Biomedical Engineering Reference
In-Depth Information
are the only animal viruses with a replication cycle
based solely on RNA to be extensively developed as
expression vectors. Sindbis virus (Xiong
et al
. 1989)
and Semliki Forest virus (SFV) (Liljestrom & Garoff
1993) have been the focus of much of this research.
These display a broad host range and cell tropism,
and mutants have been isolated with reduced cyto-
pathic effects (Agapov
et al
. 1998, Frolov
et al
. 1999;
reviewed by Schlesinger & Dubensky 1999).
The wild-type alphavirus comprises two genes: a
vectors (Berglung
et al
. 1996, Lundstrom 1997). For
example, replication-competent vectors have been
constructed in which an additional subgenomic
promoter is placed either upstream or downstream
of the capsid polyprotein gene. If foreign DNA is
introduced downstream of this promoter, the rep-
licase protein produces two distinct subgenomic
RNAs, one corresponding to the transgene. Such
insertion vectors tend to be unstable and have been
largely superseded by replacement vectors in which
the capsid polyprotein gene is replaced by the trans-
gene. The first 120 bases of both the Sindbis and the
SFV structural polyprotein genes include a strong
enhancer of protein synthesis, which significantly
increases the yield of recombinant protein (Frolov
& Schlesinger 1994, Sjoberg
et al
. 1994). This is
downstream of the translational initiation site, so in
many vectors this enhancer region is included so
that the foreign gene is expressed as an N-terminal
fusion protein. This can result in extremely high
levels of recombinant protein synthesis - up to 50%
of the total cellular protein.
Both plasmid replicon and viral transduction
vectors have been developed from the alphavirus
genome. A versatile Sindbis replicon vector, pSinRep5,
is currently marketed by Invitrogen (Fig. 10.9). The
vector is a plasmid containing bacterial backbone
elements, the Sindbis replicase genes and packaging
5
gene encod-
ing a polyprotein from which the capsid structural
proteins are autocatalytically derived. Since the
genome is made of RNA, it can act immediately as a
substrate for protein synthesis. However, because
protein synthesis in eukaryotes is dependent on
mRNAs possessing a specialized 5
′
gene encoding viral replicase and a 3
′
cap structure,
only the replicase gene is initially translated. The
replicase protein produces a negative-sense comple-
mentary strand, which in turn acts as a template
for the production of full-length daughter genomes.
However, the negative strand also contains an inter-
nal promoter, which allows the synthesis of a sub-
genomic positive-sense RNA containing the capsid
polyprotein gene. This subgenomic RNA is sub-
sequently capped and translated.
A number of different strategies have been used
to express recombinant proteins using alphavirus
′
Poly A
3' restriction sites for
linearization prior to
in vitro
transcription
Subgenomic
promoter to drive
expression of
inserted genes
G
Nonstructural
genes (
nsP1-4
)
for
in vitro
replication of
recombinant
RNA
pSinRep5
SP6 promoter
for synthesis of
in vitro
RNA
transcripts
Packaging signal
allows recombinant
RNA to be packaged
into virus
Fig. 10.9
The Sindbis virus-based
vector pSinRep5. (Reproduced with
permission from Invitrogen.)
