Necroptosis blockade prevents lung injury in severe influenza.

Severe influenza A virus (IAV) infections can result in hyper-inflammation, lung injury and acute respiratory distress syndrome1-5 (ARDS), for which there are no effective pharmacological therapies. Necroptosis is an attractive entry point for therapeutic intervention in ARDS and related inflammatory conditions because it drives pathogenic lung inflammation and lethality during severe IAV infection6-8 and can potentially be targeted by receptor interacting protein kinase 3 (RIPK3) inhibitors. Here we show that a newly developed RIPK3 inhibitor, UH15-38, potently and selectively blocked IAV-triggered necroptosis in alveolar epithelial cells in vivo. UH15-38 ameliorated lung inflammation and prevented mortality following infection with laboratory-adapted and pandemic strains of IAV, without compromising antiviral adaptive immune responses or impeding viral clearance. UH15-38 displayed robust therapeutic efficacy even when administered late in the course of infection, suggesting that RIPK3 blockade may provide clinical benefit in patients with IAV-driven ARDS and other hyper-inflammatory pathologies.

Comparison of the Epidemiological and Clinical Characteristics of Hospitalized Children With Pneumonia Caused by SARS-CoV-2, Influenza A, and Human Adenoviruses: A Case-Control Study.

Background. This case-control study aims to investigate the clinical characteristics in pediatric patients with pneumonia infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, and human adenoviruses (HAdVs). Methods. Hospitalized pediatric patients with pneumonia infected with SARS-CoV-2 at Wuhan Children's Hospital and pneumonia infected with influenza A, and HAdVs at Qilu Children's Hospital were compared. Clinical manifestations, laboratory examinations, and imaging characteristics were analyzed. Results. The proportions of hyperpyrexia (54.3%, 33.9%), cough (100%, 99.2%), wheezing (45.7%, 53.7%), diarrhea (31.4%, 14.9%), and fever (100%, 75.2%) in patients with influenza A and HAdVs were higher than those of patients with SARS-CoV-2 (9.4%, P < .001; 48.5%, P < .001; 0%, P < .001; 8.8%, P = .002; 41.5%, P < .001; respectively). Laboratory examinations revealed the proportions of leukocytosis (37.1%, 52.9%), abnormal rates of neutrophils (40%, 40.5%), and lymphocytosis (42.9%, 65.3%) in influenza A and HAdV pneumonia groups were significantly higher than coronavirus disease 2019 (COVID-19) group (0%, P < .001; 0%, P < .001; 0%, P < .001; respectively). The proportion of elevated procalcitonin (5.7%, 14%) in patients with influenza A and HAdVs was significantly lower than those in patients with SARS-CoV-2 (64%, P < .001). In chest computed tomography, ground-glass opacities near the pleura were more common in patients with COVID-19 than those in patients with influenza A and HAdVs (32.7% vs 0% vs 0%, P < .001). Conclusion. Fever, cough, and wheezing are more common in the influenza A and HAdVs groups, whereas procalcitonin and computed tomography findings are likely to be pronounced in COVID-19 pneumonia. It provides a variety of methods except polymerase chain reaction for differentiating COVID-19 pneumonia from influenza A and HAdVs pneumonia.

A virus-specific monocyte inflammatory phenotype is induced by SARS-CoV-2 at the immune-epithelial interface.

Infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) provokes a potentially fatal pneumonia with multiorgan failure, and high systemic inflammation. To gain mechanistic insight and ferret out the root of this immune dysregulation, we modeled, by in vitro coculture, the interactions between infected epithelial cells and immunocytes. A strong response was induced in monocytes and B cells, with a SARS-CoV-2-specific inflammatory gene cluster distinct from that seen in influenza A or Ebola virus-infected cocultures, and which reproduced deviations reported in blood or lung myeloid cells from COVID-19 patients. A substantial fraction of the effect could be reproduced after individual transfection of several SARS-CoV-2 proteins (Spike and some nonstructural proteins), mediated by soluble factors, but not via transcriptional induction. This response was greatly muted in monocytes from healthy children, perhaps a clue to the age dependency of COVID-19. These results suggest that the inflammatory malfunction in COVID-19 is rooted in the earliest perturbations that SARS-CoV-2 induces in epithelia.

