Deciphering the enigmatic expression mechanism of the newly discovered PIPO gene in the Potyviridae family of plant viruses

Lead Research Organisation: University of Cambridge
Department Name: Pathology

Abstract

Plant viruses are one of the major causes of crop loss world-wide, with revenue lost due to reduced yield amounting to some US$60 billion annually. Revenue is also lost due to the implementation of costly control strategies (e.g. chemical control of insects that transmit viruses between host plants, destruction of infected orchards). More importantly, virus-induced crop failure exacerbates famine and ruins livelihoods in developing nations and communities that rely on subsistence farming. Thus, providing effective control measures for plant viral diseases is a crucial component of strategies for maintaining food security both in the UK and worldwide. This is particularly important, now, as populations continue to expand and natural resources including arable land are further depleted.

The largest and most economically important group of plant viruses are the potyviruses. This virus family encompasses almost a third of known plant virus species and is responsible for around half of viral crop damage worldwide. Potyviruses that are of great agricultural significance include potato viruses Y and A, turnip mosaic virus, soybean mosaic virus, sweet potato feathery mottle virus, zucchini yellow mosaic virus, papaya ringspot virus, and plum pox virus. Plum pox, for example, is considered the most devastating viral disease of stone-fruit species such as plum and apricot (estimated costs amounting to 10 billion euro over 30 years). Turnip mosaic virus is particularly important in the UK and worldwide, infecting a huge variety of crops including many brassicas (oilseed rape, cabbage, cauliflower, turnip etc), lettuce, courgette, rhubarb and radish. Meanwhile, sweet potato feathery mottle potyvirus presents a dire threat to food security in sub-Saharan Africa.

We are interested in the mechanisms by which viruses replicate and spread within plants - a drama that unfolds at the molecular level. The central 'dogma' of molecular biology, articulated by Nobel Laureate Francis Crick in 1958, describes the transfer of information between the three major classes of information-carrying biological chemicals: genetic information passes from one generation to the next via the replication of DNA and, within an organism, genes encoded within the DNA genome are 'transcribed' into 'messenger' RNA molecules that are used ('translated') to direct the synthesis of proteins. The roles of DNA and RNA are predominantly as carriers of genetic information, while proteins can have varied roles, for example catalyzing important chemical reactions ('enzymes'), or helping to form the architecture of the cell and its contents. Remarkably, however, most plant viruses, have tiny genomes that are made up of RNA instead of DNA. In most cases, the RNA genome serves directly as a messenger RNA for translation of the viral proteins by pirating the host cell's protein synthesis machinery. Some of these virus proteins are enzymes that the virus uses to replicate its genome, while other virus proteins are used to make the protective capsids that protect the viral genome as it is ferried from one host to another.

Because most plant virus genomes serve directly as messenger RNAs, plant viruses have evolved a variety of unusual mechanisms for controlling gene expression at the level of protein translation. Some of these mechanisms are extraordinarily different from mechanisms used by host plant genes, and are therefore potential targets for virus control strategies. We aim to decipher a completely new and unsuspected translational mechanism that we recently discovered in the potyviruses. The translational mechanism is essential for potyvirus infectivity, but appears to involve completely novel mechanisms, that are not known to be used by any other virus or organism. By figuring out this mechanism, we hope to learn new ways of sustainably controlling potyviruses. We also hope to learn new mechanisms for controlling gene expression that will be useful in biotechnology.

Technical Summary

Translational control plays an integral role in the infectious cycle of all RNA viruses. RNA viruses have evolved many non-canonical translation mechanisms that are rarely or never used by cellular genes. Therefore, understanding these diverse mechanisms may provide the basis for novel strategies for virus control, but can also lead to new knowledge of the cellular translational apparatus and new gene expression tools for biotechnology. Here we propose a detailed analysis of an extraordinary but poorly understood frameshifting mechanism that occurs in probably all members of the family Potyviridae, the largest and most economically important family of plant viruses.

