Análisis molecular de la ubiquitinación de RIG-I en el contexto de la infección por el virus influenza

Comunicaciones a congreso

Archival influenza virus genomes from Europe reveal genomic variability during the 1918 pandemic

The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze both the first 1918 IAV genomes from Europe and the first from samples prior to the autumn peak. 1918 IAV genomic diversity is consistent with a combination of local transmission and long-distance dispersal events. Comparison of genomes before and during the pandemic peak shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, local molecular clock modeling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of origination through an intrasubtype reassortment

The herpesviral Fc receptor fcr-1 down-regulates the NKG2D ligands MULT-1 and H60

Members of the α- and β-subfamily of herpesviridae encode glycoproteins that specifically bind to the Fc part of immunoglobulin (Ig)G. Plasma membrane resident herpesviral Fc receptors seem to prevent virus-specific IgG from activating antibody-dependent effector functions. We show that the mouse cytomegalovirus (MCMV) molecule fcr-1 promotes a rapid down-regulation of NKG2D ligands murine UL16-binding protein like transcript (MULT)-1 and H60 from the cell surface. Deletion of the m138/fcr-1 gene from the MCMV genome attenuates viral replication to natural killer (NK) cell response in an NKG2D-dependent manner in vivo. A distinct N-terminal module within the fcr-1 ectodomain in conjunction with the fcr-1 transmembrane domain was required to dispose MULT-1 to degradation in lysosomes. In contrast, down-modulation of H60 required the complete fcr-1 ectodomain, implying independent modes of fcr-1 interaction with the NKG2D ligands. The results establish a novel viral strategy for down-modulating NK cell responses and highlight the impressive diversity of Fc receptor functions

The Novel Human Influenza A(H7N9) Virus Is Naturally Adapted to Efficient Growth in Human Lung Tissue

A novel influenza A virus (IAV) of the H7N9 subtype has been isolated from severely diseased patients with pneumonia and acute respiratory distress syndrome and, apparently, from healthy poultry in March 2013 in Eastern China. We evaluated replication, tropism, and cytokine induction of the A/Anhui/1/2013 (H7N9) virus isolated from a fatal human infection and two low-pathogenic avian H7 subtype viruses in a human lung organ culture system mimicking infection of the lower respiratory tract. The A(H7N9) patient isolate replicated similarly well as a seasonal IAV in explanted human lung tissue, whereas avian H7 subtype viruses propagated poorly. Interestingly, the avian H7 strains provoked a strong antiviral type I interferon (IFN-I) response, whereas the A(H7N9) virus induced only low IFN levels. Nevertheless, all viruses analyzed were detected predominantly in type II pneumocytes, indicating that the A(H7N9) virus does not differ in its cellular tropism from other avian or human influenza viruses. Tissue culture-based studies suggested that the low induction of the IFN-β promoter correlated with an efficient suppression by the viral NS1 protein. These findings demonstrate that the zoonotic A(H7N9) virus is unusually well adapted to efficient propagation in human alveolar tissue, which most likely contributes to the severity of lower respiratory tract disease seen in many patients

Archival influenza virus genomes from Europe reveal genomic variability during the 1918 pandemic

The 1918 influenza pandemic was the deadliest respiratory pandemic of the 20th century and determined the genomic make-up of subsequent human influenza A viruses (IAV). Here, we analyze both the first 1918 IAV genomes from Europe and the first from samples prior to the autumn peak. 1918 IAV genomic diversity is consistent with a combination of local transmission and long-distance dispersal events. Comparison of genomes before and during the pandemic peak shows variation at two sites in the nucleoprotein gene associated with resistance to host antiviral response, pointing at a possible adaptation of 1918 IAV to humans. Finally, local molecular clock modeling suggests a pure pandemic descent of seasonal H1N1 IAV as an alternative to the hypothesis of origination through an intrasubtype reassortment.Peer Reviewe

