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It is unique among the animal viruses of this order with respect to its transcription and replication in the nucleus, which provides access to the splicing machinery. BDV is noncytolytic, highly neurotropic and causes diseases of the central nervous system (CNS) in a wide range of vertebrates. As in other Mononegavirales, the BDV polymerase complex or ribonucleoprotein complex, consists of the nucleoprotein N, the phosphoprotein P, the polymerase L and viral genomic RNA. In the case of BDV another protein is involved, termed protein X. BVD N forms a homotetramer and does not spontaneously interact with cellular RNA. Each protomer consists of two helical domains and N- and C-terminal extensions, involved in domain exchange and tetramer stabilization. An open question remained how BVD N interacts with RNA, although overall structural similarities with nucleoproteins from rhabdoviruses and vesiculoviruses suggested similar modes of RNA interaction. Protein P plays an essential role in assembly and regulation of the polymerase complex via interactions with X, N, L and itself. Oligomerization of P is required for the formation of an active polymerase complex, similar to other negative strand RNA polymerase complexes. P requires an intact C-terminus for N interaction and may contact two different sites on N. Phosphoproteins from Rhabdoviruses and Sendai virus contain two different binding sites for N, one to keep N soluble and free from unspecific RNA and the other to bind to N-RNA complexes forming the polymerase complex together with the polymerase L. However, BVD N does not require P binding to prevent non-specific RNA interaction, since BDV N oligomerizes spontaneously into tetramers that do not complex RNA, thus the precise role of N-P interaction in the absence of RNA is not known. The aim of our study was to understand the interaction between the BDV nucleo- and the phosphoprotein as well as the nucleoprotein and the viral RNA. Even though, no conclusive data were obtained upon crystallographic approaches, concerning N in complex with different truncated P-constructs and BDV genomic RNA, we present data about N-P and N-RNA interactions. I show that P’, an N-terminally truncated isoform of the phosphoprotein, present in BDV infected cells, oligomerizes into tetramers. The tetrameric P’ interacts with BDV-N, thus forming hetero-octamers. The P’-N interaction requires five C-terminal amino acids of P’ to form a stable complex with a kD of 1.66 μM. Tetrameric N is destabilized in the presence of 5’ genomic BDV RNA, which leads to the formation of N-RNA polymers. Similar N-RNA polymers are formed in the presence of P’, leading to P’-N-RNA polymers. Electron microscopy analyses of N-RNA and N-P’-RNA complexes revealed large “open” ring-like and string-like assemblies with the RNA exposed and accessible for degradation. The N or N-P polymers remain intact after RNA degradation indicating that polymerization is not mainly stabilized by RNA interaction. The N-RNA interaction is mediated via recognition of basic residues within the cleft of the N-and C-terminal domains similar to the observed nucleoprotein-RNA recognition of other negative strand-RNA viruses. In conclusion, these data provide insight on the molecular interactions between the viral RNA and the nucleo- and phosphoprotein of the BDV ribonucleoprotein complex. 2009-12-18 English Virology. 