Triggering Degradation of Host Cellular Proteins for Robust Propagation of Influenza Viruses.

Following infection, influenza viruses strive to establish a new host cellular environment optimized for efficient viral replication and propagation. Influenza viruses use or hijack numerous host factors and machinery not only to fulfill their own replication process but also to constantly evade the host's antiviral and immune response. For this purpose, influenza viruses appear to have formulated diverse strategies to manipulate the host proteins or signaling pathways. One of the most effective tactics is to specifically induce the degradation of the cellular proteins that are detrimental to the virus life cycle. Here, we summarize the cellular factors that are deemed to have been purposefully degraded by influenza virus infection. The focus is laid on the mechanisms for the protein ubiquitination and degradation in association with facilitated viral amplification. The fate of influenza viral infection of hosts is heavily reliant on the outcomes of the interplay between the virus and the host antiviral immunity. Understanding the processes of how influenza viruses instigate the protein destruction pathways could provide a foundation for the development of advanced therapeutics to target host proteins and conquer influenza.

Mapping the interaction sites of human and avian influenza A viruses and complement factor H.

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects "self" cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated.

Airway epithelial CD47 plays a critical role in inducing influenza virus-mediated bacterial super-infection.

Respiratory viral infection increases host susceptibility to secondary bacterial infections, yet the precise dynamics within airway epithelia remain elusive. Here, we elucidate the pivotal role of CD47 in the airway epithelium during bacterial super-infection. We demonstrated that upon influenza virus infection, CD47 expression was upregulated and localized on the apical surface of ciliated cells within primary human nasal or bronchial epithelial cells. This induced CD47 exposure provided attachment sites for Staphylococcus aureus, thereby compromising the epithelial barrier integrity. Through bacterial adhesion assays and in vitro pull-down assays, we identified fibronectin-binding proteins (FnBP) of S. aureus as a key component that binds to CD47. Furthermore, we found that ciliated cell-specific CD47 deficiency or neutralizing antibody-mediated CD47 inactivation enhanced in vivo survival rates. These findings suggest that interfering with the interaction between airway epithelial CD47 and pathogenic bacterial FnBP holds promise for alleviating the adverse effects of super-infection.

Influenza A virus NS1 effector domain is required for PA-X-mediated host shutoff in infected cells.

Many viruses inhibit general host gene expression to limit innate immune responses and gain preferential access to the cellular translational apparatus for their protein synthesis. This process is known as host shutoff. Influenza A viruses (IAVs) encode two host shutoff proteins: nonstructural protein 1 (NS1) and polymerase acidic X (PA-X). NS1 inhibits host nuclear pre-messenger RNA maturation and export, and PA-X is an endoribonuclease that preferentially cleaves host spliced nuclear and cytoplasmic messenger RNAs. Emerging evidence suggests that in circulating human IAVs NS1 and PA-X co-evolve to ensure optimal magnitude of general host shutoff without compromising viral replication that relies on host cell metabolism. However, the functional interplay between PA-X and NS1 remains unexplored. In this study, we sought to determine whether NS1 function has a direct effect on PA-X activity by analyzing host shutoff in A549 cells infected with wild-type or mutant IAVs with NS1 effector domain deletion. This was done using conventional quantitative reverse transcription polymerase chain reaction techniques and direct RNA sequencing using nanopore technology. Our previous research on the molecular mechanisms of PA-X function identified two prominent features of IAV-infected cells: nuclear accumulation of cytoplasmic poly(A) binding protein (PABPC1) and increase in nuclear poly(A) RNA abundance relative to the cytoplasm. Here we demonstrate that NS1 effector domain function augments PA-X host shutoff and is necessary for nuclear PABPC1 accumulation. By contrast, nuclear poly(A) RNA accumulation is not dependent on either NS1 or PA-X-mediated host shutoff and is accompanied by nuclear retention of viral transcripts. Our study demonstrates for the first time that NS1 and PA-X may functionally interact in mediating host shutoff.IMPORTANCERespiratory viruses including the influenza A virus continue to cause annual epidemics with high morbidity and mortality due to the limited effectiveness of vaccines and antiviral drugs. Among the strategies evolved by viruses to evade immune responses is host shutoff-a general blockade of host messenger RNA and protein synthesis. Disabling influenza A virus host shutoff is being explored in live attenuated vaccine development as an attractive strategy for increasing their effectiveness by boosting antiviral responses. Influenza A virus encodes two proteins that function in host shutoff: the nonstructural protein 1 (NS1) and the polymerase acidic X (PA-X). We and others have characterized some of the NS1 and PA-X mechanisms of action and the additive effects that these viral proteins may have in ensuring the blockade of host gene expression. In this work, we examined whether NS1 and PA-X functionally interact and discovered that NS1 is required for PA-X to function effectively. This work significantly advances our understanding of influenza A virus host shutoff and identifies new potential targets for therapeutic interventions against influenza and further informs the development of improved live attenuated vaccines.

