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

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.

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.