2009 Nov 27. [Epub ahead of print] RNS-Viren Borna Disease Virus (BDV) ist ein Vertreter der Bornaviridae in der Ordnung Mononegavirales (MNV). Unter denjenigen Viren dieser Ordnung, die Tiere infizieren, ist es bezüglich seiner Replikation und Transkription im Nukleus, einzigartig. BDV ist nicht zytolytisch, strikt neurotrop und verursacht Erkrankungen des zentralen Nervensystems (ZNS) bei einer großen Anzahl von Vertebraten, insbesondere beim Pferd. Der aktive BDV Polymerase Komplex besteht wie bei allen MNVs, aus dem Nukleoprotein N, dem Phosphoprotein P und der Polymerase L. Bei BDV ist daran außerdem noch das Protein X beteiligt. BVD N bildet Homotetramere und assoziiert nicht, wie im Gegensatz zu Nukleoproteinen anderer MNVs, mit zellulärer RNA. Jedes N Protomer besteht aus zwei helikalen Domänen und kurzen N- und C—terminalen Fortsätzen, mit deren Hilfe das N Tetramer stabilisiert wird. Es war jedoch nicht klar, wie BDV N mit der viralen RNA interagiert, obwohl die starke strukturelle Ähnlichkeit mit den Nukleoproteinen der Rhabdoviren auf vergleichbare RNA Interaktions-Modi hinwiesen. BDV-P spielt durch Interaktionen mit X, N, L und sich selbst eine essentielle Rolle beim Aufbau und der Regulierung des Polymerase-Komplexes, wobei die Oligomerisierung ähnlich wie bei anderen MNVs, für die Bildung eines aktiven Polymerase-Komplexes notwendig ist. P benötigt einen intakten C-terminus zur Interaktion mit dem Nukleoprotein N und kontaktiert möglicherweise zwei unterschiedliche Stellen auf N. Phosphoproteine von Rhabdoviren und Sendai Virus enthalten jeweils zwei unterschiedliche Bindestellen für N. Über die eine wird die Bindung des Nukleoproteins an unspezifische RNA verhindert, über die andere binden die Phosphoproteine an N-RNA Komplexe und vermitteln so die Ausbildung eines aktiven Polymerase Komplexes. Interessanterweise benötigt das Nukleoprotein von BDV das Phosphoprotein nicht, um die Interaktion mit unspezifischer RNA zu verhindern, da das Nukleoprotein spontan Tetramere ausbildet, ohne dabei RNA zu komplexieren, was eine Ausnahme unter den Mononegavirales darstellt. Das Ziel meiner Untersuchungen war es, die Wechselwirkungen zwischen dem Nukleo- und dem Phosphoprotein, und dem Nukleoprotein und der viralen RNA mithilfe von biochemischen, biophysikalischen und strukturaufklärenden Methoden aufzuklären. Obwohl es nicht gelang, röntgenkristallographische Daten, weder von N-P, noch N-RNA Komplexen zu erhalten, konnte gezeigt werden dass P‘, eine N-terminal verkürzte und in BDV infizierten Zellen vorkommende Isoform des Phosphoproteins, zu Tetrameren oligomerisiert. Es interagiert mit N und formt mit diesem Heterooktamere, wobei die letzten 5 C-terminalen Aminosäurereste zur stabilen Komplexbildung benötigt werden. Das tetramerische Nukleoprotein wird in Anwesenheit von BDV genomischer 5’ RNA destabilisiert, was zu N-RNA Polymeren führt. Solche N-RNA Polymere, werden auch in Anwesenheit von P‘ gebildet. Elektronenmikroskopische Analysen der N-RNA und N-P‘-RNA Komplexe zeigen große “offene” Ring- und Stäbchenartige Strukturen. Die RNA innerhalb dieser Strukturen bleibt dabei ungeschützt und zugänglich für RNase. Beim enzymatischen Abbau der RNA bleiben die N oder N-P‘ Polymere jedoch intakt, was die Vermutung zulässt, dass die Polymere nicht alleine durch die RNA stabilisiert werden. Interaktionen zwischen N und der viralen RNA werden durch Erkennung basischer Aminosäurereste im Inneren einer Spalte im Nukleoprotein vermittelt. Publikationsserver der Universitätsbibliothek Marburg Universitätsbibliothek Marburg genomische Einzelstrang-RNA opus:2557 2009 https://archiv.ub.uni-marburg.de/diss/z2010/0095/cover.png ppn:225850834 Medizin Nukleoprotein doctoralThesis 2022-03-24 Borna Disease Virus https://doi.org/10.17192/z2010.0095 Nucleoprotein genomic ss-RNA ths Prof. Dr. Lingelbach Klaus Lingelbach, Klaus (Prof. Dr.) Hygiene u. Med. Mikrobiologie mit Medizinaluntersuchungsamt Phosphoprotein Phosphoprotein 115 application/pdf