urn:nbn:de:hebis:04-z2015-01592 ths Prof. Dr. Weber Friedemann Weber, Friedemann (Prof. Dr.) angeborenes Immunsystem Virus-Wirt-InteraktionFrühe Virusdetektion durch RIG-I und PKR und virale Gegenmaßnahmen Immunsystem Viruses are a constant threat to mankind causing diseases ranging from mild symptoms to fatal outcome. A rapid and efficient antiviral response is therefore crucial for the survival of the host. RIG-I-like receptors (RLR) and other immune receptors, like protein kinase R (PKR), specifically detect viral RNA species in the host cytoplasm. The sensing of virus infection triggers intracellular defense mechanisms resulting in viral alertness in the infected and surrounding cells, and forms the link to the adaptive immune system. Viruses, in turn, have evolved sophisticated countermeasures to dampen the antiviral response. The molecular mechanisms involved range from a broad shut-off of the host cell metabolism to a sensitive interference with key components of the immune system. For a better understanding, which viral RNA structures are detected by immune receptors like RIG-I and PKR and what kind of viral antagonists lead to their inhibition, it is crucial to be able to determine their activation status. Hence, limited protease digestion and native polyacrylamide gel electrophoresis (PAGE) were established to directly monitor RIG-I and PKR conformational switching and oligomerization upon activation, respectively. Various studies helped to identify RIG-I stimulating RNA structures in vitro, but the first viral structure triggering an antiviral interferon response in the natural context of virus infection remained to be resolved. We identified 5triphosphorylated (5ppp) panhandle structures packaged into nucleocapsids as physiological RIG-I agonists. Independent of virus transcription and replication, the incoming encapsidated genomes of bunyaviruses (La Crosse virus; LACV and Rift Valley fever virus; RVFV) and orthomyxovirus (influenza A virus; FLUAV) were able to stimulate RIG-I activation and an antiviral signaling cascade. Surprisingly, antiviral activity of RIG-I against FLUAV was already promoted by binding to the 5ppp panhandle and was independent of RIG-I downstream signaling ability. In addition to RIG-I, we also identified PKR as an immune sensor of incoming nucleocapsids. PKR thereby interacts with the intergenic region (IGR) of viral genome segments using ambisense coding strategy. Association of PKR with the IGR of incoming RVFV (Bunyaviridae) and arenavirus nucleocapsids promotes PKR phosphorylation and conformational switching and hence full PKR activation. To antagonize immediate recognition by RIG-I and PKR, viruses need to adapt. RIG-I activation by FLUAV nucleocapsids was altered by an adaptive mutation of the polymerase complex subunit PB2. Mammalian adaptation mutation PB2 E627K stabilizes the polymerase complex association with the nucleocapsid thereby preventing RIG-I recognition of the 5ppp panhandle. Additionally, Lassa virus (Arenaviridae) nucleoprotein interacts with PKR and promotes its degradation via the proteasomal pathway. Therefore, we identified entry of viral nucleocapsids as the first time-point of immune recognition in the natural context of virus infection and give further insights how viruses have evolved to counteract immediate recognition. Gerlach, Michaela Gerlach Michaela doctoralThesis Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg application/pdf RIG-I Medical sciences Medicine Medizin 2015-09-17 Virus-host interplay-Immediate virus recognition by RIG-I and PKR and viral counterstrategies English Medizin 2015-03-04 2015 RIG-I like receptors ppn:364106301 Philipps-Universität Marburg RIG-I Medizin https://archiv.ub.uni-marburg.de/diss/z2015/0159/cover.png https://doi.org/10.17192/z2015.0159 opus:6014 Viren stellen eine ständige Bedrohung der Menschheit dar, die Krankheiten mit milden Symptomen bis hin zu letalem