MOLECULAR AND CELLULAR BIOLOGY, JUlY 1987, p. 2538-2544
0270-7306/87/072538-07$02.00/0
Copyright C) 1987, American Society for Microbiology
Vol. 7, No. 7
Use of a Hybrid Vaccinia Virus-T7 RNA Polymerase System for
Expression of Target Genes
THOMAS R. FUERST, PATRICIA L. EARL, AND BERNARD MOSS*
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892
Received 27 January 1987/Accepted 1 April 1987
A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been
developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under
control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice
flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene
occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase
synthesis began shortly after infection, the target gene was not expressed until late times and was largely
inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel
electrophoresis and had the predicted size. With this system, Escherichia coli (-galactosidase, hepatitis B virus
surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of
synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia
virus expression system.
Molecular cloning vectors have been developed that allow
expression of heterologous genes in procaryotic and eucaryotic cells. Genetic elements carried by these vectors typically confer drug resistance, ability to replicate autonomously, and regulatory controlling elements juxtaposed to
the target gene of interest. While this approach has been
extensively used in bacterial and mammalian cell expression
systems, certain features of one system may be advantageous over another. Bacteria are easy to use and high
expression is often attainable, but synthesis of native
eucaryotic proteins is frequently not achieved. Mammalian
cells can faithfully express eucaryotic proteins, but usually
at relatively low levels. An expression system that used
procaryotic transcriptional elements in mammalian cells
might have important advantages since the catalytic activity,
induciblity, and promoter specificity are well characterized.
For these reasons, a chimeric expression system that used
favorable transcriptional components from bacteria in a
eucaryotic milieu may offer a highly specific and efficient
method for the synthesis of ecuaryotic proteins.
A eucaryotic transient-expression system based on a
recombinant vaccinia virus that synthesizes bacteriophage
T7 RNA polymerase in the cytoplasm of infected cells was
recently described (5). Plasmids containing the target genes
flanked by T7 promoter and termination sequences were
introduced into infected cells by transfection procedures.
The efficiency of the vaccinia virus-T7 transient system
relative to that of more conventional mammalian transient
expression systems was attributed to the high catalytic
activity of T7 RNA polymerase and stringent T7 promoter
specificity. Since it is possible to infect tissue culture cells
synchronously with vaccinia virus, the absolute level of
expression was probably limited by transfection of the target
gene. It would seem that the efficiency of the system would
be enhanced if vaccinia virus were used to deliver the target
gene as well as the T7 RNA polymerase. Increased copy
number of the target gene template might also result from a
10,000 to 20,000 amplification of the virus genome during
replication.
*
In this communication, we describe the construction of
several recombinant vaccinia viruses which contain DNA
coding segments for Escherichia coli ,-galactosidase (P3-gal),
hepatitis B virus surface antigen (HBsAg), and human immunodeficiency virus (HIV) envelope proteins flanked by
bacteriophage T7 promoter and termination regulatory elements. Mammalian cells coinfected with recombinant vaccinia viruses that contain the T7 RNA polymerase gene
under control of a vaccinia promoter and the target gene
under control of a T7 promoter provide higher levels of
expression than those previously obtained with recombinant
vaccinia viruses.
MATERIALS AND METHODS
Virus and cells. Vaccinia virus (strain WR) was originally
obtained from the American Type Culture Collection, propagated in HeLa cells, and purified as reported previously
(10). HeLa S3 cells were grown in Eagle medium supplemented with 5% horse serum. Human thymidine kinasenegative (TK-) 143 cells (16) were grown in Eagle medium
containing 10% fetal bovine serum (FBS) and 25 pug of
5-bromodeoxyuridine (BUdR) per ml. CV-1 and BSC-1 cells
were grown in Dulbecco modified Eagle medium containing
10% FBS. H9 cells were grown in RPMI medium with 8%
FBS.