Advancing Precision Vaccinology by Molecular and Genomic Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 in Germany, 2021

Background Comprehensive pathogen genomic surveillance represents a powerful tool to complement and advance precision vaccinology. The emergence of the Alpha variant in December 2020 and the resulting efforts to track the spread of this and other severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern led to an expansion of genomic sequencing activities in Germany. Methods At Robert Koch Institute (RKI), the German National Institute of Public Health, we established the Integrated Molecular Surveillance for SARS-CoV-2 (IMS-SC2) network to perform SARS-CoV-2 genomic surveillance at the national scale, SARS-CoV-2–positive samples from laboratories distributed across Germany regularly undergo whole-genome sequencing at RKI. Results We report analyses of 3623 SARS-CoV-2 genomes collected between December 2020 and December 2021, of which 3282 were randomly sampled. All variants of concern were identified in the sequenced sample set, at ratios equivalent to those in the 100-fold larger German GISAID sequence dataset from the same time period. Phylogenetic analysis confirmed variant assignments. Multiple mutations of concern emerged during the observation period. To model vaccine effectiveness in vitro, we employed authentic-virus neutralization assays, confirming that both the Beta and Zeta variants are capable of immune evasion. The IMS-SC2 sequence dataset facilitated an estimate of the SARS-CoV-2 incidence based on genetic evolution rates. Together with modeled vaccine efficacies, Delta-specific incidence estimation indicated that the German vaccination campaign contributed substantially to a deceleration of the nascent German Delta wave. Conclusions SARS-CoV-2 molecular and genomic surveillance may inform public health policies including vaccination strategies and enable a proactive approach to controlling coronavirus disease 2019 spread as the virus evolves.Peer Reviewe

Synopse virologischer Analysen im Nationalen Referenzzentrum für Influenzaviren während der COVID-19-Pandemie

Das Nationale Referenzzentrum für Influenzaviren gewinnt durch die fortlaufende Untersuchung von Proben aus den Sentinelpraxen der Arbeitsgemeinschaft Influenza einen umfassenden Überblick über die zirkulierenden respiratorischen Erreger in Deutschland. Dazu gehören neben SARS-CoV-2 und den Influenzaviren auch das Respiratorische Synzytialvirus, Parainfluenzaviren, humane Metapneumoviren, humane saisonale Coronaviren und humane Rhinoviren. Die Analyseergebnisse von 15.660 Sentinelproben sowie weiteren Isolaten im Zeitraum von Kalenderwoche 5/2020 bis 21/2022 werden im Epidemiologischen Bulletin 22/2022 vorgestellt. Beschrieben werden außerdem die Zirkulation respiratorischer Erreger im Vergleich zu vorpandemischen Saisons, die molekulare Charakterisierung und phylogenetische Analysen, die Überprüfung der Passgenauigkeit der eingesetzten Influenzaimpfstoffe und die Resistenzprüfung von Influenzaviren

Characterisation of expression and signal transduction of the cell adhesion molecule CEACAM1 in PC12 cells

