Untangling the processes of replication and encapsidation in Picornavirales

For a virus to be able to spread from one organism to another, it must protect its genetic material (genome) from the harsh external environment. This is particularly true of the many viruses that use ribonucleic acid (RNA) rather than the more common and more stable deoxyribonucleic acid (DNA) for their genomes. Protection is achieved by surrounding the fragile genome by a protein shell made up of many copies of one or a few coat proteins to produce virus particles. Both the viral genome and the coat proteins are produced when a virus multiplies inside a cell. The process by which the genome is surrounded by the protein shell is known as encapsidation and this also takes place within the cell. Encapsidation is highly selective, ensuring that only the genome of the virus, and not the RNA normally present in the host cell, is incorporated into virus particles.
Despite its critical importance in the viral life cycle, very little is known about the mechanism of encapsidation in one of the major families of RNA-containing viruses, the Picornavirales. This family contains viruses which can infect either animals or plants, and some members are of great medical (poliovirus, hepatitis A virus, common cold virus), veterinary (foot-and-mouth disease virus) and agricultural (rice tungro spherical virus) importance. Clearly a greater understanding of a process essential for virus spread would be of huge significance to our ability to combat these diseases. Such understanding would help to develop virus particles that can act as vehicles for the delivery of specific nucleic acid sequences to cells for a variety of medical applications.
One of the reasons that so little information is available about encapsidation in the Picornavirales is that the process appears to be intimately associated with other aspects of the replication cycle including amplification of the genome (replication) and protein synthesis (translation). However, at the John Innes Centre (JIC), we have shown that it is possible to untangle these processes in the case of the plant-infecting member of the Picornavirales, cowpea mosaic virus (CPMV) using transient expression of the various viral components in plants. Furthermore, studies at the University of Leeds (UoL) using electron microscopy have shown that it is possible to see the RNA within assembled particles of CPMV, revealing details of how the genome interacts with the coat protein subunits to form a virus particle. In this proposal, we wish to combine the expertise available at JIC and UoL to understand how the CPMV specifically encapsidated its genome within viral particles. We will investigate whether there is a size limit on RNA molecules which can be efficiently encapsidated and the linkage between encapsidation and other aspects of the viral replication cycle. The knowledge gained from these studies would be applicable not only to CPMV but to all members of the family Picornavirales and would also aid the application of these viruses in bionanotechnology.

Despite their importance as pathogens of both animals and plants, and their relatively simple, non-enveloped capsids, remarkably little is known about the mechanism of RNA packaging in members of the family Picornavirales. This proposal will make use of recent developments in plant transient expression at the John Innes Centre (JIC) and cryo-electron microscopy (cryo-EM) at the University of Leeds (UoL) to determine the mechanism of RNA encapsidation in cowpea mosaic virus (CPMV), a bipartite member of this large virus family. In particular, it will build on the recent demonstration that co-expression of the CPMV coat protein precursor (VP60) and the proteinase necessary for its processing (24K), leads to the assembly of RNA-free capsids into which replicating RNA molecules can be packaged (JIC) and the ability to observe RNA within CPMV particles by cryo-EM (UoL). Specifically, the requirement of replication for packaging will be determined and interactions between particles and replicase-associated proteins will be analysed. Further, the ability of RNAs of differing sizes and sequences to be efficiently packaged will be assessed and the structures adopted by different RNAs within particles will be determined. Finally, the roles of specific amino acids, particularly those at the C-terminus of the small (S) coat protein in the encapsidation process will be determined. As well as being important for our understanding of an important part of the replication cycle of the Picornavirales, the results of this study will also be of relevance to the general field of RNA-protein interactions. Furthermore, they will be highly significant for the development of CPMV particles as a means of delivering specific RNA molecules.

This is a "science-led" project which will utilise recent developments in the production of empty CPMV particles via transient expression in plants at JIC and high resolution cryo-EM methodology at UoL to address an outstanding problem in virology - how is an RNA genome selectively incorporated into virus particles in a very large order of viruses, the Picornavirales? Members of this order cause significant diseases of mammals (e.g. foot-and-mouth disease virus) and plants (e.g. rice tungro spherical virus) and the protection of the viral RNA through encapsidation in virus particles is essential for virus spread from host to host. Despite their importance as pathogens and many years of investigation, this problem has defied solution mainly due to the lack of tools with which to address it. The development at JIC of a method of efficiently producing empty (RNA-free) virus-like particles (eVLPs) of cowpea mosaic virus (CPMV) and the demonstration that only replication-competent RNA molecule can be encapsidated into eVLPs has opened the door to the study of the mechanism encapsidation. By coupling the availability of this packaging system with the ability to visualise RNA within particles via cryo-EM at UoL, we are finally in a position to unravel the previously intractable process whereby specific viral sequences are efficiently incorporated into virus particles. Thus the immediate impact of the research will be the elucidation of RNA packaging in the Picornavirales.

Incorporation of RNA into assembled virus particles is a vital step in the viral replication cycle not just of the Picornavirales but of all viruses as it enables the labile genetic material to be disseminated through the harsh external environment. Understanding how defined RNAs are incorporated into virus particles in the case of the Picornavirales will undoubtedly provide insights into the mechanism of encapsidation of may additional families of viruses and will thus constitute a major advance in our state of knowledge of how viruses package specific RNA molecules and are thus able to spread. Thus the impact of the proposed research will extend well beyond the realm of the Picornavirales into virology in general. Understanding the mechanism of RNA packaging will have considerable impact on those wishing to develop anti-viral strategies by interfering with the process. Since viral diseases limit both animal and plant productivity, this project is relevant to the BBSRC Research priority of Food Security.

In addition to being highly relevant to those working on the Picornavirales and other viruses as pathogens, the research proposed will have an impact on those wishing to develop virus-like particles into RNA-delivery vehicles. This is a rapidly expanding field of bionanotechnology as it offers a means of delivering therapeutic RNA molecules (e.g. siRNAs) into cells. A major challenge is to devise means for the specific incorporation of the required RNAs into particles. As a result of this project, we will understand how this can be done in the case of CPMV which will aid the development of this virus as an RNA delivery agent. Furthermore, the results will elucidate the basic principles underlying specific packaging which will relevant to many viruses of both plants and animals. This should stimulate the further development of additional RNA delivery systems.

In addition to the scientific outputs of the project, a particularly significant impact will be the training of two post-doctoral scientists in state-of-the-art techniques in plant-based expression systems (JIC) and cryo-EM (UoL). This will increase the scientific capability of the UK. Furthermore, although this is essentially an academic research project, both JIC and UoL will consider intellectual property (IP) issues at an early stage. In particular we will consider commercial opportunities that may arise from the potential use of CPMV particles to deliver specific RNA sequences to cells.