Download Semliki Forest virus-based DNA expression vector

Gene Therapy (1998) 5, 415–418
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BRIEF COMMUNICATION
Semliki Forest virus-based DNA expression vector:
transient protein production followed by cell death
A Kohno, N Emi, M Kasai, M Tanimoto and H Saito
First Department of Internal Medicine, Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466, Japan
We have constructed a novel DNA expression vector
based on Semliki Forest virus (SFV). SFV produces nonstructural proteins (nsPs) which replicate genomic RNA
and amplify the mRNA encoding the structural proteins of
SFV. A recombinant cDNA genome of SFV, in which the
SFV structural genes were replaced by a polylinker cassette to allow for insertion of heterologous DNA, was
placed under the control of a cytomegalovirus immediate–
early enhancer/promoter with a polyadenylation signal.
Transfection of mammalian cells with this SFV-based plasmid vector, pSFV3-CMV-lacZ-pA, resulted in transient
high-level expression of a ␤-galactosidase reporter gene.
The expression level of ␤-galactosidase from pSFV3-CMVlacZ-pA was more than 20-fold higher than that obtained
from the plasmid with deleted nsPs genes, pSFV3A5976lacZ, demonstrating that the nsPs genes were essential for
the high level of expression. Substantial ␤-galactosidase
activity was detected in the medium of pSFV3-CMV-lacZpA-transfected cells, suggesting that the overproduction of
␤-galactosidase caused cell death and release of the protein into the medium. We have demonstrated a high-level
expression of the exogenous ␤-galactosidase gene
from pSFV3-CMV-lacZ-pA constructed using an SFV
replication system.
Keywords: self-amplifying vector; Semliki Forest virus; plasmid vector
There are many expression vectors which can be used
in eukaryotic cells. However, transfection of conventional
plasmid vectors usually results in only a single or a few
recombinant DNA molecules reaching the nucleus.
Consequently, only a limited number of transcripts can
be generated. To increase expression levels, several selfamplifying systems of gene transfer have been
developed. Some of these self-amplifying systems use
RNA genomes of alphaviruses, such as Sindbis virus1–3
and Semliki Forest virus (SFV).4,5
SFV, a member of the Alphavirus genus, is a smallenveloped virus with a single-stranded RNA genome of
positive polarity. The 5′ two-thirds of the viral genome
encode nonstructural (replication) proteins (nsPs1–4) and
the 3′ one-third encodes structural proteins.6,7 Upon
infection, the RNA genome functions as mRNA for the
translation of nonstructural proteins. These subsequently
replicate the virus by copying the plus-strand RNA genome into minus-strand RNA and vice versa. The minusstrand RNA also serves as a template for the synthesis of
a shorter subgenomic RNA which encodes the structural
proteins. Transcription starting at the internal subgenomic promoter in the minus-strand results in the production of large amounts of subgenomic mRNA.6,7
SFV-derived vectors are based on the insertion of a
genomic SFV cDNA into an SP6 promoter plasmid, and
subsequent modification by deletion of the SFV structural
genes to allow for the insertion of heterologous DNA as
part of the SFV replicon.4 Since the in vitro transcript from
Correspondence: A Kohno
Received 8 May 1997; accepted 27 October 1997
such constructs also encodes the SFV replicase, high levels of expression of the heterologous gene can be achieved by directly transfecting the recombinant RNA into
cells. It was reported that recombinant RNA vectors
based on SFV were transfected with high efficiency into
animal tissue culture cells by means of electroporation.4
Although this system can be used, the preparation of
capped RNA vectors by in vitro transcription is necessary
before transfection and the RNA molecules are unstable
in general. Therefore, it is of great use to construct a DNA
vector based on self-amplifying systems of SFV,
especially in consideration of in vivo expression.
In this report, we describe the development of an SFVderived DNA-based expression vector which can initiate
the replication cascade in transfected mammalian cells to
produce high-level expression of a reporter gene. Furthermore, we demonstrate that the transfection of cells
with this SFV-based DNA vector results in cell death,
along with the release of large amounts of heterologous
proteins encoded in the vector.
Plasmid pSFV3 (GIBCO BRL, Grand Island, NY, USA),
an SFV-based plasmid vector, contains nsPs genes followed by a subgenomic RNA promoter and a polylinker
cassette which replaces the structural genes of SFV
(Figure 1a).4 The nsPs genes of pSFV are under the transcriptional control of the SP6 promoter; therefore, in vitro
transcription and capping of the 5′ end are required for
the expression of the gene of interest in mammalian cells.
