Virus-mediated gene transfer

Virus-mediated gene transfer

Viral genome doesnt integrate into plant genome.

Viral vectors are episomal vectors, therefore they have
high copy number per cell and they are not subjected to the
position effect. The gene product is very rapidly
accumulated.

Viral genome sequences are excellent source of
promoters, enhancers and other components useful for
designing gene vectors.

Virus is systemically spread in the plant body.

Generally have wide host range.
Most notables
1. CaMV based vectors.
2. Gemini virus based vectors.
3. TMV based vectors.
Basic features:
1. Broad host range, virulence, easy mechanical transmission
and seed transmission.

2. Carry additional genetic information within the packaging limit.
Viruses that are rod shaped or have multipartite genome or
contain a helper or satellite component offer potential for
carrying extra genetic material.
CaMV
Circular (top) and linearized (bottom) genome maps of Cauliflower mosaic virus (CaMV). ORFs or ORF
segments encoding a protein are represented by boxes shaded in different patterns according to the
different putative functions of the genes (vertical lines for the putative movement protein, slanted lines for
the capsid protein and horizontal lines for the reverse transcriptase protein). The symbols used are the
followings: movement protein active site, * RNA binding site, ? protease active site, reverse
transcriptase active site, D RNAse H consensus sequence. The map starts at the intergenic region of the
circular genome for convenience, the arrow shows the position of the promoter and the number 1 indicates
the origin of DNA replication.
CaMV
CaMV
1. dsDNA virus. Packaged as nucleosome.
2. Mechanical and aphid mediated transmission
3. Virion DNA alone or cloned CaMV DNA is infectious when simply
rubbed on leaves
4. Up to 10
6
copies per cell. 3-4 weeks for systemic infection through
plant.
I
II
III
VI
V
VI
VII
35S RNA (polycistronic)
19S RNA
8031 bp
Gene I: plasmadesmata movement
Gene IV: translation transactivation
Gene V: reverse transcriptase
Gene III/IV: assembly
Gene II/VI: inclusion bodies
transcription
nucleus
35S RNA
19S RNA
translation
Reverse
transcription
uncoating
Gene IV
Gene V
Gene III/IV
assembly
Inclusion body
(gene VI)
Gene I
CaMV activity in plant cell
Expression of a bacterial gene in plants by using a viral
vector


Several properties of the cauliflower mosaic virus (CaMV) indicate that it
could provide a useful vector for gene transfer in higher plants: (1) it has
a relatively small double-stranded genome that can be easily
manipulated in vitro; (2) cloned viral DNA is infectious when rubbed onto
healthy leaves; (3) virus spreads throughout the plant and can be found
in most cells at high copy number. Two regions of the CaMV genome
open reading frames (ORFs) II and VIIdo not seem to be essential for
infection, as both can be either deleted or expanded by small inserts of
foreign DNA. No functional genes have yet been introduced into these
ORFs. Here we report the replacement of CaMV ORF II by the R67
plasmid-encoded dihydrofolate reductase (DHFR) gene; this gene (dhfr)
confers resistance to methotrexate in Escherichia coli. The chimaeric
viral DNA can be stably propagated in turnip plants and the dhfr gene is
expressed, producing a functional enzyme.
Nature 310, 511 - 514 (09 August 1984)
Mammalian Metallothionein Functions in Plants
A recombinant cauliflower mosaic virus (Ca-MTII) was constructed
by inserting a cDNA clone of Chinese hamster metallothionein II
into the ORF II of the cloned virus pCa-BB1. Systemically-infected
Brassica campestris tissue contained metallothionein at a level of
0.5% of the soluble leaf protein. This efficient expression conferred
4X the Cd-binding capacity when compared with Ca-BB1 infected
leaves. Ca-MTII-infected leaves exposed to 1 mM CdCl2 bound all
the free Cd whereas uninfected leaves possessed 43.8 nmol free
Cd per milligram of protein. This may be responsible for Cd
resistance in the Ca-MTII plant cells. Metallothionein is the first
mammalian gene product shown to be functional in plants.
Bio/Technology 5, 1053 - 1056 (1987)
Transfection of Brassica campestris leaves with cauliflower mosaic virus
(CaMV) harboring a mammalian metallothionein (MT) cDNA at the ORFII
position lowered the glucosinolate (GS) concentration to approximately
one-half the level in leaves infected with wild-type CaMV. This
suppression was independent of the plant's sulfate status, suggesting that
the pathways for protein (MT) and GS biosynthesis were competing for S
on an equal basis. The expression of MT may have lowered the
endogenous levels of Cys, 3'-phosphoadenosine-5'-phosphosulfate, or
Met, all of which are required for GS synthesis in B. campestris. These
results indicate that the introduction of structural genes coding for high
levels of specific amino acids can be used to alter the production of non-
proteinaceous molecules within plants.
Expression of Mammalian Metallothionein Suppresses
Glucosinolate Synthesis in Brassica campestris
Plant Physiol. (1990) 93, 522-524
Challenges with CaMV vector
Small insertions (10-30 bp) in various sites abolished infectivity.
Only gene II could tolerate insertion of significant size and could
be entirely removed

But the largest insert tolerated so far is 256-531 bp.

