Molecular mechanism of transcription by influenza virus polymerase – FluTranscript

Structural biology methods including production of recombinant polymerase and reconstitution with model viral RNAs, biochemistry, biophysics, structure determination by X-ray crystallography and cryo-electron microscopy. Molecular virology in cells using the influenza minigenome system or viruses produced by reverse genetics with wild type or mutated viruses. Proteomics methods to identify host proteins involved in viral transcription.

The major result of the FluTranscript project to date is the elucidation of the detailed mechanism of transcription by influenza virus polymerase. Using high resolution cryo-electron-microscopy we have visualised the conformational dynamics of the polymerase during the complete transcription cycle from pre-initiation to termination, focussing on the template trajectory. After exiting the active site cavity after being copied, 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 whilst tightly binding to and protecting both template ends, allowing efficient production of multiple mRNAs from a single RNP.

Ongoing work aims to elucidate how influenza polymerase interacts with host polymerase II during cap-snatching at the beginning of transcriptiion
and to identify the composition of viral mRNPs.

1. A Structure-Based Model for the Complete Transcription Cycle of Influenza Polymerase. Wandzik JM, Kouba T, Karuppasamy M, Pflug A, Drncova P, Provaznik J, Azevedo N, Cusack S. Cell. 2020, 181(4):877-893.e21. PMID: 32304664.
2. Structural snapshots of actively transcribing influenza polymerase. Kouba T, Drncová P, Cusack S. Nat Struct Mol Biol. 2019, 26(6):460-470. PMID:31160782
3. Structure and Function of Influenza Polymerase. Wandzik JM, Kouba T, Cusack S. Cold Spring Harb Perspect Med. 2020 Apr 27:a038372. doi: 10.1101/cshperspect.a038372. Online ahead of print. PMID: 32341065
4. Influenza Virus RNA-Dependent RNA Polymerase and the Host Transcriptional Apparatus. Tim Krischuns, Maria Lukarska, Nadia Naffakh and Stephen Cusack. Annual Review of Biochemistry 2021:90 (In press).
5. Patent application: NUCLEIC ACID CONSTRUCT BINDING TO INFLUENZA POLYMERASE PB1 RNA SYNTHESIS ACTIVE SITE. Publication number: WO/2020/239822. Publication date: 03.12.2020.

Influenza virus poses a serious threat to global public health. Development of improved influenza prevention and therapy, whether it be the universal vaccine or novel anti-influenza drugs, will depend strongly on fundamental science enhancing our understanding of the molecular mechanisms involved in viral replication and transmission. In FluTranscript, we focus on the unique mechanism by which the influenza virus RNA-dependent RNA polymerase (FluPol) synthesises viral mRNAs in the nucleus of the infected cell. FluPol binds directly to cellular RNA polymerase II (Pol II) enabling it to pirate nascent capped transcripts as primers for viral mRNA synthesis, a process known as ‘cap-snatching’. A poly(A) tail is added to the viral transcript by stuttering of FluPol on a 5' proximal oligo-U motif at the end of the genomic template, thus bypassing the cellular machinery. The goal of the FluTranscript project is to provide a detailed, mechanistic description of viral transcription in vitro and in the cellular context. This will be achieved by combining the complementary expertise in structural biology and molecular virology of the two partners, who are both leaders in the field of influenza research. Using X-ray crystallography and state-of-the-art single particle cryo-electron microscopy we will determine structural snapshots of FluPol performing key steps in transcription including initiation, elongation and termination. We will also determine structures in which chain perturbing RNA synthesis inhibitors, such as nucleoside analogues, are bound in the FluPol active site, thus providing valuable data for optimising design of novel anti-viral molecules. To complement these in vitro studies, we aim to understand how FluPol interacts with Pol II and other transcription related factors to gain access to capped Pol II transcripts in the cellular context. We will purify ‘cap-snatching’ complexes from infected cells to identify the factors required and use this data to reconstitute the coupled Pol II-FluPol two polymerase complex for structural studies by cryo-electron microscopy. Finally, using RNA-protein cross-linking and proteomics methods, we will characterise the viral mRNA interactome, to determine how the particular process of viral mRNA synthesis impacts the RNA-binding protein composition of viral messenger ribonucleoproteins (mRNPs). Cross-validation between the in vitro structural and cellular virological data will be a critical part of the project. The expected outcomes of FluTranscript will be new insight into the functioning of the influenza replication/transcription machine and new opportunities for development of next-generation anti-influenza drugs.