Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses

first_page settings Order Article Reprints Open AccessHypothesis Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses by Markus Kastner 1,†,‡, Andreas Karner 1,†,§ [ORCID] , Rong Zhu 1,† [ORCID] , Qiang Huang 2 [ORCID] , Andreas Geissner 3,4,‖, Anne Sadewasser 5,¶, Markus Lesch 6, Xenia Wörmann 6, Alexander Karlas 6,**, Peter H. Seeberger 3,4 [ORCID] , Thorsten Wolff 5 [ORCID] , Peter Hinterdorfer 1 [ORCID] , Andreas Herrmann 7 and Christian Sieben 8,9,* [ORCID] 1 Institute for Biophysics, Johannes Kepler University Linz, 4020 Linz, Austria 2 State Key Laboratory of Genetic Engineering, Shanghai Engineering Research Center of Industrial Microorganisms, MOE Engineering Research Center of Gene Technology, School of Life Sciences, Fudan University, Shanghai 200438, China 3 Department for Biomolecular Systems, Max Planck Institute for Colloids and Interfaces, 14476 Potsdam, Germany 4 Institute of Chemistry and Biochemistry, Freie Universität Berlin, 14195 Berlin, Germany 5 Division of Influenza and other Respiratory Viruses, Robert Koch-Institute, 13353 Berlin, Germany 6 Molecular Biology Department, Max Planck Institute for Infection Biology, 10117 Berlin, Germany 7 Institut für Chemie und Biochemie, Freie Universität Berlin, Altensteinstraße 23a, 14195 Berlin, Germany 8 Nanoscale Infection Biology Group, Department of Cell Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany 9 Institute for Genetics, Technische Universität Braunschweig, 38106 Braunschweig, Germany * Author to whom correspondence should be addressed. † These authors contributed equally to this work. ‡ Current address: Materials Characterization Lab (MCL), Materials Research Institute (MRI), Pennsylvania State University, University Park, PA 16802, USA. § Current address: University of Applied Sciences Upper Austria, School of Medical Engineering and Applied Social Sciences, Garnisonstr. 21, 4020 Linz, Austria. ‖ Current address: Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada. ¶ Current address: Secarna Pharmaceuticals GmbH & Co. KG, Am Klopferspitz 19, 82152 Planegg, Germany. ** Current address: Viral Vectors and Gene Therapeutics, ProBioGen AG, 13086 Berlin, Germany. Viruses 2023, 15(7), 1507; https://doi.org/10.3390/v15071507 Received: 9 May 2023 / Revised: 21 June 2023 / Accepted: 28 June 2023 / Published: 5 July 2023 (This article belongs to the Special Issue Physical Virology - Viruses at Multiple Levels of Complexity) Download Browse Figures Review Reports Versions Notes Abstract Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA’s receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus–cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA’s receptor binding preference does not necessarily reflect virus–cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells

Activities of the influenza B virus non-structural protein 1 against the innate host defense of infected cells