Ausgang verursachen. Für das Überleben des Wirts ist daher eine schnelle und effiziente antivirale Immunantwort von entscheidender Bedeutung. Die meisten der bekannten neu auftretenden und hochpathogenen Viren besitzen ein RNA-Genom. RIG-I ähnliche Rezeptoren (RLR; RIG-I like receptors) und andere Immunrezeptoren, wie die Proteinkinase R (PKR), reagieren auf RNA-Strukturen im Zytoplasma der Wirtszelle. Die Detektion von Virusinfektionen induziert intrazelluläre Abwehrmechanismen, die einen antiviralen Zustand in der infizierten und den Nachbarzellen vermittelt und zudem das adaptive Immunsystem aktiviert. Viren wiederum haben komplexe Abwehrmaßnahmen entwickelt um die Immunreaktion zu verhindern. Die molekularen Mechanismen reichen vom unspezifischen Eingreifen in den Wirtszellmetabolismus bis hin zu einer spezifischen Inhibition von Schlüsselfaktoren der Immunantwort. Für ein besseres Verständnis welche viralen RNA-Strukturen durch Immunrezeptoren, wie RIG-I und PKR, detektiert werden und welche Art von viralen Antagonisten zu ihrer Inhibition führen, muss man den Aktivierungszustand von Immunrezeptoren genau bestimmen können. Hierfür wurde der limitierte Proteaseverdau und die native Polyacrylamid-Gelelektrophorese (PAGE), für einen direkten Nachweis der RIG-I und PKR Konformationsänderung beziehungsweise Oligomerisierung nach Aktivierung, etabliert. Verschiedene Studien haben geholfen, RIG-I stimulierende RNA-Strukturen in vitro zu identifizieren. Die erste Virusstruktur, welche die initiale antivirale Immunreaktion im natürlichen Kontext der Virusinfektion auslösen kann blieb jedoch ungeklärt. Im Rahmen dieser Studie konnten wir die 5 triphosphorylierte (5ppp) Pfannenstielstruktur viraler Nukleokapside als physiologischen RIG-I Agonisten identifizieren. Unabhängig von viraler Transkription und Replikation konnten die eintretenden enkapsidierten Genome von Bunyaviren (La Crosse Virus; LACV und Rift Valley Fieber Virus; RVFV) und Orthomyxoviren (Influenza A Viren; FLUAV) RIG-I Aktivierung und eine antivirale Signalkaskade stimulieren. Überraschenderweise wurde die antivirale Aktivität von RIG-I gegen FLUAV bereits durch die Bindung an die 5ppp Nukleokapside vermittelt, und war unabhängig von der RIG-I vermittelten Signalweiterleitung. Neben RIG-I konnte auch PKR als Immunsensor eintretender Nukleokapside identifiziert werden. PKR interagiert dabei mit der intergenischen Region (IGR; intergenic region) der viralen Genomsegmente mit Ambisense-Kodierungsstrategie. Die Assoziation von PKR mit der IGR eintretender RVFV (Bunyaviridae) und Arenavirus Nukleokapside vermittelt PKR-Phosphorylierung und Konformationsänderung und damit volle PKR Aktivierung. Um einer unmittelbaren Detektion durch RIG-I und PKR zu entgehen, müssen sich Viren anpassen. So wird die RIG-I Aktivierung durch FLUAV Nukleokapside durch eine adaptive Mutation der PB2-Polymeraseuntereinheit verändert. Die Adaptionsmutation PB2 E627K stabilisiert die Interaktion des FLUAV Polymerasekomplexes mit dem Nukleokapsid und verhindert dadurch die RIG-I vermittelte Detektion. Zudem interagiert das Lassa Virus (Arenaviridae) Nukleoprotein mit PKR und induziert dessen Abbau über das Proteasom. Somit konnte das Eintreten der viralen Nukleokapside in die Wirtszelle als erster Zeitpunkt der Immunerkennung im natürlichen Kontext der Virusinfektion nachgewiesen werden. Des Weiteren ergeben sich aus dieser Arbeit Einblicke, wie Viren sich entwickelt haben um dieser unmittelbaren Immundetektion zu entkommen. monograph RIG-I-ähnliche Rezeptoren Strahle L, Garcin D, Kolakofsky D. Sendai virus defective-interfering genomes and the activation of interferon-beta. Virology 2006;351:101–11. Wagstaff KM, Sivakumaran H, Heaton SM, Harrich D, Jans DA (2012) Ivermectin is a specific inhibitor of importin alpha/beta-mediated nuclear import able to inhibit replication of HIV-1 and dengue virus. 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