Feruloylated Oligosaccharides Prevented Influenza-Induced Lung Inflammation via the RIG-I/MAVS/TRAF3 Pathway.

Uncontrolled inflammation contributes significantly to the mortality in acute respiratory infections. Our previous research has demonstrated that maize bran feruloylated oligosaccharides (FOs) possess notable anti-inflammatory properties linked to the NF-kB pathway regulation. In this study, we clarified that the oral administration of FOs moderately inhibited H1N1 virus infection and reduced lung inflammation in influenza-infected mice by decreasing a wide spectrum of cytokines (IFN-α, IFN-ß, IL-6, IL-10, and IL-23) in the lungs. The mechanism involves FOs suppressing the transduction of the RIG-I/MAVS/TRAF3 signaling pathway, subsequently lowering the expression of NF-κB. In silico analysis suggests that FOs have a greater binding affinity for the RIG-I/MAVS signaling complex. This indicates that FOs have potential as promising targets for immune modulation. Moreover, in MAVS knockout mice, we confirmed that the anti-inflammatory function of FOs against influenza depends on MAVS. Comprehensive analysis using 16S rRNA gene sequencing and metabolite profiling techniques showed that FOs have the potential to restore immunity by modulating the gut microbiota. In conclusion, our study demonstrates that FOs are effective anti-inflammatory phytochemicals in inhibiting lung inflammation caused by influenza. This suggests that FOs could serve as a potential nutritional strategy for preventing the H1N1 virus infection and associated lung inflammation.

Exposure to PM2.5 modulate the pro-inflammatory and interferon responses against influenza virus infection in a human 3D bronchial epithelium model.

Epidemiological studies showed a positive association between exposure to PM2.5 and the severity of influenza virus infection. However, the mechanisms by which PM2.5 can disrupt antiviral defence are still unclear. From this perspective, the objective of this study was to evaluate the effects of PM2.5 on antiviral signalling in the respiratory epithelium using the bronchial Calu-3 cell line grown at the air-liquid interface. Pre-exposure to PM2.5 before infection with the influenza virus was investigated, as well as a co-exposure. Although a physical interaction between the virus and the particles seems possible, no effect of PM2.5 on viral replication was observed during co-exposure, although a downregulation of IFN-ß release was associated to PM2.5 exposure. However, pre-exposure slightly increased the viral nucleoprotein production and the pro-inflammatory response. Conversely, the level of the myxovirus resistance protein A (MxA), an interferon-stimulated gene (ISG) induced by IFN-ß, was reduced. Therefore, these results suggest that pre-exposure to PM2.5 could alter the antiviral response of bronchial epithelial cells, increasing their susceptibility to viral infection.

Early-life influenza A (H1N1) infection independently programs brain connectivity, HPA AXIS and tissue-specific gene expression profiles.

