Influenza A virus exploits transferrin receptor recycling to enter host cells.

Influenza A virus (IAV) enters host cells mostly through clathrin-dependent receptor-mediated endocytosis. A single bona fide entry receptor protein supporting this entry mechanism remains elusive. Here we performed proximity ligation of biotin to host cell surface proteins in the vicinity of attached trimeric hemagglutinin-HRP and characterized biotinylated targets using mass spectrometry. This approach identified transferrin receptor 1 (TfR1) as a candidate entry protein. Genetic gain-of-function and loss-of-function experiments, as well as in vitro and in vivo chemical inhibition, confirmed the functional involvement of TfR1 in IAV entry. Recycling deficient mutants of TfR1 do not support entry, indicating that TfR1 recycling is essential for this function. The binding of virions to TfR1 via sialic acids confirmed its role as a directly acting entry factor, but unexpectedly even headless TfR1 promoted IAV particle uptake in trans. TIRF microscopy localized the entering virus-like particles in the vicinity of TfR1. Our data identify TfR1 recycling as a revolving door mechanism exploited by IAV to enter host cells.

Identification of a short sequence motif in the influenza A virus pathogenicity factor PB1-F2 required for inhibition of human NLRP3.

Influenza A virus infection activates the NLRP3 inflammasome, a multiprotein signaling complex responsible for the proteolytic activation and release of the proinflammatory cytokine IL-1ß from monocytes and macrophages. Some influenza A virus (IAV) strains encode a short 90-amino acid peptide (PB1-F2) on an alternative open reading frame of segment 2, with immunomodulatory activity. We recently demonstrated that contemporary IAV PB1-F2 inhibits the activation of NLRP3, potentially by NEK7-dependent activation. PB1-F2 binds to NLRP3 with its C-terminal 50 amino acids, but the exact binding motif was unknown. On the NLRP3 side, the interface is formed through the leucine-rich-repeat (LRR) domain, potentially in conjunction with the pyrin domain. Here, we took advantage of PB1-F2 sequences from IAV strains with either weak or strong NLRP3 interaction. Sequence comparison and structure prediction using Alphafold2 identified a short four amino acid sequence motif (TQGS) in PB1-F2 that defines NLRP3-LRR binding. Conversion of this motif to that of the non-binding PB1-F2 suffices to lose inhibition of NLRP3 dependent IL-1ß release. The TQGS motif further alters the subcellular localization of PB1-F2 and its colocalization with NLRP3 LRR and pyrin domain. Structural predictions suggest the establishment of additional hydrogen bonds between the C-terminus of PB1-F2 and the LRR domain of NLRP3, with two hydrogen bonds connecting to threonine and glutamine of the TQGS motif. Phylogenetic data show that the identified NLRP3 interaction motif in PB1-F2 is widely conserved among recent IAV-infecting humans. Our data explain at a molecular level the specificity of NLRP3 inhibition by influenza A virus. IMPORTANCE: Influenza A virus infection is accompanied by a strong inflammatory response and high fever. The human immune system facilitates the swift clearance of the virus with this response. An essential signal protein in the proinflammatory host response is IL-1b. It is released from inflammatory macrophages, and its production and secretion depend on the function of NLRP3. We had previously shown that influenza A virus blocks NLRP3 activation by the expression of a viral inhibitor, PB1-F2. Here, we demonstrate how this short peptide binds to NLRP3 and provide evidence that a four amino acid stretch in PB1-F2 is necessary and sufficient to mediate this binding. Our data identify a new virus-host interface required to block one signaling path of the innate host response against influenza A virus.

Influenza A viruses balance ER stress with host protein synthesis shutoff.

Excessive production of viral glycoproteins during infections poses a tremendous stress potential on the endoplasmic reticulum (ER) protein folding machinery of the host cell. The host cell balances this by providing more ER resident chaperones and reducing translation. For viruses, this unfolded protein response (UPR) offers the potential to fold more glycoproteins. We postulated that viruses could have developed means to limit the inevitable ER stress to a beneficial level for viral replication. Using a relevant human pathogen, influenza A virus (IAV), we first established the determinant for ER stress and UPR induction during infection. In contrast to a panel of previous reports, we identified neuraminidase to be the determinant for ER stress induction, and not hemagglutinin. IAV relieves ER stress by expression of its nonstructural protein 1 (NS1). NS1 interferes with the host messenger RNA processing factor CPSF30 and suppresses ER stress response factors, such as XBP1. In vivo viral replication is increased when NS1 antagonizes ER stress induction. Our results reveal how IAV optimizes glycoprotein expression by balancing folding capacity.

