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ISIS Report 15/06/09

New Evidence Links CaMV 35S Promoter to HIV Transcription

The controversial promoter in all GM crops does enhance multiplication of disease-causing viruses; yet another reason why [1] GM is Dangerous and Futile (SiS 40). Dr. Mae-Wan Ho and Prof. Joe Cummins

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The CaMV 35S promoter that should never have been used

The cauliflower mosaic virus (CaMV) was the first plant virus found to contain DNA instead of RNA as genetic material [2]. The CaMV 35S promoter was exploited extensively to drive the expression of foreign genes in transgenic plants, so much so that it is present in all genetically modified (GM) crops commercially grown today.

In 2000, some six years after the first GM crop was commercialised, we drew attention to new and old findings that have been overlooked on the hazards of the CaMV 35S promoter; including its relationship to hepatitis B virus (HPV) and human immune deficiency virus (HIV); the discovery of its recombination hotspot that enhances both genomic rearrangement and the potential for horizontal gene transfer and recombination; and far from being specific for plants, the promoter is promiscuously active in all kingdoms of living organisms, including animal and human cells [3-5] (Cauliflower Mosaic Viral Promoter - A Recipe for Disaster?, Hazards of Transgenic Plants Containing the Cauliflower Mosaic Viral Promoter, CaMV 35S promoter fragmentation hotspot confirmed, and it is active in animals , ISIS scientific publications). We called for all GM crops containing the CaMV 35S promoter to be withdrawn [3]; and were met with an avalanche of criticisms, which we answered [4, 5] and abuse which we largely ignored.

Since then, at least two different research teams have confirmed that the CaMV 35S promoter is active in animal and human cells [6, 7]. And new evidence has emerged that the CaMV 35S promoter specifically induces transcription factors required for making CaMV and HIV genomes by reverse transcription [8]. (We thank ISIS member Ingrid Blank from South Africa for drawing our attention to the publication.}

The danger is that if the CaMV 35S promoter transfers into human cells, it would facilitate the transcription of HIV and activate other disease-causing viruses, including the human cytomegalovirus (HCMV) that is latent in high proportions of human populations

CaMV related to HPBV and HIV

CaMV is a pararetrovirus whose DNA genome is replicated by reverse transcription of an RNA intermediate. The CaMV genome consists of a circular double-stranded DNA molecule of ~8kb that forms a mini-chromosome in the nucleus of the host cell. Phylogenetically, CaMV belongs to a group of caulimoviruses most closely related to the hepadnaviruses of animals, which includes the human hepatitis B virus. The reverse transcriptase of CaMV, however, is most similar to that of retrotransposons belonging to the Gypsy group, and also to that of retroviruses such as HIV [9].

CaMV multiplication depends on specific host transcription factors

CaMV is transcribed by the host cell RNA polymerase II (RNAPII) into two major transcripts, the 35S and the 19SRNAs from their respective promoters. CaMV therefore, relies on host RNAPII to synthesize its viral RNA templates for reverse transcription (into more viral genomes) and translation of its coat and other proteins.

During transcription, the C-terminus of RNAPII is phosphorylated by cyclin-dependent kinases (CDKs). The CDKs and interacting cyclin T partners form the transcription elongation factor b (P-TEF-b) complexes that phosphorylate the RNAPII C-terminal domain to promote transcription elongation. In Arabidopsis thaliana, CDKC;1, CDKC;2, and their interacting cyclin T partners CyCT1:4 and CYCT1:5 are important for cauliflower mosaic virus infection.

Researchers led by Zhixiang Chen at Purdue University, West Lafayette, Indiana, in the United States used knockout mutants of the corresponding genes to investigate how the different factors affect CaMV infection [8]. They found that knockout mutants of cdkc:2 and cyct1:5 are highly resistant to CaMV infection, and the double mutant even more so. (Note: the convention is to represent the protein in capital letters and the corresponding genes in small italics.) Infection was delayed 3 to 4 days relative to wild type in the single mutants. At ~3 weeks after CaMV inoculation, almost 100 percent of the single mutants developed symptoms, but only 10 to 20 percent of the double mutant plants had symptoms, reaching 40 to 50 percent at 4 weeks.        

