RÉSUMÉ
Influenza A virus RNA synthesis produces full-length and aberrant RNA molecules, which include defective viral genomes (DVG) and mini viral RNAs (mvRNA). Sequencing approaches have shown that several hundred unique aberrant RNA species may be present during infection, and that they can vary in size, segment origin, and sequence. Moreover, a subset of aberrant RNA molecules can bind and activate host pathogen receptor retinoic acid-inducible gene I (RIG-I), leading to innate immune signaling and the expression of type I and III interferons. Understanding the kinetics and distribution of these immunostimulatory aberrant RNA sequences is important for modeling the outcomes of IAV infection. We here first show that reverse transcription and PCR steps can yield imperfect aberrant RNA quantification data in a sequence-dependent manner. Next, we developed an amplification-free LbuCas13a-based detection method to quantify mvRNA amplification kinetics and subcellular distributions. We show that our assay can quantify the copy numbers of 10 specific mvRNA sequences in total RNA from cell culture, animal tissue or clinical nasopharyngeal swab extracts. In addition, we find kinetic and distribution differences between immunostimulatory and non-immunostimulatory mvRNAs, as well as mvRNAs derived from different segments, during infection. Overall, our results reveal a hitherto hidden diversity in the behavior of IAV mvRNAs and they suggest that their production is linked to replication of the individual viral segments. Cas13 is therefore a valuable new tool in our repertoire for investigating the impact of aberrant RNAs on RNA virus infection.
RÉSUMÉ
During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid-inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response.
Sujet(s)
Grippe humaine , Lignée cellulaire , Protéine-58 à domaine DEAD/génétique , Protéine-58 à domaine DEAD/métabolisme , Humains , Immunité innée , Grippe humaine/génétique , ARN viral/génétique , ARN viral/métabolisme , Réplication virale/génétiqueRÉSUMÉ
Influenza A viruses cause a mild-to-severe respiratory disease that affects millions of people each year. One of the many determinants of disease outcome is the innate immune response to the viral infection. While antiviral responses are essential for viral clearance, excessive innate immune activation promotes lung damage and disease. The influenza A virus RNA polymerase is one of viral proteins that affect innate immune activation during infection, but the mechanisms behind this activity are not well understood. In this review, we discuss how the viral RNA polymerase can both activate and suppress innate immune responses by either producing immunostimulatory RNA species or directly targeting the components of the innate immune signalling pathway, respectively. Furthermore, we provide a comprehensive overview of the polymerase residues, and their mutations, associated with changes in innate immune activation, and discuss their putative effects on polymerase function based on recent advances in our understanding of the influenza A virus RNA polymerase structure.
Sujet(s)
Immunité innée/immunologie , Immunomodulation/immunologie , Virus de la grippe A/enzymologie , RNA replicase/génétique , Protéines virales/génétique , Humains , Virus de la grippe A/génétique , Virus de la grippe A/immunologie , Grippe humaine/immunologie , Mitochondries/métabolisme , ARN viral/génétique , Récepteurs à l'acide rétinoïque/métabolisme , Transduction du signal/physiologie , Réplication virale/génétiqueRÉSUMÉ
Influenza virus-infected cells vary widely in their expression of viral genes and only occasionally activate innate immunity. Here, we develop a new method to assess how the genetic variation in viral populations contributes to this heterogeneity. We do this by determining the transcriptome and full-length sequences of all viral genes in single cells infected with a nominally "pure" stock of influenza virus. Most cells are infected by virions with defects, some of which increase the frequency of innate-immune activation. These immunostimulatory defects are diverse and include mutations that perturb the function of the viral polymerase protein PB1, large internal deletions in viral genes, and failure to express the virus's interferon antagonist NS1. However, immune activation remains stochastic in cells infected by virions with these defects and occasionally is triggered even by virions that express unmutated copies of all genes. Our work shows that the diverse spectrum of defects in influenza virus populations contributes to-but does not completely explain-the heterogeneity in viral gene expression and immune activation in single infected cells.IMPORTANCE Because influenza virus has a high mutation rate, many cells are infected by mutated virions. But so far, it has been impossible to fully characterize the sequence of the virion infecting any given cell, since conventional techniques such as flow cytometry and single-cell transcriptome sequencing (scRNA-seq) only detect if a protein or transcript is present, not its sequence. Here we develop a new approach that uses long-read PacBio sequencing to determine the sequences of virions infecting single cells. We show that viral genetic variation explains some but not all of the cell-to-cell variability in viral gene expression and innate immune induction. Overall, our study provides the first complete picture of how viral mutations affect the course of infection in single cells.