Virus Infection, Genetic Mutations, and Prion Infection in Prion Protein Conversion.

Conformational conversion of the cellular isoform of prion protein, PrPC, into the abnormally folded, amyloidogenic isoform, PrPSc, is an underlying pathogenic mechanism in prion diseases. The diseases manifest as sporadic, hereditary, and acquired disorders. Etiological mechanisms driving the conversion of PrPC into PrPSc are unknown in sporadic prion diseases, while prion infection and specific mutations in the PrP gene are known to cause the conversion of PrPC into PrPSc in acquired and hereditary prion diseases, respectively. We recently reported that a neurotropic strain of influenza A virus (IAV) induced the conversion of PrPC into PrPSc as well as formation of infectious prions in mouse neuroblastoma cells after infection, suggesting the causative role of the neuronal infection of IAV in sporadic prion diseases. Here, we discuss the conversion mechanism of PrPC into PrPSc in different types of prion diseases, by presenting our findings of the IAV infection-induced conversion of PrPC into PrPSc and by reviewing the so far reported transgenic animal models of hereditary prion diseases and the reverse genetic studies, which have revealed the structure-function relationship for PrPC to convert into PrPSc after prion infection.

Tetraspanins: Host Factors in Viral Infections.

Tetraspanins are transmembrane glycoproteins that have been shown increasing interest as host factors in infectious diseases. In particular, they were implicated in the pathogenesis of both non-enveloped (human papillomavirus (HPV)) and enveloped (human immunodeficiency virus (HIV), Zika, influenza A virus, (IAV), and coronavirus) viruses through multiple stages of infection, from the initial cell membrane attachment to the syncytium formation and viral particle release. However, the mechanisms by which different tetraspanins mediate their effects vary. This review aimed to compare and contrast the role of tetraspanins in the life cycles of HPV, HIV, Zika, IAV, and coronavirus viruses, which cause the most significant health and economic burdens to society. In doing so, a better understanding of the relative contribution of tetraspanins in virus infection will allow for a more targeted approach in the treatment of these diseases.

Innate immune responses at the asymptomatic stage of influenza A viral infections of Streptococcus pneumoniae colonized and non-colonized mice.

Seasonal Influenza A virus (IAV) infections can promote dissemination of upper respiratory tract commensals such as Streptococcus pneumoniae to the lower respiratory tract resulting in severe life-threatening pneumonia. Here, we aimed to compare innate immune responses in the lungs of healthy colonized and non-colonized mice after IAV challenge at the initial asymptomatic stage of infection. Responses during a severe bacterial pneumonia were profiled for comparison. Cytokine and innate immune cell imprints of the lungs were analyzed. Irrespective of the colonization status, mild H1N1 IAV infection was characterized by a bi-phasic disease progression resulting in full recovery of the animals. Already at the asymptomatic stage of viral infection, the pro-inflammatory cytokine response was as high as in pneumococcal pneumonia. Flow cytometry analyses revealed an early influx of inflammatory monocytes into the lungs. Neutrophil influx was mostly limited to bacterial infections. The majority of cells, except monocytes, displayed an activated phenotype characterized by elevated CCR2 and MHCII expression. In conclusion, we show that IAV challenge of colonized healthy mice does not automatically result in severe co-infection. However, a general local inflammatory response was noted at the asymptomatic stage of infection irrespective of the infection type.

Molecular mechanisms of the influenza fusion peptide: insights from experimental and simulation studies.

A key step in infections by enveloped viruses, such as influenza, is the fusion between the viral envelope and the host cell membrane, which allows the virus to insert its genetic material into the host cell and replicate. The influenza virus fusion process is promoted by hemagglutinin (HA), a glycoprotein that contains three identical monomers composed of two polypeptide chains (HA1 and HA2). Early studies on this protein revealed that HA-mediated fusion involves the insertion of the HA2 N-terminal segment into the host membrane and that this segment, known as the fusion peptide, is a key player in the fusion process. This mini-review highlights the main findings that have been obtained by experimental and computational studies on the HA fusion peptide, which give us a glimpse of its mode of action.

Innate Immune Responses to Influenza Virus Infections in the Upper Respiratory Tract.