To place pSFV3 under the control of a RNA polymerase
II promoter, a cytomegalovirus (CMV) immediate–early
(IE) enhancer/promoter sequence was inserted upstream
of the nsP1 gene. In addition, to ensure that mRNA transcribed from the plasmid was properly terminated, a
SFV-based DNA expression vector
A Kohno et al
416
Figure 1 Construction of plasmid vectors. (a) Construction of pSFV3. The promoter sequence for the subgenomic RNA is located at the end of nsP4
gene and the BamHI–SmaI–XmaI polylinker cassette is positioned downstream of the subgenomic promoter. (b) To construct pSFV3-CMV-lacZ-pA,
the cytomegalovirus (CMV) immediate–early (IE) enhancer/promoter sequence and the simian virus (SV) 40 late polyadenylation signal sequence were
inserted upstream of the nsP1 gene and downstream of the polylinker cassette of pSFV3, respectively (pSFV3-CMV-pA), and the ␤-galactosidase gene
was positioned in the polylinker cassette. The CMV-IE enhancer/promoter sequence and the SV40 polyadenylation signal sequence were obtained by
polymerase chain reaction using the pCI Mammalian Expression Vector (Promega) as a template. Primers for the amplification of the CMV-IE
enhancer/promoter region (CI-CMV1; 5′-ACATGCATGCTCAATATTGGCCATTAGC-3′, CI-CMV2; 5′-ACATGCATGCCTGACTGCGTTAGCAATT3′) contain an SphI site, and the PCR product was ligated into the unique SphI site of pSFV3. The primers for the SV40 polyadenylation signal (CIpA1; 5′-ACTAGTCAGACATGATAAGATACA-3′, CI-pA2; 5′-ACTAGTTACCACATTTGTAGAGGT-3′) contain an SpeI site, and the PCR product
was ligated into the unique SpeI site of pSFV3. (c) To delete the nsPs genes from pSFV3-CMV-pA, pSFV3-CMV-pA was digested with AccI, and a 5976 bp
fragment was self-ligated to construct pSFV3A5976. The ␤-galactosidase gene was inserted into the polylinker cassette to construct pSFV3A5976-lacZ.
SFV-based DNA expression vector
A Kohno et al
Figure 2 Production of ␤-galactosidase in baby hamster kidney (BHK)
cells transfected with pSFV3-CMV-lacZ-pA or pSFV3A5976-lacZ.
Quantitative levels of ␤-galactosidase expression were determined in 20 ␮l
of cell lysates (total lysates, 200 ␮l) after the indicated time-periods of
culture. ␤-Galactosidase activity was assayed using the ␤-galactosidase
Enzyme Assay System (Promega) according to the manufacturer’s
instructions. Each assay included a standard curve of 1 to 5 × 10−3 units
of ␤-galactosidase. Quantitative assay of total protein in cell lysates was
performed using the Bio-Rad Protein Assay Kit (Bio-Rad, Richmond, CA,
USA) according to the manufacturer’s instructions. ␤-Galactosidase
activity was normalized to 1.0 ␮g of total protein and plotted on a logarithmic scale. The data reflect the results of three similar experiments.
simian virus (SV) 40 late polyadenylation signal sequence
was inserted downstream of the polylinker cassette
(pSFV3-CMV-pA). The CMV-IE enhancer/promoter
sequence and the SV40 polyadenylation signal sequence
were obtained by polymerase chain reaction using the
pCI Mammalian Expression Vector (Promega, Madison,
WI, USA) as a template. Plasmid pSFV3-CMV-lacZ-pA
was constructed by the insertion of the E. coli ␤-galactosidase gene (lacZ) into the polylinker cassette of pSFV3CMV-pA (Figure 1b). Plasmid pSFV3A5976-lacZ was
constructed by the deletion of the nsPs genes from
pSFV3-CMV-lacZ-pA (Figure 1c).
To compare the expression level of lacZ from pSFV3CMV-lacZ-pA and pSFV3A5976-lacZ, baby hamster kidney (BHK) cells were transfected with these plasmid vectors as follows. BHK cells were plated into a 24-well plate
at a density of 1 × 105 cells per well in 0.5 ml of Iscove’s
modified Dulbecco’s medium (IMDM; GIBCO BRL) with
10% (v/v) fetal calf serum (FCS) and incubated at 37°C in
a 5% CO2 humidified atmosphere. Eighteen hours later,
transfection of pSFV3-CMV-lacZ-pA or pSFV3A5976-lacZ
was performed. Starburst polyamidoamine dendrimers
(generation 4; Yunitika, Osaka, Japan)8 were used as
mediators of DNA transduction. Plasmid DNA was prepared by diluting either 3.0 ␮g of pSFV3-CMV-lacZ-pA
or 1.8 ␮g of pSFV3A976-lacZ (equivalent number of plasmid molecules) in 270 ␮l of plain IMDM per well. Twenty
micrograms of dendrimers were diluted in 130 ␮l of
IMDM and added drop by drop to the DNA solution.
The solution was then gently mixed and incubated at
room temperature for 15 min. The medium in each well
was removed, cells were rinsed twice with IMDM, and
400 ␮l of the DNA–dendrimer mixture were added to
each well. After 3 h of incubation at 37°C in a 5% CO2
humidified atmosphere, the DNA–dendrimer mixture
was replaced by 1 ml of IMDM with 10% FCS. After 24
and 48 h of culture, a quantitative ␤-galactosidase assay
was performed. The expression level of ␤-galactosidase
obtained following transduction of pSFV3-CMV-lacZ-pA
was more than 20-fold higher than that obtained with the
pSFV3A5976-lacZ (Figure 2).
