Periodic Reporting for period 1 - Influenza RNP (A structural and functional understanding Influenza virus mRNA production in the context of the transcriptionally functional ribonucleoprotein (RNP) particle.)
Période du rapport: 2021-07-01 au 2023-06-30
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.
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.
The first objective concerned the production of an sh-vRNP sample amenable to study using cryo-EM and functional study using mRNA synthesis assays. In the DoA two methods for production of sh-vRNP were proposed. One utilised an influenza virus minireplicon and the other in vitro reconstitutions. Unfortunately, following months of trials and optimisation neither methodology was viable to produce the amount of sh-vRNP required for structural and functional studies. In order to progress with the overall objectives of the project it was necessary to generate sh-vRNP another way.
Through a collaboration with Dr Nadia Naffakh at the Institute Pasteur, it was possible to generate virus containing a purifiable vRNP using reverse engineering. Viruses can be produced by supplying cells with the tools to produce polymerase, nucleoprotein and the RNA sequences of the complete influenza genome. In this case, one of the RNA segments was replaced with a recombinant segment. In order to ensure homogeneity in size and RNA sequence, an affinity tag for purification was incorporated into the vRNA. The sh-vRNP was found to be homogenous and the size was as expected. achi
A manuscript detailing the sh-vRNP production method, alongside low-resolution structure solution is being composed. Additionally, the methodology and expertise of the researcher is being disseminated to other institutes to aid in their research.
Through a collaboration with Dr Nadia Naffakh at the Institute Pasteur, it was possible to generate virus containing a purifiable vRNP using reverse engineering. Viruses can be produced by supplying cells with the tools to produce polymerase, nucleoprotein and the RNA sequences of the complete influenza genome. In this case, one of the RNA segments was replaced with a recombinant segment. In order to ensure homogeneity in size and RNA sequence, an affinity tag for purification was incorporated into the vRNA. The sh-vRNP was found to be homogenous and the size was as expected. achi
A manuscript detailing the sh-vRNP production method, alongside low-resolution structure solution is being composed. Additionally, the methodology and expertise of the researcher is being disseminated to other institutes to aid in their research.
The vRNP is central to the lifecycle of the influenza virus, an extremely relevant pathogen of humans, wildlife and livestock. This project has established a workflow to produce a short and homogenous vRNP. Efforts to structurally characterise the sh-vRNP are ongoing, and if successful this knowledge could be utilised to inform future efforts to generate novel anti-virals. Additionally, the sh-vRNP could be applied to study other questions about the influenza virus lifecycle and the methodology could be applied to other key RNA viruses.