The innate immune system is the host's first line of immune defence against any invading pathogen. To establish an infection in a human host the influenza virus must replicate in epithelial cells of the upper respiratory tract. However, there are several innate immune mechanisms in place to stop the virus from reaching epithelial cells. In addition to limiting viral replication and dissemination, the innate immune system also activates the adaptive immune system leading to viral clearance, enabling the respiratory system to return to normal homeostasis. However, an overzealous innate immune system or adaptive immune response can be associated with immunopathology and aid secondary bacterial infections of the lower respiratory tract leading to pneumonia. In this review, we discuss the mechanisms utilised by the innate immune system to limit influenza virus replication and the damage caused by influenza viruses on the respiratory tissues and how these very same protective immune responses can cause immunopathology.

Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

Anti-influenza A Virus Effects and Mechanisms of Emodin and Its Analogs via Regulating PPARα/γ-AMPK-SIRT1 Pathway and Fatty Acid Metabolism.

The peroxisome proliferator-activated receptor (PPAR) α/γ-adenosine 5'-monophosphate- (AMP-) activated protein kinase- (AMPK-) sirtuin-1 (SIRT1) pathway and fatty acid metabolism are reported to be involved in influenza A virus (IAV) replication and IAV-pneumonia. Through a cell-based peroxisome proliferator responsive element- (PPRE-) driven luciferase bioassay, we have investigated 145 examples of traditional Chinese medicines (TCMs). Several TCMs, such as Polygonum cuspidatum, Rheum officinale Baillon, and Aloe vera var. Chinensis (Haw.) Berg., were found to possess high activity. We have further detected the anti-IAV activities of emodin (EMO) and its analogs, a group of common important compounds of these TCMs. The results showed that emodin and its several analogs possess excellent anti-IAV activities. The pharmacological tests showed that emodin significantly activated PPARα/γ and AMPK, decreased fatty acid biosynthesis, and increased intracellular ATP levels. Pharmaceutical inhibitors, siRNAs for PPARα/γ and AMPKα1, and exogenous palmitate impaired the inhibition of emodin. The in vivo test also showed that emodin significantly protected mice from IAV infection and pneumonia. Pharmacological inhibitors for PPARα/γ and AMPK signal and exogenous palmitate could partially counteract the effects of emodin in vivo. In conclusion, emodin and its analogs are a group of promising anti-IAV drug precursors, and the pharmacological mechanism of emodin is linked to its ability to regulate the PPARα/γ-AMPK pathway and fatty acid metabolism.

Acetylation, Methylation and Allysine Modification Profile of Viral and Host Proteins during Influenza A Virus Infection.

Protein modifications dynamically occur and regulate biological processes in all organisms. Towards understanding the significance of protein modifications in influenza virus infection, we performed a global mass spectrometry screen followed by bioinformatics analyses of acetylation, methylation and allysine modification in human lung epithelial cells in response to influenza A virus infection. We discovered 8 out of 10 major viral proteins and 245 out of 2280 host proteins detected to be differentially modified by three modifications in infected cells. Some of the identified proteins were modified on multiple amino acids residues and by more than one modification; the latter occurred either on different or same residues. Most of the modified residues in viral proteins were conserved across >40 subtypes of influenza A virus, and influenza B or C viruses and located on the protein surface. Importantly, many of those residues have already been determined to be critical for the influenza A virus. Similarly, many modified residues in host proteins were conserved across influenza A virus hosts like humans, birds, and pigs. Finally, host proteins undergoing the three modifications clustered in common functional networks of metabolic, cytoskeletal, and RNA processes, all of which are known to be exploited by the influenza A virus.

The Endogenous RIG-I Ligand Is Generated in Influenza A-Virus Infected Cells.

As a result of a viral infection, viral genomes are not only recognized by RIG-I, but also lead to the activation of RNase L, which cleaves cellular RNA to generate the endogenous RIG-I ligand (eRL). The eRL was previously identified as a specific sequence derived from the internal transcribed spacer region 2, which bears a 2'3' cyclic phosphate instead of the common 5' triphosphate. By now, the generation of the eRL and its immunostimulatory effect were shown both in vitro and in reporter systems. In this work, we aimed to elucidate whether the eRL is also generated in Influenza A (IAV) and vesicular stomatitis virus (VSV) infected cells. RNA was extracted from virus-infected cells and used for immunostimulations as well as specific PCR-strategies to detect eRL cleavage. We show that the eRL is generated in IAV infected HEK293 cells, but we could not detect specific eRL fragments in VSV infected cells. Further, RIG-I mediated IFN-response depends not only on viral genomes but also on the eRL, as immunostimulatory properties remain present under 5'triphosphate degrading conditions. In summary, we prove the IAV infection induced eRL generation in HEK293 cells, amplifying the innate immune response.

