Das Cytomegalievirus IE1-Protein als Regulator des humanen Transkriptoms und Zielstruktur RNAi-basierter Therapiestrategien

Das humane Cytomegalievirus (hCMV) ist ein medizinisch hoch relevantes Herpesvirus, das bei Menschen mit geschwächtem Immunsystem (z.B. AIDS- und Transplantationspatienten) lebensbedrohliche Krankheiten auslösen kann und zudem die wichtigste infektiöse Ursache für embryonale Defekte ist. Bisher steht jedoch kein wirksamer hCMV-Impfstoff zur Verfügung und das Spektrum geeigneter Therapeutika ist äußerst begrenzt. Das immediate-early 1 Protein (IE1) ist ein viraler Schlüsselregulator, der Proteine und übergeordnete Strukturen des infizierten Wirtszellkerns in vielfältiger Weise manipuliert und in der initialen Phase der Infektion in entscheidender Weise den weiteren Verlauf der hCMV-Replikation und Pathogenese bestimmt. Ein Ziel dieser Arbeit war es, das Potential der RNA-Interferenz-Technologie als neues Therapiekonzept gegen hCMV zu untersuchen. Hierfür wurde ein Luciferase-Reportervirus hergestellt, welches das schnelle Screening antiviraler Effekte ermöglicht. Aus insgesamt 40 getesteten hCMV-gerichteten siRNAs konnten acht Moleküle identifiziert werden, die eine spezifische Inhibition der Virusvermehrung um mehr als 90% bewirkten. Die effektivsten Kandidaten wurden in Richtung einer therapeutischen Nutzung optimiert. In diesem Zusammenhang wurden IE1- und/oder IE2-spezifische siRNAs mit Zielsequenzen, die innerhalb aller annotierten hCMV-Stämme konserviert vorliegen und auch gegen klinisch relevante hCMV-Isolate antiviral aktiv waren, ausgewählt und chemisch modifiziert. Im zweiten Teil der vorliegenden Arbeit wurde der Einfluss von IE1 auf das humane Transkriptom untersucht. Dazu wurde mit einem im Rahmen dieser Arbeit etablierten induzierbaren Expressionssystem, welches die Analyse von funktionellem IE1 außerhalb der komplexen Situation einer Virusinfektion ermöglicht, eine systematische Genexpressions-Analyse durchgeführt. Dadurch konnten 25 IE1-induzierbare humane Gene identifiziert werden, die zugleich überwiegend in Zusammenhang mit der zellulären Typ II Interferon (IFN)-Antwort stehen. Mit Hilfe verschiedener Techniken wie der quantitativen reversen Transkriptase-PCR, Immunfluoreszenz-Analysen, zellulären Fraktionierungen, Chromatin-Immunpräzipitationen sowie der Herstellung transgener Zellen, konnte ein zweiphasiger Mechanismus der IE1-vermittelten Genregulation mit Beteiligung löslicher Faktoren ermittelt werden. Des Weiteren wurde gezeigt, dass die IE1-abhängige Induktion der normalerweise durch Typ II IFN regulierten Gene interessanterweise IFN-unabhängig erfolgt. Sie ist aber dennoch abhängig vom aktivierten (Y701-phosphorylierten) signal transducer and activator of transcription 1 (STAT1), einem kritischen zellulären Mediator der IFN-Antwort. In Korrelation dazu konnte eine IE1-vermittelte nukleäre Akkumulation sowie eine spezifische Assoziation des STAT1-Proteins mit den Promotoren Typ II IFN-stimulierter Gene beobachtet werden. Aufgrund der gewonnenen Daten ließ sich schließlich ein vorläufiges Modell für die IE1-induzierte Typ II IFN-artige Antwort entwickeln. Die Ergebnisse dieser Arbeit identifizieren IE1 als vielversprechende neue Zielstruktur für die hCMV-Therapie und offenbaren eine bisher noch unbekannte Funktion des viralen Proteins, die im Zusammenhang mit der hCMV-vermittelten Pathogenese oder der Virusreaktivierung stehen könnte

Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNγ-like response

MN and CP were supported by the Wellcome Trust (www.wellcome.ac.uk) Institutional Strategic Support Fund and CP was supported by the Deutsche Forschungsgemeinschaft (PA 815/2-1; www.dfg.de).The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication.Publisher PDFPeer reviewe