0\. Titelblatt 1\. Einleitung 4 1.1. Die Zelladhäsion 4 1.2. Das Carcinoembryonale Antigen (CEA) und die CEA-Familie 9 1.3. CEACAM1: Struktur und Expression 12 1.4. Funktionen des CEACAM1 14 1.4.1. Zelladhäsion 14 1.4.2. Rezeptor für Bakterien und Viren 15 1.4.3. Tumorsuppression 16 1.4.4. Regulation der Zellproliferation und der Differenzierung 16 1.5. Signaltransduktion 19 1.5.1. Signaltransduktion des CEACAM1 20 1.6. Das Zytoskelett 23 1.7. Zielsetzung 26 2\. Ergebnisse 27 2.1. CEACAM1-Isoformen in PC12-Zellen 27 2.1.1. Transmembranäre Isoformen 27 2.1.2. Sekretierte Isoformen 29 2.2. CEACAM1-Tyrosinphosphorylierung 34 2.2.1. Stimulation von CEACAM1 induziert dessen Tyrosin-Dephosphorylierung 36 2.3. CEACAM1 bindet die Tyrosinphosphatase SHP2 38 2.4. CEACAM1-Stimulation aktiviert die Mitogen-aktivierten Protein-Kinasen (MAPK) ERK1 und ERK2 39 2.5. Clustern induziert die Bindung von CEACAM1 an das Aktin-Zytoskelett 41 2.5.1. CEACAM1 wird durch Quervernetzen nicht in Lipid-Rafts oder Endosomen rekrutiert 44 2.5.2. Modulation der CEACAM1-Aktin-Interaktion 46 2.5.3. Die Rolle der zytoplasmatischen Domäne des CEACAM1 bei der Cluster-induzierten Interaktion mit dem Aktin-Zytoskelett 47 2.5.4. Interaktion von CEACAM1-L und Aktin in Detergens-resistenten Strukturen an Zellkontakten in Rattenhirn-Endothel-Zellen 48 2.6. Funktionelle Hierarchie der Cluster-induzierten Effekte am CEACAM1 50 2.7. Einfluß der neuronalen Differenzierung der PC12-Zellen auf die CEACAM1-Stimulation 53 3\. Diskussion 56 3.1. CEACAM1-Expression in PC12-Zellen 56 3.1.1. Transmembranäre CEACAM1-Isoformen 56 3.1.2. Neue sekretierte CEACAM1-Isoformen 57 3.2. CEACAM1-Tyrosinphosphorylierung und ?Signaltransduktion 60 3.3. Die Interaktion von CEACAM1 mit dem Aktin-Zytoskelett 68 3.4. Einfluss der neuronalen Differenzierung auf die CEACAM1-Stimulation 72 3.5. Ausblick 73 4\. Zusammenfassung 75 4.1. CEACAM1-Isoformen 75 4.2. Signaltransduktion des CEACAM1 75 4.3. Die Interaktion von CEACAM1 mit dem Aktin-Zytoskelett 76 4\. Summary 77 4.1. CEACAM1 Isoforms 77 4.2. CEACAM1-mediated Signal Transduction 77 4.3. Interaction of CEACAM1 with the Actin Cytoskeleton 78 5\. Material und Methoden 79 5.1. Material 79 5.1.1. Zelllinien 79 5.1.2. Bakterien 79 5.1.3. Plasmide 79 5.1.4. Primer 79 5.1.5. Antikörper, Marker, Kits, Chemikalien 80 5.1.6. Enzyme 81 5.1.7. Nährmedien 82 5.1.8. Chemikalien 83 5.1.9. Lösungen 83 5.1.10. Geräte 87 5.2. Methoden 88 5.2.1. Zellbiologische Methoden 88 5.2.1.1. Kultivierung von Zellen 88 5.2.1.2. Auftauen und Einfrieren von Zelllinien 88 5.2.1.3. Stimulation von Zellen 88 5.2.1.4. Herstellung von Natriumorthovanadat- und -Pervanadat -Lösung 89 5.2.1.5. Solubilisierung von Zellen 89 5.2.1.6. Quantifizierung der Detergenslöslichkeit von CEACAM1 89 5.2.1.7. Quantifizierung der CEACAM1-Tyrosinphosphorylierung 89 5.2.1.8. Transfektion von DNA in Eukaryonten-Zellen 90 5.2.2. Proteinchemische Methoden 90 5.2.2.1. Proteinbestimmung 90 5.2.2.2. SDS-Polyacrylamidgelelektrophorese (SDS-PAGE) 90 5.2.2.3. Coomassie-Färbung von Proteingelen 91 5.2.2.4. Silberfärbung von Proteingelen 91 5.2.2.5. Western-Blotting 91 5.2.2.6. Dichtegradientenzentrifugation zu Isolierung von Lipid-Rafts 91 5.2.3. Immunchemische Methoden 92 5.2.3.1. Reinigung von Antikörpern durch Affinitätschromatographie 92 5.2.3.2. Immunblot 92 5.2.3.3. Immunpräzipitation 93 5.2.3.4. FACS-Analyse 93 5.2.3.5. Immunfluoreszenzfärbung und