Early-life adversity covers a range of physical, social and environmental stressors. Acute viral infections in early life are a major source of such adversity and have been associated with a broad spectrum of later-life effects outside the immune system or "off-target". These include an altered hypothalamus-pituitary-adrenal (HPA) axis and metabolic reactions. Here, we used a murine post-natal day 14 (PND 14) Influenza A (H1N1) infection model and applied a semi-holistic approach including phenotypic measurements, gene expression arrays and diffusion neuroimaging techniques to investigate HPA axis dysregulation, energy metabolism and brain connectivity. By PND 56 the H1N1 infection had been resolved, and there was no residual gene expression signature of immune cell infiltration into the liver, adrenal gland or brain tissues examined nor of immune-related signalling. A resolved early-life H1N1 infection had sex-specific effects. We observed retarded growth of males and altered pre-stress (baseline) blood glucose and corticosterone levels at PND42 after the infection was resolved. Cerebral MRI scans identified reduced connectivity in the cortex, midbrain and cerebellum that were accompanied by tissue-specific gene expression signatures. Gene set enrichment analysis confirmed that these were tissue-specific changes with few common pathways. Early-life infection independently affected each of the systems and this was independent of HPA axis or immune perturbations.

C1QTNF5 is a novel attachment factor that facilitates the entry of influenza A virus.

Influenza A virus (IAV) binds sialic acid receptors on the cell surface to enter the host cells, which is the key step in initiating infection, transmission and pathogenesis. Understanding the factors that contribute to the highly efficient entry of IAV into human cells will help elucidate the mechanism of viral entry and pathogenicity, and provide new targets for intervention. In the present study, we reported a novel membrane protein, C1QTNF5, which binds to the hemagglutinin protein of IAV and promotes IAV infection in vitro and in vivo. We found that the HA1 region of IAV hemagglutinin is critical for the interaction with C1QTNF5 protein, and C1QTNF5 interacts with hemagglutinin mainly through its N-terminus (1-103 aa). In addition, we further demonstrated that overexpression of C1QTNF5 promotes IAV entry, while blocking the interaction between C1QTNF5 and IAV hemagglutinin greatly inhibits viral entry. However, C1QTNF5 does not function as a receptor to mediate IAV infection in sialic acid-deficient CHO-Lec2 cells, but promotes IAV to attach to these cells, suggesting that C1QTNF5 is an important attachment factor for IAV. This work reveals C1QTNF5 as a novel IAV attachment factor and provides a new perspective for antiviral strategies.

Unveiling the role of host kinases at different steps of influenza A virus life cycle.

Influenza viruses remain a major public health concern causing contagious respiratory illnesses that result in around 290,000-650,000 global deaths every year. Their ability to constantly evolve through antigenic shifts and drifts leads to the emergence of newer strains and resistance to existing drugs and vaccines. To combat this, there is a critical need for novel antiviral drugs through the introduction of host-targeted therapeutics. Influenza viruses encode only 14 gene products that get extensively modified through phosphorylation by a diverse array of host kinases. Reversible phosphorylation at serine, threonine, or tyrosine residues dynamically regulates the structure, function, and subcellular localization of viral proteins at different stages of their life cycle. In addition, kinases influence a plethora of signaling pathways that also regulate virus propagation by modulating the host cell environment thus establishing a critical virus-host relationship that is indispensable for executing successful infection. This dependence on host kinases opens up exciting possibilities for developing kinase inhibitors as next-generation anti-influenza therapy. To fully capitalize on this potential, extensive mapping of the influenza virus-host kinase interaction network is essential. The key focus of this review is to outline the molecular mechanisms by which host kinases regulate different steps of the influenza A virus life cycle, starting from attachment-entry to assembly-budding. By assessing the contributions of different host kinases and their specific phosphorylation events during the virus life cycle, we aim to develop a holistic overview of the virus-host kinase interaction network that may shed light on potential targets for novel antiviral interventions.

Citric Acid Promotes Immune Function by Modulating the Intestinal Barrier.