Until recently, potyviruses were believed to express all of their proteins via a single polyprotein that is post-translationally cleaved to produce ~10 mature proteins. However, we recently discovered a novel coding sequence, PIPO, that overlaps the P3 region of the polyprotein in the -1/+2 reading frame. PIPO is essential for virus infectivity and is expressed as a transframe fusion protein via some form of frameshifting (Chung et al, 2008, PNAS 105:5897-902). Many viruses - including HIV, SARS-CoV and PRRSV - utilize programmed -1 ribosomal frameshifting to express their polymerase. In these viruses, frameshifting occurs at an 'X_XXY_YYZ' slippery heptanucleotide motif that is closely followed by a frameshift-stimulating RNA secondary structure. However, in the vast majority of potyviruses, no such elements are present. Instead a completely novel but currently uncharacterized type of frameshifting appears to occur at a highly conserved GAA_AAA_A motif.

Our research will address the questions:

1) What is the mechanism of frameshifting in potyviruses?

2) Why do potyviruses use frameshifting motifs that are so radically different from canonical -1 frameshift-stimulating motifs?

3) Do potyviruses modify the translational machinery in a manner that promotes frameshifting on GAA_AAA_A motifs?

Planned Impact

The research is relevant to one of the BBSRC's strategic priorities, namely Food Security (crop science). Potyviruses form the most numerous and economically devastating plant virus family. Turnip mosaic virus, which infects 100s of species, was ranked as the second most important virus infecting field-grown vegetables in a survey of virus diseases in 28 countries and regions. Plum pox virus is considered the most devastating viral pathogen of Prunus stone-fruit trees such as plum, peach and apricot. Potato Virus Y is rapidly becoming the most economically important potato virus worldwide and causes severe yield losses (10-80%). Zucchini yellow mosaic virus is a major pathogen of a wide variety of cucurbits. Papaya ringspot virus nearly eliminated the papaya industry in Hawaii prior to the successful deployment of transgenic resistance. One of the potyviruses that we are particularly interested in is Sweet potato feathery mottle virus (SPFMV). SPFMV is the most common virus infecting sweet potatoes worldwide. In mixed infections with sweet potato chlorotic stunt crinivirus, SPFMV is associated with severe sweet potato disease (SPVD), a devastating disease of sweet potato, with diseased plants producing almost no usable yield. SPVD is particularly important in sub-Saharan Africa, where sweet potato is the second most important root crop after cassava.

These are just some of the many species of agriculturally important potyviruses. Worldwide, total losses amount to some tens of billions of pounds per year. Thus, finding new ways to control potyviruses is vital for food security.

Potyviruses are transmitted by aphids in a non-circulative, non-persistent manner, and also by vegetative propagation, grafting and seed. The nonpersistent mode of aphid transmission makes control of the disease by insecticides difficult. Indeed there are examples of their use exacerbating virus outbreaks by causing increased probing activity and movement of aphids. Instead, control of virus spread in the field can only be achieved via virus-resistant cultivars. In order to engineer or breed resistance, it is important to have a good understanding of the molecular biology of potyviruses. PIPO clearly represents a new paradigm in our understanding of the compact set of genes and regulatory elements that the Potyviridae use to infect and damage crops with such great success. Moreover the translational mechanism of PIPO appears to be largely conserved throughout the entire family and is likely unique to potyviruses. Further, PIPO is an essential gene. Thus the frameshifting mechanism is potentially a very powerful target for wide-scale control of potyviruses. Additionally, unravelling this new, very important aspect of potyvirus molecular biology will greatly increase our knowledge of these viruses which will have benefits for virus-control strategies.

Thus the main beneficiaries will be farmers (all types of crops including vegetables, grain and fruit), worldwide, and through them the general public. Biotech industries are also potential beneficiaries (see Academic Beneficiaries section).

Part of the research builds upon and extends an international collaboration with Prof Jari Valkonen (see attached letter). Prof Valkonen is a world leader in plant virus diseases and the ability of plants to resist such diseases. He has developed new strains with higher resistance to viruses, particularly for the potato. In his work, Prof Valkonen has combined the techniques of modern molecular biology with traditional plant cultivation and processing.

The post-doctoral research associate funded by this grant would acquire new expertise in molecular biological and virological research which would be widely applicable in the UK biotech industry. In addition, we would expect to have a number of short- and long-term students pass through the lab in the same time period and acquire skills of broad relevance to the UK's economy and well-being.