Construction of vaccinia virus recombinants. Recombinant
viruses were prepared by infecting CV-1 cells with vaccinia
virus (strain WR) and transfecting them with calcium phosphate-precipitated plasmid DNA (9, 10). The cells were
harvested, and TK- recombinant viruses were isolated by
plaque assay on TK- cells in the presence of BUdR and
identified by DNA dot blot hybridization. Isolated virus was
then plaque purified once more in TK- cells with BUdR
selection, and large stocks were prepared under nonselective
conditions in HeLa S3 cells. To confirm the predicted
structures, DNA from recombinant virus was routinely
examined by restriction endonuclease analysis and DNA
hybridization.
Immunoblot analysis. To analyze polypeptide synthesis,
infected-cell monolayers were collected by centrifugation
and lysed in 0.5 ml of a solution containing 50 mM Tris
Corresponding author.
2538
VOL. 7, 1987
hydrochloride (pH 7.5), 0.15 M NaCl, 0.1% sodium dodecyl
sulfate (SDS), 1% Trition X-100, and 1% sodium deoxycholate. After 15 min at 0C, the lysate was centrifuged, and the
supernatant was transferred to a new tube. A 50-plA portion
was added to 25 RI of a solution containing 0.12 M Tris
hydrochloride (pH 6.8), 20o glycerol, 6% SDS, and 10%o
P-mercaptoethanol. The sample was heated at 100°C for 1
min and applied to an SDS-polyacrylamide gel. After separation, the proteins were electrophoretically transferred to
nitrocellulose (1) and visualized by staining with the indicated antiserum and 1251-labeled protein A, followed by
autoradiography.
RNA isolation and analysis. Total cellular RNA was isolated by the following procedure. CV-1 cells (Ca. 8.5 x 106
cells) were grown to confluence in petri dishes (100 by 20
mm) and coinfected with recombinant vaccinia viruses at a
total multiplicity of 20 PFU/cell. The virus was allowed to
adsorb for 2 h at 37°C with occasional rocking of the plate,
overlaid with 8 ml of medium containing 2.5% FBS, and
incubated at 37°C for various lengths of time. Cells were
washed three times with ice-cold phosphate-buffered saline,
collected by centrifugation, and lysed in 1.5 ml of RSB buffer
(10 mM Tris hydrochloride, pH 7.6, 10 mM NaCl, and 1.5
mM MgCl2) containing 0.5% Nonidet P-40 and 5 mM EDTA
on ice for 5 min. The cytoplasmic fraction was separated
from nuclei and insoluble material by centrifugation, 0.15 ml
of 10% SDS was added, and the material was extracted with
an equal volume of phenol saturated with TE (10 mM Tris
hydrochloride, pH 7.6, 2 mM EDTA) and then with chloroform. The aqueous layer was made 0.3 M in sodium acetate
and precipitated with 2.2 volumes of ice-cold ethanol. The
pellet was suspended in 0.3 ml of 40 mM Tris hydrochloride-10 mM NaCl-6 mM MgCl2 containing 0.1 ,ug of RQ1
DNase (Promega Biotec) and incubated for 10 min at 37°C.
This mixture was extracted with phenol and chloroform and
ethanol precipitated as before. The RNA pellet was suspended in 50 ,u1 of TE. Samples (5 pul) were fractionated on
formaldehyde-agarose gels by the method of Lehrach et al.
(8). RNA was transferred to nitrocellulose and hybridized to
32P-labeled probes as described by Thomas (21). Probes
were prepared and nick translated by the method described
by Maniatis et al. (11). RNA bands were then visualized by
autoradiography.
Construction of plasmids. Plasmid pGS50, a gift of Geoffrey Smith, was constructed by cleaving the HindIII J
fragment from the Wyeth strain of vaccinia virus with PvuII
and isolating a 1.8-kilobase-pair (kbp) HindIII-PvuII fragment. This fragment was then ligated to pUC13 that had
been cleaved with EcoRI, treated with S1 nuclease and
Klenow DNA polymerase to remove protruding ends, and
digested with HindIII.
Plasmid pTF7-5 was constructed by inserting a 200-basepair (bp) BgIII fragment from pAR2529 (received from F. W.