Optimizing the Live Attenuated Influenza A Vaccine Backbone for High-Risk Patient Groups.

Together with inactivated influenza vaccines (IIV), live attenuated influenza vaccines (LAIV) are an important tool to prevent influenza A virus (IAV) illnesses in patients. LAIVs present the advantages to have a needle-free administration and to trigger a mucosal immune response. LAIV is approved for healthy 2- to 49-year old individuals. However, due to its replicative nature and higher rate of adverse events at-risk populations are excluded from the benefits of this vaccine. Using targeted mutagenesis, we modified the nonstructural protein 1 of the currently licensed LAIV in order to impair its ability to bind the host cellular protein CPSF30 and thus its ability to inhibit host mRNA poly-adenylation. We characterized our optimized LAIV (optiLAIV) in three different mouse models mimicking healthy and high-risk patients. Using a neonatal mouse model, we show faster clearance of our optimized vaccine compared to the licensed LAIV. Despite lower replication, optiLAIV equally protected mice against homosubtypic and hetesubtypic influenza strain challenges. We confirmed the safer profile of optiLAIV in Stat1-/- mice (highly susceptible to viral infections) by showing no signs of morbidity compared to a 50% mortality rate observed following LAIV inoculation. Using a human nasal 3D tissue model, we showed an increased induction of ER stress-related genes following immunization with optiLAIV. Induction of ER stress was previously shown to improve antigen-specific immune responses and is proposed as the mechanism of action of the licensed adjuvant AS03. This study characterizes a safer LAIV candidate in two mouse models mimicking infants and severely immunocompromised patients and proposes a simple attenuation strategy that could broaden LAIV application and reduce influenza burden in high-risk populations. IMPORTANCE Live attenuated influenza vaccine (LAIV) is a needle-free, mucosal vaccine approved for healthy 2- to 49-year old individuals. Its replicative nature and higher rate of adverse events excludes at-risk populations. We propose a strategy to improve LAIV safety and explore the possibility to expand its applications in children under 2-year old and immunocompromised patients. Using a neonatal mouse model, we show faster clearance of our optimized vaccine (optiLAIV) compared to the licensed LAIV. Despite lower replication, optiLAIV equally protected mice against influenza virus challenges. We confirmed the safer profile of optiLAIV in Stat1-/- mice (highly susceptible to viral infections) by showing no signs of morbidity compared to a 50% mortality rate from LAIV. OptiLAIV could expand the applications of the current LAIV and help mitigate the burden of IAV in susceptible populations.

Influenza A viruses limit NLRP3-NEK7-complex formation and pyroptosis in human macrophages.

Pyroptosis is a fulminant form of macrophage cell death, contributing to release of pro-inflammatory cytokines. In humans, it depends on caspase 1/4-activation of gasdermin D and is characterized by the release of cytoplasmic content. Pathogens apply strategies to avoid or antagonize this host response. We demonstrate here that a small accessory protein (PB1-F2) of contemporary H5N1 and H3N2 influenza A viruses (IAV) curtails fulminant cell death of infected human macrophages. Infection of macrophages with a PB1-F2-deficient mutant of a contemporary IAV resulted in higher levels of caspase-1 activation, cleavage of gasdermin D, and release of LDH and IL-1ß. Mechanistically, PB1-F2 limits transition of NLRP3 from its auto-repressed and closed confirmation into its active state. Consequently, interaction of a recently identified licensing kinase NEK7 with NLRP3 is diminished, which is required to initiate inflammasome assembly.

RIG-I Signaling Is Critical for Efficient Polyfunctional T Cell Responses during Influenza Virus Infection.