Publications

10 25 50
 
Description Potyvirids form the largest and most economically important family of plant viruses. Several years ago, we discovered a previously overlooked gene in the potyvirid genome, comprising a short overlapping open reading frame (ORF), termed pipo, "hidden" within the P3-encoding region of the viral polyprotein ORF in an alternative reading frame. Previously we showed that pipo is conserved throughout the entire Potyviridae family, and is essential for virus infectivity. We showed that PIPO is expressed as part of a larger protein and suggested that this might involve some form of translational or transcriptional frameshifting. However the actual mechanism and stimulators remained completely unknown. The main objective of the grant was to decipher the P3N-PIPO expression mechanism, giving new insight into the molecular biology of this most important group of plant viruses.

We verified that the potyvirid pipo ORF is expressed as a frameshift fusion with the N-terminal part of the polyprotein-encoded P3 protein, giving the "transframe" product P3N-PIPO. We showed that the efficiency of frameshifting is ~1-2% (depending on species). We demonstrated that frameshifting involves polymerase (rather than ribosomal) slippage, resulting in the insertion of an extra A into a highly conserved GAAAAAA sequence in ~2% of transcripts, allowing ribosomes access to the pipo ORF when these transcripts are translated. We confirmed that slippage was specific to the viral polymerase. As the first example of polymerase slippage being functionally utilized for gene expression in positive-sense RNA viruses, this now opens up the possibility that other such viruses also utilize polymerase slippage to this end. In parallel we also confirmed a predicted second site of polymerase slippage in one subgroup of sweet-potato-infecting potyviruses (P1N-PISPO protein). The work has been published in PMIDs 26113364 and 26757490, and has led to several exciting new leads which we are currently pursuing.

Essentially all of the original objectives were completed with the sole exception of generating an infectious clone for sweet potato feathery mottle virus (SPFMV). This proved unnecessary for the completion of the other work and, due to the emergence of another group working in a similar direction, to reduce competition and duplicated effort we veered away from this minor subproject.

2017 update: We have further characterized the polymerase slippage mechanism in potyvirids, finding that it differs in several important respects from previous examples of polymerase slippage (in cellular organisms and negative-sense RNA viruses). This work is published in PMID 27185887.
Exploitation Route We are currently doing further follow-up work in-house but the details of this are confidential and will be added in the next reporting period.
Sectors Agriculture, Food and Drink

URL http://www.firthlab.path.cam.ac.uk/index.html
 
Description Potyvirids form the largest and most economically important family of plant viruses. Several years ago, we discovered a previously overlooked gene in the potyvirid genome, comprising a short overlapping open reading frame (ORF), termed pipo, "hidden" within the P3-encoding region of the viral polyprotein ORF in an alternative reading frame. Previously we showed that pipo is conserved through out the entire Potyviridae family, and is essential for virus infectivity (others subsequently showed that it is essential for virus cell-to-cell movement), making it an attractive target for breeding or engineering broad-spectrum resistence to potyvirids. PIPO is expressed as part of a "frameshift fusion" protein, P3N-PIPO. However the frameshift mechanism remained completely unknown. The main objective of the grant was to decipher the P3N-PIPO expression mechanism (giving new insight into the molecular biology of this most important group of plant viruses) and also assess whether it could be used as an anti-viral target and/or as a biotechnological tool. Although initially (based on previous indications) we expected P3N-PIPO expression to involve translational frameshifting, this turned out to be incorrect. In fact, P3N-PIPO is expressed through frameshifting at the level of transcription, with the "high" (~2%) levels of slippage at the slip site being a specific feature of the viral polymerase. This unexpected result reduced the possibilities of using the P3N-PIPO expression mechanism as a target for breeding resistence or as a biotechnological tool. While, in principal, slippage by the viral polymerase could be a target for small molecule inhibitors, this type of approach would be more relevant for human/animal viruses than plant viruses, and, in any case, would probably not be a more attractive target than simply inhibiting all viral polymerase activity. On the other hand, we made great progress in our primary aim of gaining new insight into the molecular biology of the largest and most diverse group of plant viruses. Moreover, this is the first time that polymerase slippage has been shown to be functionally utilized for gene expression in a positive-sense RNA virus. Positive-sense RNA viruses are the largest and most diverse group of eukaryote-infecting viruses, and the group includes many human pathogens such as dengue, Japanese encephalitis, yellow fever, chikungunya, hepatitis E, hepatitis C, hepatitis A, and polio viruses. It is possible that other such viruses also use polymerase slippage to access short overlapping "hidden" ORFs (i.e. previously overlooked genes). Thus, our findings potentially have a broader impact in human and animal virology, and this is something that we are investigating further. There are also many interesting new mechanistic and virus-host interaction questions to address. Our new interest in polymerase fidelity also provoked some interesting discussions about the fidelity of protein synthesis at a BioProNET academia-industry network meeting. We also investigated another frameshift fusion protein, P1N-PISPO (overlapping ORF pispo, in the P1 region of the polyprotein ORF), that we (and others) had identified in one subgroup of related sweet-potato-infecting potyviruses. We showed that P1N-PISPO is expressed via a polymerase slippage mechanism identical to that used for P3N-PIPO expression. Meanwhile, collaborators working with us showed that P1N-PISPO is involved in the suppression of silencing. These discoveries may be relevant for breeding resistence to sweet potato virus disease (SPVD) which causes heavy yield losses in sweet potato - the second most important root crop in sub-Saharan Africa. In the immediate term, we need to understand more thoroughly how P1N-PISPO affects the synergistic interaction between these potyviruses and sweet potato chlorotic stunt virus (a crinivirus) that is associated with severe SPVD. Related to this and other projects in our lab, we have developed (and continue to expand) an online resource of virus gene expression data (http://www.firthlab.path.cam.ac.uk/vindex.html) which is linked-to from ViralZone and NCBI, and of use to the wider virology community.
First Year Of Impact 2014
Sector Agriculture, Food and Drink
Impact Types Economic

