ジャーナル: Nat Commun / 年: 2022 タイトル: Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription. 著者: Benoît Arragain / Quentin Durieux Trouilleton / Florence Baudin / Jan Provaznik / Nayara Azevedo / Stephen Cusack / Guy Schoehn / Hélène Malet / 要旨: Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse ...Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize its conformational dynamics by cryo-electron microscopy, unveiling the precise molecular mechanics underlying its essential activities. We find that replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that prompt primer cleavage prior to primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The uncovered detailed mechanics should be helpful for the future design of antivirals counteracting bunyaviral life threatening pathogens.
名称: RNA (5'-R(P*AP*CP*GP*AP*GP*UP*GP*UP*CP*GP*UP*AP*CP*C)-3') タイプ: rna / ID: 1 詳細: 5prime vRNA of La Crosse M segment. Mutation of nucleotides G2, U3, A9 and C10 into C2, G3, C9 and G10 コピー数: 1
凍結剤: ETHANE / チャンバー内湿度: 100 % / チャンバー内温度: 293 K / 装置: FEI VITROBOT MARK IV
詳細
1.3 uM LACV-LCItag_H34K were sequentially incubated for 1h at 4 degree with (i) 1.9 uM 5prime 1-17 BPm, (ii) 1.9 uM 3prime vRNA 1-25. LACV-LCItag_H34K bound to vRNAs was incubated with 100 uM ATP/GTP/UTP and 5mM MgCl2 for 4h at 30 degree