konfokale Laser-Scanning Mikroskopie 93 5.2.4. Mikrobiologische Methoden 94 5.2.4.1. Kultivierung von E. coli 94 5.2.4.2. Herstellung kompetenter Bakterien 94 5.2.5. Molekularbiologische Methoden 95 5.2.5.1. RNA-Präparation 95 5.2.5.2. cDNA-Synthese 95 5.2.5.3. Polymerasekettenreaktion (PCR) 95 5.2.5.4. Agarose-Gelelektrophorese 95 5.2.5.5. Isolierung von DNA-Fragmenten mittels Gelelution 95 5.2.5.6. Klonierung und Ligation von PCR-Produkten 96 5.2.5.7. Transformation von Plasmid-DNA in E. coli 97 5.2.5.8. Plasmid-Schnell-Präparation 97 5.2.5.9. Midi- und Maxi-Plasmidpräparationen 97 5.2.5.10. Spaltung von DNA durch Restriktiosendonuklesaen 98 5.2.5.11. Sequenzierung 98 6\. Literatur 99 7\. Anhang 126 Abkkürzungen 126 Veröffentlichungen 128 Lebenslauf 130 Danksagung 131CEACAM1-Isoformen In PC12-Zellen der Ratte wurden die Spleißvarianten CEACAM1-4L und CEACAM1-4S nachgewiesen. Zusätzlich wurden zwei neue Isoformen, CEACAM1-4C1 und CEACAM1-4C2, identifiziert. Beide Formen sind sekretierte Proteine. Das CEACAM1-4C2 weist einen zu CEACAM1-4L identischen C-Terminus auf. Mittels eines Antiserums gegen die zytoplasmatische Domäne von CEACAM1-4L konnte CEACAM1-4C2 deshalb auf Proteinebene sowohl in vitro in konditioniertem Medium von PC12-Zellen als auch in vivo in Rattenserum nachgewiesen werden. Im Serum von Hepatom-tragenden Tieren war CEACAM1-4C2 verstärkt nachzuweisen. Signaltransduktion des CEACAM1 Die CEACAM1-Tyrosinphosphorylierung konnte nach Inhibition zellulärer Tyrosinphos-phatasen mit dem Phosphataseinhibitor Pervanadat dargestellt werden. Die Modulation der makromolekularen Organisation des CEACAM1 durch Gabe von Antikörpern wurde angewendet, um einen CEACAM1-spezifischen Reiz zu erzeugen. Die Stimulation mit dem anti-CEACAM1 mAk Be 9.2 in Kombination mit einem sekundären Antikörper bewirkte dabei die Erzeugung großer CEACAM1-Cluster in der Plasmamembran. Die Stimulation von CEACAM1 durch Clustern hatte seine schnelle und reversible Tyrosin- Dephosphorylierung zur Folge. Eine direkte Auswirkung dieser Dephosphorylierung bestand in der Modulation der Bindung der Tyrosinphosphatase SHP2 an CEACAM1: Diese Interaktion war von der CEACAM1-Tyrosinphosphorylierung abhängig und wurde deshalb nach Stimulation verringert. Das an der Membran initiierte Signal bewirkte im Zytoplasma die temporäre und spezifische Aktivierung der MAP-Kinasen ERK1 und ERK2. Die verwandten MAP-Kinasen JNK und p38 wurden dagegen nicht aktiviert. Nach der durch NGF induzierten neuronalen Differenzierung der PC12-Zellen war das konstitutive Niveau der CEACAM1-Tyrosinphosphorylierung reduziert und die Stimulation von CEACAM1 führte nicht mehr zu einer weiteren Dephosphorylierung. Die Interaktion von CEACAM1 mit dem Aktin-Zytoskelett Die Stimulation des CEACAM1 durch Clustern bewirkte seine Bindung an das Aktin- Zytoskelett. Es wurde ein Versuchssystem etabliert, bei dem die Extrahierbarkeit von CEACAM1 aus Zellen mit Detergens Triton X-100 als Maß für die Interaktion mit dem Aktin-Kortex diente. Die F-Aktin-destabilisierenden Reagenzien Cytochalasin D und Latrunculin A konnten die Cluster-induzierte Unlöslichkeit des CEACAM1 deutlich verringern. Die CEACAM1-Aktin-Interaktion war abhängig vom Zustand der Zellen: Sowohl die Erhöhung der Zelldichte als auch die neuronale Differenzierung der PC12-Zellen bewirkte eine verstärkte Interaktion. Die CEACAM1-Tyrosinphosphorylierung hatte keinen Einfluß auf