The mutants were not resistant to tobacco mosaic virus (a RNA virus) or cabbage leaf curl virus, a single-stranded DNA virus, neither of which replicates through reverse transcription.

CaMV 35S promoter depends on the same transcription factors

To test whether CDKC:2 and CYCT1:5 are important for the viral promoter activity, the researchers transformed the cdkc:2 and cyct1:5 mutants with a construct containing a b -glucuronidase (GUS) reporter gene driven by the CaMV 35S promoter. As controls, the same reporter gene construct was transformed into the wild type and also the cyct:2-1 mutant, which responds normally to CaMV. They looked for GUS gene expression and transcripts in 10 to 20 percent of independent wild-type or mutant transformants. The wild type and cyct;2-1 mutant had an average of ~265 units of GUS activity, and accumulated high levels of GUS transcripts. The single cdkc:2 and cyct1:5 mutants had ~66 units and correspondingly reduced levels of GUS transcripts. In the double cdkc:2 and cyct1:5 mutant, GUS activity was further reduced to ~35 units, and the reduced GUS activity was correlated with very low levels of GUS transcripts. Thus, CDKC;2 and CYCT1:5 are required for the high CaMV 35S promoter activity, and furthermore, they are induced by the CaMV35S promoter.

CaMV 35S promoter induce transcription factors for HIV and other pathogenic viruses

In humans, P-TEFb is required by HIV-1 for its transcription and replication [10]. The long terminal repeat of HIV-1 has minimal promoter activity in the absence of the viral Tat protein. The CaMV 35S promoter, on the other hand, is strongly active in plant cells in the absence of any viral protein [11]. Thus, the presence of CaMV 35S promoter effectively facilitates the transcription of HIV and other viruses. A more recent study reported that human T-lymphotropic virus type 1, another complex retrovirus, recruits P-TEFb to stimulate viral gene transcription [12]. No such close link of P-TEFb has been reported with other animal DNA viruses that also depend on RNAPII for transcription.

Thus, P-TEFb appears to be an evolutionary conserved target of complex retroviruses and pararetroviruses for transcription activation. Although human P-TEFb is not known to play a crucial role in the transcription of any human DNA virus, its over-expression in human cells can greatly activate the in vivo activity of the cytomegalovirus promoter [13]. Recently, it has been reported that replication of human cytomegalovirus is dependent on the cellular protein kinase CDK9 and cyclin T1 proteins [14]; which are similar respectively to the CDKC;2 and CYCT1:5 induced by the CaMV 35S promoter.

Within crop plants, the CaMV promoter is well known to alter the level and patterns of activity of adjacent tissue and organ-specific gene promoters [15]. In the absence of the 35S promoter sequence, the AAP2 promoter is active only in vascular tissue as indicated by the expression of the AAP2:Gus gene. With the 35S promoter sequence in the same T-plasmid used to transform tobacco plants, the resultant transgenic plants exhibit 2-fold to five-fold increase in AAP2 promoter activity and the promoter became active in all tissue types. Similar effects were found on the ovary specific AGL5:iaaM gene, and ovule- and early embryo-specific PAB5:barnase gene. In contrast, the NOS promoter did not have such effects. Thus, the 35S promoter sequence can convert an adjacent tissue and organic specific gene into a globally active promoter.

Furthermore, a 60-nucleotide region (S1) downstream of the transcription start site of the cauliflower mosaic virus 35S RNA was found to enhance gene expression [16]. The region contains sequence motifs with enhancer function that re normally masked by the powerful upstream enhancers of the promoter. A repeated CT-rich motif is involved both in enhancer function and interaction with plant nuclear proteins. The SI region can also enhance expression from heterologous promoters, and the researchers speculated that this could guarantee a “minimal basal activity of the promoter under every possible circumstance,” and could reflect a fundamental survival strategy for the virus.