␤-Galactosidase expressing cells were visualized by
staining with X-gal (5-bromo-4-chloro-3-indolyl-␤-dgalactopyranoside) 24 h after transfection. Briefly, after
the medium was removed, cells were washed twice with
phosphate-buffered saline (PBS), fixed with 1.25% glutaraldehyde in PBS, and stained with X-gal solution.
Approximately 30 min following the addition of X-gal
solution, about 20% of the cells incubated with pSFV3-
Figure 3 Expression of ␤-galactosidase in transfected BHK cells. Cytochemical staining for ␤-galactosidase activity was performed for 30 min at 37°C
on BHK cells transfected with pSFV3-CMV-lacZ-pA (a) and with pSFV3A5976-lacZ (b).
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SFV-based DNA expression vector
A Kohno et al
418
Figure 4 Time-course of ␤-galactosidase activity in cell lysates and culture medium of pSFV3-CMV-lacZ-pA-transfected cells. The medium in
each culture was replaced by 1 ml of fresh Iscove’s modified Dulbecco’s
medium (IMDM) with 10% fetal calf serum (FCS) at 12 h for a continued
36 h of culture and every 24 h for longer cultures. ␤-Galactosidase activity
in cell lysates and culture medium was measured after the indicated timeperiods of culture. Similar results were obtained in two other experiments.
CMV-lacZ-pA showed a dense blue color, while only a
limited number of cells incubated with pSFV3A5976-lacZ
demonstrated a very faint color (Figure 3). However,
after overnight staining there was no difference in the
frequency of positively stained cells, and it was estimated
that 25–35% of the cells were positively stained in both
pSFV3-CMV-lacZ-pA- and pSFV3A5976-lacZ-transfected
cells. Unexpectedly, differences in appearance were
observed between the cells transfected with pSFV3-CMVlacZ-pA and pSFV3A5976-lacZ. Most of the cells transfected with pSFV3A5976-lacZ maintained the same shape
as untransfected cells, while the cells transfected with
pSFV3-CMV-lacZ-pA showed a condensed round form.
Figure 4 shows the time-course of ␤-galactosidase
activity in cell lysates and culture medium of pSFV3CMV-lacZ-pA-transfected cells. Transfection was performed as described above except that the initial number of
BHK cells was 2 × 104 per well. ␤-Galactosidase activity
in the lysates of the cells transfected with pSFV3-CMVlacZ-pA showed a transient increase with a peak 36 h
after transfection. In contrast, ␤-galactosidase activity in
the medium began to increase 36 h after transfection and
showed a delayed peak at 72 h. In the medium of
pSFV3A5976-lacZ-transfected cells, measurable activity
of ␤-galactosidase was not detected (data not shown). ␤Galactosidase is an intracellular protein and is not
released from cells. The leakage of ␤-galactosidase from
pSFV3-CMV-lacZ-pA-transfected cells suggests that the
overproduction of ␤-galactosidase resulted in cell death
and release of the protein into the medium. Another comparative study of transfection using pSFV3-CMV-lacZ-pA
and pSFV3A5976-lacZ was done in human 293 cell line.
Although X-gal staining of transfected cell cultures and
␤-galactosidase assay in cell lysates revealed lower
expression levels in 293 cells than in BHK cells, the level
of expression with pSFV3-CMV-lacZ-pA was higher than
with pSFV3A5976-lacZ (data not shown).
This is the first article demonstrating a high level of
expression of an exogenous gene by a plasmid vector
constructed using a SFV replication system. Recently,
Herweijer et al9 and Dubensky et al10 independently
reported that they constructed plasmid vectors from
Sindbis virus, a subgroup III Alphavirus with two envelope proteins, as compared with SFV which has three
envelope proteins. In their systems as well, they showed
10- to 30-fold higher expression compared with conventional methods.
Comparison of ␤-galactosidase reporter gene expression
between pSFV3-CMV-lacZ-pA and pSFV3A5976-lacZ plasmids showed that the nsPs genes were essential for highlevel expression of ␤-galactosidase. After transfection of
pSFV3-CMV-lacZ-pA, an initial plus-strand full-length
RNA is transcribed in the nucleus, translocated into the
cytoplasm, and then translated into the nsPs of SFV. This
initiates the replication cascade, and consequently, the highlevel expression of ␤-galactosidase is achieved. However,
protein synthesis in the transfected cell is probably
inhibited due to competition for translational machinery
with self-amplified RNA derived from the plasmid. Protein
synthesis in host cells infected by wild-type SFV is inhibited
by the competitive production of viral proteins,7 consequently leading to cell death.
A major advantage of the SFV-derived plasmid vector,
pSFV3-CMV-pA, is a high-level expression of an exogenous gene using the self-amplifying systems of SFV. In
addition, this vector is transfected into cells as doublestranded DNA. Therefore, there is no need for in vitro transcription and mRNA capping as required for the transfection of previously described SFV-derived RNA vectors.
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