Studier) made blunt with Klenow polymerase into pGS50
which had been cleaved with EcoRI and ClaI and the ends
made flush with Klenow polymerase. The BglII fragment
contains the T7 promoter (410) and terminator (T4)), corresponding to bacteriophage 17 DNA nucleotide positions
22,880 to 22,928 and 24,106 to 24,228, respectively, joined by
a synthetic linker (CGGGATCCGG), generating a unique
BamHI site in the plasmid (4). The +10 promoter is directed
clockwise, opposite to transcription from the ampicillin
promoter in pUC19.
A 3.5-kbp SstI fragment containing the envelope gene
(gpl60) of human T-cell lymnphotropic virus type III (HTLVIII) clone BH8 (15) was inserted into M13mpl8. The se-
VACCINIA VIRUS-T7 HYBRID EXPRESSION SYSTEM
2539
quence immediately upstream of the initiating ATG was
changed to GATATCCACCATG by oligonucleotide mutagenesis of single-stranded M13 phage DNA (23). This generated an EcoRV site for ease of subcloning and a translation
initiation codon with a eucaryotic consensus sequence (7).
An additional EcoRV site was inserted following the termination codon of the gpl60 gene. The resulting clone, mpPEenv, contained the entire gp160 coding sequence flanked by
EcoRV sites which could be easily isolated as a 2.5-kb
fragment and inserted into vaccinia virus recombination
vectors.
RESULTS
Construction of recombinant vaccinia viruses and comparison of coinfection and transient-expression systems. Previous
studies (5) indicated that transient expression was obtained
from a plasmid containing the E. coli a-gal gene (1acZ)
placed next to a T7 promoter in cells infected with a
recombinant vaccinia virus expressing T7 RNA polymerase.
We now wished to determine whether better expression
would occur if the T7 promoter-target gene template was
inserted into vaccinia virus. As a target we again chose the
E. coli lacZ gene so that direct comparisons could be made.
Moreover, the assay for 3-gal is simple and quantitative and
there is no detectable background in mammalian cells (2). An
expression cassette containing the lacZ gene flanked by T7
promoter (410) and terminator (T4)) regulatory elements was
inserted into the middle of the vaccinia virus tk gene to form
a recombination vector (Fig. 1). This plasmid was
transfected into cells that were infected with vaccinia virus,
and a TK- recombinant virus containing the lacZ target
gene, designated vTF7LZ-1, was isolated.
3-Gal expression was measured after coinfecting CV-1
cells with vTF7LZ-1 and a second recombinant virus expressing T7 RNA polymerase (vTF7-3). The data (Table 1)
indicated that lacZ expression was about 14-fold higher with
the vaccinia virus-T7 coinfection system than with the
related transient-expression system. Control experiments
showed that p-gal activity was not detected when cells were
infected individually with vTF7-3 or vTF7LZ-1.
Dependence of ,-gal expression on virus multiplicities.
Since the T7 RNA polymerasee and the target gene were
carried on different viruses, we wished to determine the
amounts of each needed for optimal expression. CV-1 cells
were coinfected with recombinant viruses vTF7-3 and
vTF7LZ-1 at a range of multiplicities. In general, equal
amounts of each recombinant virus were best, and multiplicities of 10 resulted in the highest level of a-gal expression
(Table 2). Increasing or decreasing the relative amount of
either recombinant virus resulted in a diminution of total
p-gal expression.
Comparison of
amount of 3-gal produced from vaccinia
virus and bacteriophage T7 promoters. The experiment described above indicated that a substantial increase in a-gal
expression could be achieved with the vaccinia virus-T7
coinfection system compared with transient expression of
the target gene from a plasmid. We also compared the
coinfection system with a straight vaccinia virus recombinant system in which expression of lacZ is under control of
a vaccinia virus promoter. The vaccinia virus promoter
used, P7.5, was the same as that regulating expression of the
T7 RNA polymerase gene (T7 gene 1) in recombinant virus
vTF7-3. Promoter P7.5 contains early and late regulatory
signals (3), which permits continuous expression of foreign
genes. A TK- recombinant virus containing P7.5 fused to
2540
FUERST ET AL.
MOL. CELL. BIOL.
the lacZ gene inserted into the tk locus by homologous
recombination was isolated and referred to as vTFLZ.