Infektionen durch Influenza A oder B Viren können schwere oder letale Krankheitsverläufe zur Folge haben. Der multifunktionelle Virulenzfaktor NS1 spielt dabei für den Verlauf der Infektion aufgrund einer Blockade von angeborenen antiviralen Abwehrreaktionen der Zelle eine entscheidende Rolle. Die in silico-Identifikation des in über 300 Influenza B Virusstämmen konservierten NS1-Sequenzmotivs 121-Y-P-x-x-P-x-(K/R)-127 mit Homologie zum Klasse II-SH3-Bindemotiv (PxxPxK/R) wies auf bisher unbekannte Funktionen des NS1-Proteins hin, deren Bedeutung für die Interaktionen von Virus und Wirt, sowie den viralen Vermehrungsverlauf in der vorliegenden Arbeit analysiert wurde. Zur Untersuchung des NS1-Sequenzmotivs wurden Virusmutanten mit Aminosäuresubstitutionen in dem hochkonservierten Bereich durch Methoden der reversen Genetik hergestellt. Überraschenderweise führte die vollständige Substitution des 122-P-x-x-P-x-(K/R)-127-Motivs durch 122-G-g-g-G-g-G-127 (xSH3G) nicht zu einer Reduktion des Virustiters, sondern zu einer gesteigerten Virusreplikation von bis zu drei Log-Stufen in humanen Zellen. Diese Steigerung ging mit verstärkter viraler Genexpression im Vergleich zum Wildtyp einher, wobei die IFN-beta-Expression der infizierten Zellen nicht beeinflusst wurde. In einem globalen Ansatz konnte mittels quantitativer Massenspektrometrie-basierter Proteomik gezeigt werden, dass die xSH3G- Substitution zur Bindung des B/NS1-Proteins an das Chaperon HSP90beta führt. Sowohl diese Interaktion als auch die Manipulation der Crk/JNK/ATF2-, Akt- und PKR-vermittelten zellulären Signalwege korrelierten mit einer verstärkten Apoptoseinduktion in den xSH3G-Virus-infizierten Zellen. Zudem konnte gezeigt werden, dass die NS1-xSH3G-Mutation eine Interaktion des HSP90beta-Chaperons mit der viralen Polymeraseuntereinheit PB2 zur Folge hat. Dieses Ergebnis deutet in Analogie zu Untersuchungen der Influenza A Virusreplikation darauf hin, dass infolge der HSP90-PB2-Bindung eine Steigerung der Viruspolymeraseassemblierung im Zellkern die erhöhte Virusvermehrung der xSH3G-Virusvariante indiziert. Die vorliegende Arbeit präsentiert eines von wenigen bisher bekannten Beispielen, bei dem die Substitution einer hochkonservierten Sequenz in einem viralen Protein zu verstärkter Replikationsfähigkeit der Virusmutante führt. Die Ergebnisse der durchgeführten Untersuchungen verdeutlichen, dass durch Mutationsanalyse eines viralen Proteins nicht nur die ursprünglichen Funktionen der substituierten Aminosäuren verloren gehen, sondern auch unerwartete Protein-Protein- Interaktionen und -aktivitäten auftreten können. Eine Überexpression von HSP90 könnte in Zukunft bei der Vakzinherstellung genutzt werden, um die Influenzavirusproduktion in Zellkultur zu optimieren.Infections with influenza A or B virus can lead to serious or lethal courses of disease. The multifunctional virulence factor NS1 plays a crucial role for the course of infection by blocking the innate antiviral immune response of infected cells. The in silico identification of the highly conserved motif 121-Y-P-x-x-P-x-(K/R)-127 with homology to the class II SH3 binding motif (PxxPxK/R) within the NS1 sequence of more than 300 influenza B virus strains indicated previously unknown protein functions. The importance of these protein functions for interactions of virus and host cell as well as for viral replication was analyzed in the presented study. To investigate the NS1 protein motif virus mutants with amino acid substitution within the highly conserved region were generated by methods of reverse genetics. Surprisingly, the entire substitution of 122-P-x-x-P-x-(K/R)-127 to 122-G-g-g-G-g-G-127 (xSH3G) did not lead to a reduction of virus titer, but to increased virus replication up to three log levels in human cells. This enhanced virus propagation involved increased gene expression compared to wildtype, but had no effect on IFN-beta expression levels of infected cells. In a global approach using quantitative mass spectrometry based proteomics it could be shown that xSH3G substitution leads to binding of the B/NS1 protein to the chaperon HSP90beta. Both, this interaction and manipulation of Crk/JNK/ATF2-, Akt- and PKR-mediated cellular signaling pathways correlated with increased apoptosis induction in xSH3G-infected cells. It was also demonstrated that the NS1-xSH3G mutations lead to interaction of HSP90beta with the viral polymerase subunit PB2. This result indicates that HSP90-PB2 binding might induce increased virus propagation by the enhancement of virus polymerase assembly in the cell nucleus by analogy with investigations of influenza A virus replication. The presented study shows one of only few known examples where substitution of a highly conserved sequence within a viral protein leads to increased replication of the virus mutant. The results illustrate that mutations within a viral protein may not only lead to a loss of the natural functions, but can also result in the occurrence of unexpected protein-protein interactions and activities. In future, overexpression of HSP90 might be used to optimize influenza virus production in cell culture for vaccine generation