Amidst increasing concern about antibiotic resistance resulting from the overuse of antibiotics, there is a growing interest in exploring alternative agents. One such agent is citric acid, an organic compound commonly used for various applications. Our research findings indicate that the inclusion of citric acid can have several beneficial effects on the tight junctions found in the mouse intestine. Firstly, the study suggests that citric acid may contribute to weight gain by stimulating the growth of intestinal epithelial cells (IE-6). Citric acid enhances the small intestinal villus-crypt ratio in mice, thereby promoting intestinal structural morphology. Additionally, citric acid has been found to increase the population of beneficial intestinal microorganisms, including Bifidobacterium and Lactobacillus. It also promotes the expression of important protein genes such as occludin, ZO-1, and claudin-1, which play crucial roles in maintaining the integrity of the tight junction barrier in the intestines. Furthermore, in infected IEC-6 cells with H9N2 avian influenza virus, citric acid augmented the expression of genes closely associated with the influenza virus infection. Moreover, it reduces the inflammatory response caused by the viral infection and thwarted influenza virus replication. These findings suggest that citric acid fortifies the intestinal tight junction barrier, inhibits the replication of influenza viruses targeting the intestinal tract, and boosts intestinal immune function.

Accurate calculation of affinity changes to the close state of influenza A M2 transmembrane domain in response to subtle structural changes of adamantyl amines using free energy perturbation methods in different lipid bilayers.

Experimental binding free energies of 27 adamantyl amines against the influenza M2(22-46) WT tetramer, in its closed form at pH 8, were measured by ITC in DPC micelles. The measured Kd's range is ~44 while the antiviral potencies (IC50) range is ~750 with a good correlation between binding free energies computed with Kd and IC50 values (r = 0.76). We explored with MD simulations (ff19sb, CHARMM36m) the binding profile of complexes with strong, moderate and weak binders embedded in DMPC, DPPC, POPC or a viral mimetic membrane and using different experimental starting structures of M2. To predict accurately differences in binding free energy in response to subtle changes in the structure of the ligands, we performed 18 alchemical perturbative single topology FEP/MD NPT simulations (OPLS2005) using the BAR estimator (Desmond software) and 20 dual topology calculations TI/MD NVT simulations (ff19sb) using the MBAR estimator (Amber software) for adamantyl amines in complex with M2(22-46) WT in DMPC, DPPC, POPC. We observed that both methods with all lipids show a very good correlation between the experimental and calculated relative binding free energies (r = 0.77-0.87, mue = 0.36-0.92 kcal mol-1) with the highest performance achieved with TI/MBAR and lowest performance with FEP/BAR in DMPC bilayers. When antiviral potencies are used instead of the Kd values for computing the experimental binding free energies we obtained also good performance with both FEP/BAR (r = 0.83, mue = 0.75 kcal mol-1) and TI/MBAR (r = 0.69, mue = 0.77 kcal mol-1).

Antigen-specific memory NK cell responses against HIV and influenza use the NKG2/HLA-E axis.

Multiple studies have broadened the roles of natural killer (NK) cells functioning as purely innate lymphocytes by demonstrating that they are capable of putative antigen-specific immunological memory against multiple infectious agents including HIV-1 and influenza. However, the mechanisms underlying antigen specificity remain unknown. Here, we demonstrate that antigen-specific human NK cell memory develops upon exposure to both HIV and influenza, unified by a conserved and epitope-specific targetable mechanism largely dependent on the activating CD94/NKG2C receptor and its ligand HLA-E. We validated the permanent acquisition of antigen specificity by individual memory NK cells by single-cell cloning. We identified elevated expression of KLRG1, α4ß7, and NKG2C as biomarkers of antigen-specific NK cell memory through complex immunophenotyping. Last, we uncovered individual HLA-E-restricted peptides that may constitute the dominant NK cell response in HIV-1- and influenza-infected persons in vivo. Our findings clarify the mechanisms contributing to antigen-specific memory NK cell responses and suggest that they could be potentially targeted therapeutically for vaccines or other therapeutic interventions.