Retinoic acid inducible gene-I (RIG-I) is an innate RNA sensor that recognizes the influenza A virus (IAV) RNA genome and activates antiviral host responses. Here, we demonstrate that RIG-I signaling plays a crucial role in restricting IAV tropism and regulating host immune responses. Mice deficient in the RIG-I-MAVS pathway show defects in migratory dendritic cell (DC) activation, viral antigen presentation, and priming of CD8+ and CD4+ T cell responses during IAV infection. These defects result in decreased frequency of polyfunctional effector T cells and lowered protection against heterologous IAV challenge. In addition, our data show that RIG-I activation is essential for protecting epithelial cells and hematopoietic cells from IAV infection. These diverse effects of RIG-I signaling are likely imparted by the actions of type I interferon (IFN), as addition of exogenous type I IFN is sufficient to overcome the defects in antigen presentation by RIG-I deficient BMDC. Moreover, the in vivo T cell defects in RIG-I deficient mice can be overcome by the activation of MDA5 -MAVS via poly I:C treatment. Taken together, these findings demonstrate that RIG-I signaling through MAVS is critical for determining the quality of polyfunctional T cell responses against IAV and for providing protection against subsequent infection from heterologous or novel pandemic IAV strains.

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.

Human Dendritic Cell Response Signatures Distinguish 1918, Pandemic, and Seasonal H1N1 Influenza Viruses.

UNLABELLED: Influenza viruses continue to present global threats to human health. Antigenic drift and shift, genetic reassortment, and cross-species transmission generate new strains with differences in epidemiology and clinical severity. We compared the temporal transcriptional responses of human dendritic cells (DC) to infection with two pandemic (A/Brevig Mission/1/1918, A/California/4/2009) and two seasonal (A/New Caledonia/20/1999, A/Texas/36/1991) H1N1 influenza viruses. Strain-specific response differences included stronger activation of NF-κB following infection with A/New Caledonia/20/1999 and a unique cluster of genes expressed following infection with A/Brevig Mission/1/1918. A common antiviral program showing strain-specific timing was identified in the early DC response and found to correspond with reported transcript changes in blood during symptomatic human influenza virus infection. Comparison of the global responses to the seasonal and pandemic strains showed that a dramatic divergence occurred after 4 h, with only the seasonal strains inducing widespread mRNA loss. IMPORTANCE: Continuously evolving influenza viruses present a global threat to human health; however, these host responses display strain-dependent differences that are incompletely understood. Thus, we conducted a detailed comparative study assessing the immune responses of human DC to infection with two pandemic and two seasonal H1N1 influenza strains. We identified in the immune response to viral infection both common and strain-specific features. Among the stain-specific elements were a time shift of the interferon-stimulated gene response, selective induction of NF-κB signaling by one of the seasonal strains, and massive RNA degradation as early as 4 h postinfection by the seasonal, but not the pandemic, viruses. These findings illuminate new aspects of the distinct differences in the immune responses to pandemic and seasonal influenza viruses.

The nucleoprotein of newly emerged H7N9 influenza A virus harbors a unique motif conferring resistance to antiviral human MxA.

UNLABELLED: Interferon-induced Mx proteins show strong antiviral activity against influenza A viruses (IAVs). We recently demonstrated that the viral nucleoprotein (NP) determines resistance of seasonal and pandemic human influenza viruses to Mx, while avian isolates retain Mx sensitivity. We identified a surface-exposed cluster of amino acids in NP of pandemic A/BM/1/1918 (H1N1), comprising isoleucine-100, proline-283, and tyrosine-313, that is essential for reduced Mx sensitivity in cell culture and in vivo. This cluster has been maintained in all descendant seasonal strains, including A/PR/8/34 (PR/8). Accordingly, two substitutions in the NP of PR/8 [PR/8(mut)] to the Mx-sensitive amino acids (P283L and Y313F) led to attenuation in Mx1-positive mice. Serial lung passages of PR/8(mut) in Mx1 mice resulted in a single exchange of tyrosine to asparagine at position 52 in NP (in close proximity to the amino acid cluster at positions 100, 283, and 313), which partially compensates loss of Mx resistance in PR/8(mut). Intriguingly, the NP of the newly emerged avian-origin H7N9 virus also contains an asparagine at position 52 and shows reduced Mx sensitivity. N52Y substitution in NP results in increased sensitivity of the H7N9 virus to human Mx, indicating that this residue is a determinant of Mx resistance in mammals. Our data strengthen the hypothesis that the human Mx protein represents a potent barrier against zoonotic transmission of avian influenza viruses. However, the H7N9 viruses overcome this restriction by harboring an NP that is less sensitive to Mx-mediated host defense. This might contribute to zoonotic transmission of H7N9 and to the severe to fatal outcome of H7N9 infections in humans. IMPORTANCE: The natural host of influenza A viruses (IAVs) are aquatic birds. Occasionally, these viruses cross the species barrier, as in early 2013 when an avian H7N9 virus infected humans in China. Since then, multiple transmissions of H7N9 viruses to humans have occurred, leaving experts puzzled about molecular causes for such efficient crossing of the species barrier compared to other avian influenza viruses. Mx proteins are known restriction factors preventing influenza virus replication. Unfortunately, some viruses (e.g., human IAV) have developed some resistance, which is associated with specific amino acids in their nucleoproteins, the target of Mx function. Here, we demonstrate that the novel H7N9 bird IAV already carries a nucleoprotein that overcomes the inhibition of viral replication by human MxA. This is the first example of an avian IAV that is naturally less sensitive to Mx-mediated inhibition and might explain why H7N9 viruses transmitted efficiently to humans.

