Imaging Poliovirus Entry in Live Cells

Viruses initiate infection by transferring their genetic material across a cellular membrane and into the appropriate compartment of the cell. The mechanisms by which animal viruses, especially nonenveloped viruses, deliver their genomes are only poorly understood. This is due in part to technical difficulties involved in direct visualization of viral gene delivery and to uncertainties in distinguishing productive and nonproductive pathways caused by the high particle-to–plaque forming unit ratio of most animal viruses. Here, we combine an imaging assay that simultaneously tracks the viral capsid and genome in live cells with an infectivity-based assay for RNA release to characterize the early events in the poliovirus (PV) infection. Effects on RNA genome delivery from inhibitors of cell trafficking pathways were probed systematically by both methods. Surprisingly, we observe that genome release by PV is highly efficient and rapid, and thus does not limit the overall infectivity or the infection rate. The results define a pathway in which PV binds to receptors on the cell surface and enters the cell by a clathrin-, caveolin-, flotillin-, and microtubule-independent, but tyrosine kinase- and actin-dependent, endocytic mechanism. Immediately after the internalization of the virus particle, genome release takes place from vesicles or tightly sealed membrane invaginations located within 100–200 nm of the plasma membrane. These results settle a long-lasting debate of whether PV directly breaks the plasma membrane barrier or relies on endocytosis to deliver its genome into the cell. We expect this imaging assay to be broadly applicable to the investigation of entry mechanisms for nonenveloped viruses

Development and Assessment of a Pooled Serum as Candidate Standard to Measure Influenza A Virus Group 1 Hemagglutinin Stalk-Reactive Antibodies

The stalk domain of the hemagglutinin has been identified as a target for induction of protective antibody responses due to its high degree of conservation among numerous influenza subtypes and strains. However, current assays to measure stalk-based immunity are not standardized. Hence, harmonization of assay readouts would help to compare experiments conducted in different laboratories and increase confidence in results. Here, serum samples from healthy individuals (n = 110) were screened using a chimeric cH6/1 hemagglutinin enzyme-linked immunosorbent assay (ELISA) that measures stalk-reactive antibodies. We identified samples with moderate to high IgG anti-stalk antibody levels. Likewise, screening of the samples using the mini-hemagglutinin (HA) headless construct #4900 and analysis of the correlation between the two assays confirmed the presence and specificity of anti-stalk antibodies. Additionally, samples were characterized by a cH6/1N5 virus-based neutralization assay, an antibody-dependent cell-mediated cytotoxicity (ADCC) assay, and competition ELISAs, using the stalk-reactive monoclonal antibodies KB2 (mouse) and CR9114 (human). A “pooled serum” (PS) consisting of a mixture of selected serum samples was generated. The PS exhibited high levels of stalk-reactive antibodies, had a cH6/1N5-based neutralization titer of 320, and contained high levels of stalk-specific antibodies with ADCC activity. The PS, along with blinded samples of varying anti-stalk antibody titers, was distributed to multiple collaborators worldwide in a pilot collaborative study. The samples were subjected to different assays available in the different laboratories, to measure either binding or functional properties of the stalk-reactive antibodies contained in the serum. Results from binding and neutralization assays were analyzed to determine whether use of the PS as a standard could lead to better agreement between laboratories. The work presented here points the way towards the development of a serum standard for antibodies to the HA stalk domain of phylogenetic group 1

Zell permeable Nukleokapside als neues Werkzeug für einen effizienten Gentransfer und die HBV Biologie

