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ORIGINAL ARTICLE

New evidence links CaMV 35S promoter to HIV transcription

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Pages 172-174 | Received 06 Jul 2009, Accepted 04 Nov 2009, Published online: 26 Dec 2009

Abstract

New evidence raises the possibility that the CaMV 35S promoter in practically all transgenic crops grown commercially may enhance multiplication of disease-associated viruses including HIV through induction of proteins required for their transcription.

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 (Citation1). 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 grown commercially today.

In 2000, some 6 years after the first GM crop was commercialized, 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 (Citation2–4). We called for all GM crops containing the CaMV 35S promoter to be withdrawn (Citation2) and were met with an avalanche of criticisms, which we answered (Citation3,Citation4), and abuse, which we largely ignored.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 (Citation5,Citation6). And new evidence has emerged that the CaMV 35S promoter specifically induces transcription factors required for making CaMV and HIV genomes by reverse transcription (Citation7).

The danger is that if the CaMV 35S promoter transfers into human cells, it may 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 ∼8 kb 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 hepadna-viruses 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 (Citation8).

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 19S RNAs 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 (Citation7). They found that knockout mutants of cdkc:2 and cyct1:5 are highly resistant to CaMV infection, and the double mutant even more so. Infection was delayed by 3–4 days relative to wild type in the single mutants. At ∼3 weeks after CaMV inoculation, almost 100% of the single mutants developed symptoms, but only 10–20% of the double mutant plants had symptoms, reaching 40–50% at 4 weeks.

The mutants were not resistant to tobacco mosaic virus (an 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 β-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 10–20% 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 induces transcription factors for HIV and other pathogenic viruses

In humans, P-TEFb is required by HIV-1 for its transcription and replication (Citation9). 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 (Citation10). 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 (Citation11). 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 pararetro-viruses 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 (Citation12). Recently, it has been reported that replication of human cytomegalovirus is dependent on the cellular protein kinase CDK9 and cyclin T1 proteins (Citation13), 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 (Citation14). 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 twofold to fivefold 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 organ-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 (Citation15). The region contains sequence motifs with enhancer function that are normally masked by the powerful upstream enhancers of the promoter. A repeated CT-rich motif is involved in both 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 suggest 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.

Acknowledgments

We thank Ingrid Blank, South African member of the Institute of Science in Society, for drawing our attention to the publication by Cui et al. (Citation7).

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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