Cauliflower Mosaic (CaMV)

Dr.Ramesh C.K Cauliflower Mosaic Virus (CaMV) • Cauliflower mosaic virus (CaMV) is a member of the genus , one of the six genera in the family which are pararetroviruses that infect . • Pararetroviruses group due to its mode of replication via reverse transcription of a pre- genomic RNA intermediate. just like but the viral particles contain DNA instead of RNA. • True retroviruses are not known in plants; however, pararetroviruses (caulimoviridae) share many retroviral properties, replicating by transcription in the nucleus followed by reverse transcription in the . • Pararetroviruses have circular DNA genomes that do not integrate into the host genome, and display several unique expression strategies. • CaMV infects mostly plants of the family (such as cauliflower and turnip) but some CaMV strains are also able to infect species of the genera and .

• CaMV induces a variety of systemic symptoms such as mosaic, necrotic lesions on leaf surfaces, stunted growth, and deformation of the overall plant structure.

• CaMV is transmitted by aphid species such as . Once introduced within a plant host cell, virions migrate to the nuclear envelope of the plant cell. Structure • The CaMV particle is an icosahedron with a diameter of 52 nm built from 420 protein (CP), which surrounds a solvent-filled central cavity. • In addition to capsid proteins, caulimoviruses are also surrounded by virus associated proteins. These proteins are responsible for assisting in the binding of the virus to DNA • CaMV contains a circular double-stranded DNA molecule of about 8.0 kilobases, interrupted by nicks that result from the actions of RNAse H during reverse transcription. • After entering the host cell, these single stranded "nicks" in the viral DNA are repaired, forming a supercoiled molecule that binds to . For this purpose the virus has two promoters (35S and 19S) in front of its genes,which the plant cell believes to be its own.Furthermore, these promoters override the plant's own regulatory system, as theyare constitutive, i.e. they are constantly switched on and can't be regulated orswitched off by the plant. • This DNA is transcribed into a full length, 35S RNA and a subgenomic 19S RNA.

• Cauliflower mosaic virus is well known for its strong constitutive 35S . This promoter runs transcription for the entire cauliflower mosaic virus genome. It is because of its efficiency, it is commonly used for studying transgenic plants in relation to gene transfer vectors. • The 600 nucleotide leader sequence of the 35S promoter consists of 8 open reading frames that each possess different functions . • In biology, an ORF or coding sequence of a gene begins with the start codon, continues with the amino acid codons, and ends at a termination codon. However, a gene is more than the respective ORF, with sequences upstream of the start codon and sequences downstream of the stop codon. • There are 6 major coding regions and 2 minor coding regions.

• I functions to produce movement proteins to assist progeny to pass through plasmodesmata into uninfected cells. • Open reading frame II produces aphid transmission factors which is required for the transmission of the virus from the aphid to the plant. • Open reading frame III, which previously had no assigned function and is a minor coding region, is believed to promote DNA binding as well as produce structural proteins. • ORF IV is responsible for the production of capsid proteins that surround and protect viral genomes. • ORF V produces proteins that have proteinases as well as participates in the process. • Open reading frame VI produces trans activator proteins that promote formation of inclusion bodies. Inclusion bodies are places of viral multiplication needed to produce many progeny virions. • The second minor coding region, open reading frame VII, has an unknown function but is speculated to have some function relating to targeting new viruses and bringing them to inclusion bodies . • ORF1 – Movement Protein • ORF2 – Aphid/Insect Transmission Factor • ORF3 – Virion-associated protein Structural protein, DNA- binding capabilities • ORF4 – Capsid Protein • ORF5 – pro-pol Protease, bifunctional Reverse Transcriptase and RNaseH • ORF6 – Transactivator/viroplasmin Inclusion Body Formation/Trafficking; Possibly more functions • ORF7/8 – Unknown (Appears to not be required for infection) Replication • The viral dsDNA genome is introduced via an aphid bite. • Once inside the host, the viral genome is un-encapsulated and its DNA is transcribed by the host RNA polymerase to make a copy of viral RNA. Host reverse transcriptase then transcribes copies viral RNA back to DNA. • Viral RNA also is used to produce gene products such as new capsid proteins and movement proteins in combination with viral DNA to assemble progeny virions • Capsid proteins house genetic material of viruses and offer protection. • Movement proteins interact with plasmodesmata in order to allow for transport that otherwise would not occur. • Movement proteins then facilitate movement of progeny virions into uninfected cells of the host plant via plasmodesmata. Promoter of the 35S RNA • The promoter of the 35S RNA is a very strong constitutive promoter responsible for the transcription of the whole CaMV genome.

• 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 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.

• Recent study has indicated that the CaMV 35S promoter is also functional in some animal cells, although the promoter elements used are different from those in plants.

• The 35S RNA is particularly complex, containing a highly structured 600 nucleotide long leader sequence with six to eight short open reading frames (ORFs). Use of CaMV in gene transfer

• For effective transmission of CaMV the foreign DNA must be encapsulated in viral protein and the inserted gene should not interfere with native assembly of virus CaMV does not contain any non coding region wherein foreign DNA can be inserted • But there a are two genes namely gene II and gene VII have no essential functions for the virus So the gene of interest can be replaced in the place of these two genes • Attractive feature is infection is systemic • Use of CaMV as a vector pertains to the bacterial plasmid dhfr (dihydroxy folatereductase) gene inserted in the place of gene II and successfully expressed in plants • This dhfr gene is needed for providing resistance to • methotrexate( inhibitor of dhfr and extremely toxic to plants) • Cauliflower mosaic virus has a very important function in biotechnology. It is because of its efficient promoter that is used to produce cloned genes, that it can be used to create transgenic plants that can take use of this efficiency. Limitation of CaMV

• Limited capacity for insertion • The small margin for error when inserting genes only in open reading frame II and open reading frame VII. • Infective capacity is lost if few hundreds of nucleotides are introduced • Because of its exceeding natural genome size they are not effectively suitable for gene transfer