The traditional herbal formulation, Jianpiyifei II, reduces pulmonary inflammation induced by influenza A virus and cigarette smoke in mice.

Chronic obstructive pulmonary disease (COPD) is a worldwide chronic inflammatory lung disease, and influenza A virus (IAV) infection is a common cause of acute exacerbations of COPD (AECOPD). Therefore, targeting viral infections represents a promising strategy to prevent the occurrence and development of inflammatory flare ups in AECOPD. Jianpiyifei II (JPYFII) is a traditional herbal medicine used in China to treat patients with COPD, and its clinical indications are not well understood. However, investigation of the anti-inflammatory effects and underlying mechanism using an animal model of smoking have been reported in a previous study by our group. In addition, some included herbs, such as Radix astragali and Radix aupleuri, were reported to exhibit antiviral effects. Therefore, the aim of the present study was to investigate whether JPYFII formulation relieved acute inflammation by clearing the IAV in a mouse model that was exposed to cigarette smoke experimentally. JPYFII formulation treatment during smoke exposure and IAV infection significantly reduced the number of cells observed in bronchoalveolar lavage fluid (BALF), expression of proinflammatory cytokines, chemokines, superoxide production, and viral load in IAV-infected and smoke-exposed mice. However, JPYFII formulation treatment during smoke exposure alone did not reduce the number of cells in BALF or the expression of Il-6, Tnf-a, and Il-1ß. The results demonstrated that JPYFII formulation exerted an antiviral effect and reduced the exacerbation of lung inflammation in cigarette smoke (CS)-exposed mice infected with IAV. Our results suggested that JPYFII formulation could potentially be used to treat patients with AECOPD associated with IAV infection.

IFITM Proteins That Restrict the Early Stages of Respiratory Virus Infection Do Not Influence Late-Stage Replication.

Interferon-induced transmembrane (IFITM) proteins inhibit a broad range of enveloped viruses by blocking entry into host cells. We used an inducible overexpression system to investigate if IFITM1, IFITM2, and IFITM3 could modulate early and/or late stages of influenza A virus (IAV) or parainfluenza virus 3 (PIV-3) infection in human A549 airway epithelial cells. IAV and PIV-3 represent respiratory viruses which utilize distinct cellular entry pathways. We verify entry by endocytosis for IAV, whereas PIV-3 infection was consistent with fusion at the plasma membrane. Following induction prior to infection, all three IFITM proteins restricted the percentage of IAV-infected cells at 8 hours postinfection. In contrast, prior induction of IFITM1 and IFITM2 did not inhibit PIV-3 infection, although a modest reduction was observed with IFITM3. Small interfering RNA (siRNA)-mediated knockdown of endogenous IFITM1, IFITM2, and IFITM3 expression, in the presence or absence of pretreatment with type I interferon, resulted in increased IAV, but not PIV-3, infection. This finding suggests that while all three IFITMs display antiviral activity against IAV, they do not restrict the early stages of PIV-3 infection. IAV and PIV-3 infection culminates in viral egress through budding at the plasma membrane. Inducible expression of IFITM1, IFITM2, or IFITM3 immediately after infection did not impact titers of infectious virus released from IAV- or PIV-3-infected cells. Our findings show that IFITM proteins differentially restrict the early stages of infection of two respiratory viruses with distinct cellular entry pathways but do not influence the late stages of replication for either virus. IMPORTANCE Interferon-induced transmembrane (IFITM) proteins restrict the initial stages of infection for several respiratory viruses; however, their potential to modulate the later stages of virus replication has not been explored. In this study, we highlight the utility of an inducible overexpression system to assess the impact of IFITM proteins on either early- or late-stage replication of two respiratory viruses. We demonstrate antiviral activity by IFITM1, IFITM2, and IFITM3 against influenza A virus (IAV) but not parainfluenza virus 3 (PIV-3) during the early stages of cellular infection. Furthermore, IFITM induction following IAV or PIV-3 infection does not restrict the late stages of replication of either virus. Our findings show that IFITM proteins can differentially restrict the early stages of infection of two viruses with distinct cellular entry pathways and yet do not influence the late stages of replication for either virus.

