Untersuchungen zu Eintrittsmechanismen des Herpes simplex Virus Typ 1 in Epithelzellen

Primäre Eintrittspforte für die Herpes simplex Virus Typ1 (HSV-1) Infektion ist das Schleimhautepithel. Der zelluläre Eintrittsprozess von HSV-1 bedingt eine Interaktion verschiedener viraler Glykoproteine mit unterschiedlichen Zelloberflächen-Rezeptoren, die gefolgt ist von der Penetration der Plasmamembran und dem Transport zum Zellkern. Das zentrale Interesse der vorliegende Arbeit bestand darin, zum Verständnis des viralen Eintritts in vivo beizutragen. Der HSV-1 Eintritt in individuelle Zellen wurde durch Immunfärbung gegen ICP0, einem Produkt sehr früher viraler Genexpression, sichtbar gemacht. Infektionsstudien in der epithelialen MDCKII Zellinie und in primären Keratinozyten deuten auf einen polaren Eintritt von HSV-1 unter Nutzung basolateraler Membrandomänen hin. Wenn humane Vorhautepidermis infiziert wurde, wurde HSV-1 Eintritt ausschließlich über basale Zellen beobachtet. Diese Ergebnisse deuten darauf hin, dass effizienter viraler Eintritt den Zugang zu basolateralen Membrandomänen bedingt. Zur weiteren Charakterisierung der HSV-1 Eintrittsmechanismen in Epithelzellen wurden potentielle molekulare Determinanten untersucht. Immunfärbung gegen den potentiellen Ko-Rezepor Nectin-1 zeigte keine Korrelation der Rezeptor-Lokalisierung und den Virus-Eintrittsstellen in Keratinozyten-Kulturen. Die Beteiligung des HSV-1 Glykoproteins D am Viruseintritt wurde durch Blockierungsexperimente bestätigt. Erste pharmakologische Inhibierungsexperimente in MDCKII Zellen ergaben keine Hinweise auf endozytotische Aufnahme verbunden mit pH-abhängiger Membranfusion von HSV-1. Da infizierte MDCKII Zellen und Keratinozyten Eigenschaften von migrierenden Zellen zeigten, könnten Prozesse der Zellmigration viralen Eintritt fördern. Die Reorganisation des Aktin-Zytoskeletts während der Zellmigration wird durch die Rhoähnlichen GTPasen Cdc42, Rac1 und RhoA kontrolliert. Nach transienter Expression dominanter GTPase Isoformen in MDCKII Zellen wurde eine signifikante Reduktion der HSV-1 Infizierbarkeit beobachtet, falls die dominant aktiven Isoformen von Cdc42 und Rac1 sowie die dominant inaktive Isoform von Cdc42 exprimiert wurden. Im Gegensatz dazu zeigten weitere Isoformen und die Wildtyp Proteine keinen Effekt auf die Infizierbarkeit. Ko-Expressionsexperimente zeigten, dass dieser inhibierende Einfluss auf die Infizierbarkeit nicht über Hemmung des ICP0 Promotors vermittelt wird. Zusätzlich wurden Infektionsstudien von stabil transfizierten MDCKII Zellen durchgeführt, die dominante Isoformen von Cdc42 und Rac1 nach Induktion exprimierten. Die Expression von dominant aktivem Rac1 und dominant inaktivem Cdc42 nicht aber von dominant aktivem Cdc42 führte zu einer signifikanten Reduktion der HSV-1 Infizierbarkeit. Ein unterschiedlicher Verlauf der Aktivierungskaskaden von Rho GTPasen in transient und stabil transfizierten MDCKII Zellen könnte für den unterschiedlichen Effekt von dominant aktivem Cdc42 auf die HSV-1 Infizierbarkeit verantwortlich sein. Untersuchungen zum Einfluss der Cdc42/Rac1 Signaltransduktionswege auf die HSV-1 Infizierbarkeit ergaben keine Hinweise auf eine direkte Beteiligung von PAK-1 oder N-WASP in diesem Prozess. Diese Ergebnisse ergaben erste Hinweise auf eine Rolle der Cdc42/Rac1 Signaltransduktionswege und möglicherweise der Dynamik des Aktin-Zytoskeletts im Prozess des HSV-1 Eintritts

Virus-like particle size and molecular weight/mass determination applying gas-phase electrophoresis (native nES GEMMA)