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

Annexin V incorporated into influenza virus particles inhibits gamma interferon signaling and promotes viral replication

International audienceDuring the budding process, influenza A viruses (IAVs) incorporate multiple host cell membrane proteins. However, for most of them, their significance in viral morphogenesis and infectivity remains unknown. We demonstrate here that the expression of annexin V (A5) is upregulated at the cell surface upon IAV infection and that a substantial proportion of the protein is present in lipid rafts, the site of virus budding. Western blotting and immunogold analysis of highly purified IAV particles showed the presence of A5 in the virion. Significantly, gamma interferon (IFN-gamma)-induced Stat phosphorylation and IFN-gamma-induced 10-kDa protein (IP-10) production in macrophage-derived THP-1 cells was inhibited by purified IAV particles. Disruption of the IFN-gamma signaling pathway was A5 dependent since downregulation of its expression or its blockage reversed the inhibition and resulted in decreased viral replication in vitro. The functional significance of these results was also observed in vivo. Thus, IAVs can subvert the IFN-gamma antiviral immune response by incorporating A5 into their envelope during the budding process. IMPORTANCE Many enveloped viruses, including influenza A viruses, bud from the plasma membrane of their host cells and incorporate cellular surface proteins into viral particles. However, for the vast majority of these proteins, only the observation of their incorporation has been reported. We demonstrate here that the host protein annexin V is specifically incorporated into influenza virus particles during the budding process. Importantly, we showed that packaged annexin V counteracted the antiviral activity of gamma interferon in vitro and in vivo. Thus, these results showed that annexin V incorporated in the viral envelope of influenza viruses allow viral escape from immune surveillance. Understanding the role of host incorporated protein into virions may reveal how enveloped RNA viruses hijack the host cell machinery for their own purposes

ANGPTL4 silencing via antisense oligonucleotides reduces plasma triglycerides and glucose in mice without causing lymphadenopathy

Angiopoietin-like 4 (ANGPTL4) is an important regulator of plasma triglyceride (TG) levels and an attractive pharmacological target for lowering plasma lipids and reducing cardiovascular risk. Here, we aimed to study the efficacy and safety of silencing ANGPTL4 in the livers of mice using hepatocyte-targeting GalNAc-conjugated antisense oligonucleotides (ASOs). Compared with injections with negative control ASO, four injections of two different doses of ANGPTL4 ASO over 2 weeks markedly downregulated ANGPTL4 levels in liver and adipose tissue, which was associated with significantly higher adipose LPL activity and lower plasma TGs in fed and fasted mice, as well as lower plasma glucose levels in fed mice. In separate experiments, injection of two different doses of ANGPTL4 ASO over 20 weeks of high-fat feeding reduced hepatic and adipose ANGPTL4 levels but did not trigger mesenteric lymphadenopathy, an acute phase response, chylous ascites, or any other pathological phenotypes. Compared with mice injected with negative control ASO, mice injected with ANGPTL4 ASO showed reduced food intake, reduced weight gain, and improved glucose tolerance. In addition, they exhibited lower plasma TGs, total cholesterol, LDL-C, glucose, serum amyloid A, and liver TG levels. By contrast, no significant difference in plasma alanine aminotransferase activity was observed. Overall, these data suggest that ASOs targeting ANGPTL4 effectively reduce plasma TG levels in mice without raising major safety concerns