Inhibition of p38 mitogen-activated protein kinase impairs influenza virus-induced primary and secondary host gene responses and protects mice from lethal H5N1 infection.

Highly pathogenic avian influenza viruses (HPAIV) induce severe inflammation in poultry and men. One characteristic of HPAIV infections is the induction of a cytokine burst that strongly contributes to viral pathogenicity. This cell-intrinsic hypercytokinemia seems to involve hyperinduction of p38 mitogen-activated protein kinase. Here we investigate the role of p38 MAPK signaling in the antiviral response against HPAIV in mice as well as in human endothelial cells, the latter being a primary source of cytokines during systemic infections. Global gene expression profiling of HPAIV-infected endothelial cells in the presence of the p38-specific inhibitor SB 202190 revealed that inhibition of p38 MAPK leads to reduced expression of IFNß and other cytokines after H5N1 and H7N7 infection. More than 90% of all virus-induced genes were either partially or fully dependent on p38 signaling. Moreover, promoter analysis confirmed a direct impact of p38 on the IFNß promoter activity. Furthermore, upon treatment with IFN or conditioned media from HPAIV-infected cells, p38 controls interferon-stimulated gene expression by coregulating STAT1 by phosphorylation at serine 727. In vivo inhibition of p38 MAPK greatly diminishes virus-induced cytokine expression concomitant with reduced viral titers, thereby protecting mice from lethal infection. These observations show that p38 MAPK acts on two levels of the antiviral IFN response. Initially the kinase regulates IFN induction and, at a later stage, p38 controls IFN signaling and thereby expression of IFN-stimulated genes. Thus, inhibition of MAP kinase p38 may be an antiviral strategy that protects mice from lethal influenza by suppressing excessive cytokine expression.

A single amino acid substitution in the novel H7N9 influenza A virus NS1 protein increases CPSF30 binding and virulence.

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than the parental virus. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern.

Divergent H7 immunogens offer protection from H7N9 virus challenge.

