Plant virus based

vectors
Lecture by:
Dr. Navami Pai
INTRODUCTION-
EXPRESSION VECTORS

• An expression vector, is usually a plasmid or virus designed for gene

expression in cells. The vector is used to introduce a specific gene
into a target cell, and can commandeer the cell's mechanism for
protein synthesis to produce the protein encoded by the gene.
Expression vectors are the basic tools in biotechnology for the
production of proteins.
• The vector is engineered to contain regulatory sequences that act as
promoter regions and lead to efficient transcription of the gene
carried on the expression vector.
• The goal of a well-designed expression vector is the efficient
production of protein, and this may be achieved by the production
of significant amount of stable messenger RNA, which can then be
translated into protein.
• The expression of a protein may be tightly controlled, and the
protein is only produced in significant quantity when necessary
through the use of an inducer, in some systems, however the protein
may be expressed constitutively
Expression cassette

• A typical gene expression cassette has the following region beginning with the 5’end:

i).Promoter: For transcription initiation

ii). Enhancer/silencer: Concerned with regulation of gene
iii). Transcriptional start site or cap site: From here initiation of transcription take place
iv). Leader sequence: It is untranslated region
v). Initiation codon
vi). Exons
vii). Introns
viii). The stop codon
ix). A second untranslated region, and
x). Poly A tail
• Promoter is a region of DNA sequence which helps in the
transcription of a particular gene. This contains specific DNA
sequences as well as response elements which provide a secure initial
binding site for RNA polymerase. These proteins called transcription
factors that recruit RNA polymerase.
• The CAAT and TATA boxes represent consensus sequences within
promoter for RNA polymerase II.
• The CAP site on a DNA template is where transcription
begins. It corresponds to the nucleotide at the 5'-end of the
RNA transcript which accepts the 7-methylguanine cap.
• ATG (AUG in mRNA) is initiation codon for mRNA translation, and
mark the beginning of coding sequence of the gene. A sequence
between the cap site and ATG is not translated and form the 5'-leader
sequence of mRNA.
• Codon TAG/TAA/TGA are chain terminating codon and it is followed
by a stretch of nontranslated region.
• At the end, poly-adenylation site is present which denotes the end of
transcription.
Plant expression systems

• DNA Vectors
1. Cauliflower mosaic virus (CMV)-double stranded DNA viruses
2. Gemini viruses- single stranded DNA viruses
• RNA Vectors
1. Tobacco mosaic virus (TMV)-single stranded RNA virus
Geminiviruses

• Gemini viruses are small circular DNA viruses that replicate in

plant nuclei.
• Geminiviruses are being used as convenient
autonomously replicating vectors for foreign gene
amplification in plants.
• Using tissue culture techniques, they have been
adapted for the analysis of the regulation of gene
expression in a wide range of hosts, including both
mono- and dicotyledonous species.
• In monocotyledonous plants that are particularly
recalcitrant to transformation, geminivirus symptom-
induction has been used as a sensitive marker for DNA
• Geminiviruses have a small genome of ∼2.8 Kb.
• They are transmitted via insect vectors like
whitefly Bemisia tabaci and leaf hoppers.
Features of geminiviruses

(1)geminiviruses are able to infect a wide range of host

plant species making these as efficient vectors for
multiple hosts at once;
(2)require only one protein, Rep (replication associated
protein; RepA in case of mastreviruses), to initiate
replication inside the host cell.
• replicate inside host cells via homologous
recombination-dependent replication, in addition to
rolling circle replication.
• replicate efficiently inside host cell and produce high
amounts of replicons, in turn producing a lot of SSNs
and target sequence.
Geminiviral vectors and their
functional relevance.

• Virulence factors like the viral movement protein (MP)

and coat protein (CP) are deleted and replaced with the
donor DNA which give rise to “geminivectors.”
• These vectors have multidirectional functionalities in
which they primarily act as:
• (A) gene transfer vectors, where vectors are used for the
transient expression of genes of interest, production of
specific proteins, and participation and annotation of unknown
gene characteristics, among others;
• (B) CRISPR/Cas9-based genome repair: where they are used
for template repair in the host genome, while also essentially
participating in genome editing; and
• (C) biopharmaceuticals: where production of important
vaccines has been successfully conducted using
geminivectors. Monoclonal antibodies have been prepared for
the deadly Ebola virus, and antigen production for hepatitis B
and HIV has been successful.
Cauliflower mosaic virus