Time courses of 3-gal synthesis in CV-1 cells infected only
with vTFLZ or coinfected with vTF7-3 and vTF7LZ are
shown in Fig. 2. The infected cells were harvested at various
times, and the proteins were separated by polyacrylamnide
Bam Hi
BgIl
Bgl
11
(Xbal)
Smal
ATG
TAA
bacZ
laenov Polyi
Cla
Eco
TABLE 1.
Comparison of p-gal expression from plasmid
and virus templates
ViruSa
Plasmid
,B-Gal (nmol/3 x
106 cells)
pTF7LZ-lb
vTF7-3
vTF7LZ-1
+
-
-
-
+
-
<10
<10
+
+
+
-
+
28,250
2,315
a CV-1 cells (approximately 3 x 106 cells) were infected (+) or not infected
(-) with the indicated recombinant vaccinia viruses. When cells were infected
or coinfected with recombinant vaccinia viruses, a multiplicity of 10 PFU/cell
for each virus was used. At 2 h, virus inoculum was replaced with 5 ml of
Eagle medium containing 2.5% FBS. Cells were collected at 24 h and
separated from culture medium by centrifugation. Cell pellets were suspended
in 1.0 ml of phosphate-buttered saline, freeze-thawed three times, and
dispersed by sonication. The cellular debris was removed by centrifugation,
and the supernatant was assayed for 1-gal activity as described by Fuerst et
al. (5). Expression is given as nanomoles of product formed in 30 minutes per
3.0 x 106 cells.
b Transient assay conditions were used following the procedures of Fuerst
et al. (5). CV-1 cells were infected with vaccinia virus recombinant vTF7-3
(+) at a multiplicity of 30 PFU/cell for 30 min at 37°C. The virus inoculum was
replaced with 10 l.g of calcium phosphate-precipitated pTF7LZ-1 DNA (+).
After 30 min, 5 ml of Eagle medium containing 2.5% FBS was added. Cells
were collected at 24 h and treated and assayed for 13-gal as stated above.
TK
antiserum
gel electrophoresis and immunoblotted with
(see Materials and Methods). A polypeptide of approxi-
P-gal
N
s
\lUld Type Vaccinii a
Virus
>
Recor Tbinadon
i
VI
kif*ction
Transfecion
mately 120,000 daltons, corresponding to the molecular size
of bona fide E. coli ,-gal, was detected in both expression
systems. P-Gal synthesis was observed 3 h after infection
with vTFLZ, reflecting the "early" character of the P7.5
promoter. However, P-gal was barely detected 6 h after
infection in cells coinfected with vTF7-3 and vTF7LZ-1. The
relative amount of P-gal protein produced was quantitated
by densitometry: 48 h after infection, the hybrid vaccinia
virus-T7 system produced p-gal at a fivefold higher level
than the straight single vaccinia virus recombinant system.
Absolute levels of p-gal produced over a 48-h time period
in cells infected with vTFLZ or coinfected with vTF7-3 and
vTF7LZ-1 are shown in Table 3. These values were calculated
CV-1 Cells
Homologous
Recombination
TK Recombinant Virus
FIG. 1. Construction of vaccinia virus recombinants conttaining
the E. coli lacZ gene flanked by T7 promoter and termiinator
sequences. A 3.2-kbp DNA segment containing the lacZ geniLe with
translation and termination codons was obtained by cleavEage of
pWS61 (provided by A. Majmdar, National Institutes of HIealth)
with XbaI, filling in the protruding end with the Klenow fragmient of
DNA polymerase I and deoxynucleotide triphosphates, and clheaving
with SmaI. The fragment was blunt-end-ligated to pAR2529 (.'5) that
had been treated with Klenow fragment. The resulting pkasmid,
pTF7LZ-1, has the lacZ coding sequence flanked by the T7 promoter (4r10) and terminator (TO0). The expression cassette , 4101acZ-T+, was excised from pTF7LZ-1 with BgIII, its staggerei d ends
were filled in with Klenow fragment, and it was inserted into IpGS50
cleaved within the tk gene with ClaI and EcoRI and made blunit. The
resulting recombination vector, plasmid pTF7ILZ-1, conttaining
flanking DNA sequences comprising the left (tkL) and righit (tkR)
parts of the tk gene, was transfected into cells that were infected
with wild-type vaccinia virus. After homologous recombinatic mn was
allowed to occur, TK- recombinant virus was selected.