Sujet(s)
Variation génétique , Immunité innée , Sous-type H1N1 du virus de la grippe A , Analyse de séquence d'ARN , Analyse sur cellule unique , Protéines virales non structurales , Cellules A549 , Humains , Sous-type H1N1 du virus de la grippe A/génétique , Sous-type H1N1 du virus de la grippe A/immunologie , Protéines virales non structurales/génétique , Protéines virales non structurales/immunologieRÉSUMÉ
INTRODUCTION: S. aureus is a pathogen to which individuals are exposed shortly after birth, with immune responses to S. aureus increasing during childhood. There is marked heterogeneity between the anti- S. aureus immune responses of different humans, the basis of which is not fully understood. METHODS: To investigate development of anti-S. aureus immune responses, we studied S. aureus colonised mice under controlled conditions. Mice were either acquired colonised from breeding colonies, or experimentally colonised by exposure to a cage environment which had been sprayed with a S. aureus suspension. Colonisation was monitored by sequential stool sampling, and immunoglobulin levels against both whole fixed S. aureus and individual S. aureus antigens quantified. The immunological impact of colonisation on subsequent vaccination was investigated. RESULTS: Colonised BALB/c and BL/6 mice develop serum anti- S. aureus cell surface IgG1 antibodies. Responses were proportional to the cumulative S. aureus bioburden in the mice, and were higher in BALB/c mice, which have higher colonisation levels, than in C57BL/6 animals. We observed marked variation in the induction of anti-cell surface antibodies, even in genetically identical mice experimentally colonised with the same S. aureus clone. Heterogeneity was also evident when monitoring immune responses to the secreted S. aureus protein EapH2. Approximately 50% of colonised mice developed anti-EapH2 responses (responders); in other mice, responses were not significantly different to those in uncolonised mice (non-responders). Following vaccination with a replication deficient adenovirus expressing EapH2, less anti-EapH2 antibody was generated in non-responder than responder animals. CONCLUSIONS: In genetically identical mice, S. aureus colonisation results in all-or-nothing antibody responses against some antigens, including EapH2. For antigens involved in colonisation success by microbes, apparently stochastic early immune responses may impact both vaccine responses and the establishment of an animal-specific microbiome.
Sujet(s)
Anticorps antibactériens/sang , Antigènes bactériens/immunologie , État de porteur sain/immunologie , État de porteur sain/microbiologie , Tube digestif/microbiologie , Vaccins antistaphylococciques/administration et posologie , Animaux , Fèces/microbiologie , Femelle , Immunoglobuline G/sang , Souris , Souris de lignée BALB C , Souris de lignée C57BL , Staphylococcus aureus/immunologie , Staphylococcus aureus/physiologie , VaccinationRÉSUMÉ
INTRODUCTION: There is a need for an efficacious vaccine reducing infections due to Staphylococcus aureus, a common cause of community and hospital infection. Infecting organisms originate from S. aureus populations colonising the nares and bowel. Antimicrobials are widely used to transiently reduce S. aureus colonisation prior to surgery, a practice which is selecting for resistant S. aureus isolates. S. aureus secretes multiple proteins, including the protease inhibitors extracellular adhesion protein homologue 1 and 2 (EapH1 and EapH2). METHODS: Mice were vaccinated intramuscularly or intranasally with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins, or with control viruses. Using murine S. aureus colonisation models, we monitored S. aureus colonisation by sequential stool sampling. Monitoring of S. aureus invasive disease after intravenous challenge was performed using bacterial load and abscess numbers in the kidney. RESULTS: Intramuscular vaccination with Adenovirus serotype 5 and Modified Vaccinia Ankara viral vectors expressing EapH1 and EapH2 proteins significantly reduces bacterial recovery in the murine renal abscess model of infection, but the magnitude of the effect is small. A single intranasal vaccination with an adenoviral vaccine expressing these proteins reduced S. aureus gastrointestinal (GI) tract colonisation. CONCLUSION: Vaccination against EapH1 / EapH2 proteins may offer an antibiotic independent way to reduce S. aureus colonisation, as well as contributing to protection against S. aureus invasive disease.
