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 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.

A quantitative model for virus uncoating predicts influenza A infectivity.

For virus infection of new host cells, the disassembly of the protective outer protein shell (capsid) is a critical step, but the mechanisms and host-virus interactions underlying the dynamic, active, and regulated uncoating process are largely unknown. Here, we develop an experimentally supported, multiscale kinetics model that elucidates mechanisms of influenza A virus (IAV) uncoating in cells. Biophysical modeling demonstrates that interactions between capsid M1 proteins, host histone deacetylase 6 (HDAC6), and molecular motors can physically break the capsid in a tug-of-war mechanism. Biochemical analysis and biochemical-biophysical modeling identify unanchored ubiquitin chains as essential and allow robust prediction of uncoating efficiency in cells. Remarkably, the different infectivity of two clinical strains can be ascribed to a single amino acid variation in M1 that affects binding to HDAC6. By identifying crucial modules of viral infection kinetics, the mechanisms and models presented here could help formulate novel strategies for broad-range antiviral treatment.

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.

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.

Silent neonatal influenza A virus infection primes systemic antimicrobial immunity.

Infections with influenza A viruses (IAV) cause seasonal epidemics and global pandemics. The majority of these infections remain asymptomatic, especially among children below five years of age. Importantly, this is a time, when immunological imprinting takes place. Whether early-life infections with IAV affect the development of antimicrobial immunity is unknown. Using a preclinical mouse model, we demonstrate here that silent neonatal influenza infections have a remote beneficial impact on the later control of systemic juvenile-onset and adult-onset infections with an unrelated pathogen, Staphylococcus aureus, due to improved pathogen clearance and clinical resolution. Strategic vaccination with a live attenuated IAV vaccine elicited a similar protection phenotype. Mechanistically, the IAV priming effect primarily targets antimicrobial functions of the developing innate immune system including increased antimicrobial plasma activity and enhanced phagocyte functions and antigen-presenting properties at mucosal sites. Our results suggest a long-term benefit from an exposure to IAV during the neonatal phase, which might be exploited by strategic vaccination against influenza early in life to enforce the host's resistance to later bacterial infections.

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.

A single respiratory tract infection early in life reroutes healthy microbiome development and affects adult metabolism in a preclinical animal model.

In adult animals, acute viral infections only temporarily alter the composition of both respiratory and intestinal commensal microbiota, potentially due to the intrinsic stability of this microbial ecosystem. In stark contrast, commensal bacterial communities are rather vulnerable to perturbation in infancy. Animal models proved that disruption of a balanced microbiota development e.g., by antibiotics treatment early in life, increases the probability for metabolic disorders in adults. Importantly, infancy is also a phase in life with high incidence of acute infections. We postulated that acute viral infections in early life might pose a similarly severe perturbation and permanently shape microbiota composition with long-term physiological consequences for the adult host. As a proof of concept, we infected infant mice with a sub-lethal dose of influenza A virus. We determined microbiota composition up to early adulthood (63 days) from small intestine by 16S rRNA gene-specific next-generation sequencing. Infected mice underwent long-lasting changes in microbiota composition, associated with increase in fat mass. High-fat-high-glucose diet promoted this effect while co-housing with mock-treated animals overwrote the weight gain. Our data suggest that in the critical phase of infancy even a single silent viral infection could cast a long shadow and cause long-term microbiota perturbations, affecting adult host physiology.

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.

Visualization of Respiratory Commensal Bacteria in Context of Their Natural Host Environment.

Commensal microbes are an integral component of mammalian physiology. 16S rRNA gene-specific next generation sequencing from DNA of total organs, swabs or lavages has revolutionized the characterization of bacterial communities in virtually every ecological niche of the body. Culturomics, next allowed the isolation and characterization of commensal bacteria in the lab and the establishment of artificial communities of bacteria, which were eventually reintroduced in model organisms. Spatial organization of microbiota within a given host environment is critical to the physiological or pathological phenotypes provoked by commensal microbiota. In situ hybridization (ISH) is a complementary technique to sequencing and culturing to visualize the presence of individual bacterial operational taxonomic unit (OTUs) in context of the colonized organ. We recently applied highly sensitive in situ RNA hybridization to detection of commensal bacteria in low abundance respiratory tract samples of mice housed under specific pathogen free conditions. This technique allows species-specific detection of living bacteria using RNAScopeTM technology, while preserving the natural environment of the organ. We here provide a detailed step-by-step protocol describing the detection of commensal lung bacteria in respiratory tissue.

