Strukturelle Analysen der Nukleokapside negativsträngiger RNA-Viren als Zielobjekte des antiviralen Dynamin-verwandten Myxovirus-Resistenzproteins A
Zusammenfassung der Projektergebnisse
Nucleoproteins (Ns/NPs) of negative strand RNA viruses encapsidate viral RNA in ribonucleoprotein complexes (RNPs) and assist the viral RNA dependent RNA polymerase in the transcription and replication of the viral genome. NPs also constitute the target of the interferon-induced antiviral Myxovirus resistance (Mx) proteins. These dynamin-like GTPases were suggested to oligomerize around the viral RNPs thereby interfering with viral replication. The molecular basis of how the divergent RNPs of negative-strand RNA viruses assemble around viral RNA and the molecular determinants for Mx activities on NPs are, however, not well understood. This project has aimed to address these open questions by using a structure-function approach. To this end, we determined N structures of two medically relevant negative-strand RNA bunyaviruses, e.g. Toscana virus (genus Phlebovirus) and Hantaan virus (genus Hantavirus) in the first funding periods. Based on a comprehensive biochemical and cell-based analysis, we proposed a molecular model of how RNA binding mediates the oligmerization of these Ns into a helical RNP. We also analyzed the mechanism of GTP hydrolysis in the MxA protein and the role of nucleotide binding and hydrolysis for its interaction with viral RNPs and cellular membranes. In the second funding period, we continued our analysis on MxA and characterized the functional consequences of allelic MxA variations to fight influenza virus infection. We determined the high resolution crystal structure of Thogoto virus NP, a model orthomyxovirus that is restricted in an Mx-dependent manner. Based on the structure, we characterized its assembly and RNA binding mode. Docking studies with an inhibitor library yielded a small molecule that interferes with THOV replication. Furthermore, initial cryoEM studies on the THOV NP suggested a similar arrangement of the helical RNP as seen in influenza RNPs. In this way, our studies shed new light on the mechanism of RNA encapsidation in negative-strand RNA viruses as a basis to understand the antiviral action of Mx proteins.
Projektbezogene Publikationen (Auswahl)
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Structural insights into RNA encapsidation and helical assembly of the Toscana virus nucleoprotein. Nucleic Acids Res. 42 (9): 6025-37 (2014)
Olal D., Dick A., Woods V.L. Jr, Liu T., Li S., Devignot S., Weber F., Saphire E.O., Daumke O.
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Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity. J Biol Chem. 290 (20):12779-92. (2015)
Dick A., Graf L., Olal D., von der Malsburg A., Gao S., Kochs G., Daumke O.
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Structure of the hantavirus nucleoprotein provides insights into the mechanism of RNA encapsidation. Cell Rep. (2016) 14(9):2092-2099
Olal D., Daumke O.
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Effects of allelic variations in the human myxovirus resistance protein A on its antiviral activity. J Biol Chem. (2018), 293(9):3056-3072
Graf L., Dick A., Sendker F., Barth E., Marz M., Daumke O., Kochs G.