Human Cytomegalovirus IE1 Protein Elicits a Type II Interferon-Like Host Cell Response That Depends on Activated STAT1 but Not Interferon-γ

Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity

Brackish water algal reefs – facies analysis as a tool to identify palaeoenvironmental variations in Miocene deposits (Mainz‐Weisenau, Germany)

Brackish‐water carbonates are far less studied than their marine or limnic counterparts. However, their association with few, specialized species enables the documentation of fine‐scale changes in the depositional environment. The Cenozoic Mainz Basin (Germany) was only sporadically connected to the North Sea and the Paratethys, exposing several transitions from marine to fresh water influence. Focusing on one outcrop of the Rüssingen Formation of Mainz‐Weisenau (Aquitanian, Miocene), we present a detailed analysis of the faunal and sedimentological responses to changing salinities and water depth, including algal reef growth and facies development. The deposits include allochthonous limestones surrounding an autochthonous reef complex and several smaller reef patches. The allochthonous facies is dominated by the gastropod Hydrobia inflata, and the reef facies is mainly made up by the green alga Cladophorites sp. The algal thalli are overgrown by cryptocrystalline, organic precipitations, and laminated, chemical precipitations. Locally, quiver‐shaped structures of Trichoptera sp. protective cases occur. The depositional setting was a shallow, low energy, and brackish environment supersaturated by carbonate. We could not confirm a general trend of reducing salinities as reported for the Rüssingen Formation. Our results question previously reported episodic desiccation events, because apparent caliche horizons actually represent thin beds of increased Cladophorites growth. Set‐up, distribution of the reef facies, and reef debris indicate short‐time variations of temperature, salinity and water depth. We conclude that these variations are based on the geographic position at the edge of an algal reef barrier, separating the Mainz Basin from the Rhine Rift Valley.This study investigates the record of small‐scale changes in the depositional environment of Miocene brackish‐water deposits from the Mainz Basin (Germany) by facies analysis. Set‐up, distribution of the reef facies, and reef debris indicate short‐time variations of temperature, salinity and water depth. The apparent caliche horizons actually represent thin beds of increase Cladophorites growth. The example demonstrates that these brackish deposits are a sensitive recorder of palaeoenvironmental change.Open access funding enabled and organized by Projekt DEAL. WOA Institution: FRIEDRICH‐ALEXANDER‐UNIVERSITAET ERLANGEN‐NURNBERG Blended DEAL: ProjektDEA

Human cytomegalovirus IE1 protein elicits a type II interferon-like host cell response that depends on activated STAT1 but not interferon-γ

Human cytomegalovirus (hCMV) is a highly prevalent pathogen that, upon primary infection, establishes life-long persistence in all infected individuals. Acute hCMV infections cause a variety of diseases in humans with developmental or acquired immune deficits. In addition, persistent hCMV infection may contribute to various chronic disease conditions even in immunologically normal people. The pathogenesis of hCMV disease has been frequently linked to inflammatory host immune responses triggered by virus-infected cells. Moreover, hCMV infection activates numerous host genes many of which encode pro-inflammatory proteins. However, little is known about the relative contributions of individual viral gene products to these changes in cellular transcription. We systematically analyzed the effects of the hCMV 72-kDa immediate-early 1 (IE1) protein, a major transcriptional activator and antagonist of type I interferon (IFN) signaling, on the human transcriptome. Following expression under conditions closely mimicking the situation during productive infection, IE1 elicits a global type II IFN-like host cell response. This response is dominated by the selective up-regulation of immune stimulatory genes normally controlled by IFN-γ and includes the synthesis and secretion of pro-inflammatory chemokines. IE1-mediated induction of IFN-stimulated genes strictly depends on tyrosine-phosphorylated signal transducer and activator of transcription 1 (STAT1) and correlates with the nuclear accumulation and sequence-specific binding of STAT1 to IFN-γ-responsive promoters. However, neither synthesis nor secretion of IFN-γ or other IFNs seems to be required for the IE1-dependent effects on cellular gene expression. Our results demonstrate that a single hCMV protein can trigger a pro-inflammatory host transcriptional response via an unexpected STAT1-dependent but IFN-independent mechanism and identify IE1 as a candidate determinant of hCMV pathogenicity