Influenza RNP · A structural and functional understanding Influenza virus mRNA production in the context of the transcriptionally functional ribonucleoprotein (RNP) particle.
Horizon 2020 — Marie Skłodowska-Curie Actions
- Duration
- 2021-07-01 → 2023-06-30
- EU contribution
- €184,708
- Participants
- 1
- Scheme
- MSCA-IF
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Results in brief
A structural and functional understanding Influenza virus mRNA production in the context of the transcriptionally functional ribonucleoprotein (RNP) particle.
Seasonal disease caused by influenza virus is a major cause of morbidity and mortality worldwide. Additionally, the threat of pandemic potential influenza emerging remains present, especially as Europe continues to grappling with major outbreaks in wild bird populations and poultry. In order to design new influenza anti-viral treatments, it is necessary to understand the molecular biology of influenza virus replication. The influenza genome is split across eight segments which are packaged as viral ribonucleoprotein particles (vRNPs). Each vRNP consists of influenza virus RNA bound by multiple copies of the nucleoprotein and the polymerase, the enzyme responsible for copying the RNA strand. Upon infection of a host cell, the vRNPs are delivered to the host cell nucleus. Once there the polymerase of the vRNP enacts the process of messenger RNA (mRNA) synthesis, otherwise known as transcription, using the viral RNA (vRNA) as a template. The mRNA is then used by the host cell to produce more viral protein, which are used during influenza virus replication to produce further copies of the viral genome in the form of eight unique vRNPs. In this action, the aim was to understand the molecular organisation of the influenza virus replication machinery, termed the viral ribonucleoprotein (vRNP), during the process of messenger RNA (mRNA) production. A structural understanding of this dynamic protein/RNA complex could inform potential drug design approaches, and also provide a methodology to study other aspects of the influenza lifecycle involving the vRNP. In order to study the vRNP it was necessary to generate a homogenous and short vRNP sample, with an editable vRNA, termed the sh-vRNP. Typically, the vRNP is a long and flexible molecule which makes it hard to study structurally. In order to aid characterisation using cryo-electron microscopy (cryo-EM), a shorter sh-vRNP was generated. To study the dynamic process of transcription it was necessary to produce molecules that have identical and editable RNA sequences, that can be stalled at specified points of RNA synthesis. Unfortunately, due to issues relating to the production of sh-vRNP, the project was delayed. Sh-vRNP can now be successfully produced and studies of the structural and functional characteristics of this molecule are in the preliminary stage. Research utilising this tool will be continued beyond the end of this action. In addition to aiding research concerning influenza transcription, the sh-vRNP tool could be useful for future studies of the influenza vRNP during different stages of the virus lifecycle. Furthermore, this methodology could be applied to study similar viruses. A manuscript detailing the production of the sh-vRNP and subsequent preliminary structural analyses is being produced.
Data: CORDIS, © European Union
Project objective
Influenza is a major public health burden, with seasonal outbreaks contributing significantly to mortality worldwide, and the emergence of pandemic strains remaining an ever-present threat. Influenza drug and vaccine conception efforts are aided by a thorough understanding of its molecular biology.A key aspect of the influenza lifecycle is the production of capped and poly-adenylated messenger RNA by the heterotrimeric influenza polymerase (FluPol). Ground-breaking work performed by the Cusack lab, has described with residue-resolution detail, the FluPol structures that form during transcription of short, non-nucleoprotein (NP) bound viral RNAs (vRNAs). However, influenza transcription in vivo occurs within the ribonucleoprotein (RNP) particle and does not utilise naked genome segments. The viral RNP (vRNP) is a super-helical complex composed FluPol bound at the conserved 3′ and 5′ ends of a vRNA, which is coated with NP. The current low-resolution structures provide little information about the molecular details of vRNP function, particularly, how NPs interact with FluPol and the vRNA template.Via an inter-disciplinary approach, I will utilise cryo-electron microscopy methods, transcription assays and single-molecule fluorescence, to obtain the first high resolution structure of a dynamic influenza vRNP, with a particular focus on the spatial organisation of NPs relative to FluPol. In addition to this work facilitating future influenza drug research, it will provide a basis to investigate the vRNP during other lifecycle stages and act as proof-of-principle for study of other viral protein-RNA complexes, such as those from corona-, arena- and bunyaviruses.Work will be performed in the groups of Stephen Cusack and Olivier Duss based at EMBL Grenoble and Heidelberg, respectively. Here, I will have access to world-leading facilities and training opportunities, supporting my growth as an independent researcher and an expert in RNA virus structural biology.
Original text from CORDIS.
Participants
- EUROPEAN MOLECULAR BIOLOGY LABORATORY · HeidelbergCoordinatorGermany
Links
Data: CORDIS, © European Union