Biomedical Engineering Reference
In-Depth Information
and autonomously replicating episomal copies can
be detected. Transgenic tobacco plants have also
been produced carrying an integrated copy of DNA
B (Hayes
et al.
1988, 1989). In the presence of re-
plication functions supplied by DNA A, the DNA-B
sequence is rescued from the transgene and can
replicate episomally. DNA B can then provide move-
ment functions for DNA A, facilitating the systemic
spread of the vector.
copies. Some of these viruses have been extensively
developed as vectors for foreign gene expression
(see comprehensive reviews by Scholthof
et al.
1996,
Porta & Lomonossoff 2001). Two examples are dis-
cussed below.
Tobacco mosaic virus
TMV is one of the most extensively studied plant
viruses and was thus a natural choice for vector
development. The virus has a monopartite RNA
genome of 6.5 kb. At least four polypeptides are pro-
duced, including a movement protein and a coat
protein, which are translated from subgenomic
RNAs (Fig. 12.16). The first use of TMV as a vector
was reported by Takamatsu
et al.
(1987). They re-
placed the coat-protein gene with
cat
, and obtained
infected plants showing high-level CAT activity at
the site of infection. However, the recombinant
virus was unable to spread throughout the plant,
because the coat protein is required for systemic
infection.
Since there should be no packaging constraints
with TMV, Dawson
et al.
(1989) addressed the
deficiencies of the TMV replacement vector by gen-
erating a replication-competent
addition vector
, in
which the entire wild-type genome was preserved.
Dawson and colleagues added the bacterial
cat
gene,
controlled by a duplicated coat-protein subgenomic
promoter, between the authentic movement and
coat-protein genes of the TMV genome. In this case,
systemic infection occurred in concert with high-
level CAT activity, but recombination events in
infected plants resulted in deletion of the transgene
and the production of wild-type TMV RNA. Homo-
logous recombination can be prevented by replacing
the TMV coat-protein gene with the equivalent
sequence from the related
Odontoglossum
ringspot
virus (Donson
et al.
1991). This strategy has been
used to produce a range of very stable expression
vectors, which have been used to synthesize a vari-
ety of valuable proteins in plants, such as ribosome-
inactivating protein (Kumagai
et al.
1993) and
single-chain Fv (scFv) antibodies (McCormick
et al.
1999). It has also been possible to produce complete
monoclonal antibodies by coinfecting plants with
separate TMV vectors expressing the heavy and
light immunoglobulin chains (Verch
et al.
1998).
RNA viruses as expression vectors
Most plant RNA viruses have a filamentous mor-
phology, so the packaging constraints affecting the
use of DNA viruses, such as CaMV, should not pre-
sent a limitation in vector development. However,
investigation into the use of RNA viruses as vectors
lagged behind research on DNA viruses, awaiting
the advent of robust techniques for the manipula-
tion of RNA genomes.
A breakthrough was made in 1984, when a full-
length clone corresponding to the genome of brome
mosaic virus (BMV) was obtained. Infectious RNA
could be produced from this cDNA by
in vitro
transcription (Ahlquist & Janda 1984, Ahlquist
et al.
1984). The BMV genome comprises three segments:
RNA1, RNA2 and RNA3. Only RNA1 and RNA2
are necessary for replication. RNA3, which is
dicistronic, encodes the viral coat protein and move-
ment protein. During BMV infection, a subgenomic
RNA fragment is synthesized from RNA3, con-
taining the coat-protein gene alone. It is therefore
possible to replace the coat-protein gene with for-
eign DNA and still generate a productive infection
(Ahlquist
et al.
1987). This was demonstrated by
French
et al.
(1986) in an experiment where the
coat-protein gene was substituted with the
cat
reporter
gene. Following the introduction of recombinant
RNA3 into barley protoplasts, along with the essen-
tial RNA1 and RNA2 segments, high-level CAT
activity was achieved.
This experiment showed that BMV was a poten-
tially useful vector for foreign gene expression. How-
ever, to date, BMV has been used solely to study the
function of genes from other plant viruses. Following
the demonstration that infectious BMV RNA could
be produced by
in vitro
transcription, the genomes of
many other RNA viruses have prepared as cDNA
