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

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.

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?

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.