Quinazolin-derived myeloperoxidase inhibitor suppresses influenza A virus-induced reactive oxygen species, pro-inflammatory mediators and improves cell survival.

Superoxide radicals and other reactive oxygen species (ROS) are implicated in influenza A virus-induced inflammation. In this in vitro study, we evaluated the effects of TG6-44, a novel quinazolin-derived myeloperoxidase-specific ROS inhibitor, on influenza A virus (A/X31) infection using THP-1 lung monocytic cells and freshly isolated peripheral blood mononuclear cells (PBMC). TG6-44 significantly decreased A/X31-induced ROS and virus-induced inflammatory mediators in THP-1 cells (IL-6, IFN-γ, MCP-1, TNF-α, MIP-1ß) and in human PBMC (IL-6, IL-8, TNF-α, MCP-1). Interestingly, TG6-44-treated THP-1 cells showed a decrease in percent cells expressing viral nucleoprotein, as well as a delay in translocation of viral nucleoprotein into the nucleus. Furthermore, in influenza A virus-infected cells, TG6-44 treatment led to suppression of virus-induced cell death as evidenced by decreased caspase-3 activation, decreased proportion of Annexin V+PI+ cells, and increased Bcl-2 phosphorylation. Taken together, our results demonstrate the anti-inflammatory and anti-infective effects of TG6-44.

Transient Excursions to Membrane Core as Determinants of Influenza Virus Fusion Peptide Activity.

Fusion of viral and host cell membranes is a critical step in the life cycle of enveloped viruses. In the case of influenza virus, it is mediated by subunit 2 of hemagglutinin (HA) glycoprotein whose N-terminal fragments insert into the target membrane and initiate lipid exchange. These isolated fragments, known as fusion peptides (HAfp), already possess own fusogenic activity towards liposomes. Although they have long been studied with the hope to uncover the details of HA-mediated fusion, their actual mechanism of action remains elusive. Here, we use extensive molecular dynamics simulations combined with experimental studies of three HAfp variants to fully characterize their free energy landscape and interaction with lipid bilayer. In addition to customary assumed peptides localization at lipid-water interface, we characterize membrane-spanning configurations, which turn out to be metastable for active HAfps and unstable for the fusion inactive W14A mutant. We show that, while the degree of membrane perturbation by surface peptide configurations is relatively low and does not show any mutation-related differences, the effect of deeply inserted configurations is significant and correlates with insertion depth of the N-terminal amino group which is the highest for the wild type HAfp. Finally, we demonstrate the feasibility of spontaneous peptide transition to intramembrane location and the critical role of strictly conserved tryptofan residue 14 in this process.

Influenza Virus-Induced Novel miRNAs Regulate the STAT Pathway.

MicroRNAs (miRNAs) are essential regulators of gene expression in humans and can control pathogenesis and host-virus interactions. Notably, the role of specific host miRNAs during influenza virus infections are still ill-defined. The central goal of this study was to identify novel miRNAs and their target genes in response to influenza virus infections in airway epithelium. Human airway epithelial cells exposed to influenza A virus (IAV) induced several novel miRNAs that were identified using next-generation sequencing (NGS) and their target genes by biochemical methods. NGS analysis predicted forty-two RNA sequences as possible miRNAs based on computational algorithms. The expression patterns of these putative miRNAs were further confirmed using RT-PCR in human bronchial epithelial cells exposed to H1N1, H9N1(1P10), and H9N1 (1WF10) strains of influenza virus. A time-course study showed significant downregulation of put-miR-34 in H1N1 and put-miR-35 in H9N1(1P10)-infected cells, which is consistent with the NGS data. Additionally, put-miR-34 and put-miR-35 showed a high fold enrichment in an argonaute-immunoprecipitation assay compared to the controls, indicating their ability to form a complex with argonaute protein and RNA-induced silencing complex (RISC), which is a typical mode of action found with miRNAs. Our earlier studies have shown that the replication and survival of influenza virus is modulated by certain transcription factors such as NF-ĸB. To identify the target(s) of these putative miRNAs, we screened 84 transcription factors that have a role in viral pathogenesis. Cells transfected with mimic of the put-miR-34 showed a significant decrease in the expression of Signal Transducers and Activators of Transcription 3 (STAT3), whereas the inhibitor of put-miR-34 showed a significant increase in STAT3 expression and its phosphorylation. In addition, put-miR-34 had 76% homology to the untranslated region of STAT3. NGS and PCR array data submitted to the Gene Ontology project also predicted the role of transcription factors modulated by put-miR-34. Our data suggest that put-miR-34 may be a good target for antiviral therapy.

