Cryo-electron microscopic structure of the nucleoprotein-RNA complex of the European filovirus, Lloviu virus

ヨーロッパに分布するエボラウイルス近縁ウイルスの増殖機構を解明 --広範囲の抗フィロウイルス療法の開発に期待--. 京都大学プレスリリース. 2023-04-10.Lloviu virus (LLOV) is a novel filovirus detected in Schreiber’s bats in Europe. The isolation of the infectious LLOV from bats has raised public health concerns. However, the virological and molecular characteristics of LLOV remain largely unknown. The nucleoprotein (NP) of LLOV encapsidates the viral genomic RNA to form a helical NP-RNA complex, which acts as a scaffold for nucleocapsid formation and de novo viral RNA synthesis. In this study, using single-particle cryo-electron microscopy, we determined two structures of the LLOV NP-RNA helical complex, comprising a full-length and a C-terminally truncated NP. The two helical structures were identical, demonstrating that the N-terminal region determines the helical arrangement of the NP. The LLOV NP-RNA protomers displayed a structure similar to that in the Ebola and Marburg virus, but the spatial arrangements in the helix differed. Structure-based mutational analysis identified amino acids involved in the helical assembly and viral RNA synthesis. These structures advance our understanding of the filovirus nucleocapsid formation, and provide a structural basis for the development of anti-filoviral therapeutics

Structural insights into the agonists binding and receptor selectivity of human histamine H₄ receptor

慢性アレルギー疾患に関わるヒスタミン受容体の構造解明 --新規アトピー性皮膚炎・喘息治療薬の開発に貢献--. 京都大学プレスリリース. 2023-10-23.Histamine is a biogenic amine that participates in allergic and inflammatory processes by stimulating histamine receptors. The histamine H₄ receptor (H₄R) is a potential therapeutic target for chronic inflammatory diseases such as asthma and atopic dermatitis. Here, we show the cryo-electron microscopy structures of the H₄R-Gq complex bound with an endogenous agonist histamine or the selective agonist imetit bound in the orthosteric binding pocket. The structures demonstrate binding mode of histamine agonists and that the subtype-selective agonist binding causes conformational changes in Phe344[7.39], which, in turn, form the “aromatic slot”. The results provide insights into the molecular underpinnings of the agonism of H₄R and subtype selectivity of histamine receptors, and show that the H₄R structures may be valuable in rational drug design of drugs targeting the H₄R

Migration of Influenza Virus Nucleoprotein into the Nucleolus Is Essential for Ribonucleoprotein Complex Formation

Influenza A virus double-helical ribonucleoprotein complex (RNP) performs transcription and replication of viral genomic RNA (vRNA). Although RNP formation occurs in the nuclei of virus-infected cells, the nuclear domains involved in this process remain unclear. Here, we show that the nucleolus is an essential site for functional RNP formation. Viral nucleoprotein (NP), a major RNP component, temporarily localized to the nucleoli of virus-infected cells. Mutations in a nucleolar localization signal (NoLS) on NP abolished double-helical RNP formation, resulting in a loss of viral RNA synthesis ability, whereas ectopic fusion of the NoLS enabled the NP mutant to form functional double-helical RNPs. Furthermore, nucleolar disruption of virus-infected cells inhibited NP assembly into double-helical RNPs, resulting in decreased viral RNA synthesis. Collectively, our findings demonstrate that NP migration into the nucleolus is a critical step for functional RNP formation, showing the importance of the nucleolus in the influenza virus life cycle

Structural insight into Marburg virus nucleoprotein–RNA complex formation

致死的な出血熱を引き起こすマールブルグウイルスの増殖機構を解明 --エボラ・マールブルグウイルスの創薬に期待--. 京都大学プレスリリース. 2022-03-07.Viruses of a feather: Similar structures in Marburg and Ebola viruses provide clues for antivirals. 京都大学プレスリリース. 2022-07-25.The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP–RNA complex. The NP–RNA complex constitutes the core structure for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP–RNA complex determined using cryo-electron microscopy at a resolution of 3.1 Å. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for helical assembly and subsequent viral RNA synthesis. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV