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ISIS Report 28/01/13

Potentially Dangerous Virus Gene Hidden in Commercial GM Crops

The European Food Safety Authority has just discovered a virus gene in GM crops it has been approving over the past twenty years; a thorough independent risk assessment based on existing data shows that the only reasonable course of action is a total recall of all affected GM crops. Dr Jonathan Latham and Dr Allison Wilson

A belated discovery with serious ramifications for safety

The European Food Safety Authority (EFSA) has belatedly discovered that the most common genetic regulatory sequence in commercial GMOs also encodes a significant fragment of a viral gene. The finding – published quietly in a new journal [1] - has serious ramifications for crop biotechnology and its regulation, but possibly even more so for consumers and farmers. There are clear indications that this viral gene (called Gene VI) might not be safe for human consumption, although the authors fall short of stating that. It also may disturb the normal functioning of crops, including their natural pest resistance.

The authors, Nancy Podevin from the European Food Safety Authority (EFSA) and Patrick du Jardin at University of Liege in Belgium, discovered that of the 86 different transgenic events (unique insertions of foreign DNA) commercialized to-date in the United States 54 contain portions of Gene VI within them. They all have the regulatory sequence called the CaMV 35S promoter (from the cauliflower mosaic virus CaMV), the most commonly used for driving gene expression in GMOs. The events therefore include some of the most widely grown GM crops all over the world such as Roundup Ready soybeans (40-3-2) and MON810 maize. Also included is the controversial NK603 maize recently reported to cause tumours in rats [2] (see also [3] GM Cancer Warning Can No Longer Be Ignored, SiS 56).

The researchers themselves concluded that the presence of segments of Gene VI “might result in unintended phenotypic changes.” They reached this conclusion because similar fragments of Gene VI have already been shown to be active on their own [4].

In general, viral genes expressed in plants raise both agronomic and human health concerns [5, 6]. That is because many viral genes function to disable their host in order to facilitate pathogen invasion, often by incapacitating specific anti-pathogen defences. Incorporating such genes could clearly lead to undesirable and unexpected outcomes in agriculture. Furthermore, viruses that infect plants are often not that different from viruses that infect humans. For example, sometimes the genes of human and plant viruses are interchangeable, while on other occasions inserting plant viral fragments as transgenes has caused the genetically altered plant to become susceptible to an animal virus [7]. Thus, in various ways, inserting viral genes accidentally into crop plants and the food supply confers a significant potential for harm. (Editor’s comment: there is also evidence that the CaMV 35S promoter may actually induce transcription factors for HIV and other pathogenic viruses [8] New Evidence Links CaMV 35S Promoter to HIV Transcription, ISIS scientific publication.)

Regulatory options

The discovery of Gene VI in commercial GMO crops by a scientist within EFSA must have presented regulators with sharply divergent options. They could recall all CaMV Gene VI-containing crops (in Europe that would mean revoking importation and planting approvals) or undertake a retrospective risk assessment of the CaMV promoter and its Gene VI sequences and hope to give it a clean bill of health. They clearly took the latter option as the easy way out. Recalling all GMOs would be a massive political and financial decision and would also be a huge embarrassment to the regulators themselves. But it would leave very few GMO crops on the market and might even mean the end of crop biotechnology.

Regulators, if they had been doing their proper job, would have had a third option to gauge the seriousness of any potential GMO hazard. GMO monitoring - required by EU regulations - ought to have enabled them to find out if deaths, illnesses, or crop failures have been reported by farmers or health officials that could be correlated with the Gene VI sequence. Unfortunately, not one country has carried through on promises to officially and scientifically monitor any hazardous consequences of GMOs. EFSA regulators might now be regretting their failure to implement meaningful GMO monitoring. It would be a good question for European politicians to ask EFSA and for the board of EFSA to ask the GMO panel - whose job it is to implement monitoring - why monitoring was not carried out.

Searching the database for allergens, the main thrust of Podevin and du Jardin’s paper [1], is just a distraction, perhaps to reassure the public that Gene VI does not contain a known allergen, while the more serious potential hazards are buried in the false reassurance.

Let us look in more detail at Gene VI and its known properties, and their safety implications.

The many functions of Gene VI

Gene VI, like most plant viral genes, produces a protein that is multifunctional. It has four known roles in the viral infection cycle. The first is to participate in the assembly of virus particles. The second is to suppress anti-pathogen defenses by inhibiting a general cellular system called RNA silencing [9]. Third, Gene VI has the highly unusual function of transactivating (described below) the long RNA (the 35S RNA) produced by CaMV [10]. Fourth, unconnected to these other mechanisms, Gene VI has very recently been shown to make plants highly susceptible to a bacterial pathogen [11]; it does this by interfering with a common anti-pathogen defence mechanism in plants. We shall concentrate on those functions of Gene VI that have important safety implications.

Gene VI inhibits RNA silencing

RNA silencing is a mechanism for controlling gene expression at the level of RNA abundance [12]. It is also an important antiviral defence mechanism in both plants and animals, and therefore most viruses have evolved genes (like Gene VI) that disable it [13].

This attribute of Gene VI raises two obvious biosafety concerns: it will lead to aberrant gene expression in GMO crop plants, with unknown consequences, and it will interfere with the ability of plants to defend themselves against viral pathogens. There are numerous experiments showing that, in general, viral proteins that disable gene silencing enhance infection by a wide spectrum of viruses [6].

Gene VI is a unique transactivator of gene expression

Multicellular organisms make proteins by a mechanism in which only one protein is produced by each passage of a ribosome along a messenger RNA (mRNA). Once that protein is completed the ribosome dissociates from the mRNA. However, in a CaMV-infected plant cell, or as a transgene, Gene VI directs the ribosome to get back on an mRNA (reinitiate) and produce the next protein in line on the mRNA, if there is one. This property of Gene VI enables CaMV to produce multiple proteins from a single long RNA (the 35S RNA). Importantly, this function of Gene VI (which is called transactivation) is not limited to the 35S RNA. Gene VI seems able to transactivate cellular mRNA [14, 15]. There are likely to be thousands of mRNA molecules having a short or long protein coding sequence following the primary one. These secondary coding sequences could be expressed in cells where Gene VI is expressed. The result will presumably be production of numerous random proteins within cells. The biosafety implications of this are difficult to assess. These proteins could be allergens, plant or human toxins, or they could be harmless. Moreover, the answer will differ for each commercial crop species into which Gene VI has been inserted.

Gene VI interferes with host defences

A very recent finding, not known to Podevin and du Jardin [1], is that Gene VI has a second mechanism by which it interferes with plant anti-pathogen defences [11], the result is to make plants carrying Gene VI more susceptible to certain pathogens, and less susceptible to others. Obviously, this could impact farmers. Furthermore, the discovery of an entirely new function for gene VI while EFSA’s paper was in press, also makes clear that a full appraisal of all the likely effects of Gene VI is not currently achievable.

Is there a direct human toxicity issue?

When Gene VI is intentionally expressed in transgenic plants, they become chlorotic (yellow), deformed, and less fertile in a dose-dependent manner [16]. Plants expressing Gene VI also show gene expression abnormalities. These results indicate that the protein produced by Gene VI is functioning as a toxin and is harmful to plants [17]. As the known targets of Gene VI - ribosomes and gene silencing - are also present in human cells, it is reasonable to be concerned that the protein produced by Gene VI might be a human toxin. This is a question that can only be answered by future experiments.

Is Gene VI protein produced in GM crops?

There are two aspects to this question. One is the length of Gene VI accidentally introduced by developers. This appears to vary but most of the 54 approved transgenes contain the same 528 base pairs of the CaMV 35S promoter sequence, corresponding to approximately the final third of Gene VI. Deleted fragments of Gene VI are active when expressed in plant cells, and all functions of Gene VI are believed to reside in this final third. Thus, there is clear potential for unintended effects if this fragment is expressed, as some researchers have commented [4, 15, 18].

The second aspect of this question is what quantity of Gene VI could be produced in GMO crops? This can ultimately only be resolved by direct quantitative experiments. Nevertheless, we can expect the amount of Gene VI product will be specific to each independent insertion event. That is because significant Gene VI expression probably would require specific sequences, such as the presence of a gene promoter and an ATG (protein start codon). Commercial transgenic crop varieties can also contain supernumery copies of the transgene, including those that are incomplete or rearranged [5], and those could be important additional sources of Gene VI protein. The decision of regulators to allow such multiple and complex insertion events was always highly questionable, but the realization that the CaMV 35S promoter contains Gene VI sequences provides yet another reason to believe that complex insertion events increase the likelihood of a biosafety problem.

Even direct quantitative measurements of Gene VI protein in individual crop would not fully resolve the scientific questions, however. No-one knows, for example, what quantity, location or timing of protein production would be of significance for risk assessment, and so answers necessary to perform science-based risk assessment are unlikely to emerge soon.

Big lessons for biotechnology

It is perhaps the most basic assumption in all of risk assessment that the developer of a new product should provide regulators with accurate information about what is being assessed. Perhaps the next most basic assumption is that regulators independently verify this information. We now know, however, that for over twenty years neither of those simple expectations has been met. Major public universities, biotech multinationals, and government regulators everywhere, seemed not to have appreciated the possibility that the DNA constructs they were responsible for could harbour an extra viral gene.

This lapse occurred despite the fact that Gene VI was not truly hidden; the relevant information on the existence of Gene VI has been freely available in the scientific literature since well before the first biotech approval in the nucleotide sequence of CaMV published in 1980 [19]. We ourselves have offered specific warnings that viral sequences could contain unsuspected genes [6]. The persistent inability of regulatory risk assessment to incorporate longstanding and repeated scientific findings is highly worrying; all the more so that this is not an isolated event.

There exist other examples of commercially approved viral sequences having overlapping genes that were never subjected to risk assessment. These include numerous commercial GMOs containing promoter regions of the closely related virus figwort mosaic virus (FMV) not considered by Podevin and du Jardin [1]. Inspection of commercial sequence data shows that the commonly used FMV promoter overlaps its own Gene VI [20]. A third example is the virus-resistant potato NewLeaf Plus (RBMT-22-82). This transgene contains approximately 90 % of the P0 gene of potato leaf roll virus. The known function of this gene, whose existence was discovered only after US approval, is to inhibit the anti-pathogen defences of its host [21]. Fortunately, this potato variety was never actively marketed.

A further key point relates to the biotech industry and their campaign to secure public approval aided and abetted by a permissive regulatory environment. This has led to the repeated claim that GMO technology is precise and predictable; and further, their own competence and self-interest would prevent them from ever bringing potentially harmful products to the market, and only well studied and fully understood transgenes are commercialized. All of these claims have been exposed to be false in the revelations surrounding Gene VI.

What regulators should do now

Even now that EFSA's own researchers have belatedly considered the risk, no one can say whether the public has been harmed, though harm appears a clear scientific possibility. From the perspective of professional and scientific risk assessment, this situation represents a complete and catastrophic system failure.

The saga of Gene VI is not yet over. We have now carried out the retrospective risk assessment in full, something that EFSA has failed to do [1]. We show that the existing data clearly indicate a potential for significant harm. The only course of action consistent with protecting the public and respecting the science is for EFSA, and other jurisdictions, to order a total recall. This recall should also include GMOs containing the FMV promoter and its own overlapping Gene VI.

A slightly different version of this report was first published on Independent Science News, 21 January 2013, (http://independentsciencenews.org/commentaries/regulators-discover-a-hidden-viral-gene-in-commercial-gmo-crops/)

References

1. Podevin N and du Jardin P. Possible consequences of the overlap between the CaMV 35S promoter regions in plant transformation vectors used and the viral gene VI in transgenic plants. GM Crops and Food 2012, 3, 1-5.

2. Séralini G-E, Clair E, Mesnage R, Gress S, Defarge N, Malatesta M, Hennequin D, de Vendômois J-S. Long term toxicity of a Roundup herbicide and a Roundup-tolerant genetically modified maize. Food and Chemical Toxicology published online September 2012. http://dx.doi.org/10.1016/j.fct.2012.08.005

3. Saunders PT and Ho MW. GM cancers can no longer be ignored. Science in Society 56, 2-4, 2012.

4. De Tapia M, Himmelbach A, and Hohn T (1993) Molecular dissection of the cauliflower mosaic virus translation transactivator. EMBO J 12: 3305-14.

5. Wilson AK, Latham and RA Steinbrecher (2006) Transformation-induced mutations in transgenic plants: Analysis and biosafety implications. Biotechnology and Genetic Engineering Reviews 23: 209-234.

6. Latham JR, and AK Wilson (2008) Transcomplementation and Synergism in Plants: Implications for Viral Transgenes? Molecular Plant Pathology 9: 85-103.

7. Dasgupta R , Garcia BH,  Goodman RM (2001) Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes. Proc. Natl. Acad. Sci. USA 98: 4910-4915.

8. Ho MW and Cummins J. New evidence links CaMV 35S promoter to HIV transcription. Microb Ecol Health Dis 2009, 21, 172-4.

9. Haas G, Azevedo J, Moissiard G, Geldreich A, Himber C, Bureau M et al. Nuclear import of CaMV P6 is required for infection and suppression of the RNA silencing factor DRB4. EMBO J 2008, 27, 2102-12.