GP96 Drives Exacerbation of Secondary Bacterial Pneumonia following Influenza A Virus Infection.

Influenza A virus (IAV) infection predisposes the host to secondary bacterial pneumonia, known as a major cause of morbidity and mortality during influenza virus epidemics. Analysis of interactions between IAV-infected human epithelial cells and Streptococcus pneumoniae revealed that infected cells ectopically exhibited the endoplasmic reticulum chaperone glycoprotein 96 (GP96) on the surface. Importantly, efficient pneumococcal adherence to epithelial cells was imparted by interactions with extracellular GP96 and integrin αV, with the surface expression mediated by GP96 chaperone activity. Furthermore, abrogation of adherence was gained by chemical inhibition or genetic knockout of GP96 as well as addition of RGD peptide, an inhibitor of integrin-ligand interactions. Direct binding of extracellular GP96 and pneumococci was shown to be mediated by pneumococcal oligopeptide permease components. Additionally, IAV infection induced activation of calpains and Snail1, which are responsible for degradation and transcriptional repression of junctional proteins in the host, respectively, indicating increased bacterial translocation across the epithelial barrier. Notably, treatment of IAV-infected mice with the GP96 inhibitor enhanced pneumococcal clearance from lung tissues and ameliorated lung pathology. Taken together, the present findings indicate a viral-bacterial synergy in relation to disease progression and suggest a paradigm for developing novel therapeutic strategies tailored to inhibit pneumococcal colonization in an IAV-infected respiratory tract. IMPORTANCE Secondary bacterial pneumonia following an influenza A virus (IAV) infection is a major cause of morbidity and mortality. Although it is generally accepted that preceding IAV infection leads to increased susceptibility to secondary bacterial infection, details regarding the pathogenic mechanism during the early stage of superinfection remain elusive. Here, we focused on the interaction of IAV-infected cells and Streptococcus pneumoniae, which revealed that human epithelial cells infected with IAV exhibit a cell surface display of GP96, an endoplasmic reticulum chaperon. Notably, extracellular GP96 was shown to impart efficient adherence for secondary infection by S. pneumoniae, and GP96 inhibition ameliorated lung pathology of superinfected mice, indicating it to be a useful target for development of therapeutic strategies for patients with superinfection.

Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine.

Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.

TAP dysfunction in dendritic cells enables noncanonical cross-presentation for T cell priming.

Classic major histocompatibility complex class I (MHC-I) presentation relies on shuttling cytosolic peptides into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Viruses disable TAP to block MHC-I presentation and evade cytotoxic CD8+ T cells. Priming CD8+ T cells against these viruses is thought to rely solely on cross-presentation by uninfected TAP-functional dendritic cells. We found that protective CD8+ T cells could be mobilized during viral infection even when TAP was absent in all hematopoietic cells. TAP blockade depleted the endosomal recycling compartment of MHC-I molecules and, as such, impaired Toll-like receptor-regulated cross-presentation. Instead, MHC-I molecules accumulated in the ER-Golgi intermediate compartment (ERGIC), sequestered away from Toll-like receptor control, and coopted ER-SNARE Sec22b-mediated vesicular traffic to intersect with internalized antigen and rescue cross-presentation. Thus, when classic MHC-I presentation and endosomal recycling compartment-dependent cross-presentation are impaired in dendritic cells, cell-autonomous noncanonical cross-presentation relying on ERGIC-derived MHC-I counters TAP dysfunction to nevertheless mediate CD8+ T cell priming.