Relevance of Host Cell Surface Glycan Structure for Cell Specificity of Influenza A Viruses

Influenza A viruses (IAVs) initiate infection via binding of the viral hemagglutinin (HA) to sialylated glycans on host cells. HA’s receptor specificity towards individual glycans is well studied and clearly critical for virus infection, but the contribution of the highly heterogeneous and complex glycocalyx to virus–cell adhesion remains elusive. Here, we use two complementary methods, glycan arrays and single-virus force spectroscopy (SVFS), to compare influenza virus receptor specificity with virus binding to live cells. Unexpectedly, we found that HA’s receptor binding preference does not necessarily reflect virus–cell specificity. We propose SVFS as a tool to elucidate the cell binding preference of IAVs, thereby including the complex environment of sialylated receptors within the plasma membrane of living cells.Science, Faculty ofNon UBCChemistry, Department ofReviewedFacultyResearche

The dynamic proteome of influenza A virus infection identifies M segment splicing as a host range determinant

Pandemic influenza A virus (IAV) outbreaks occur when strains from animal reservoirs acquire the ability to infect and spread among humans. The molecular basis of this species barrier is incompletely understood. Here we combine metabolic pulse labeling and quantitative proteomics to monitor protein synthesis upon infection of human cells with a human- and a bird-adapted IAV strain and observe striking differences in viral protein synthesis. Most importantly, the matrix protein M1 is inefficiently produced by the bird-adapted strain. We show that impaired production of M1 from bird-adapted strains is caused by increased splicing of the M segment RNA to alternative isoforms. Strain-specific M segment splicing is controlled by the 3′ splice site and functionally important for permissive infection. In silico and biochemical evidence shows that avian-adapted M segments have evolved different conserved RNA structure features than human-adapted sequences. Thus, we identify M segment RNA splicing as a viral host range determinant.Peer Reviewe

Development and characterization of DNAzyme candidates demonstrating significant efficiency against human rhinoviruses

BACKGROUND: Infections with human rhinoviruses (RVs) are responsible for millions of common cold episodes and the majority of asthma exacerbations, especially in childhood. No drugs specifically targeting RVs are available. OBJECTIVE: We sought to identify specific anti-RV molecules based on DNAzyme technology as candidates to a clinical study. METHODS: A total of 226 candidate DNAzymes were designed against 2 regions of RV RNA genome identified to be sufficiently highly conserved between virus strains (ie, the 5'-untranslated region and cis-acting replication element) by using 3 test strains: RVA1, RVA16, and RVA29. All DNAzymes were screened for their cleavage efficiency against in vitro-expressed viral RNA. Those showing any catalytic activity were subjected to bioinformatic analysis of their reverse complementarity to 322 published RV genomic sequences. Further molecular optimization was conducted for the most promising candidates. Cytotoxic and off-target effects were excluded in HEK293 cell-based systems. Antiviral efficiency was analyzed in infected human bronchial BEAS-2B cells and ex vivo-cultured human sinonasal tissue. RESULTS: Screening phase-generated DNAzymes characterized by either good catalytic activity or by high RV strain coverage but no single molecule represented a satisfactory combination of those 2 features. Modifications in length of the binding domains of 2 lead candidates, Dua-01(-L12R9) and Dua-02(-L10R11), improved their cleavage efficiency to an excellent level, with no loss in eminent strain coverage (about 98%). Both DNAzymes showed highly favorable cytotoxic/off-target profiles. Subsequent testing of Dua-01-L12R9 in BEAS-2B cells and sinonasal tissue demonstrated its significant antiviral efficiency. CONCLUSIONS: Effective and specific management of RV infections with Dua-01-L12R9 might be useful in preventing asthma exacerbations, which should be verified by clinical trials

Average ERPs of N170 and LPC component topographies across three conditions.

<p>Average ERPs of N170 and LPC component topographies across three conditions.</p