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.

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.

Evolution of the Bunyamwera virus polymerase to accommodate deletions within genomic untranslated region sequences.

UNLABELLED: The untranslated regions (UTR) present at the ends of bunyavirus genome segments are required for essential steps in the virus life cycle and provide signals for encapsidation by nucleocapsid protein and the promoters for RNA transcription and replication as well as for mRNA transcription termination. For the prototype bunyavirus, Bunyamwera virus (BUNV), only the terminal 11 nucleotides (nt) of the segments are identical. Thereafter, the UTRs are highly variable both in length and in sequence. Furthermore, apart from the conserved termini, the UTRs of different viruses are highly variable. We previously generated recombinant BUNV carrying the minimal UTRs on all three segments that were attenuated for growth in cell culture. Following serial passage of these viruses, the viruses acquired increased fitness, and amino acid changes were observed to accumulate in the viral polymerase (L protein) of most mutant viruses, with the vast majority of the amino acid changes occurring in the C-terminal region. The function of this domain within L remains unknown, but by using a minigenome assay we showed that it might be involved in UTR recognition. Moreover, we identified an amino acid mutation within the polymerase that, when introduced into an otherwise wild-type BUNV, resulted in a virus with a temperature-sensitive phenotype. Viruses carrying temperature-sensitive mutations are good candidates for the design of live attenuated vaccines. We suggest that a combination of stable deletions of the UTRs together with the introduction of temperature-sensitive mutations in both the nucleocapsid and the polymerase could be used to design live attenuated vaccines against serious pathogens within the family Bunyaviridae. IMPORTANCE: Virus growth in tissue culture can be attenuated by introduction of mutations in both coding and noncoding sequences. We generated attenuated Bunyamwera viruses by deleting sequences within both the 3' and 5' untranslated regions (UTR) on each genome segment and showed that the viruses regained fitness following serial passage in cell culture. The fitter viruses had acquired amino acid changes predominantly in the C-terminal domain of the viral polymerase (L protein), and by using minigenome assays we showed that the mutant polymerases were better adapted to recognizing the mutant UTRs. We suggest that deletions within the UTRs should be incorporated along with other specific mutations, including deletion of the major virulence gene encoding the NSs protein and introduction of temperature-sensitive mutations, in the design of attenuated bunyaviruses that could have potential as vaccines.

Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin.

UNLABELLED: Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043. IMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.

Attenuation of bunyamwera orthobunyavirus replication by targeted mutagenesis of genomic untranslated regions and creation of viable viruses with minimal genome segments.

Bunyamwera virus (BUNV) is the prototype virus for both the genus Orthobunyavirus and the family Bunyaviridae. BUNV has a tripartite, negative-sense RNA genome. The coding region of each segment is flanked by untranslated regions (UTRs) that are partially complementary. The UTRs play an important role in the virus life cycle by promoting transcription, replication, and encapsidation of the viral genome. Using reverse genetics, we generated recombinant viruses that contained deletions within the 3' and/or 5' UTRs of the L or M segments to determine the minimal UTRs competent for virus viability. We then generated viruses carrying deleted UTRs in all three segments. These viruses were grossly attenuated in tissue culture, being significantly impaired in their ability to produce plaques in BHK cells, and had a reduced capacity to cause host cell protein shutoff. After serial passage in tissue culture, some viruses partially recovered fitness, generating higher titers and producing larger plaques. We determined the complete nucleotide sequence for each virus. The deleted UTR sequences were maintained, and no amino acid changes were observed in the nonstructural proteins (NSs and NSm), the nucleocapsid protein (N), or the Gn glycoprotein. One virus had a single amino acid substitution in Gc. Three viruses contained amino acid changes in the viral polymerase that mostly occurred in the C-terminal domain of the L protein. Although the role of this domain remains unknown, we suggest that those changes might be involved in the evolution of the polymerase to recognize the deleted UTRs more efficiently.

Antiviral properties of trans-δ-viniferin derivatives against enveloped viruses.

Over the last century, the number of epidemics caused by RNA viruses has increased and the current SARS-CoV-2 pandemic has taught us about the compelling need for ready-to-use broad-spectrum antivirals. In this scenario, natural products stand out as a major historical source of drugs. We analyzed the antiviral effect of 4 stilbene dimers [1 (trans-δ-viniferin); 2 (11',13'-di-O-methyl-trans-δ-viniferin), 3 (11,13-di-O-methyl-trans-δ-viniferin); and 4 (11,13,11',13'-tetra-O-methyl-trans-δ-viniferin)] obtained from plant substrates using chemoenzymatic synthesis against a panel of enveloped viruses. We report that compounds 2 and 3 display a broad-spectrum antiviral activity, being able to effectively inhibit several strains of Influenza Viruses (IV), SARS-CoV-2 Delta and, to some extent, Herpes Simplex Virus 2 (HSV-2). Interestingly, the mechanism of action differs for each virus. We observed both a direct virucidal and a cell-mediated effect against IV, with a high barrier to antiviral resistance; a restricted cell-mediated mechanism of action against SARS-CoV-2 Delta and a direct virustatic activity against HSV-2. Of note, while the effect was lost against IV in tissue culture models of human airway epithelia, the antiviral activity was confirmed in this relevant model for SARS-CoV-2 Delta. Our results suggest that stilbene dimer derivatives are good candidate models for the treatment of enveloped virus infections.

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.

Nucleolar localization of influenza A NS1: striking differences between mammalian and avian cells.

In mammalian cells, nucleolar localization of influenza A NS1 requires the presence of a C-terminal nucleolar localization signal. This nucleolar localization signal is present only in certain strains of influenza A viruses. Therefore, only certain NS1 accumulate in the nucleolus of mammalian cells. In contrast, we show that all NS1 tested in this study accumulated in the nucleolus of avian cells even in the absence of the above described C-terminal nucleolar localization signal. Thus, nucleolar localization of NS1 in avian cells appears to rely on a different nucleolar localization signal that is more conserved among influenza virus strains.

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.

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.

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.