seine Bindung an Aktin, umgekehrt aber war ein intaktes Zytoskelett für die Regulation der CEACAM1-Tyrosinphosphorylierung von Bedeutung. Der zytoplasmatische Teil des CEACAM1 war nicht nötig für die Cluster-induzierte Bindung an das Aktin-Zytoskelett, wie durch Verwendung der Mutante CEACAM1-DC ohne zytoplasmatischen Teil gezeigt wurde. Die Kolokalisation von CEACAM1 und Aktin an Zellkontakten in Barbe-Endothelzelen war dagegen nur für CEACAM1-4L nachweisbar.CEACAM1-Isoforms In rat PC12 cells, the CEACAM1-4L and CEACAM1-4S splice variants were detected. Additionally, two novel isoforms, CEACAM1-4C1 and CEACAM1-4C2 were identified. Both are secreted proteins. The C-terminus of CEACAM1-4C2 is identical to that of CEACAM1-4L, which allowed the specific detection of CEACAM1-4C2 on the protein level by an antiserum directed against the CEACAM1-4L cytoplasmic part. CEACAM1-4C2 was found both in vitro in conditioned cell culture medium from PC12 cells and in vivo in rat serum. In serum of animals with a growing Morris hepatoma, the CEACAM1-4C2 level was elevated. CEACAM1-mediated Signal Transduction CEACAM1 tyrosine phosphorylation was detectable after inhibition of cellular tyrosine phosphatases with the phosphatase inhibitor pervanadate. The modulation of CEACAM1 macromolecular organisation by addition of antibodies was applied in order to induce a CEACAM1-specific stimulus. Treatment with the anti-CEACAM1 mAb Be 9.2 in combination with a secondary antibody caused the formation of large CEACAM1-clusters in the plasma membrane. Stimulation of CEACAM1 by clustering induced its fast and reversible tyrosine dephosphorylation. The interaction of CEACAM1 with the tyrosine phosphatase SHP2 was directly influenced by this dephosphorylation: the interaction, which is dependent on CEACAM1 tyrosine phosphorylation, was reduced after stimulation. The signal initiated at the membrane caused the reversible and specific activation of the MAP kinases ERK1 und ERK2. In contrast, the activity of the related kinases JNK and p38 remained unchanged. Neuronal differentiation of PC12 cells with NGF reduced the constitutive level of CEACAM1 tyrosine phosphorylation and abolished further dephosphorylation upon stimulation of CEACAM1. Interaction of CEACAM1 with the Actin Cytoskeleton Stimulation by clustering caused CEACAM1 to bind to the actin cytoskeleton. An assay was established, in which the degree of insolubility of CEACAM1 after extraction of cells with the detergent Triton X-100 was calculated as a measure for its interaction with the actin cortex. The F-actin-destabilizing reagents cytochalasin D and latrunculin A significantly reduced the level of clustering-induced CEACAM1 detergent insolubility. The CEACAM1-actin-interaction was dependent on several aspects of the cellular state: both an increase in cell density as well as neuronal differentiation of PC12 cells induced a stronger interaction. The level of CEACAM1 tyrosine phosphorylation had no influence on its interaction with actin. Contrary, an intact cytoskeleton was important for the regulation of CEACAM1 tyrosine phosphorylation. The cytoplasmic part of CEACAM1 was dispensable for the clustering-induced binding to the actin cytoskeleton, demonstrated with a deletion mutant lacking the cytoplasmic tail. Adversly, the colocalization of CEACAM1 and actin at cell contacts in Barbe endothelial cells was only detected for CEACAM1-4L

