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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 . 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
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
. (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 .
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 . 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
In humans, P-TEFb is required by HIV-1 for its transcription and replication
. 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 .
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 . 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 . Recently, it has been reported that replication
of human cytomegalovirus is dependent on the cellular protein kinase CDK9 and
cyclin T1 proteins ; 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 . 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 . 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
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