Exploitation of plant viruses as transformation vectors by massive infection may be harmful and even deleterious to the target plants. It is still however able to express and produce foreign proteins.
Plant viruses must exhibit some of the exemplary features before they are considered as vectors. They should extend their broader host-range, spread of seed transmission and carry additional copies of gene of interest.
Several viral vectors require suitable modification in order to accommodate extra nucleic acid and also aggressive in infection process. Although several groups of viruses have been identified, some moderate progresses have been made only in two groups. These two groups are Caulimo virus and Gemini virus, which have DNA genome as genetic material.
Cauliflower Mosaic Virus (Caulimovirus):
Cauliflower mosaic virus (CamV) belongs to the group caulimovirus, can be used as potential candidate to deliver foreign gene into the plant. It is perhaps the best studied viruses among plant virus, which infects several members belonging to Cruciferae family. Cauliflower mosaic virus contains circular double helical DNA as genetic material.
As an infective agent, can cause disease in wide range of commercially important cultivated crops. Cauliflower mosaic DNA has been subjected to a wide range of manipulation. This was the only and first virus to be manipulated and used as a favourable choice for genetic engineering work. Elucidation of 8 kb CamV reveals that, it contains six major and two minor reading frames (Fig. 14.12).
Presence of ribonucleotide in DNA leads to the conclusion that CamV replication involves the synthesis of negative DNA strand by employing reverse transcriptase and followed by synthesis of positive DNA strand. Once the synthesis of double strand DNA completes, it is then packed into viral particles and continue the cycles of transcription and translation.
Cauliflower mosaic virus can be used as a potential vector due to the infective nature of its genetic material. This could be proved by applying viruses on the leaf rubbed with abrasive material. The CamV cannot accommodate foreign DNA, if the size exceeds its normal size. The inserted DNA may destabilize infectious nature of the virus.
Other constraints are the packaging of genome and limitation of the insertion of foreign DNA. Despite the marginal constraints, CamV genome can be packed in nucleosome and is able to undergo transcription by plant RNA polymerase II. The genome of CamV consists of six major and two minor open reading frames (ORF), in tightly packed arrangements.
The two ORF regions, one (ORFII) codes for insect transmission factor and other (PRF VII) with unknown functions can be replaced with gene of interest.
It is a DNA virus, known to infect wide range of economically important and agriculture crops of monocotyledonous and dicotyledonous plants. Several diseases such as maize streak virus and curly top virus are caused by Gemini virus. The genome is single Stranded Circular DNA and its replication takes place by DNA immediately.
Tobacco Mosaic Virus Based Expression Vector:
Tobacco mosaic virus (TMV) is a RNA virus and shows several advantages by designing expression vector. TMV was the first virus to be purified and sequenced. As far as biohazard is considered TMV could be used as a comparatively safe recombinant virus in the field.
The coat protein of the TMV is one of the most accumulated proteins in plants reachable upto 10% of the dry weight of infected plant. Approximately, under ideal condition, 2000 kg tobacco protein can be produced per acre per year. Moreover, TMV can be purified in crystalline form in substantial quantity by simple methodology.
The single stranded RNA genetic material of TMV encloses 6300 nucleotides with four open reading frames (Fig. 14.13). The filamentous nature of the RNA virus is determined by the length of the viral nucleic acid. Both 183 kD read through protein and 126 kD coat proteins are translated from the 5′ end of the genomic RNA.
These two proteins form replicase complex. In addition to these two proteins, the 30-kD movement protein and 17.5-kD capsid proteins are translated at 3′ region of sub-genomic mRNA during replication.
TMV can be subjected to a wide range of manipulation by replacing the viral coat protein with a foreign protein, for example, replacement of coat protein with reporter gene chloramphenical acetyl transferase (CAT) resulted in a free-RNA virus that generated high CAT activity. Improvisation of TMV vector was achieved by placing CAT gene under the control of a coat protein sub-genomic promoter of TMV into the entire TMV genome.
This recombinant virus produced third sub-genomic mRNA and this was translated into CAT enzyme with high activity. Similarly, another TMV hybrid expression vector TB2 was designed in which coat protein gene and neomycin phosphotransferase marker gene was placed under the control of sub-genomic promoters of TMV coat protein and ORSV, respectively.
This was referred as extra gene vector. TB2 effectively produced the foreign protein without any major constraints. Another extra gene in TMV based vector, 4GD-PL, was developed from tomato green mosaic virus. The 4GD-PL vector was able to express foreign proteins systematically throughout plants.
All these studies demonstrated that proximity of genes to the 3′-untranslated region of the genome increases efficiency of their translation. Another improved TMV vector, 30B, was designed in which the start codon (AUG) of the capsid protein was mutated to AGA, and restriction cloning sites were engineered (40 nucleotide) to provide a full-size sub-genomic RNA promoter.
Possibility of satellite RNA to be used as vector has been considered. They vary in their size between 0.27 and 1.6 kb. They are not indispensable for virus replication. However, their functions can alter pathogenecity of virus.
Cow Pea Mosaic Virus Expression Vector:
Cow pea mosaic virus (CpmV) is also a RNA virus and infects species of legumes. There are two separate positive strand-RNA molecules present in the genetic material of CpmV. The number of nucleotides present in the RNA I and RNA II strand is 5889 and 3480, respectively. Although RNA I alone can replicate on its own but both RNAs are indispensable for infectivity.
The proteins involved in the replication of the virus are encoded by RNA I whereas movement proteins are encoded by RNA II. CpmV capside of both large (L) and small (S) coat protein of 30 copies each are in isohedral symmetry. The two capsid proteins are folded into three antiparallel β-barrel structures.
In the construction of CpmV expression vector, preference was given to the replacement of stable chimeras by insertion of foreign sequences rather than replacement for native residues.
Therefore, in the construction of viable and well refined CpmV vector, precise site of insertion of foreign sequence was given a prime choice by introducing foreign DNA sequence into βB-βC loop of the S protein for most chimeras foreign sequences inserts immediately upstream of proline 23 of the S protein. In view of propagating the chimeras, engineered pCP2 and pCP1 are linearised and inoculated on cow pea plants. (Fig. 14.14)