Understanding RNA packaging signals in foot-and-mouth disease virus (FMDV) for improved vaccine production

Lead Research Organisation: The Pirbright Institute
Department Name: Picornavirus Molecular Biology

Abstract

Foot-and-mouth disease virus (FMDV) causes an economically devastating disease of livestock affecting both the developed and developing world. One of the world's biggest challenges is to meet a growing demand for food. Animal viruses such as FMDV have a significant negative impact on productivity of the livestock industries. Thus improved control of FMD could sustainably improve the performance of global livestock industries and have enormous economic and social value worldwide especially in developing regions where livestock are seen as a means to raise millions from poverty.

FMDV is composed of a 'blueprint' for making new virus, enclosed within a protective protein shell or capsid. The blueprint is a single piece of RNA, similar to the DNA genetic code in humans. When FMDV replicates, it uses the RNA blueprint and host cell machinery to make new proteins and new copies of the RNA. The proteins assemble together to make new virus capsids. The RNA is a single linear molecule, like a long piece of string but it all ends up miraculously 'packaged' inside the capsid in a neatly folded ball. How this happens is not understood. We have discovered that there are parts of the RNA that might act as hooks to help the RNA grab hold of the virus proteins. This might guide the folding of the RNA into the correct shape for it to be enclosed by the assembling proteins. This is exciting because people have been wondering about how this works for decades and now we are getting close to finding out.

We would like to determine what the hooks are on the RNA and how they grab onto the proteins. This is a critical part of the virus life cycle for FMDV and many other related viruses. We believe the work we propose will provide detailed new understanding of the genome packaging process so that we will be able to reprogramme the viral blueprint to improve on the production of FMD vaccines by increasing yield and/or stability of vaccine.

Technical Summary

Genome packaging in non-enveloped RNA viruses involves direct interactions between capsid proteins and RNA secondary structures or 'packaging signals' as exemplified by the bacteriophage MS2. More recently, such interactions have also been demonstrated for many other non-enveloped RNA viruses of plants, animals and humans, including viruses in the picornavirus family, such as the livestock pathogen foot-and-mouth disease virus (FMDV).

In recent years, we and others have obtained novel cryo-EM structures of picornaviruses which showed direct contacts between the genome and the inside of the capsid. We have also identified packaging signals (PS) in diverse picornavirus genomes using deep sequencing, RNA-capsid interaction studies and in silico approaches. The role of such PS in virus assembly has been confirmed by mutagenesis and reverse genetics.

We will apply these methods and preliminary findings to confirm and characterise in detail the involvement of packaging signals in the assembly of FMDV. We will use mutagenesis of virus genomes and trans-encapsidation assays to understand the requirements for functional packaging signals. We will characterise the affinity and structure of interactions between RNA packaging signals and the capsid. We will use in silico re-coding, synthetic biology and reverse genetics to engineer optimised PS into the FMDV genome to improve efficiency of assembly. Finally, we will explore the potential of such engineered viruses to increase the yield and stability of FMD vaccines.

For many non-enveloped RNA viruses, packaging has been poorly understood for decades. This proposal will provide novel understanding of this neglected part of the virus life cycle and will apply this directly to improving vaccines for FMD.
 
Description Genome packaging, whereby viral RNA is incorporated into protective protein capsids to produce more virus particles, is a crucial step in the FMDV life cycle. It is a stringent process as only viral RNA is encapsidated, while cellular RNA is excluded. We have revealed the essential role of RNA packaging signals in FMDV packaging, specifically those in the pseudoknot region and in a region that contains up to 200 cytosines, termed the poly-(C) tract. We have demonstrated that the length of the poly-(C) tract significantly affects packaging, where genomes containing longer poly-(C) tracts are favoured. This is the first role that has been identified for the poly-(C) tract in FMDV. We have also found an essential packaging signal in the pseudoknot region, which is complemented by other packaging signals located throughout the genome, that together help encapsidate FMDV RNA. These results provide compelling evidence for the involvement of packaging signals in FMDV packaging. Based on this, we have proposed a simple model for FMDV packaging, which involves a switch from genome replication to genome packaging, and is controlled by packaging signals.
Exploitation Route We are investigating the engineering of packaging signals to increase the yield and stability of FMD vaccines
Sectors Agriculture

Food and Drink

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description Leeds FBS 
Organisation University of Leeds
Department Faculty of Biological Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Research
Collaborator Contribution Research and student supervision
Impact Research
Start Year 2009
 
Description York 
Organisation University of York
Country United Kingdom 
Sector Academic/University 
PI Contribution Collaboration
Collaborator Contribution Collaboration
Impact Collaboration
Start Year 2015
 
Description Pirbright Pride Day - picornavirus group 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Pirbright open day for family/friends of staff or students and local community. Excellent engagement with the public and sparked new ideas for outreach to improve other events.
Year(s) Of Engagement Activity 2023