Complicated polycistronic design (ATG of cloned DNA must not
interfere with the termination of gene I).

CaMV genome was inserted into Ti vector to integrate into
genome.

Due to these limitations, CaMV vectors have not be widely used.
Gemini viruses
Gemini viruses: smallest viral genome ~2.7 kb
WDV, TGV and MSV: ssDNA genome replicates as dsDNA intermediate
V1
V2
C1
C2
WDV
AV1
TGMV

DNA A
AC1
AC2
AC3
TGMV

DNA B
BV1
BV1
Monopartite
Bipartite
V2: capsid protein
V1: systemic spread
C1 and C2: replication
AC1: replication
AV1: Coat protein
AC2: transactivation of sense genes
AC3: delayed or attenuated symptoms
BC1 and BV1: spread
Gemini viruses
Most have insect mediated transmission. No mechanical transmission.
Overcome by cloning viral genome into Ti plasmid and carrying out
Agroinfection
WDV genome allows insertion of up to 3 kb foreign sequence
pWDV2 V2 deletion vector
MCS (multi-cloning site)
C1
C2
V1
dsDNA form of virus genome is infectious and coat protein gene is not
required for systemic infection
The use of plant DNA viruses as vectors for the transfer of foreign genes to plants offers
two potential advantages over other, existing methods of producing transgenic plants.
First, these viruses systemically infect whole plants, thus obviating the need for the
difficult and time-consuming step of regeneration from transformed single cells or
protoplasts. Second, the viruses replicate as separate, autonomous entities within the
plant's cells so that any gene cloned in a plant DNA-virus vector would be amplified to
high copy number, a feature that differs from methods that produce transgenic plants by
the chromosomal integration of foreign DNA. To date, attention has focused on the
development of vectors based on the cauliflower mosaic virus (CaMV), but their use is
hampered by the narrow range of plants infected by CaMV, and by practical limitations
on inserting foreign DNA that are imposed by the biology of CaMV. Here we describe the
use of vectors based on the gemini tomato golden mosaic virus (TGMV) to introduce the
neomycin phosphotransferase (neo gene) into plants. Our results indicate that
geminivirus-derived vectors should be useful not only for amplification of gene
expression by the systemic infection of plants, but also for heritable gene amplification
by the integration of stable master copies of the vector into the plant chromosomal DNA.
Gene amplification and expression in plants by a replicating geminivirus vector
Nature 334, 179 - 182 (14 July 1988)

RNA virus for foreign gene expression
The vast majority of plants viruses consist of plus strand RNA.
Engineering gene expression vectors based on RNA viruses has awaited
the successful cloning of the viral genome into in vitro transcription
vectors from which infectious transcripts can be obtained.

cDNA cloning of Brome Mosaic Virus (1984) and Tobacco Mosaic Virus
(1986) were reported. In these cases CP gene was replaced with cat
gene and the resulting vectors were expressed in barley or tobacco
protoplasts.
TMV
ssRNA virus of 6395 bases with 4 ORFs
126 / 183 kDa
30 kDa CP
126 / 183 kDa: replicase complex translated from a single frame
(183 kDa is generated by readthrough of a leaky amber termination
codon of 130 kDa gene

