A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase

• Published in: Cell Vol. 181; no. 4; pp. 877 - 893.e21
• Main Authors: Wandzik, Joanna M., Kouba, Tomas, Karuppasamy, Manikandan, Pflug, Alexander, Drncova, Petra, Provaznik, Jan, Azevedo, Nayara, Cusack, Stephen
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 14.05.2020
• Subjects: cap-snatching; Catalytic Domain; Computer Simulation; cryo-electron microscopy; Cryoelectron Microscopy - methods; end binding site; Genome, Viral - genetics; Humans; Influenza A virus - genetics; Influenza A virus - metabolism; Influenza virus; Influenza, Human - genetics; Influenza, Human - virology; Nucleotidyltransferases - metabolism; poly-adenylation; RNA, Messenger - metabolism; RNA, Viral - metabolism; RNA-dependent RNA polymerase; RNA-Dependent RNA Polymerase - genetics; RNA-Dependent RNA Polymerase - metabolism; secondary 3; Structure-Activity Relationship; transcription; Transcription, Genetic - genetics; Virus Replication - genetics; end binding site; transcription; Influenza virus; poly-adenylation; RNA-dependent RNA polymerase; cap-snatching; secondary 3; cryo-electron microscopy
• ISSN: 0092-8674; 1097-4172
• DOI: 10.1016/j.cell.2020.03.061

Influenza polymerase uses unique mechanisms to synthesize capped and polyadenylated mRNAs from the genomic viral RNA (vRNA) template, which is packaged inside ribonucleoprotein particles (vRNPs). Here, we visualize by cryoelectron microscopy the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focusing on the template trajectory. After exiting the active site cavity, the template 3′ extremity rebinds into a specific site on the polymerase surface. Here, it remains sequestered during all subsequent transcription steps, forcing the template to loop out as it further translocates. At termination, the strained connection between the bound template 5′ end and the active site results in polyadenylation by stuttering at uridine 17. Upon product dissociation, further conformational changes release the trapped template, allowing recycling back into the pre-initiation state. Influenza polymerase thus performs transcription while tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single vRNP. [Display omitted] •Cryo-EM snapshots of the transcription elongation, termination, and recycling states•After being copied, the template 3′ end rebinds the polymerase in a secondary site•Mechanism of viral mRNA poly(A) tail formation by stuttering elucidated•Efficient reformation of the promoter allows multiple transcripts from one RNP Influenza polymerase transcribes the negative sense viral RNA genome into mRNA in the nucleus of infected cells. This work by Cusack and colleagues reports high-resolution cryo-EM structures of the polymerase at various stages of transcription providing a molecular basis for the complete transcription cycle, which should enable improved inhibitor design.