Differential effects of microRNAs miR-21, miR-99 and miR-145 on lung regeneration and inflammation during recovery from influenza pneumonia.

In a mouse model of influenza pneumonia, we previously documented that proliferating alveolar type II (AT2) cells are the major stem cells involved in early lung recovery. Profiling of microRNAs revealed significant dysregulation of specific ones, including miR-21 and miR-99a. Moreover, miR-145 is known to exhibit antagonism to miR-21. This follow-up study investigated the roles of microRNAs miR-21, miR-99a, and miR-145 in the murine pulmonary regenerative process and inflammation during influenza pneumonia. Inhibition of miR-21 resulted in severe morbidity, and in significantly decreased proliferating AT2 cells due to impaired transition from innate to adaptive immune responses. Knockdown of miR-99a culminated in moderate morbidity, with a significant increase in proliferating AT2 cells that may be linked to PTEN downregulation. In contrast, miR-145 antagonism did not impact morbidity nor the proliferating AT2 cell population, and was associated with downregulation of TNF-alpha, IL1-beta, YM1, and LY6G. Hence, a complex interplay exists between expression of specific miRNAs, lung regeneration, and inflammation during recovery from influenza pneumonia. Inhibition of miR-21 and miR-99a (but not miR-145) can lead to deleterious cellular and molecular effects on pulmonary repair and inflammatory processes during influenza pneumonia.

T cell-specific P2RX7 favors lung parenchymal CD4+ T cell accumulation in response to severe lung infections.

CD4+ T cells are key components of the immune response during lung infections and can mediate protection against tuberculosis (TB) or influenza. However, CD4+ T cells can also promote lung pathology during these infections, making it unclear how these cells control such discrepant effects. Using mouse models of hypervirulent TB and influenza, we observe that exaggerated accumulation of parenchymal CD4+ T cells promotes lung damage. Low numbers of lung CD4+ T cells, in contrast, are sufficient to protect against hypervirulent TB. In both situations, lung CD4+ T cell accumulation is mediated by CD4+ T cell-specific expression of the extracellular ATP (eATP) receptor P2RX7. P2RX7 upregulation in lung CD4+ T cells promotes expression of the chemokine receptor CXCR3, favoring parenchymal CD4+ T cell accumulation. Our findings suggest that direct sensing of lung eATP by CD4+ T cells is critical to induce tissue CD4+ T cell accumulation and pathology during lung infections.

Variant-dependent oxidative and cytokine responses of human neutrophils to SARS-CoV-2 spike protein and anti-spike IgG1 antibodies.

Introduction: Severe forms of COVID-19, the disease caused by SARS-CoV-2, are characterized by acute respiratory distress syndrome, robust lung inflammation and death in some patients. Strong evidence has been accumulating that polymorphonuclear neutrophilic granulocytes (PMN) play an important role in the pathophysiology of severe COVID-19. SARS-CoV-2 directly induces in vitro PMN activation, mainly the release of neutrophil extracellular traps (NETs). However, the viral components inducing this PMN response remain unclear. Methods: In this work human PMN responses were assessed in vitro in response to the spike (S) protein of two different SARS-CoV-2 variants, anti-S IgG1 antibodies or immune complexes formed by them. Production of reactive oxygen species (ROS) was measured by Diogenes-based chemiluminescence. Release of myeloperoxidase (MPO) was assessed by ELISA while secretion of a list of cytokines and growth factors was determined by high-performance multiplex cytokine assay. Results and discussion: We show that the SARS-CoV-2 Omicron variant S protein and anti-spike IgG1, either alone or together, stimulate ROS production in human PMNs. We also observed that the SARS-CoV-2 Wuhan S protein and anti-S IgG1 antibody together trigger MPO release from PMNs. Based on the relevance of SARS-CoV-2 and influenza co-infections, we have also investigated the impact of influenza virus infection on the previous PMN responses to S proteins or anti-S antibodies. We did not detect any significant effect of influenza co-infection on ROS generation in PMNs. Our data also show that PMN stimulation by S proteins induced the release of different chemokines, growth factors, regulatory and proinflammatory cytokines. Overall, our findings show that the SARS-CoV-2 S protein, an anti-spike IgG1 antibody or their immune complex, promote oxidative responses of PMNs in a variant-dependent manner, contributing to a better understanding of the role of PMN responses during SARS-CoV-2 infection.