10. Park H-S, Himmelbach A, Browning KS, Hohn T, and Ryabova LA (2001). A plant viral ‘‘reinitiation’’ factor interacts with the host translational machinery. Cell 106: 723–733.

11. Love AJ , Geri C, Laird J, Carr C, Yun BW, Loake GJ et al. Cauliflower mosaic virus Protein P6 iInhibits Signaling responses to salicylic acid and regulates innate immunity. PLoS One 2012, 7(10), e47535.

12. Bartel P. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell 2004, 116, 281-297.

13. Dunoyer P and Voinnet O. The complex interplay between plant viruses and host RNA-silencing pathways. Curr Opinion in Plant Biology 2006, 8, 415–423.

14. Futterer J, and Hohn T. Translation of a polycistronic mRNA in presence of the cauliflower mosaic virus transactivator protein. EMBO J 1991, 10, 3887-3896.

15. Ryabova LA , Pooggin MH and Hohn T. Viral strategies of translation initiation: Ribosomal shunt and reinitiation. Progress in Nucleic Acid Research and Molecular Biology 2002, 72, 1-39.

16. Zijlstra C, Schärer-Hernández N, Gal S, Hohn T. Arabidopsis thaliana expressing the cauliflower mosaic virus ORF VI transgene has a late flowering phenotype. Virus Genes 1996, 13, 5-17.

17. Takahashi H, Shimamoto K, Ehara Y. Cauliflower mosaic virus gene VI causes growth suppression, development of necrotic spots and expression of defence-related genes in transgenic tobacco plants. Molecular and General Genetics 1989, 216, 188-194.

18. Kobayashi K and Hohn T. Dissection of cauliflower mosaic virus transactivator/viroplasmin reveals distinct essential functions in vasic virus Rreplication. J Virol 2003, 77, 8577–8583.

19. Franck A, Guilley H, Jonard G, Richards K and Hirth L . Nucleotide sequence of cauliflower mosaic virus DNA. Cell 1980, 2, 285-294.

20. Richins R, Scholthof H, Shepherd RJ. Sequence of figwort mosaic virus DNA (caulimovirus group). NAR 1987, 15, 8451-8466.

21. Pfeffer S, Dunoyer P, Heim F, Richards KE, Jonard G, Ziegler-Graff V. P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing. J Virol 2002, 76, 6815–6824.

There are 3 comments on this article so far. Add your comment
Rory Short Comment left 28th January 2013 18:06:30
This call for a total recall is a logically reasoned one. Unfortunately it conflicts with all sorts of vested interests and is therefore sadly unlikely to happen.The consumer must just stay away from GM foods.
John Fryer Comment left 28th January 2013 20:08:38
JUST DISCOVERED Virus fragments and bacteria frgaments have been in GMO crops since the inception in 1971. Professor Robert Pollack complained back then that using bacteria and virus parts in genetic engineering constituted the most dangerous experiment man could ever do. After a heated argument with the originators of this new technology we finally and more than 40 years on get a glimmer of recognition that this technology is not the same as paleolithic food diets and normal plant breeding methods. Have EFSA just discovered they have their own brains or will they rely on industry EXPERTS as normal?
David Llewellyn Foster Comment left 9th February 2013 09:09:58
Dr Ho ~ I have just been reading in Forbes, the all guns blazing dismissal by the self-possessed founder of the Genetic Literacy Project , Jon Entine http://blogs.forbes.com/jonentine/ to whom I addressed the following comment: As you are such a strong advocate for modified food, I'd be interested in your own diet, and whether you would be prepared to test your confident claims by consuming measured quantities of GMO's for say, a year and monitoring the effects, to document any alleged benefits or evident negative consequences. Also, I am curious to know why GMO's are so unwelcome in Europe, if the risks as you maintain are so completely negligible. I mean, it is not as though the entire European Union is composed of fanatical anthroposophists. Rudolph Steiner by the way, who was a student of Goethe, may well have been considered "lunatic" in some people's estimation, but he apparently escaped certification. Be that as it may, I disagree with P L Borst's contention, since Steiner developed his ideas about biodynamics independently, whereas Sir Albert Howard who is generally acknowledged as introducing organic principles to the UK, learned about soil health in India. Should you require more information about these traditional methods, I'm sure you would benefit from Dr Vandana Shiva's expertise. The British soil association is not composed of lunatics to the best of my knowledge, and I believe some Indians are even thought to be wise.

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