by comparing the specific
enzyme activities of
p-gal
in
both cell extract and culture medium with that of purified
enzyme. Approximately 15 p.g of enzyme was synthesized
per 3 x 106 cells with the vaccinia virus-T7 system, compared with 3 pug for the straight vaccinia virus system. The
time course of enzyme activity paralleled that obtained by
immunoblotting, as shown above.
Effect of araC on ,-gal expression. The delayed onset of
p-gal synthesis observed with the vaccinia virus-T7 system
TABLE 2. Dependence of
vTF7-3 MOI
13-Gal (nmol/3
0.1
0.1
1
1-gal
26,800
7,330
10
1,400
30
450
a CV-1 cells
(approximately 3
expression
on
virus multiplicitya
106 cells) at vTF7LZ-1 MOI of:
1
10
Jo
x
15,000
41,700
1,700
14,800
14,950
3,900
80,800
17,950
1,150
8,520
34,950
13,650
x 106 cells) were coinfected with recombinant viruses vTF7-3 and vTF7LZ-1 at the indicated multiplicity of infection
(MOI) for each virus. At 2 h, virus inoculum was replaced with 5 ml of Eagle
medium (no phenol red) containing 2.5% FBS. Cells were collected at 24 h and
separated from culture medium by centrifugation, and cell pellets were
prepared. Both cell pellet and culture medium were assayed for 1-gal activity,
and the combined values are expressed as nanomoles of product formed in 30
minutes per 3 x 106 cells.
VACCINIA VIRUS-T7 HYBRID EXPRESSION SYSTEM
VOL. 7, 1987
2 0 0-
-
-
TABLE 4. Effect of araC on 3-gal expressiona
1-Gal expression (nmol/3 x 106 cells)
VACCINIA
AT7
VACCINIA
-3
_
95-
Time
postinfection
(h)
1
3
6
9
43 -
3
6
12 24 48 1
3
6 12 24 48
12
24
HOURS AFTER INFECTION
FIG. 2. Comparison of synthesis of P-gal from vaccinia virus and
T7 promoters. Immunoblot analysis of p-gal synthesis. CV-1 cells
were infected with 10 PFU of vTFLZ (vaccinia) or coinfected with
10 PFU of vTF7-3 and vTF7LZ-1 (vaccinia/T7) per cell and harvested at various times after infection. Cell samples were lysed,
separated by SDS-polyacrylamide gel electrophoresis, and transferred to nitrocellulose, and p-gal was detected immunochemically
(see Materials and Methods). The sizes of marker polypeptides (in
kilodaltons) are indicated on the left.
may reflect a requirement for replication and concomitant
genomic amplification of the recombinant virus for expression of either the T7 RNA polymerase or the T7 promotertarget gene template. To test this, cells were coinfected with
vTF7-3 and vTF7LZ-1 in the presence or absence of
cytosine arabinoside (araC), an inhibitor of vaccinia virus
DNA replication (3). In cells infected with the vaccinia
virus-T7 hybrids, araC inhibited expression of p-gal by 80 to
90% throughout infection (Table 4). In contrast, with the
single vaccinia virus recombinant vTFLZ, araC had no effect
on P,-gal expression early during infection but inhibited
expression by about 50% at later times. The latter results are
consistent with the early/late P7.5 promoter used for construction of vTFLZ.