Respiratory tissue-associated commensal bacteria offer therapeutic potential against pneumococcal colonization.

Under eubiotic conditions commensal microbes are known to provide a competitive barrier against invading bacterial pathogens in the intestinal tract, on the skin or on the vaginal mucosa. Here, we evaluate the role of lung microbiota in Pneumococcus colonization of the lungs. In eubiosis, the lungs of mice were dominantly colonized by Lactobacillus murinus. Differential analysis of 16S rRNA gene sequencing or L. murinus-specific qPCR of DNA from total organ homogenates vs.broncho alveolar lavages implicated tight association of these bacteria with the host tissue. Pure L. murinus conditioned culture medium inhibited growth and reduced the extension of pneumococcal chains. Growth inhibition in vitro was likely dependent on L. murinus-produced lactic acid, since pH neutralization of the conditioned medium aborted the antibacterial effect. Finally, we demonstrate that L. murinus provides a barrier against pneumococcal colonization in a respiratory dysbiosis model after an influenza A virus infection, when added therapeutically.

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.

Author Correction: Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microbiome disturbance and resilience dynamics of the upper respiratory tract during influenza A virus infection.

Infection with influenza can be aggravated by bacterial co-infections, which often results in disease exacerbation. The effects of influenza infection on the upper respiratory tract (URT) microbiome are largely unknown. Here, we report a longitudinal study to assess the temporal dynamics of the URT microbiomes of uninfected and influenza virus-infected humans and ferrets. Uninfected human patients and ferret URT microbiomes have stable healthy ecostate communities both within and between individuals. In contrast, infected patients and ferrets exhibit large changes in bacterial community composition over time and between individuals. The unhealthy ecostates of infected individuals progress towards the healthy ecostate, coinciding with viral clearance and recovery. Pseudomonadales associate statistically with the disturbed microbiomes of infected individuals. The dynamic and resilient microbiome during influenza virus infection in multiple hosts provides a compelling rationale for the maintenance of the microbiome homeostasis as a potential therapeutic target to prevent IAV associated bacterial co-infections.

Ménage à trois: Virus, Host, and Microbiota in Experimental Infection Models.

Infections of mammals with pathogenic viruses occur mostly in the polymicrobial environment of mucosal surfaces or the skin. In recent years our understanding of immune modulation by the commensal microbiota has increased dramatically. The microbiota is today accepted as the prime educator and maintainer of innate and adaptive immune functions. It became further apparent that some viral pathogens profit from the presence of commensal bacteria and their metabolites, especially in the intestinal tract. We further learned that the composition and abundance of the microbiota can change as a consequence of acute and chronic viral infections. Here we discuss recent developments in our understanding of the triangular relationship of virus, host, and microbiota under experimental infection settings.

Phosphoproteomic-based kinase profiling early in influenza virus infection identifies GRK2 as antiviral drug target.

Although annual influenza epidemics affect around 10% of the global population, current treatment options are limited and development of new antivirals is needed. Here, using quantitative phosphoproteomics, we reveal the unique phosphoproteome dynamics that occur in the host cell within minutes of influenza A virus (IAV) infection. We uncover cellular kinases required for the observed signaling pattern and find that inhibition of selected candidates, such as the G protein-coupled receptor kinase 2 (GRK2), leads to decreased IAV replication. As GRK2 has emerged as drug target in heart disease, we focus on its role in IAV infection and show that it is required for viral uncoating. Replication of seasonal and pandemic IAVs is severely decreased by specific GRK2 inhibitors in primary human airway cultures and in mice. Our study reveals the IAV-induced changes to the cellular phosphoproteome and identifies GRK2 as crucial node of the kinase network that enables IAV replication.

Influenza A Virus Genetic Tools: From Clinical Sample to Molecular Clone.

Implementation of reverse genetics for influenza A virus, that is, the DNA-based generation of infectious viral particles in cell culture, opened new avenues to investigate the function of viral proteins and their interplay with host factors on a molecular level. This powerful technique allows the introduction, depletion, or manipulation of any given sequence in the viral genome, as long as it gives rise to replicating virus progeny. Reverse genetics can be used to generate targeted reassortant viruses by mixing segments of different viral strains, thus providing insight into phenotypes of potentially pandemic viruses arising from natural reassortment. It was further instrumental for the development of novel vaccine strategies, allowing rapid and targeted exchange of viral surface antigens on a well-replicating genetic backbone of cell culture-adapted or cold-adapted/attenuated viral strains. Establishment of reverse genetics and rescue of molecular clones of influenza A virus have been extensively described before. Here we give a detailed stand-alone protocol encompassing clinical sampling of influenza A virus specimens and subsequent plasmid-based genetics to rescue, manipulate, and confirm a fully infectious molecular clone. This protocol is based on the combined techniques and experience of a number of influenza laboratories, which are credited and referenced whenever appropriate.