Transcriptomics profile of human bronchial epithelial cells exposed to ambient fine particles and influenza virus (H3N2).

Fine particulate matter (PM2.5) pollution remains a major threat to public health. As the physical barrier against inhaled air pollutants, airway epithelium is a primary target for PM2.5 and influenza viruses, two major environmental insults. Recent studies have shown that PM2.5 and influenza viruses may interact to aggravate airway inflammation, an essential event in the pathogenesis of diverse pulmonary diseases. Airway epithelium plays a critical role in lung health and disorders. Thus far, the mechanisms for the interactive effect of PM2.5 and the influenza virus on gene transcription of airway epithelial cells have not been fully uncovered. In this present pilot study, the transcriptome sequencing approach was introduced to identify responsive genes following individual and co-exposure to PM2.5 and influenza A (H3N2) viruses in a human bronchial epithelial cell line (BEAS-2B). Enrichment analysis revealed the function of differentially expressed genes (DEGs). Specifically, the DEGs enriched in the xenobiotic metabolism by the cytochrome P450 pathway were linked to PM2.5 exposure. In contrast, the DEGs enriched in environmental information processing and human diseases, such as viral protein interaction with cytokines and cytokine receptors and epithelial cell signaling in bacterial infection, were significantly related to H3N2 exposure. Meanwhile, co-exposure to PM2.5 and H3N2 affected G protein-coupled receptors on the cell surface. Thus, the results from this study provides insights into PM2.5- and influenza virus-induced airway inflammation and potential mechanisms.

Neuraminidase-dependent entry of influenza A virus is determined by hemagglutinin receptor-binding specificity.

IMPORTANCE: Influenza A viruses (IAVs) contain hemagglutinin (HA) proteins involved in sialoglycan receptor binding and neuraminidase (NA) proteins that cleave sialic acids. While the importance of the NA protein in virion egress is well established, its role in virus entry remains to be fully elucidated. NA activity is needed for the release of virions from mucus decoy receptors, but conflicting results have been reported on the importance of NA activity in virus entry in the absence of decoy receptors. We now show that inhibition of NA activity affects virus entry depending on the receptor-binding properties of HA and the receptor repertoire present on cells. Inhibition of entry by the presence of mucus correlated with the importance of NA activity for virus entry, with the strongest inhibition being observed when mucus and OsC were combined. These results shed light on the importance in virus entry of the NA protein, an important antiviral drug target.

Endothelial AHR activity prevents lung barrier disruption in viral infection.

Disruption of the lung endothelial-epithelial cell barrier following respiratory virus infection causes cell and fluid accumulation in the air spaces and compromises vital gas exchange function1. Endothelial dysfunction can exacerbate tissue damage2,3, yet it is unclear whether the lung endothelium promotes host resistance against viral pathogens. Here we show that the environmental sensor aryl hydrocarbon receptor (AHR) is highly active in lung endothelial cells and protects against influenza-induced lung vascular leakage. Loss of AHR in endothelia exacerbates lung damage and promotes the infiltration of red blood cells and leukocytes into alveolar air spaces. Moreover, barrier protection is compromised and host susceptibility to secondary bacterial infections is increased when endothelial AHR is missing. AHR engages tissue-protective transcriptional networks in endothelia, including the vasoactive apelin-APJ peptide system4, to prevent a dysplastic and apoptotic response in airway epithelial cells. Finally, we show that protective AHR signalling in lung endothelial cells is dampened by the infection itself. Maintenance of protective AHR function requires a diet enriched in naturally occurring AHR ligands, which activate disease tolerance pathways in lung endothelia to prevent tissue damage. Our findings demonstrate the importance of endothelial function in lung barrier immunity. We identify a gut-lung axis that affects lung damage following encounters with viral pathogens, linking dietary composition and intake to host fitness and inter-individual variations in disease outcome.