1 Index and Abstract 2 Introduction 3 Results 4 Discussion 5 Materials and Methods 6 References ans AppendixGENE TRANSFER In order to combine the advantages of viral gene transfer (efficacy and protection of the nucleic acid) and of non-viral gene transfer (safety) a novel tool in gene transfer technology was developed. This new system is based on cell permeable HBV nucleocapsids which are loaded with non- viral or viral nucleic acids. Cell permeability is mediated by a short peptide (12aa, TLM). The TLM was inserted in the spike tip or fused to the N-terminus of the hepatitis B virus (HBV) core protein (HBc). HBV capsids expressed by E. coli or insect cells were isolated by affinity chromatography. To separate fully assembled capsids from HBcAg oligomers size exclusion chromatography was performed. Electron microscopy revealed that insertion of the TLM in the HBcAg molecule does not affect the assembly to capsids. The cell permeability of fully assembled TLM capsids was verified in cells by using confocal and electron microscopy as well as a life cell assay with fluorophore-labeled capsids. To exclude an endocytotic uptake of capsids, several endocytosis inhibitors were used in the presence of a FITC-labeled transferrin control. It was demonstrated that cell permeable TLM capsids translocate directly in an endocytosis-independent manner into the cytoplasm of different cell types. In order to package DNA for gene transfer into the capsids, two different protocols were developed. For the in vitro packaging, the purified bacterial expressed capsids were subjected to partial denaturation to dissociate them. The HBcAg oligomers were renaturated and thereby reassembled by stepwise dialysis in the presence of the plasmid DNA to be packaged (SHBs or eGFP served as marker genes). According to the life cycle of HBV a second, an eukaryotic in vivo expression system for packaging of nucleic acids into capsids was developed. Spodoptera frugiperda cells were triple infected with baculoviruses encoding HBV polymerase, HBc and the reporter gene construct (harboring two encapsidation signals). Inside of the insect cells the polymerase first interacts with encapsidation signals of the mRNA to be packaged and then this complex was recognizes and packaged by the core protein. The amount of packaged DNA was quantified by TaqMan PCR. The capacity of these TLM nucleocapsids to successful transfer genes into poorly transfectable primary human hepatocytes was analyzed by specific ELISA and immunofluorescence, monitoring the expression of reporter genes. These results indicate that cell permeable HBV-derived nucleocapsids provide a novel tool for safe and efficient gene transfer. HBV BIOLOGY The post entry intracellular trafficking of the HBV nucleocapsid and transport of the viral genome is still poorly understood. The current models to investigate this affect the integrity of the cell by using digitonin or microinjection. To establish a physiological model system cell permeable HBV nucleocapsids were used in this study. Without affecting the integrity of cells it is possible to investigate intracellular trafficking of these particles. TLM nucleocapsids rapidly translocate across the plasma membrane as intact particles (already after 5 minutes) from the medium into almost all cells (e.g. HuH7 and primary human hepatocytes), whereas no significant internalization was seen in case of wild type nucleocapsids, lacking the TLM. To distinguish between complete particles and HBcAg dimers by confocal microscopy a monoclonal antibody that recognizes complete particles selectively and a rabbit-derived serum that recognizes particles as well as HBcAg dimers were used. Initial results suggest that internalized nucleocapsids move as complete particles directed by the cytoskeleton towards the nuclear membrane and nuclear pore complex (NPC) with a speed of ~0,15 μm/min. A colocalisation of fully assembled nucleocapsids with the microtubule organization center as well as with F-actin and a significant reorganization of F-actin during trafficking can be observed. A specific binding of assembled HBV capsids at β-tubulin was found. There were not any complete capsids or HBcAg dimers detectable inside of the nucleus (up to 48 hours post incubation). However, if the intracellular localization of wt or TLM nucleocapsids was analyzed by using digitonin-permeabilized cells, in contrast to the described new model HBcAg dimers were detected after 120 minutes inside of the nucleus. The results presented in this study suggest that TLM nucleocapsids allow a detailed analysis of HBV post entry processes. This makes it possible to study the intracellular trafficking of HBV nucleocapsid, the interaction with subcellular structures (e.g. cytoskeleton and NPC) and the disassembly without affecting the integrity of the cell.GENTRANSFER Um die Vorteile des viralen Gentransfers (Effizienz und Schutz der Nukleinsäure) mit denen des nicht viralen Gentransfers (Sicherheit) zu kombinieren, wurde eine neue Methode entwickelt. Sie basiert auf zellpermeablen HBV Nukleokapsiden (5000 kDa), welche mit nicht-viralen oder viralen Nukleinsäuren beladen