for antiviral intervention.

Impact of Staphylococcus aureus Small Colony Variants on Human Lung Epithelial Cells with Subsequent Influenza Virus 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.

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.

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.

Immunomodulatory Nonstructural Proteins of Influenza A Viruses.

Influenza epidemics and pandemics still represent a severe public health threat and cause significant morbidity and mortality worldwide. As intracellular parasites, influenza viruses are strongly dependent on the host cell machinery. To ensure efficient production of progeny viruses, viral proteins extensively interfere with cellular signalling pathways to inhibit antiviral responses or to activate virus-supportive functions. Here, we review various functions of the influenza virus nonstructural proteins NS1, PB1-F2, and PA-X in infected cells and how post-transcriptional modifications of these proteins affect the viral life cycle. Furthermore, we discuss newly discovered interactions between these proteins and the antiviral interferon response.

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.

Targeting a metabolic pathway to fight the flu.

Our antiviral arsenal to fight influenza viruses is limited and we need novel anti-flu drugs. Recently, cellular drug targets came into focus and omics analysis were instrumental to suggest candidate factors. In this issue of The FEBS Journal, Kainov and colleagues used transcriptome data to investigate virus-induced changes in tryptophan metabolism that may serve as immunomodulatory approach against viruses.

Vemurafenib Limits Influenza A Virus Propagation by Targeting Multiple Signaling Pathways.

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.