CAULIFLOWER MOSAIC VIRUS(CamV)

Cauliflower mosaic virus

Cauliflower mosaic virus
Virus classification
Group:	Group VII (dsDNA-RT)
Family:	Caulimoviridae
Genus:	Caulimovirus
Species:	Cauliflower mosaic virus

Cauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of the six genera in the Caulimoviridae family, pararetroviruses that infect plants (Pringle, 1999). Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA

Structure

The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 capsid protein (CP) subunits arranged with a triangulation T = 7, which surrounds a solvent-filled central cavity (Cheng et al., 1992). It contains a circular double-stranded DNA molecule of about 8.0 kB, interrupted by sitespecific discontinuities resulting from its replication by reverse transcription. After entering the host, the single stranded nicks in the viral DNA are repaired, forming a supercoiled molecule that binds to histones. This DNA is transcribed into a full length, terminally redundant 35S RNA and a subgenomic 19S RNA.

Genome

The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome. It is well known for its use in plant transformation. It causes high levels of gene expression in dicot plants. However, it is less effective in monocots, especially in cereals. The differences in behavior are probably due to differences in quality and/or quantity of regulatory factors. The promoter was named CaMV 35S promoter ("35S promoter") because the coefficient of sedimentation of the viral transcript, whose expression is naturally driven by this promoter, is 35S. It is one of the most widely used, general-purpose constitutive promoters. It was discovered at the beginning of the 1980s, by Chua and collaborators at the Rockefeller University.
The 35S RNA is particularly complex, containing a highly structured 600 nucleotide long leader sequence with six to eight short open reading frames This leader is followed by seven tightly arranged longer ORFs that encode all the viral proteins (reviewed by Hohn and Fütterer, 1997). The mechanism of expression of these proteins is very special. The ORF VI protein (encoded by the 19S RNA) controls translation reinitiation of major open reading frames on the polycistronic 35S RNA, which is normally only happens on bacterial mRNA's. TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 (Park et al., 2001).

Genomic map of CaMV

• ORF I - Movement protein
• ORF II - Insect transmission factor
• ORF III
• ORF IV - Capsid protein
• ORF V - Protease, reverse transcriptase and RNaseH
• ORF VI - Translational activator / Inclusion body protein
• ORF VII - Unknown (dispensable)

Replication

CaMV replicates by reverse transcription. Initially all the gaps present in the genome are sealed and the covalently closed DNA then associates with host histones to form a super coiled mini chromosome. Transcription of this mini chromosome produces 35s RNA which translates protein as well as forming dsDNA by the process of reverse transcription. New viral particles are produced which are targeted to inclusion bodies & are released outside.
The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is inserted into transgenic plants in a form which is different to that found when it is present in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.
CaMV contains about 8 kb double-strand DNA genome and produces spherical particles. CaMV infections are systemic, and even its DNA is infectious when inoculated on abraded plant surfaces. The CaMV genome has 8 tightly packed genes, of which only two small genes, genes II and VII, are nonessential; as a result, only these two genes can be replaced/deleted without a loss of infectivity. In addition, modified CaMV genomes exceeding the natural genome size (8024 bp) by even a few hundred bp are not packaged into virions. These two factors seriously limit the size of DNA insert clonable in CaMV. Bacterial dhfr (dihydrofolate reductase) gene was inserted in the CaMV genome in place of gene II, and was successfully expressed in plants. EncyclopediaCauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of the six genera in the Caulimoviridae family, pararetroviruses that infect plants Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA .Structure Cauliflower mosaic virus (CaMV) is the type member of the caulimoviruses, one of the six genera in the Caulimoviridae family, pararetroviruses that infect plants. Pararetroviruses replicate through reverse transcription just like retroviruses, but the viral particles contain DNA instead of RNA
Structure
The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 capsid protein (CP) subunits arranged with a triangulation T = 7, which surrounds a solvent-filled central cavity (Cheng et al., 1992). It contains a circular double-stranded DNA molecule of about 8.0 kB, interrupted by sitespecific discontinuities resulting from its replication by reverse transcription. After entering the host, the single stranded nicks in the viral DNA are repaired, forming a supercoiled molecule that binds to histones. This DNA is transcribed into a full length, terminally redundant 35S RNA and a subgenomic 19S RNA.Genome
The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome. It is well known for its use in plant transformation. It causes high levels of gene expression in dicot plants. However, it is less effective in monocots, especially in cereals. The differences in behavior are probably due to differences in quality and/or quantity of regulatory factors. The promoter was named CaMV 35S promoter ("35S promoter") because the coefficient of sedimentation of the viral transcript, whose expression is naturally driven by this promoter, is 35S. It is one of the most widely used, general-purpose constitutive promoters. It was discovered at the beginning of the 1980s, by Chua and collaborators at the Rockefeller University.
The 35S RNA is particularly complex, containing a highly structured 600 nucleotide long leader sequence with six to eight short open reading frames (ORFs)
 This leader is followed by seven tightly arranged longer ORFs that encode all the viral proteins . The mechanism of expression of these proteins is very special. The ORF VI protein (encoded by the 19S RNA) controls translation reinitiation of major open reading frames on the polycistronic 35S RNA, which is normally only happens on bacterial mRNA's. TAV function depends on its association with polysomes and eukaryotic initiation factor eIF3 .
 ORF I Movement protein ORF II Insect transmission factor ORF III ORF IV Capsid protein ORF V Protease, reverse transcriptase and RNaseH ORF VI Translational activator / Inclusion body protein ORF VII Unknown (dispensable)Replication
CaMV replicates by reverse transcription. Initially all the gaps present in the genome gets sealed. The covalently closed DNA associates with host histones to form a super coiled mini chromosome. Transcription of the former produces 35s RNA which translates protein as well as forms dsDNA by the process of reverse transcription. New viral particles are produced which gets targeted to inclusion body & is released outside. The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant.
It is inserted into transgenic plants in a form which is different to its naturally occurring state arising in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible. Cauliflower Mosiac Virus (CaMV) -
CaMV contains about 8 kb double-strand DNA genome and produces Spherical particles. CaMV infections are systemic, and even its DNA is infectious when inoculated on abraded plant surfaces.