Sujet(s)
Protéines bactériennes/immunologie , État de porteur sain/prévention et contrôle , Infections à staphylocoques/prévention et contrôle , Vaccins antistaphylococciques/immunologie , Staphylococcus aureus/immunologie , Adenoviridae/génétique , Administration par voie nasale , Animaux , Charge bactérienne , Protéines bactériennes/génétique , État de porteur sain/microbiologie , Femelle , SourisRÉSUMÉ
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.
Sujet(s)
Antiviraux/pharmacologie , Kinase-2 associée au récepteur couplé à une protéine G/antagonistes et inhibiteurs , Grippe humaine/métabolisme , Grippe humaine/virologie , Thérapie moléculaire ciblée , Phosphoprotéines/métabolisme , Protein kinases/métabolisme , Protéomique/méthodes , Séquence d'acides aminés , Animaux , Cellules cultivées , Activation enzymatique/effets des médicaments et des substances chimiques , Kinase-2 associée au récepteur couplé à une protéine G/métabolisme , Humains , Poumon/anatomopathologie , Poumon/virologie , Souris , Infections à Orthomyxoviridae/métabolisme , Infections à Orthomyxoviridae/virologie , Phosphoprotéines/composition chimique , Phosphorylation/effets des médicaments et des substances chimiques , Pénétration virale/effets des médicaments et des substances chimiques , Réplication virale/effets des médicaments et des substances chimiquesRÉSUMÉ
Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are enteric bacterial pathogens of worldwide importance. Most EPEC and non-O157 EHEC strains express lymphostatin (also known as LifA), a chromosomally encoded 365-kDa protein. We previously demonstrated that lymphostatin is a putative glycosyltransferase that is important in intestinal colonization of cattle by EHEC serogroup O5, O111, and O26 strains. However, the nature and consequences of the interaction between lymphostatin and immune cells from the bovine host are ill defined. Using purified recombinant protein, we demonstrated that lymphostatin inhibits mitogen-activated proliferation of bovine T cells and, to a lesser extent, proliferation of cytokine-stimulated B cells, but not NK cells. It broadly affected the T cell compartment, inhibiting all cell subsets (CD4, CD8, WC-1, and γδ T cell receptor [γδ-TCR]) and cytokines examined (interleukin 2 [IL-2], IL-4, IL-10, IL-17A, and gamma interferon [IFN-γ]) and rendered T cells refractory to mitogen for a least 18 h after transient exposure. Lymphostatin was also able to inhibit proliferation of T cells stimulated by IL-2 and by antigen presentation using a Theileria-transformed cell line and autologous T cells from Theileria-infected cattle. We conclude that lymphostatin is likely to act early in T cell activation, as stimulation of T cells with concanavalin A, but not phorbol 12-myristate 13-acetate combined with ionomycin, was inhibited. Finally, a homologue of lymphostatin from E. coli O157:H7 (ToxB; L7095) was also found to possess comparable inhibitory activity against T cells, indicating a potentially conserved strategy for interference in adaptive responses by attaching and effacing E. coli.