 
Description European Research Council Consolidators Grant
Amount € 1,780,000 (EUR)
Funding ID 646891 
Organisation European Research Council (ERC) 
Sector Public
Country European Union (EU)
Start 09/2015 
End 08/2020
 
Description Wellcome Trust Senior Research Fellowship
Amount £1,131,431 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2015 
End 03/2020
 
Title Turnip rosette virus infectious clones 
Description Turnip rosette virus infectious clones (first infectious clones for a Sobemovirus species able to infect Arabidopsis) 
Type Of Material Biological samples 
Year Produced 2014 
Provided To Others? Yes  
Impact Receiving labs haven't yet published. 
 
Title ViRAD database 
Description The Virus Re-Annotation Database (ViRAD) covers all RNA viruses and contains in-house reannotation of the coding regions based on homology, comparative genomics, expert review, and our own experimental analyses. The database also includes results of applying our in-house developed comparative genomic software to all RNA virus genomes. It is useful to the community for the identification of novel features in RNA virus genomes, which can be used to guide experimental analyses. 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact NA 
URL http://www.firthlab.path.cam.ac.uk/virad.html
 
Description Analysis of transcriptional slippage mechanisms in RNA viruses 
Organisation Tallinn University of Technology
Country Estonia 
Sector Academic/University 
PI Contribution Intellectual input. Experimental work. High-thoughput sequencing.
Collaborator Contribution Intellectual input. Data processing. Experimental work.
Impact Multi-disciplinary: Bioinformatics + Plant Biology + Virology. PMID 27185887. Grant funding for collaborator.
Start Year 2015
 
Description Extended studies of potyvirus PIPO expression 
Organisation Iowa State University
Country United States 
Sector Academic/University 
PI Contribution Sharing of research strategies, plasmids, information.
Collaborator Contribution Sharing of research strategies, plasmids, information.
Impact Publications; conference talks; media releases; grant funding. Joining of experimental (molecular virology) and bioinformatic expertise.
Start Year 2006
 
Description PISPO function and expression mechanism 
Organisation International Potato Center
Country Peru 
Sector Charity/Non Profit 
PI Contribution Proposed research hypothesis and initiated project. Bioinformatic analysis. High-throughput sequencing and analysis. Contributed to writing manuscript. (PI and postdoc time, reagents, sequencing costs.)
Collaborator Contribution PhD student working on the project for ~4 years; also intellectual input and PhD supervision from the two PIs; some technical support; reagents.
Impact PMID 26757490 Some conference presentations by collaborators.
Start Year 2010
 