UNLABELLED: The emergence of avian H7N9 viruses in humans in China has renewed concerns about influenza pandemics emerging from Asia. Vaccines are still the best countermeasure against emerging influenza virus infections, but the process from the identification of vaccine seed strains to the distribution of the final product can take several months. In the case of the 2009 H1N1 pandemic, a vaccine was not available before the first pandemic wave hit and therefore came too late to reduce influenza morbidity. H7 vaccines based on divergent isolates of the Eurasian and North American lineages have been tested in clinical trials, and seed strains and reagents are already available and can potentially be used initially to curtail influenza-induced disease until a more appropriately matched H7N9 vaccine is ready. In a challenge experiment in the mouse model, we assessed the efficacy of both inactivated virus and recombinant hemagglutinin vaccines made from seed strains that are divergent from H7N9 from each of the two major H7 lineages. Furthermore, we analyzed the cross-reactive responses of sera from human subjects vaccinated with heterologous North American and Eurasian lineage H7 vaccines to H7N9. Vaccinations with inactivated virus and recombinant hemagglutinin protein preparations from both lineages raised hemagglutination-inhibiting antibodies against H7N9 viruses and protected mice from stringent viral challenges. Similar cross-reactivity was observed in sera of human subjects from a clinical trial with a divergent H7 vaccine. Existing H7 vaccine candidates based on divergent strains could be used as a first line of defense against an H7N9 pandemic. In addition, this also suggests that H7N9 vaccines that are currently under development might be stockpiled and used for divergent avian H7 strains that emerge in the future. IMPORTANCE: Sporadic human infections with H7N9 viruses started being reported in China in the early spring of 2013. Despite a significant drop in the number of infections during the summer months of 2013, an increased number of cases has already been reported for the 2013-2014 winter season. The high case fatality rate, the ability to bind to receptors in the human upper respiratory tract in combination with several family clusters, and the emergence of neuraminidase inhibitor-resistant variants that show no loss of pathogenicity and the ability to transmit in animal models have raised concerns about a potential pandemic and have spurred efforts to produce vaccine candidates. Here we show that antigen preparations from divergent H7 strains are able to induce protective immunity against H7N9 infection.

Harnessing host enhancers of SARS-CoV-2 entry as novel targets for antiviral therapy.

The WHO declared the official end of the SARS-CoV-2 caused public health emergency on May 5th, 2023, after two years in which the virus infected approximately 750 Mio individuals causing estimated up to 7 Mio deaths. Likely, the virus will continue to evolve in the human population as a seasonal respiratory pathogen. To now prevent severe infection outcomes in vulnerable individuals, effective antivirals are urgently needed to complement the protection provided by vaccines. SARS-CoV-2 enters its host cell via ACE2 mediated membrane fusion, either at the plasma membrane, if the protease TMPRSS2 is present or via the endosome, in a cathepsin dependent fashion. A small number of positive regulators of viral uptake were described in the literature, which are potentially useful targets for host directed antiviral therapy or biomarkers indicating increased or diminished susceptibility to infection. We identified here by cell surface proximity ligation novel proteins, required for efficient virion uptake. Importantly, chemical inhibition of one of these factors, SLC3A2, resulted in robust reduction of viral replication, to that achieved with a TMPRSS2 inhibitor. Our screen identified new host dependency factors for SARS-CoV-2 entry, which could be targeted by novel antiviral therapies.

Influenza A virus infection alters the resistance profile of gut microbiota to clinically relevant antibiotics.

IMPORTANCE: Influenza virus infection affects both lung and intestinal bacterial community composition. Most of the published analyses focus on the characterization of the microbiota composition changes. Here we assess functional alterations of gut microbiota such as nutrient and antibiotic resistance changes during an acute respiratory tract infection. Upon influenza A virus (IAV) infection, cecal microbiota drops accompanied by a decrease in the ability to metabolize some common nutrients under aerobic conditions. At the same time, the cecal community presents an increase in resistance against clinically relevant antibiotics, particularly cephalosporins. Functional characterization of complex communities presents an additional and necessary element of analysis that nowadays is mainly limited to taxonomic description. The consequences of these functional alterations could affect treatment strategies, especially in multimicrobial infections.

Influenza virus sequence feature variant type analysis: evidence of a role for NS1 in influenza virus host range restriction.

Genetic drift of influenza virus genomic sequences occurs through the combined effects of sequence alterations introduced by a low-fidelity polymerase and the varying selective pressures experienced as the virus migrates through different host environments. While traditional phylogenetic analysis is useful in tracking the evolutionary heritage of these viruses, the specific genetic determinants that dictate important phenotypic characteristics are often difficult to discern within the complex genetic background arising through evolution. Here we describe a novel influenza virus sequence feature variant type (Flu-SFVT) approach, made available through the public Influenza Research Database resource (www.fludb.org), in which variant types (VTs) identified in defined influenza virus protein sequence features (SFs) are used for genotype-phenotype association studies. Since SFs have been defined for all influenza virus proteins based on known structural, functional, and immune epitope recognition properties, the Flu-SFVT approach allows the rapid identification of the molecular genetic determinants of important influenza virus characteristics and their connection to underlying biological functions. We demonstrate the use of the SFVT approach to obtain statistical evidence for effects of NS1 protein sequence variations in dictating influenza virus host range restriction.

