Untangling the processes of replication and encapsidation in Picornavirales

Lead Research Organisation: John Innes Centre
Department Name: Biological Chemistry

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description Through the use of state-of-the-art near atomic resolution cryo-electron microscopy (cryo-EM), the structure of cowpea mosaic virus empty virus-like particles (CPMV eVLPs) has been determined. This has shown that the architecture of eVLPs is identical to that of CPMV particles produced via infection. Furthermore, the cryo-EM studies revealed details of the structure of the C-terminal region of the S coat protein not previously resolved by crystallography. This has enabled a model for the assembly of CPMV capsids to proposed. The finding that synthetic capsids are accurate surrogates for the original virus particles has led to the technology being deployed to produce VLPs of viruses of currently unknown structure. The structure of these can then be solved by high resolution Cryo-EM. The use of VLPs to encapsidate specific RNA molecules has been investigated and has established a clear link between virus replication and RNA packaging.
Exploitation Route The studies will be of great value for those wishing to use CPMV eVLPs as nanoshells for such purposes as drug delivery and bioimaging. In addition the approaches adopted will enable to determination of the structures of viruses for which there is currently no data.The ability to package specific RNA molecules could also be of interest to the pharmaceutical sector.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description The demonstration that CPMV VLPs have a structure very similar to those of the native virus particles means that they can be used as surrogates for development of novel diagnostic reagents. This opens the way to the development of methods for development of such reagents without the need to handle infectious material.
First Year Of Impact 2017
Sector Agriculture, Food and Drink,Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description EC H2020
Amount € 8,286,008 (EUR)
Funding ID 774078 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 11/2017 
End 10/2021
 
Description MRC Zika Grant
Amount £40,000 (GBP)
Funding ID MC/PC/15085 
Organisation Medical Research Council (MRC) 
Sector Academic/University
Country United Kingdom
Start 03/2016 
End 09/2017
 
Description Responsive mode
Amount £450,429 (GBP)
Funding ID BB/R001669/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2017 
End 09/2020
 
Description UKVRDN Grant
Amount £1,988,841 (GBP)
Funding ID GHR Project:16/107/04 
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 11/2016 
End 08/2018
 
Title Encapsidated mimics 
Description A method for producing in-tube positive control reagents for diagnosis or proficiency testing. The method is based on using capsids of cowpea mosaic virus (CPMV) to protect RNAs from degradation in field samples, The encapsidated mimic RNA consists of a version of CPMV RNA-2 containing a deletion in the region encoding the viral movement protein (48K protein) plus sequences from the target pathogen which can be detected by PCR-based methods. The deletion in the 48K protein prevents spread of the virus in the environment. The method can be adapted as detection method for a wide variety of pathogens. 
Type Of Material Technology assay or reagent 
Year Produced 2014 
Provided To Others? Yes  
Impact The detection of foot and mouth disease virus (FMDV) and proficiency testing in sub-Saharan Africa 
 
Description Collaboration with Leaf expression Systems, NRP 
Organisation Leaf Expression Systems
PI Contribution We are collaborating with Leaf expression Systems (LES) to scale-up the production of several VLPs which have previously made on a small scale at JIC. these include eVLPs from CPMV and poliovirus. The constructs originally produced at JIC have been transferred to LES.
Collaborator Contribution The use of facilities and technology for the scaled-up production of VLPs with particular emphasis on process development and quality assurance.
Impact The collaboration has been active for less than 1 year so there are no outcomes to report yet.
Start Year 2017
 
Description Encapsiidate viral mimics 
Organisation The Pirbright Institute
Country United Kingdom 
Sector Academic/University 
PI Contribution The team at JIC produced the virus particles containing deleted versions of cowpea mosaic virus (CPMV) RNA-2. The RNA within these particles is also modified to contain test sequences derived from animal virus pathogens that can be detected using PCR-based methods. The encapsidated mimic RNAs can act as in-tube positive controls for diagnostic and proficiency testing applications.
Collaborator Contribution The Pirbright Instituted tested encapsidated mimics containing sequences specific for foot and mouth disease virus (FMDV) and showed that they were effective in-tube positive controls for the diagnosis of FMDV in crude samples.
Impact Publication (Madi et al., 2015) Design of further mimic constructs based on the technology developed during this project. Use of the FMDV construct for diagnosis of infection and proficiency testing in sub-Saharan Africa.
Start Year 2014
 
Description Packaging signals - Leeds 
Organisation Imperial College London
Department Imperial College Trust
Country United Kingdom 
Sector Charity/Non Profit 
PI Contribution The JIC team have made a variety of constructs based on satellite tobacco necrosis virus (STNV) to examine the ability of variants to be packaged by the the coat protein.
Collaborator Contribution University of Leeds: design of mutant STNV RNA molecules Imperial College and UoH: Determination of the specificity of packaging
Impact A multi-disciplinary collaboration aimed and dveloping new methods of controlling plant viruses.
Start Year 2014
 