These findings indicate that the CaMV 35S promoter, if transferred to human cells, could up-regulate specific transcription factors that will multiply and activate a number of common viruses that cause diseases including cancer.

References

  1. Ho MW. GM is dangerous and futile, we need organic sustainable food and energy systems now. Science in Society 40, 4-8, 2008.
  2. Zaitlin M, Palukaitis P. Advances in understanding plant viruses and viral diseases Annu Rev Phytopathol. 2000;38:117-143.
  3. Ho MW, Ryan A and Cummins J. Cauliflower mosaic viral promoter – a recipe for Disaster? Microbial Ecology in Health and Disease 1999. 11, 194-7. http://www.i-sis.org.uk/onlinestore/papers2.php#section5
  4. Ho MW, Ryan A and Cummins J. Hazards of transgenic plants with the cauliflower mosaic viral promoter. Microbial Ecology in Health and Disease 2000, 12, 6-11. http://www.i-sis.org.uk/onlinestore/papers2.php#section5
  5. Ho MW, Ryan A and Cummins J. CaMV35S promoter fragmentation hotspot confirmed and it is active in animals. Microbial Ecology in Health and Disease 2000, 12,: 189. http://www.i-sis.org.uk/onlinestore/papers2.php#section5
  6. Myhre MR, Fenton KA, Eggert J, Nielsen KM and Traavik T. The 35S CaMV plant virus promoter is active in human enterocyte-like cells. European food Research and Technology 2006, 222, 185-93.
  7. Tepfer M, Gaubert S, Leroux-Coyau M, Prince S and Houdebine L-M. Transient expression in mammalian cells of transgenes transcribed from the cauliflower mosaic virus 35S promoter. Environ Biosafety Res 2004, 3, 91-7.
  8. Cui X, Fan B, Scholz J and Chen Z. Roles of Arabidopsis cyclin-dependent kinase C complexes in cauliflower mosaic virus infection, plant growth and development. The Plant Cell 2007, 19, 1388-1402.
  9. Xiong Y. and Eickbush TH.. Origin and evolution of retroelements based upon their reverse transcriptase sequences. EMBO J. 1990. 9, 3353-62.
  10. Mancebo HS, Lee G, Flygare J, Tomassini J, Luu P, Zhu Y, Peng J, Blau C, Hazuda D, Prcie D and Flores O. P-TEFb kinase is required for HIV Tat transcriptional activation in vivo and in vitro. Genes Dev 1997, 11, 2633-44.
  11. Odell JU, Nagy F and Chua NJ. Identificaqtion of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 1985, 313, 810-12.
  12. Zhou M, Lu H, Park H, Wilson-Chiru J, Linton R and Brady JN. Tax interacts with P-TEFb in a novel manner to stimulate human T-lymphotropic virus type 1 transcription. J. Virol 2006, 80, 4781-91.
  13. Peng J, Zhu Y, Milton JT and Price DH. Identification of multiple cyclin subunits of human P-TEFb. Genes Dev 1998, 12, 755-62.
  14. Rechter S, Scott GM, Eickhoff J, Zielke K, Anerochs S, Müller R, Stamminger T, Rawlinson WD and Marschall M. Cyclin-dependent kinases phosphorylate the cytomegalovirus RNS export protein pUL69 and Modulate its nuclear localization and activity. J Biol Chem 2009, 284, 8605-13.
  15. Zheng X, Deng W, Luo K, Duan H, Chen Y, McAvoy R, Song S, Pei Y, Li Y.The cauliflower mosaic virus (CaMV) 35S promoter sequence alters the level and patterns of activity of adjacent tissue- and organ-specific gene promoters. Plant Cell Rep 2007, 26.1195-203.
  16. Pauli S, Rothnie HM, Chen G, He X, and Hohn T. The cauliflower mosaic virus 35S promoter extends into the transcribed region. J Virol 2004, 78, 12120-8.

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