Structural Studies of RNA Packaging by Phlebovirus Nucleocapsid Proteins and RNA Capping by Flavivirus Methyltransferases.

RNA-binding Proteins;Rift Valley Fever Virus;Structural Virology;Biological Chemistry;Health Sciences;Biological Chemistry

Raymond, Donald DamianNinfa, Alexander J. ;

University of Michigan

关键词: RNA-binding Proteins;    Rift Valley Fever Virus;    Structural Virology;    Biological Chemistry;    Health Sciences;    Biological Chemistry;   

Others  :  https://deepblue.lib.umich.edu/bitstream/handle/2027.42/95947/donal_1.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Arboviruses are maintained in nature through a complex life cycle involving blood-sucking insects and susceptible vertebrate hosts. Arboviruses have evolved mechanisms to conceal their RNA genomes from the cellular antiviral response. In this study, I investigated genome packaging in phleboviruses and RNA capping in flaviviruses. Crystal structures of phlebovirus N-RNA complexes reveal a surprising diversity of binding geometries where variation in the RNA length and the number of nucleotides per N subunit gives rise to tetrameric, pentameric and hexameric structures. RVFV N binds RNA by sequestering all nucleotide bases in an RNA-binding slot with the sugar-phosphate backbone facing the solvent. The structures reveal multiple conformations of an N-terminal helical arm. The flexibility of the arm allows for the asymmetry of the N-RNA multimers and the asymmetric architecture of the RNP. The crystal structures reveal a common building block consisting of the core domain of an N subunit, four RNA nucleotides, and the helical arm of an adjacent subunit (RNA-Ncore-arm). Together with direct binding measurements showing that N binds nucleic acids with equal affinity regardless of length or sequence, these results provide a model for sequence-independent base sequestration by N, explain the observed structure of phlebovirus RNP, and elucidate how phlebovirus N protects the RNA genome from the cellular antiviral response. High resolution structures of Langat virus methyltransferase (LVMT) in apo form and in complex with GTP and GpppA cap analogues provide a detailed view of GTP and RNA cap binding to the MTase GTP-binding site and suggest possible interactions that confer guanine specificity. An RNA-cap bound structure provides the first view of an RNA cap fully extended in the active site cleft and shows the correct orientation of the RNA cap prior to ribose 2′-O methylation at the first nucleotide. Biochemical characterization of the LVMT guanylyltransferase activity using radiolabeled GTP showed the formation of the MTase-GMP adduct, which is required for the capping reaction. These results enhance our overall understanding of the cap formation process in flaviviruses.