Overcoming the Neonatal Limitations of Inducing Germinal Centers through Liposome-Based Adjuvants Including C-Type Lectin Agonists Trehalose Dibehenate or Curdlan.

Neonates and infants are more vulnerable to infections and show reduced responses to vaccination. Consequently, repeated immunizations are required to induce protection and early life vaccines against major pathogens such as influenza are yet unavailable. Formulating antigens with potent adjuvants, including immunostimulators and delivery systems, is a demonstrated approach to enhance vaccine efficacy. Yet, adjuvants effective in adults may not meet the specific requirements for activating the early life immune system. Here, we assessed the neonatal adjuvanticity of three novel adjuvants including TLR4 (glucopyranosyl lipid adjuvant-squalene emulsion), TLR9 (IC31®), and Mincle (CAF01) agonists, which all induce germinal centers (GCs) and potent antibody responses to influenza hemagglutinin (HA) in adult mice. In neonates, a single dose of HA formulated into each adjuvant induced T follicular helper (TFH) cells. However, only HA/CAF01 elicited significantly higher and sustained antibody responses, engaging neonatal B cells to differentiate into GCs already after a single dose. Although antibody titers remained lower than in adults, HA-specific responses induced by a single neonatal dose of HA/CAF01 were sufficient to confer protection against influenza viral challenge. Postulating that the neonatal adjuvanticity of CAF01 may result from the functionality of the C-type lectin receptor (CLR) Mincle in early life we asked whether other C-type lectin agonists would show a similar neonatal adjuvanticity. Replacing the Mincle agonist trehalose 6,6'-dibehenate by Curdlan, which binds to Dectin-1, enhanced antibody responses through the induction of similar levels of TFH, GCs and bone marrow high-affinity plasma cells. Thus, specific requirements of early life B cells may already be met after a single vaccine dose using CLR-activating agonists, identified here as promising B cell immunostimulators for early life vaccines when included into cationic liposomes.

Influenza A virus infection impacts systemic microbiota dynamics and causes quantitative enteric dysbiosis.

BACKGROUND: Microbiota integrity is essential for a growing number of physiological processes. Consequently, disruption of microbiota homeostasis correlates with a variety of pathological states. Importantly, commensal microbiota provide a shield against invading bacterial pathogens, probably by direct competition. The impact of viral infections on host microbiota composition and dynamics is poorly understood. Influenza A viruses (IAV) are common respiratory pathogens causing acute infections. Here, we show dynamic changes in respiratory and intestinal microbiota over the course of a sublethal IAV infection in a mouse model. RESULTS: Using a combination of 16S rRNA gene-specific next generation sequencing and qPCR as well as culturing of bacterial organ content, we found body site-specific and transient microbiota responses. In the lower respiratory tract, we observed only minor qualitative changes in microbiota composition. No quantitative impact on bacterial colonization after IAV infection was detectable, despite a robust antimicrobial host response and increased sensitivity to bacterial super infection. In contrast, in the intestine, IAV induced robust depletion of bacterial content, disruption of mucus layer integrity, and higher levels of antimicrobial peptides in Paneth cells. As a functional consequence of IAV-mediated microbiota depletion, we demonstrated that the small intestine is rendered more susceptible to bacterial pathogen invasion, in a Salmonella typhimurium super infection model. CONCLUSION: We show for the first time the consequences of IAV infection for lower respiratory tract and intestinal microbiobiota in a qualitative and quantitative fashion. The discrepancy of relative 16S rRNA gene next-generation sequencing (NGS) and normalized 16S rRNA gene-specific qPCR stresses the importance of combining qualitative and quantitative approaches to correctly analyze composition of organ associated microbial communities. The transiently induced dysbiosis underlines the overall stability of microbial communities to effects of acute infection. However, during a short-time window, specific ecological niches might lose their microbiota shield and remain vulnerable to bacterial invasion.

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.