Description PISPO function and expression mechanism 
Organisation University of Helsinki
Country Finland 
Sector Academic/University 
PI Contribution Proposed research hypothesis and initiated project. Bioinformatic analysis. High-throughput sequencing and analysis. Contributed to writing manuscript. (PI and postdoc time, reagents, sequencing costs.)
Collaborator Contribution PhD student working on the project for ~4 years; also intellectual input and PhD supervision from the two PIs; some technical support; reagents.
Impact PMID 26757490 Some conference presentations by collaborators.
Start Year 2010
 
Description Studies of potyvirus translation 
Organisation University of Wisconsin-Madison
Country United States 
Sector Academic/University 
PI Contribution Sharing of ideas, plasmids, research findings. Provision of bioinformatic analysis.
Collaborator Contribution Sharing of ideas, research findings.
Impact NA
Start Year 2011
 
Title synplot2 comparative genomics software 
Description New comparative genomics software for mapping out functional RNA elements overlapping protein coding regions, focusing on, but not limited to, virus genomics. Source code and more recently (2014) a webserver interface made available to the community. 
Type Of Technology Software 
Year Produced 2012 
Open Source License? Yes  
Impact Has been used extensively in our lab for identifying novel functional elements encoded within the genomes of economically and medically important viruses such as influenza A virus, porcine reproductive and respiratory syndrome, West Nile virus and potyviruses. Many virus functional elements only exhibit a phenotype in animal models. By first identifying and characterizing features computationally, experimental follow-up can be targetted efficiently thus vastly reducing the need for any animal experiments. 
URL http://www.firthlab.path.cam.ac.uk/virad.html
 
Description American Society for Virology (ASV) conference, 11-15 July 2015, London, Canada 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Conference talk by postdoc Allan Olspert.
Year(s) Of Engagement Activity 2015
URL http://www.asv2015.uwo.ca/
 
Description BioProNet workshop on Protein Authenticity, London, 2015 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Invited workshop talk by PI Andrew Firth, and followup discussion/interactions.
Year(s) Of Engagement Activity 2015
URL http://biopronetuk.org/
 
Description EMBO workshop - Green viruses, from gene to landscape 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Type Of Presentation poster presentation
Geographic Reach International
Primary Audience Other audiences
Results and Impact Poster presentation by post-doc Allan Olspert. Lively discussion of our findings with many interested parties. Networking.

NA
Year(s) Of Engagement Activity 2013
URL http://events.embo.org/13-plant-virus/
 
Description EMBO workshop on Recoding: Reprogramming genetic decoding, Killarney, Ireland, 13 - 18 May 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact PI Andrew Firth was a co-organizer of this workshop on Genetic Recoding. Much lively discussion between participants and forging of new networks and collaborations.

Hugely positive feedback from participants. Increased communication and knowledge sharing between practitioners of many different aspects of this growing field.
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-recoding/
 
Description EMBO workshop on Recoding: Reprogramming genetic decoding, Killarney, Ireland, 13 - 18 May 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Conference talk by PI Andrew Firth. Lively discussion of our findings with many interested parties. Networking.

NA
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-recoding/
 
Description EMBO workshop on Recoding: Reprogramming genetic decoding, Killarney, Ireland, 13 - 18 May 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Conference talk by postdoc Allan Olspert. Lively discussion of our findings with many interested parties. Networking.

NA
Year(s) Of Engagement Activity 2014
URL http://events.embo.org/14-recoding/
 
Description European Virus Bioinformatics Centre 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited talk
Year(s) Of Engagement Activity 2017
URL http://evbc.uni-jena.de/
 
Description European Virus Bioinformatics Centre discussion session 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Co-chairing round-table discussion session on priorities in virus bioinformatics.
Year(s) Of Engagement Activity 2017
URL http://evbc.uni-jena.de/
 
Description Institut Pasteur - C3BI 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited seminar
Year(s) Of Engagement Activity 2017
 
Description Ribosome Profiling Workshop (London) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Presented and led a round table discussion session at a Biochemical Society workshop on Ribosome Profiling. This is a relatively new technique and the workshop was to share experiences between the different practitioners.
Year(s) Of Engagement Activity 2016
URL http://data.plantsci.cam.ac.uk/ribosome-footprinting/
 
Description University College Cork 2017 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact Invited seminar
Year(s) Of Engagement Activity 2017