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

Abstract

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 0 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 0 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. ll

Phosphorylation controls RNA binding and transcription by the influenza virus polymerase

“…B-C. Phospho-sites on PB1 surround the catalytic core and template entry. The location of PB1 phospho-sites characterized in this study are modeled in yellow on (B) a pre-initiation complex (PDB 4WSB [ 44 ]) or (C) a transcriptional elongation complex (PDB 6T0V [ 36 ]). The motif C residues D445/D446 in the catalytic site are in orange, 5’ vRNA is light blue, 3’ vRNA is magenta, and transcription product is dark blue.…”

“…The potential impact of phosphorylation at each site was proposed based on polymerase structures ( S1 Table ). In addition to evaluating conservation of identified phosphorylated residues, we calculated the surface accessibility of each residue in multiple polymerase states: the monomeric polymerase bound to either vRNA [ 34 ] or cRNA [ 21 ], dimerized polymerase bound to cRNA [ 35 ], and polymerase during transcription elongation[ 36 ] ( S2 Table ). Residues corresponding to WSN PB1 T223, T385, S478 and T570 were inaccessible in all conformations analyzed, PB1 S384 was accessible in all conformations, and S673 was buried in the vRNA-bound structure but may be accessible in the monomeric.…”

“…The different functions are achieved in part by discrete conformations of the viral polymerase [ 39 , 41 ]. All of these require appropriate binding and positioning of the template [ 21 , 34 , 36 , 42 ]. Two of our PB1 phospho-mutants may affect this process.…”

Combining high throughput and high quality for cryo-electron microscopy data collection

“…It is also of primary importance to carefully select the acquisition parameters (pixel size and defocus range) according to the expected/desired resolution in order to get the maximum out of the data-collection session. We now routinely collect SPA data sets using active beam-tilt compensation and fringe-free imaging at a pixel size of $0.8 Å (Wandzik et al, 2020;Qi, Di Minin et al, 2019;Qi, Sorrentino et al, 2019;Cannac et al, 2020;Flaugnatti et al, 2020;Zivanov et al, 2018;Muir et al, 2020;Oosterheert et al, 2018) or even smaller (Weis et al, 2019). The ability to collect more/better data in a shorter time allows increasingly dynamic, flexible and heterogeneous complexes to be imaged (time-resolved studies, mechanistic studies etc.…”

Local computational methods to improve the interpretability and analysis of cryo-EM maps

“…Cryo-electron microscopy (cryo-EM) has become a mainstream technique for determining the structures of macromolecular complexes at close-to-atomic resolution and ultimately for building an atomic model (Ge, Scholl et al 2020, Wandzik, Kouba et al 2020. With its unique ability to reconstruct multiple conformations and compositions of the macromolecular complexes, cryo-EM allows the understanding of the structural and assembly dynamics of macromolecular complexes in their native conditions (Davis, Tan et al 2016, Plaschka, Lin et al 2017, Razi, Davis et al 2019).…”