sind. Die Zellpermeabilität wird von einem kurzen Peptid - dem TLM (12 AS) - vermittelt. Das TLM wurde sowohl in den Spike-tip inseriert als auch mit dem N-Terminus des Hepatitis B Virus (HBV) Core Proteins (HBc) fusioniert. Die HBV Kapside wurden von E. coli oder Insekten Zellen exprimiert und mittels Affinitäts-Chromatographie isoliert. Vollständig assemblierte Kapside wurden von HBcAg Oligomeren mittels Größenausschluss-Chromatographie getrennt. Elektronenmikroskopische Aufnahmen bestätigten, dass die Modifikation durch das TLM, die Fähigkeit der Moleküle sich zu Kapsiden zusammen zulagern, nicht beeinträchtigt. Die Zellpermeabilität vollständig assemblierter TLM Kapside konnte durch die Untersuchungen mit Konfokaler- und Elektronen-Mikroskopie in verschiedenen Zellen sowie durch Lebend-Zell Experimente mit Fluorophor markierten Kapsiden nachgewiesen werden. Um eine endozytotische Aufnahme der Kapside ausschließen zu können, wurden verschiedene Endozytose-Inhibitoren in Gegenwart einer FITC markierten Transferrin Kontrolle verwendet. Es konnte nachgewiesen werden, dass zellpermeable TLM Kapside in einer Endozytose unabhängigen Translokation direkt in das Zytoplasma der Zellen gelangen können. Um die für den Gentransfer nötige DNA in die Kapside zu verpacken, wurden zwei unterschiedliche Protokolle entwickelt. Für die in vitro Verpackung wurden die gereinigten, bakteriell exprimierten Kapside denaturiert und dadurch disassembliert. Die HBcAg Moleküle wurden renaturiert und dabei durch schrittweise Dialyse in Gegenwart von Plasmid DNA reassembliert (SHBs oder eGFP dienten als Marker Gene). Dies führt zur Verpackung der DNA in die Kapside. In Anlehnung an den Lebenszyklus des HBV wurde ein zweites, ein eukaryotisches in vivo System zum Verpacken von Nukleinsäuren in Kapside entwickelt. Spodoptera frugiperda Zellen wurden gleichzeitig mit Baculoviren infiziert, die für die HBV Polymerase, das Kapsid und ein Reportergen Konstrukt (zwei Enkapsidierungs-Signale besitzend) codieren. Innerhalb der Insektenzellen interagiert zu erst die Polymerase mit der zu verpackenden Nukleinsäure und dieser Komplex wird von den Kapsidproteinen erkannt und verpackt. Die Menge an, in Kapsiden verpackter DNA wurde mittels TaqMan PCR quantifiziert. Die Kapazität dieser TLM Nukleokapside, Gene erfolgreich in schwer transfizierbare primäre humane Hepatozyten zu transferieren wurde mit Hilfe spezifischer ELISA und Immunfluoreszenzen analysiert. Diesen Ergebnissen folgend, stellen die von HBV abgeleiteten zellpermeablen Nukleokapside ein neues Werkzeug zum effizienten und sicheren Gentransfer dar. HBV BIOLOGIE Nur wenige Einzelheiten der intrazellulären Fortbewegung des HBV Nukleokapsides nach dem Eintritt des Virus in die Zelle sind bekannt. Durch Mikroinjektionen oder die Vorbehandlung mit Digitonin wird zur Untersuchung dieser Fortbewegung in den derzeitig verwendeten Modellen die Integrität der Zellen beeinträchtigt. In dieser Arbeit wurden zellpermeable Nukleokapside verwendet, um ein Modelsystem der HBV Infektion zu etablieren, welches mehr der physiologischen Situation entspricht. Mit den zellpermeablen HBV Nukleokapsiden ist es möglich, deren intrazelluläre Fortbewegung zu untersuchen ohne die Integrität der Zelle zu beeinträchtigen. TLM Nukleokapside translozieren als intakte Partikel rasch (schon nach 5 Minuten) vom Medium durch die Plasmamembran in nahezu alle Zellen (z.B. primäre humane Hepatozyten). Im Falle der kein TLM besitzenden Wildtyp Kapside konnte hingegen keine signifikante Internalisierung beobachtet werden. Um mit Hilfe der konfokalen Mikroskopie komplette Partikel von HBcAg Dimeren unterscheiden zu können, wurden monoklonale Antikörper verwendet, die ausschließlich vollständig assemblierte Partikel erkennen, sowie polyklonale Antikörper die sowohl komplette Kapside als auch HBcAg Dimere erkennen. Die Ergebnisse lassen vermuten, dass sich internalisierte Nukleokapside als komplette Partikel entlang des Zytoskeletts zur Kernmembran und den Kernporen-Komplexen mit einer Geschwindigkeit von etwa 0,15 μm/min bewegen. Es konnte eine Kolokalisierung der vollständig assemblierten Nukleokapside während ihrer intrazellulären Wanderung mit dem Mikrotubuliorganisationszentrum als auch mit F-Actin sowie eine signifikante Reorganisation des F-Actins beobachtet werden. Es konnte weiterhin gezeigt werden, dass β-Tubulin spezifisch an assemblierte Kapside bindet. Zu keinem Zeitpunkt konnten (bis zu 48 Stunden nach Inkubation) komplette zellpermeable Kapside im Kern der Zellen detektiert werden. Wurde jedoch die Lokalisierung der Kapside in Digitonin permeabilisierten Zellen untersucht, konnten im Gegensatz zum neu beschriebenen Model, HBcAg Dimere nach 120 Minuten im Kern detektiert werden. Die in dieser Arbeit dargelegten Ergebnisse zeigen, dass TLM Nukleokapside eine detaillierte Analyse des intrazellulären Transportes nach dem Eintritt von HBV in die Zelle erlauben. Dies macht es möglich, Interaktion des Nukleokapsides mit subzellulären Strukturen (wie dem Zytoskelett oder dem Kernporen-Komplex) sowie die Dissoziation der Kapside in Zellen zu beobachten ohne deren Integrität zu beeinträchtigen