The influenza virus protein PB1-F2 inhibits the induction of type I interferon at the level of the MAVS adaptor protein.

PB1-F2 is a 90 amino acid protein that is expressed from the +1 open reading frame in the PB1 gene of some influenza A viruses and has been shown to contribute to viral pathogenicity. Notably, a serine at position 66 (66S) in PB1-F2 is known to increase virulence compared to an isogenic virus with an asparagine (66N) at this position. Recently, we found that an influenza virus expressing PB1-F2 N66S suppresses interferon (IFN)-stimulated genes in mice. To characterize this phenomenon, we employed several in vitro assays. Overexpression of the A/Puerto Rico/8/1934 (PR8) PB1-F2 protein in 293T cells decreased RIG-I mediated activation of an IFN-ß reporter and secretion of IFN as determined by bioassay. Of note, the PB1-F2 N66S protein showed enhanced IFN antagonism activity compared to PB1-F2 wildtype. Similar observations were found in the context of viral infection with a PR8 PB1-F2 N66S virus. To understand the relationship between NS1, a previously described influenza virus protein involved in suppression of IFN synthesis, and PB1-F2, we investigated the induction of IFN when NS1 and PB1-F2 were co-expressed in an in vitro transfection system. In this assay we found that PB1-F2 N66S further reduced IFN induction in the presence of NS1. By inducing the IFN-ß reporter at different levels in the signaling cascade, we found that PB1-F2 inhibited IFN production at the level of the mitochondrial antiviral signaling protein (MAVS). Furthermore, immunofluorescence studies revealed that PB1-F2 co-localizes with MAVS. In summary, we have characterized the anti-interferon function of PB1-F2 and we suggest that this activity contributes to the enhanced pathogenicity seen with PB1-F2 N66S- expressing influenza viruses.

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species.

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism.

Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor.

A chemical genetics approach was taken to identify inhibitors of NS1, a major influenza A virus virulence factor that inhibits host gene expression. A high-throughput screen of 200,000 synthetic compounds identified small molecules that reversed NS1-mediated inhibition of host gene expression. A counterscreen for suppression of influenza virus cytotoxicity identified naphthalimides that inhibited replication of influenza virus and vesicular stomatitis virus (VSV). The mechanism of action occurs through activation of REDD1 expression and concomitant inhibition of mammalian target of rapamycin complex 1 (mTORC1) via TSC1-TSC2 complex. The antiviral activity of naphthalimides was abolished in REDD1(-/-) cells. Inhibition of REDD1 expression by viruses resulted in activation of the mTORC1 pathway. REDD1(-/-) cells prematurely upregulated viral proteins via mTORC1 activation and were permissive to virus replication. In contrast, cells conditionally expressing high concentrations of REDD1 downregulated the amount of viral protein. Thus, REDD1 is a new host defense factor, and chemical activation of REDD1 expression represents a potent antiviral intervention strategy.

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.

Wnt-Independent SARS-CoV-2 Infection in Pulmonary Epithelial Cells.

The Wnt signaling pathway within host cells regulates infections by several pathogenic bacteria and viruses. Recent studies suggested that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection depends on ß-catenin and can be inhibited by the antileprotic drug clofazimine. Since clofazimine has been identified by us as a specific inhibitor of Wnt/ß-catenin signaling, these works could indicate a potential role of the Wnt pathway in SARS-CoV-2 infection. Here, we show that the Wnt pathway is active in pulmonary epithelial cells. However, we find that in multiple assays, SARS-CoV-2 infection is insensitive to Wnt inhibitors, including clofazimine, acting at different levels within the pathway. Our findings assert that endogenous Wnt signaling in the lung is unlikely required or involved in the SARS-CoV-2 infection and that pharmacological inhibition of this pathway with clofazimine or other compounds is not a universal way to develop treatments against the SARS-CoV-2 infection. IMPORTANCE The development of inhibitors of the SARS-CoV-2 infection remains a need of utmost importance. The Wnt signaling pathway in host cells is often implicated in infections by bacteria and viruses. In this work, we show that, despite previous indications, pharmacological modulation of the Wnt pathway does not represent a promising strategy to control SARS-CoV-2 infection in lung epithelia.