30 kDa: movement protein

17.5 kDa: capsid protein
tRNA like
5 cap
TMV based vectors
126 / 183 kDa
30 kDa CP
3
Insert (size limit)
TB2
Foreign gene is inserted into 3 end of MP in such a
way that native CP promoter drives the expression of
foreign gene, and a related virus (ORSV:
Odontoglossom ring spot virus) CP promoter drives the
expression of native CP ORF
TMV based
vectors
BSMV genome mediated expression of a foreign gene in dicot and
monocot plant cells.
Joshi RL, Joshi V, Ow DW. (1990) EMBO J. 9:2663-9.
Barley stripe mosaic virus (BSMV) possesses a tripartite genome composed of RNAs
alpha, beta and gamma that have been cloned into in vitro transcription vectors from
which infectious transcripts can be obtained. The BSMV genome has been engineered
here to serve as an expression vector in plant protoplasts. Open reading frame (ORF) b
of RNA beta, encoding a non-structural protein, was replaced by the firefly luciferase
(luc) reporter gene to yield RNA beta 2-luc. In the presence of both RNAs alpha and
gamma, RNA beta 2-luc mediated efficient expression of the luc gene upon transfection
into tobacco and maize protoplasts. This expression ranged from 20- to 123-fold higher
than the luciferase activity obtained from transfection with a luc gene mRNA. Replication
of RNA beta and its derivatives i.e. 'minus' strand synthesis, was confirmed by Northern
analysis, indicating that the high level of luc gene expression using RNA beta 2-luc
resulted from RNA amplification. ORFa of RNA beta, encoding the coat protein, was also
replaced by the luc gene to yield RNA beta 1-luc. Although transfection of RNA beta 1-
luc alone produces luciferase efficiently, neither 'minus' strand synthesis nor further
increase of luciferase activity was observed in the presence of RNAs alpha and gamma,
or RNAs alpha, beta and gamma, suggesting that the deleted sequences within ORFa
are cis-acting for replication of RNA beta. Our results demonstrate that a foreign gene
can be expressed by replacement of a viral non-structural protein gene that is essential
for virus multiplication in plants, leading to a potential strategy for virus 'containment' with
use of 'disarmed' plant viral vectors.
RNA
RNA
RNA
ORFa
ORFd
ORFc ORFb
Organization of
BSMV genome
RNA 1
ORFd
ORFc ORFb
RNA 2
ORFa
ORFd
ORFc
RNA 1-luc
ORFd
ORFc ORFb
luc
BSMV based vectors
ORFb encodes a non-
structural protein that
is essential for virus
multiplication in whole
plant.
Joshi RL, Joshi V, Ow DW. (1990) EMBO J. 9:2663
GENEWARE is a unique and extremely versatile technology, used by
LSBC to test the function of novel genes and proteins they encode (gene
prototyping), and to manufacture complex soluble proteins in bulk.
GENEWARE is one of three types of viral vectors within LSBCs viral
vector technology platform. This form of transient, plant viral-vector-based
gene expression utilizes a modified Tobacco Mosaic Virus (TMV), or
similar RNA viral replicon, carrying its normal housekeeping genes plus an
introduced new RNA promoter and structural gene to encode a new
protein of commercial interest. LSBCs dual subgenomic promoter system
is designed for the coordinated expression of a wide range of soluble
(free) protein products with high efficiency. GENEWARE's basic principle
is to use a safe vector modified from a virus to place any gene (or a large
number of genes) within a test organism. The organism then
manufactures the gene's protein product, which can be collected and
purified efficiently in LSBC's pilot and commercial bioprocessing facilities.
Kilogram amounts of a wide array of complex biopharmaceuticals can be
produced rapidly with low capital investment relative to conventional
biotechnology manufacturing operations.
http://www.lsbc.com/
Coat Protein Fusion and Virus-Like Particle Vectors
LSBCs second type of viral vector technology for gene expression and protein
manufacturing converts plant viruses, such as Tobacco Mosaic Virus (TMV) and relatives,
into a carrier and display vehicle for peptides. By inserting the gene sequence(s) for small
proteins (peptides) onto selected regions of TMVs coat protein (CP) gene, the virus can be
made to display the new peptide on the CP surface upon assembly. The CP promoter is
very strong and thus the yield of peptides can be very high. Because upon assembly each
TMV virion particle consists of over 2,100 CP subunits, the resulting product is a virus
displaying over 2,100 copies of a protein product per particle. The peptides on the viral CP
can be fused through a labile amino acid bridge, so the peptide product can be recovered
and separated from the rest of the virus post translationally. More often, the viral particle
acts as a carrier or display vehicle for peptides of interest. This technology finds major utility
in the field of vaccine development, in which case TMV becomes a viral antigen display
vehicle. The semicrystalline array of a very high number of repeating antigens can stimulate
the immune system to respond with potent cellular and humoral immunity to these vaccines.
TMV rod-shaped particles can also be coated with peptides and proteins chemically or
biochemically, to achieve the same type of effects as described with the genetic CP fusion
approach. Because more than one sequence of peptide can be displayed on the viral
surface, this type of vector enables the development of multivalent vaccines, which are
useful in the prevention and control of complex current and emerging diseases. LSBC and
its collaborators have successfully constructed several candidate vaccines using this
CP/VLP vector technology. Representative examples of such products include a feline
parvovirus (panleukopenia) vaccine, a bovine foot and mouth disease vaccine, and an
experimental human papillomavirus vaccine.
Hybrid TMV Vectors for Transient RNA Vaccination
LSBCs third type of viral vector technology for gene expression consists of a hybrid viral
replicon that integrates several separate features of viral architecture and virus-host biology.
Because TMV and related plus-strand RNA viruses are members of the Alphavirus superfamily,
consisting of RNA replicons, LSBC has designed a composite system that is manufacturable in
commercial quantities cost effectively and has a limited capacity to effect gene expression in
mammalian tissue. However, these vectors cannot integrate into the mammalian chromosome
and thus do not persist in the body. These vectors, still under development, could form the core
for a new type of transient RNA vaccination. Instead of producing protein, which becomes the
product, these new vectors would encode protein in situ only after injection as a vaccine. These
hybrid viral vectors are constructed by growing TMV or related viruses in Nicotiana plants,
purifying them to homogeneity, and then isolating their coat protein (CP) subunits away from
their native RNA genome. The CP subunits can be native, or carry on them fused surface
antigens-essentially as LSBCs CP/VLP vectors do. But the hybrid system then coalesces the
CP subunits around a plus strand of non-native, synthetic RNA containing a mammalian
Alphavirus replicating element. This element is important because native TMV RNA cannot
replicate in mammalian tissue. The TMV virion structure now consists of a repetitive antigen
either monovalent or multivalentcarried and displayed to the immune system on the CP, plus
the information to replicate and translate new protein transiently in mammalian antigen-
presenting cells, such as Dendritic cells in the skin.
While this system is earlier in development relative to LSBCs other viral vector
technologies, initial feasibility studies in vivo using model antigens have been highly
encouraging and suggest that this hybrid virus technology could form the basis of a new
multivalent, bi-functional (gene/protein) vaccine strategy to combat cancers and many infectious
diseases.
McCormick et al. PNAS Vol. 96, Issue 2, 703-708, January 19, 1999