CaMV genome has 8 tightly packed genes, of which only two small genes, genes II and VII, are nonessential; as a result, only these two genes can be replaced/deleted without a loss of infectivity. In addition, CaMV genomes exceeding the natural genome size (8024 bp) by even a few hundred bp are not packaged into virions. These two features seriously limit the size of DNA insert clonable in CaMV. Bacterial dhfr (dihydrofolate reductase) gene was inserted in the CaMV genome in place of gene II, and was successfully expressed in plants.

The CaMV 35S Promoter

The CaMV 35S promoter is being used in almost all GM crops currently grown or tested, especially GM maize. It is the promoter of choice for plant genetic engineering, as it is a strong and constitutive promoter. Failure to recognise or to ignore its capacity to be universally active in almost any organism is irresponsible and careless and shows a serious lack of scientific rigour and commitment to safety.
Any safety assessment can be expected to be flawed that does not resort to actual laboratory test of the capacity of bacteria and fungi to utilise the particular genes and their promoters.

Risks Associated with the Use of the Cauliflower Mosaic Virus Promoter in Transgenic Crops

The Cauliflower Mosaic Virus promoter (CaMV 35S) is used in most transgenic crops to activate foreign genes which have been artificially inserted into the host plant. It is potentially dangerous.

            It is inserted into transgenic plants in a form which is different to its naturally ocurring state arising in its natural brassica plant hosts. This enables it to operate in a wide range of host-organism environments which would otherwise not be possible.

The Use of Cauliflower Mosaic Virus

The majority of crop plant constructions for herbicide or disease resistance employ a Promoter from cauliflower mosaic virus (CaMV). Regardless of the gene transferred, all transfers require a promoter, which is like a motor driving production of the genes' message. Without a promoter, the gene is inactive, but replicated, CaMV is used because it is a powerful motor which drives replication of the retrovirus and is active in both angiosperms and gymnosperms. The CaMV pararetrovirus replication cycle involves production vegetative virus containing RNA which is reverse transcribed to make DNA similar to HIV, Human Leukemia Virus and Human hepatitis B.

The CaMV promoter is preferred above other potential promoters because it is a more powerful promoter than others and is not greatly influenced by environmental conditions or tissue types. CaMV has two Promoters 19S and 35S, of these two the 35S promoter is most frequently used in biotechnology because it is most powerful. The 35S promoter is a DNA (or RNA) sequence about 400 base pairs in length. The use of the CaMV promoter in plants is analogous to the use of retrovirus LTR promoters in retrovirus vectors used in human gene therapy. The majority of human gene therapy trials employ LTR promoters to provide motors to activate genes.