Reconstitution of gene expression from a regulatory-protein-deficient hepatitis B virus genome by cell-permeable HBx protein

Various functions are ascribed to the HBx regulatory protein of the hepatitis B virus (HBV). Due to the low expression level of HBx, it has been difficult to correlate spatial and temporal HBx expression levels with specific functions. Based on a novel cell-permeable peptide, known as the translocation motif (TLM), cell-permeable HBx fusion proteins were generated. The TLM–HBx fusion protein is rapidly internalized from the medium into almost all cells, whereas no significant internalization was seen with wild-type HBx. The major fraction of internalized HBx protein moves from the cytoplasm to the nucleus. The cytosolic fraction, however, activates c-RAF1/extracellular-signal-related kinase 2 signalling and causes activation of activator protein 1 (AP1) and nuclear factor-κB. The TLM–HBx protein rescues HBV gene expression from an activator-deficient HBV genome. These results indicate that cell-permeable regulatory proteins provide a novel, efficient tool for a clearly defined, dose-dependent analysis of regulatory protein function, without affecting the integrity of the cell, and can be used for the safe reconstitution of virus production from a regulatory-protein-deficient virus genome

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemifusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated action

Relating influenza virus membrane fusion kinetics to stoichiometry of neutralizing antibodies at the single-particle level

The ability of antibodies binding the influenza hemagglutinin (HA) protein to neutralize viral infectivity is of key importance in the design of next-generation vaccines and for prophylactic and therapeutic use. The two antibodies CR6261 and CR8020 have recently been shown to efficiently neutralize influenza A infection by binding to and inhibiting the influenza A HA protein that is responsible for membrane fusion in the early steps of viral infection. Here, we use single-particle fluorescence microscopy to correlate the number of antibodies or antibody fragments (Fab) bound to an individual virion with the capacity of the same virus particle to undergo membrane fusion. To this end, individual, infectious virus particles bound by fluorescently labeled antibodies/Fab are visualized as they fuse to a planar, supported lipid bilayer. The fluorescence intensity arising from the virus-bound antibodies/Fab is used to determine the number of molecules attached to viral HA while a fluorescent marker in the viral membrane is used to simultaneously obtain kinetic information on the fusion process. We experimentally determine that the stoichiometry required for fusion inhibition by both antibody and Fab leaves large numbers of unbound HA epitopes on the viral surface. Kinetic measurements of the fusion process reveal that those few particles capable of fusion at high antibody/Fab coverage display significantly slower hemi-fusion kinetics. Overall, our results support a membrane fusion mechanism requiring the stochastic, coordinated action of multiple HA trimers and a model of fusion inhibition by stem-binding antibodies through disruption of this coordinated actio