(Bio-)nanoparticle analysis employing a nano-electrospray gas-phase electrophoretic mobility molecular analyzer (native nES GEMMA) also known as nES differential mobility analyzer (nES DMA) is based on surface-dry analyte separation at ambient pressure. Based on electrophoretic principles, single-charged nanoparticles are separated according to their electrophoretic mobility diameter (EMD) corresponding to the particle size for spherical analytes. Subsequently, it is possible to correlate the (bio-)nanoparticle EMDs to their molecular weight (MW) yielding a corresponding fitted curve for an investigated analyte class. Based on such a correlation, (bio-)nanoparticle MW determination via its EMD within one analyte class is possible. Turning our attention to icosahedral, non-enveloped virus-like particles (VLPs), proteinaceous shells, we set up an EMD/MW correlation. We employed native electrospray ionization mass spectrometry (native ESI MS) to obtain MW values of investigated analytes, where possible, after extensive purification. We experienced difficulties in native ESI MS with time-of-flight (ToF) detection to determine MW due to sample inherent characteristics, which was not the case for charge detection (CDMS). nES GEMMA exceeds CDMS in speed of analysis and is likewise less dependent on sample purity and homogeneity. Hence, gas-phase electrophoresis yields calculated MW values in good approximation even when charge resolution was not obtained in native ESI ToF MS. Therefore, both methods-native nES GEMMA-based MW determination via an analyte class inherent EMD/MW correlation and native ESI MS-in the end relate (bio-)nanoparticle MW values. However, they differ significantly in, e.g., ease of instrument operation, sample and analyte handling, or costs of instrumentation.Leibniz AssociationEU Horizon 2020Indiana University Graduate Training Program in Quantitative and Chemical Biolog

Entry of Human Papillomavirus Type 16 by Actin-Dependent, Clathrin- and Lipid Raft-Independent Endocytosis

Infectious endocytosis of incoming human papillomavirus type 16 (HPV-16), the main etiological agent of cervical cancer, is poorly characterized in terms of cellular requirements and pathways. Conflicting reports attribute HPV-16 entry to clathrin-dependent and -independent mechanisms. To comprehensively describe the cell biological features of HPV-16 entry into human epithelial cells, we compared HPV-16 pseudovirion (PsV) infection in the context of cell perturbations (drug inhibition, siRNA silencing, overexpression of dominant mutants) to five other viruses (influenza A virus, Semliki Forest virus, simian virus 40, vesicular stomatitis virus, and vaccinia virus) with defined endocytic requirements. Our analysis included infection data, i.e. GFP expression after plasmid delivery by HPV-16 PsV, and endocytosis assays in combination with electron, immunofluorescence, and video microscopy. The results indicated that HPV-16 entry into HeLa and HaCaT cells was clathrin-, caveolin-, cholesterol- and dynamin-independent. The virus made use of a potentially novel ligand-induced endocytic pathway related to macropinocytosis. This pathway was distinct from classical macropinocytosis in regards to vesicle size, cholesterol-sensitivity, and GTPase requirements, but similar in respect to the need for tyrosine kinase signaling, actin dynamics, Na+/H+ exchangers, PAK-1 and PKC. After internalization the virus was transported to late endosomes and/or endolysosomes, and activated through exposure to low pH

Human Papillomavirus Type 16 Entry: Retrograde Cell Surface Transport along Actin-Rich Protrusions

The lateral mobility of individual, incoming human papillomavirus type 16 pseudoviruses (PsV) bound to live HeLa cells was studied by single particle tracking using fluorescence video microscopy. The trajectories were computationally analyzed in terms of diffusion rate and mode of motion as described by the moment scaling spectrum. Four distinct modes of mobility were seen: confined movement in small zones (30–60 nm in diameter), confined movement with a slow drift, fast random motion with transient confinement, and linear, directed movement for long distances. The directed movement was most prominent on actin-rich cell protrusions such as filopodia or retraction fibres, where the rate was similar to that measured for actin retrograde flow. It was, moreover, sensitive to perturbants of actin retrograde flow such as cytochalasin D, jasplakinolide, and blebbistatin. We found that transport along actin protrusions significantly enhanced HPV-16 infection in sparse tissue culture, cells suggesting a role for in vivo infection of basal keratinocytes during wound healing

Actin dynamics in host-pathogen interaction.

The actin cytoskeleton and Rho GTPase signaling to actin assembly are prime targets of bacterial and viral pathogens, simply because actin is involved in all motile and membrane remodeling processes, such as phagocytosis, macropinocytosis, endocytosis, exocytosis, vesicular trafficking and membrane fusion events, motility, and last but not least, autophagy. This article aims at providing an overview of the most prominent pathogen-induced or -hijacked actin structures, and an outlook on how future research might uncover additional, equally sophisticated interactions

Glycans Controlling Virus Infections: Meeting Report on the 1st International Symposium on Glycovirology Schöntal, Germany, 02–04 May 2018

Glycans are, with nucleic acids, proteins and lipids, one of the four founding structures of cellular life. Due to their non-template synthesis, they are inherently heterogeneous and difficult to study with regards to their structure and function. Since 2016, the research group ViroCarb, funded by the German Research Foundation, has investigated the role of glycans in non-enveloped virus infections with a highly interdisciplinary approach. The core idea was to bring together scientists and students from various disciplines such as structural biology, cell biology, virology and chemistry to advance research by an interdisciplinary means. In 2018, ViroCarb hosted the 1st International Symposium on Glycovirology in Schöntal, Germany, with a similar aim. Scientists from various disciplines gathered to discuss their area of study, present recent findings, establish or strengthen collaborations, and mentor the next generation of glycovirologists through formal presentations and informal discussions. The secluded meeting at the monastery of Schöntal gave ample time for in-depth discussions. On behalf of ViroCarb, this report summarizes the reports and highlights advances in the field