Antisense genes are genes constructed to have a complementary sequence to a target gene, thus producing a product that combines with a gene message to inactivate it. Antisense is analogous to an antibody which combines with an antigen like a key fitting a lock. Antisense is being used to treat human cancer and HIV infection. Antisense is used to prevent spoilage in tomatos, either by targeting an enzyme degrading cell walls (polygalacturonase), or production of ethylene a hormone promoting ripening (P. Oeller et al. Genetic Engineering 49, 1989; R. Fray and D. Grierson, Trends Genetics 9, 438, 1993). Most frequently antisense targets production of a chemical metabolite producing ethylene. The antisense gene also influenced polyamines spermine and spermidine production through S-adenosylmethionine. The implication is that the plant antisense gene product should be tested in animals to ensure that critical functions including gene replication, sperm activity and gene imprinting are not disrupted.

The perceived hazards of CaMV in crop plants include the consequences of recombination and pseudo recombination. Recombination is the exchanges of parts of genes or blocks of genes between chromosomes. Pseudorecombination is a situation in which gene components of one virus are exchanged with the protein coats of another. Frequently viruses may incorporate cellular genes by recombination or pseudorecombination, it has been noted that such recombinants have selective advantages

It has been shown that the CaMV genes incorporated into the plant (canola) chromosome recombine with infecting virus to produce more virulent new virus diseases. The designers of the experiment questioned the safety of transgenic plants containing viral genes. Recombination between CaMV viruses involves the promoter and may take place either between DNA and DNA or RNA and RNA and frequently creates more severe Infections than either parent. Recently related experiments suggest altered plants may breed deadlier diseases . DNA copies of RNA Viruses are frequently propagated using the CaMV 35S promoter to drive RNA virus production . In conclusion CaMV promoters recombine with the infecting viruses to produce virulent new diseases. CaMV viruses and promoter may incorporate genes from the host creating virulent new diseases.

CaMV can recombine with insect viruses and propagated in insect cells . Thus it is likely that as large numbers of humans consume CaMV modified tomatos recombination between CaMV and hepatitis B viruses will take place creating a supervirus propagated in plants, insects and humans.

Plant biotechnology has grown out of recombinant DNA research that began in the early 1970's. The special nature of recombination has been debated since that time. In recent years, government regulators on the American and European continents, under pressure from well-funded lobby representing the biotechnology industry, have chosen to ignore the special nature of recombination. They have chosen instead to base regulations on existing frameworks for toxic chemicals and pathogenic organisms. Ignoring the special nature of recombination is likely to have costly, if not terminal, environmental consequences. A worst-case example includes the complete cloning of Human Immunodeficiency Virus (HIV) on an E. coli plasmid. When the plasmid is used to transform animal cells, intact HIV viruses are released from the cells. A careless (but legal) release of HIV bacteria to the environment would allow the plasmid to transfer to Salmonella as well as E. coli. Thus, numerous mammals and birds could contain HIV bacteria which could transform the animals, which would in turn produce HIV particles unable to target the animals T-cell receptors but easily transmitted to humans. When all the animals are HIV carriers, human survival would be marginal. The special concerns of recombination in plant biotechnology include the viruses and bacteria used in crop plant construction and gene flow between related crop plants and weeds in the field.

Currently most experts agree that virus diseases such as influenza gain strength for epidemics by alternating between animal hosts (pigs and ducks) and man. Epidemics begin when rare combinations appear in large closely associated populations such as in asia. CaMV can propagate in plant and insect hosts following recombination. It may not be outlandish to predict that CaMV may recombine with related Hepatitis B or for that matter HIV to create a most powerful disease. The salient feature being large number of people or animals consuming large numbers of virus genes incorporated into crop plants making up a major part of human and animal diet.

The use of CaMV promoter is seldom an issue in reviews of safety of gene tinkered crops. Few people have raised the important issue and more often than not their concerns are ignored by government officials "protecting" public safety. This omission may be a fatal one because it has potentially the most damaging impact, and the one perceived at the beginning of gene splicing.