The RNA Helicase DDX6 Associates with RIG-I to Augment Induction of Antiviral Signaling

Virus infections induce sensitive antiviral responses within the host cell. The RNA helicase retinoic acid-inducible gene I (RIG-I) is a key sensor of influenza virus RNA that induces the expression of antiviral type I interferons. Recent evidence suggests a complex pattern of RIG-I regulation involving multiple interactions and cellular sites. In an approach employing affinity purification and quantitative mass spectrometry, we identified proteins with increased binding to RIG-I in response to influenza B virus infection. Among them was the RIG-I related RNA helicase DEAD box helicase 6 (DDX6), a known component of cytoplasmic mRNA-ribonucleoprotein (mRNP) granules like P-bodies and stress granules (SGs). RIG-I and DDX6 both localized to the cytosol and were detected in virus-induced SGs. Coimmunoprecipitation assays detected a basal level of complexes harboring RIG-I and DDX6 that increased after infection. Functionally, DDX6 augmented RIG-I mediated induction of interferon (IFN)-β expression. Notably, DDX6 was found to bind viral RNA capable to stimulate RIG-I. These findings imply a novel function for DDX6 as an RNA co-sensor and signaling enhancer for RIG-I.Peer Reviewe

Highly Pathogenic H5N1 Influenza A Virus Strains Provoke Heterogeneous IFN-α/β Responses That Distinctively Affect Viral Propagation in Human Cells

<div><p>The fatal transmissions of highly pathogenic avian influenza A viruses (IAV) of the H5N1 subtype to humans and high titer replication in the respiratory tract indicate that these pathogens can overcome the bird-to-human species barrier. While type I interferons (IFN-α/β) are well described to contribute to the species barrier of many zoonotic viruses, current data to the role of these antiviral cytokines during human H5N1 IAV infections is limited and contradictory. We hypothesized an important role for the IFN system in limiting productive infection of avian H5N1 strains in human cells. Hence, we examined IFN-α/β gene activation by different avian and human H5N1 isolates, if the IFN-α/β response restricts H5N1 growth and whether the different strains were equally capable to regulate the IFN-α/β system via their IFN-antagonistic NS1 proteins. Two human H5N1 isolates and a seasonal H3N2 strain propagated efficiently in human respiratory cells and induced little IFN-β, whereas three purely avian H5N1 strains were attenuated for replication and provoked higher IFN secretion. Replication of avian viruses was significantly enhanced on interferon-deficient cells, and exogenous IFN potently limited the growth of all strains in human cells. Moreover, IFN-α/β activation by all strains depended on retinoic acid-inducible gene I excluding principal differences in receptor activation between the different viruses. Interestingly, all H5N1 NS1 proteins suppressed IFN-α/β induction comparably well to the NS1 of seasonal IAV. Thus, our study shows that H5N1 strains are heterogeneous in their capacity to activate human cells in an NS1-independent manner. Our findings also suggest that H5N1 viruses need to acquire adaptive changes to circumvent strong IFN-α/β activation in human host cells. Since no single amino acid polymorphism could be associated with a respective high- or low induction phenotype we propose that the necessary adaptations to overcome the human IFN-α/β barrier involve mutations in multiple H5N1 genes.</p> </div