Structural and functional studies of negative-strand RNA virus nucleoproteins as target of the dynamin-like Myxovirus Resistance Protein A

• Applicant: Professor Dr. Oliver Daumke
• Subject Area: Structural Biology
• Term: from 2012 to 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 226824050

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 proposal aims to address these questions by using a structure-function approach. In the first funding period, we determined N structures of two medically relevant negative-strand RNA bunyaviruses, e.g. Toscana virus (genus Phlebovirus) and Hantaan virus (genus Hantavirus). Based on a comprehensive biochemical and cell-based analysis, we proposed a molecular model of how RNA binding mediates the transition of the planar hexameric N into a helical RNP. Additionally, we crystallized and collected diffraction data of the Thogoto virus NP, a model orthomyxovirus that is restricted in an Mx-dependent manner in host cells. Finally, we 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 coming funding period, we will continue our structural and functional investigations on viral NPs and their interactions with Mx proteins. In particular, we will address the molecular mechanisms of RNA binding for selected Ns/NPs using biochemical and structural studies. With Thogoto virus NP as a model, we will employ our structural information to characterize the molecular mechanisms of how MxA interacts with the viral machinery. Finally, we will identify cellular interaction partners of MxA and/or the NPs to understand how MxA exerts its antiviral function. Results of this work will shed new light on the mechanism of RNA encapsidation in negative-strand RNA viruses and will lead to molecular models of how Mx proteins can restrict a variety of negative-strand RNA viruses.

• DFG Programme: Research Grants