Description Packaging signals - Leeds 
Organisation University of Hertfordshire
Country United Kingdom 
Sector Academic/University 
PI Contribution The JIC team have made a variety of constructs based on satellite tobacco necrosis virus (STNV) to examine the ability of variants to be packaged by the the coat protein.
Collaborator Contribution University of Leeds: design of mutant STNV RNA molecules Imperial College and UoH: Determination of the specificity of packaging
Impact A multi-disciplinary collaboration aimed and dveloping new methods of controlling plant viruses.
Start Year 2014
 
Description Packaging signals - Leeds 
Organisation University of Leeds
Country United Kingdom 
Sector Academic/University 
PI Contribution The JIC team have made a variety of constructs based on satellite tobacco necrosis virus (STNV) to examine the ability of variants to be packaged by the the coat protein.
Collaborator Contribution University of Leeds: design of mutant STNV RNA molecules Imperial College and UoH: Determination of the specificity of packaging
Impact A multi-disciplinary collaboration aimed and dveloping new methods of controlling plant viruses.
Start Year 2014
 
Description PBVAB Conference Presentation 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation "Combining transient expression and high resolution cryo-electron microscopy to produce novel virus-like particles" by Walden M., Meshcheriakova Y., Marsian J., Hesketh E., Ranson N., Lomonossoff G. P. PBVAB 2017, Albufeira, Portugal, ,5-7th June 2017
Year(s) Of Engagement Activity 2012,2014,2017
 
Description Geminivirus talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Talk entitled "The Structural Features of a Begomovirus Particle Revealed by Cryo-Electron Microscopy" Saunders K., Hesketh E., Fisher C., Ranson N., Lomonossoff GP. The 2017 meeting of the American Society for Virology meeting at the University of Wisconsin-Madison, USA. 24th - 28h June 2017.
Year(s) Of Engagement Activity 2017
 
Description Glycosylation, Manchester 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact Presentation entitled "The mechanism of glycosylation and the consequences of glycoform on therapeutic protein functionality" by G.P.Lomonossoff. Manchester, UK,.Establishing International Partnerships in Industrial Biotechnology and Bioenergy - Improved glycoform-based biopharmaceutical production in plants. 18-20th Sept. 2017
Year(s) Of Engagement Activity 2017
 
Description JIC ASM 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Professional Practitioners
Results and Impact Talk entitled "Well, we Built it - now will they come?" by GP Lomonossoff, JIC Annual Science Meeting 2017, 12 Oct 2017
Year(s) Of Engagement Activity 2017
 
Description Meeting on Norwich Research Park 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Professional Practitioners
Results and Impact Presentation "Transient expressions of synthetic biology in plants" at Earlham Institute, Norwich Research Park, Meeting "Decoding and Recoding Biological Systems", 19/05/17
Year(s) Of Engagement Activity 2017
 
Description NanoBioMater 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Lectured on "Plant viruses and virus-like particles as building blocks for bionanotechnology" at the NanoBioMater summer school, Bad Herrenalb, Germany, 22/06/15
Year(s) Of Engagement Activity 2015
 
Description OpenPlant Forum 2016, JIC, Norwich 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Postgraduate students
Results and Impact I delivered a talk entitled "Making all kinds of everything - The joy of plant-based transient expression" at OpenPlant Forum 2016, JIC, Norwich on 26th July 2016.
Year(s) Of Engagement Activity 2015,2016
URL http://www.openplant.org
 
Description Oral presentation at AAB Conference "Advances in Plant virology", 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Peyret H., Saxena P., Kruse I., Lomonossoff G. P.(2018) Studies on cowpea mosaic virus show that RNA encapsidation is dependent on replication: implications for the assembly of members of the order Picornavirales. AAB Conference "Advances in Plant virology", Jury's Inn, Birmingham UK 12-13th April 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at AAB Conference "Advances in Plant virology", 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P.(2018) Synthetic virus-like particles and how to make them in plants AAB meeting "Advances in Plant Virology", Jury's Inn, Birmingham 12-13th April 2018
Year(s) Of Engagement Activity 2018
 
Description Oral presentation at VLPNPV, Bern 2018 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Lomonossoff G. P., Walden M., Steele J., Meshcheriakova Y., Marsian J., Saunders K., Thuenemann E., Peyret H., Hesketh E., Thompson R.(2018) Plants as a highly effective platform for VLP production VLPNPV Meeting Bern, Switzerland, 25-27th Sept. 2018
Year(s) Of Engagement Activity 2018
 
Description Seminar at NUIG 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Synthetic virus-like particles and how to make them in plants Seminar at National University of Ireland - Galway (NUIG), Galway, Republic of Ireland.
Year(s) Of Engagement Activity 2018
 
Description Talk at Norwich Science Festival 2016 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact This talk, entitled "The Green Vaccine Machine" was delivered as part of the Norwich Science Festival on 24/10/16 at The Forum in Norwich.
Year(s) Of Engagement Activity 2013,2016
 
Description Vienna Meeting 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Presentation "Transient Expressions of Synthetic Biology in Plants" by G.P. Lomonossoff, 4th International Conference Plant Transformation & Biotechnology, Vienna, Austria 30/06/17
Year(s) Of Engagement Activity 2017