• Caulimoviruses are a group of plant viruses which have double-

stranded DNA.
• These viruses have a very narrow host range. One such virus is
Cauliflower Mosaic Virus (CaMV).
• This virus infects plants of the family Cruciferae.
• The caulimoviruses are isometric particles and are 50 nm in
diameter.
• The cells of the infected plants accumulate virus-containing
proteinaceous inclusion bodies (i.e., viroplasma).
• These infected regions are regarded as the sites of virus
assembly.
• In order to get itself and its DNA replicated (multiplied) within a plant cell,
the virus must trick the plant's own molecular ‘machinery' to do this
task.
• 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 they are constitutive, i.e. they are constantly switched on and can't be
regulated or switched off by the plant.
• The CaMV 35S well known promoter is being used in almost all GM
crops currently grown or tested, especially GM maize. It is the promoter
of selection for plant genetic engineering, as it is a strong and constitutive
promoter.
CaMV genome

• The DNA of the CaMV particles is a circular, double-

stranded molecule which is 8024 bp long.
• It has three discontinuities (gaps) at specific sites on
each strand.
• Two gaps are present on one strand whereas other
strand has only one gap.
• CaMV is transferred in nature by insects but its DNA can infect
plants by simply rubbing onto their leaves. The free genome of
CaMV is infectious and it has no tissue restriction. It has six genes,
• Gene I produces the factors needed for cell to cell spread of the
virus.
• Gene II is responsible for the production of the aphid transmission
factor. (gene that can be replaced)
• Gene III encodes a virion structural protein
• Gene IV produces the coat of protein.
• Gene V produces the enzyme reverse transcriptase.
• Gene VI produces the inclusion body protein.
• Schematic representation of (A) a genetically modified
(GM) plant construct and (B) the Cauliflower mosaic
virus (CaMV) genome, highlighting the shared P35S
promoter sequence.
• (A) In a GM plant, a gene conferring a specific trait (in blue) is
inserted, and this transgene is expressed via the 35S promoter,
P35S (in yellow) and the Nos terminator, TNOS (in orange).
• (B) The coding sequences of the seven CaMV genes (I-VII)
encoding the seven proteins (Pl-P7) are represented with blue
arrows. The CaMV 35S promoter (P35S) is shown in yellow and the
19S promoter (P19S) is shown in orange. P35S overlaps with the
end of gene VI. Gene IV overlaps with the end of gene III and the
beginning of gene V.
Advantages

• The promoter sequences of the DNA of CaMV are very

efficient.
• These promoters are isolated and are used to construct
vector systems in which foreign genes are efficiently
expressed.
Disadvantages

• The envelope (=capsid) of CaMV does not

accommodate larger DNA than the normal size.
• Genes of CaMV are closely packaged. CaMV lacks long
non-essential sequences that could be replaced by
foreign genes.
• If DNA longer than few hundred nucleotides is
inserted, the infectivity of vector is destroyed.
Tobacco Mossaic Virus (TMV)

• TMV have single-stranded RNA genome which also serves as

mRNA. It encodes at least four proteins in three open reading
frames.
• The 4 genes encode a replicase (with methyltransferase [MT] and
RNA helicase [Hel] domains), an RNA-dependent
RNA polymerase, a so-called movement protein (MP) and a
capsid protein (CP).
• Of these the coat protein (cp) gene and MP gene seems to be
nonessential and can be site of integration of transgene.
• TMV was the first virus that has been engineered as a deconstructed
vector system.
Creating the TMV vector construct

• This vector consist of two steps, first, is the use of cDNA copy of
viral genome for cloning in E. coli and, second, is in vitro
transcription of the recombinant viral genome cDNA to produce
infectious RNA copies to be used for plant infection.
• Icon Genetics, a biotechnology company based in Germany, developed
a technique for transfecting plants with these recombinant virus vector
modules, known as Magnifection.
• Magnifection combines agroinfiltration with the delivery of a
deconstructed vector that lacks the ability to spread to other plants.
• Schematic diagram of a tobacco mosaic virus based
launch vector system for production of vaccine
antigens in non transgenic plants.
• Arrows indicate positions of sub genomic mRNA
promoters.
• The target antigen replaces the coat protein coding
sequence.
• LB and RB refer to left border and right border,
respectively, of the T-DNA in the Agrobacterium
tumefaciens binary vector; this T-DNA is transferred
into the nuclei of the plant cells following
agroinfiltration;
• 35S: 35S promoter from cauliflower mosaic virus (a
plant DNA virus) that drives transcription of the
transgene;
• MP: Movement protein required for cell-to-cell
movement;
• nosT: nos transcriptional terminator from A.
tumefaciens nopaline synthase gene;
• 126/183kDa: replicase proteins of tobacco mosaic
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