Replication-competent influenza virus with a protein-responsive multiplication ability.

Applications of influenza A viruses (IAV) for virotherapy and biotechnology have accelerated substantially with the development of reverse genetic technology and advances in the understanding of packaging signals. While the use of a replication-competent IAV is particularly promising, owing to its efficient transmission to organ depths with high infectivity, there is also a risk that its multiplication cannot be controlled in a cell-type-specific manner, causing an infectious disease. Therefore, here a simple and effective replication-competent IAV-based cell-targeting system has been developed. It was demonstrated that the activity of the ribonucleoprotein complex (RNP) of IAV could be regulated by the interaction between the endogenous protein and a nanobody fused to the subunit of RNA-dependent RNA polymerase (RdRp). To validate the feasibility of the method, it was demonstrated that RNP containing RdRp fused with Nb139, a nanobody against p53, is inactive in HEK293T cells expressing endogenous p53, but active in p53-defective Saos-2 cells. Finally, a replication-competent IAV was successfully generated that multiplies only in p53-defective tumor cells and an IAV vector was developed that can deliver a foreign gene in cell type-specific manner. The method is flexible because the nanobody can be easily altered to target a different cell type, offering a valuable platform for virotherapy and biotechnology.

RIG-I agonist SLR10 promotes macrophage M1 polarization during influenza virus infection.

Rationale: A family of short synthetic, triphosphorylated stem-loop RNAs (SLRs) have been designed to activate the retinoic-acid-inducible gene I (RIG-I) pathway and induce a potent interferon (IFN) response, which may have therapeutic potential. We investigated immune response modulation by SLR10. We addressed whether RIG-I pathway activation with SLR10 leads to protection of nonsmoking (NS) and cigarette smoke (CS)-exposed mice after influenza A virus (IAV) infection. Methods: Mice were given 25 µg of SLR10 1 day before IAV infection. We compared the survival rates and host immune responses of NS and CS-exposed mice following challenge with IAV. Results: SLR10 significantly decreased weight loss and increased survival rates in both NS and CS-exposed mice during IAV infection. SLR10 administration repaired the impaired proinflammatory response in CS-exposed mice without causing more lung injury in NS mice as assessed by physiologic measurements. Although histopathologic study revealed that SLR10 administration was likely to result in higher pathological scores than untreated groups in both NS and CS mice, this change was not enough to increase lung injury evaluated by lung-to-body weight ratio. Both qRT-PCR on lung tissues and multiplex immunoassay on bronchoalveolar lavage fluids (BALFs) showed that most IFNs and proinflammatory cytokines were expressed at lower levels in SLR10-treated NS mice than control-treaded NS mice at day 5 post infection (p.i.). Remarkably, proinflammatory cytokines IL-6, IL-12, and GM-CSF were increased in CS-exposed mice by SLR10 at day 5 p.i. Significantly, SLR10 elevated the ratio of the two chemokines (CXCL9 and CCL17) in BALFs, suggesting macrophages were polarized to classically activated (M1) status. In vitro testing also found that SLR10 not only stimulated human alveolar macrophage polarization to an M1 phenotype, but also reversed cigarette smoke extract (CSE)-induced M2 to M1 polarization. Conclusions: Our data show that SLR10 administration in mice is protective for both NS and CS-exposed IAV-infected mice. Mechanistically, SLR10 treatment promoted M1 macrophage polarization in the lung during influenza infection. The protective effects by SLR10 may be a promising intervention for therapy for infections with viruses, particularly those with CS-enhanced susceptibility to adverse outcomes.