Rapid production of specific vaccines for lymphoma by expression of
the tumor-derived single-chain Fv epitopes in tobacco plants

Abstract
Rapid production of protein-based tumor-specific vaccines for the treatment of
malignancies is possible with the plant-based transient expression system described
here. We created a modified tobamoviral vector that encodes the idiotype-specific
single-chain Fv fragment (scFv) of the immunoglobulin from the 38C13 mouse B cell
lymphoma. Infected Nicotiana benthamiana plants contain high levels of secreted
scFv protein in the extracellular compartment. This material reacts with an anti-
idiotype antibody by Western blotting, ELISA, and affinity chromatography,
suggesting that the plant-produced 38C13 scFv protein is properly folded in solution.
Mice vaccinated with the affinity-purified 38C13 scFv generate >10 g/ml anti-
idiotype immunoglobulins. These mice were protected from challenge by a lethal
dose of the syngeneic 38C13 tumor, similar to mice immunized with the native 38C13
IgM-keyhole limpet hemocyanin conjugate vaccine. This rapid production system for
generating tumor-specific protein vaccines may provide a viable strategy for the
treatment of non-Hodgkin's lymphoma.
Schematic diagram of the viral expression vector used to produce murine 38C13
scFv NHL antigen in Nicotiana benthamiana host plants. Shown are the sequences
encoding rice -amylase signal peptide fused in-frame to the SphI site
(underlined), introduced by PCR, 5' of the 38C13 heavy chain. Also indicated in the
linear map are the positions of the SP6 transcription start site, the 126-kDa, 183-
kDa, and 30-kDa proteins from TMV, and the tomato mosaic virus coat protein, as
well as pBR322 sequences for bacterial propagation.
Protein analysis of N. benthamiana plants 2 weeks after inoculation. Lanes 1 and
2, Coomassie-stained SDS/PAGE of IF extract [secreted proteins were isolated by
vacuum infiltration, centrifugation, and sterile filtration (IF extracts)] from viral constructs
expressing rice -amylase protein (lane 1) or of 38C13 scFv IF extract (lane 2) run under
nonreducing conditions. Lanes 3 and 4, Western blot of a duplicate gel probed with anti-
38C13-specific monoclonal antibody S1C5 containing the control extract (lane 3) or 38C13
scFv (lane 4). Lanes 5 and 6 show S1C5 Western blots of 38C13 scFv-containing IF
prepared under reducing conditions (but separated by PAGE under nonreducing
conditions) or IF containing an 38C13 scFv protein that has no cysteine at amino acid
3. Lanes 7 and 8, Coomassie stain of the starting IF extract (lane 7) and of single-pass,
S1C5 affinity-purified 38C13 scFv protein (lane 8). Lane 9, silver stain of affinity-purified
protein.