We measured the levels of T7 RNA polymerase to determine whether inhibition of P-gal expression by araC was due
to the kinetics of polymerase synthesis or to inhibition of
template virus replication. Cells were infected with recombinant virus vTF7-3 in the presence and absence of araC,
and lysates were prepared at various times after infection
and assayed in vitro for T7 RNA polymerase with a DNA
template containing a T7 promoter (Fig. 3). In the absence of
inhibitor, T7 RNA polymerase activity was detected within 3
h and continued to increase for more than 12 h. AraC had
little or no effect on T7 RNA polymerase activity during the
early phase of infection but inhibited activity by approximately 50% after 12 h, consistent with the use of the 7.5-kDa
No
araC
With
araC
<10
495
1,134
1,794
2,453
5,760
<10
525
1,240
1,710
1,861
2,528
Time postinfection (h)
Vaccinia
1
3
6
12
24
48
<10
96
255
596
1,378
3,185
x
.'
a
CV-1 cell monolayers were infected with vaccinia virus recombinant
VTFLZ (Vaccinia) or coinfected with recombinants VTF7-3 and vTF7LZ-1
(Vaccinia-T7). Both cell extracts and culture medium were assayed for 13-gal,
and the amount of protein (based on activity) was extrapolated from a
standard activity curve established for purified 1-gal (Sigma) under identical
reaction conditions.
<10
<10
394
959
5,064
28,500
<10
<10
70
215
722
2,439
82
78
86
91
5
0
oD(a
4
_
Y In
o
,-3
x
E
< 0
z_
cr
N
5,358
15,463
%
Inhibition
6
0
<10
<10
82
821
5
24
56
With
araC
>1
%
106 cells)
Vaccinia-T7
0
0
No
araC
promoter. Thus, the severe reduction in p-gal in coinfected
cells in the presence of araC was probably not due to the
absence of T7 RNA polymerase.
Northern blot analysis of lacZ transcripts. The late expression of 3-gal could be due to either an early block in
transcription or translation of the mRNAs. To distinguish
between these possibilities, P-gal mRNA was analyzed.
CV-1 cells were coinfected with vTF7-3 and vTF7LZ-1, and
at various times after infection, bulk cytoplasmic RNA was
isolated, separated by agarose gel electrophoresis, blotted to
nitrocellulose, and probed with 32P-labeled pTF7LZ-1 (Fig.
4). A 3.3-kb lacZ transcript was only observed from 6 h after
infection. The size of the lacZ transcript corresponded to
that predicted (33-nucleotide 410 leader, 3.2-kb lacZ coding
sequence, and 110-nucleotide TX sequence), suggesting that
correct initiation and termination had occurred. Accumulation of the transcript over a 24-h period indicated that it was
relatively stable in vaccinia virus-infected cells. Since the
pattern of transcription corresponded with the kinetics of
p-gal protein synthesis, we concluded that template was
either inaccessible to the T7 RNA polymerase or present at
too low a copy number at early times after infection.
E
1-Gal (ng/3
%
Inhibition
a
CV-1 cells were infected with 10 PFU of recombinant virus vTFLZ
(Vaccinia) or coinfected with 10 PFU each of recombinant viruses vTF7-3 and
vTF7LZ-1 (Vaccinia-T7) per cell. Cultures were incubated in the absence or
presence of araC at 40 p.g/ml. At the indicated times after infection, cells were
harvested and assayed for 1-gal. Activity is expressed as nanomoles of
product formed in 30 minutes per 3.0 x 106 cells. Percent inhibition is
expressed as the percent decrease of activity in the presence of araC.
0
TABLE 3. Determination of a-gal activitya
Vacciniatl7
Vaccinia
57 -
1
2541
1
0
0
10
20
Time after infection (hr)
30
FIG. 3. Synthesis of T7 RNA polymerase. CV-1 cells were
infected at 20 PFU/cell with recombinant virus vTF7-3 in the
absence (l) or presence (R) of araC at 40 ,ug/ml or with wild-type
vaccinia virus (A). At the indicated times after infection, cells were
harvested and assayed for T7 RNA polymerase by an in vitro
transcription assay as described by Fuerst et al. (5). Incorporation of
[a-32P]GTP into RNA that bound to DEAE-cellulose filters was
measured.
2542
FUERST ET AL.
MOL. CELL. BIOL.
Expression of HBsAg and HIV envelope proteins. To demonstrate the utility of the hybrid system, we expressed the
DNA coding segments for HBsAg and HIV gpl60 following
a procedure similar to that illustrated in Fig. 1. The HBsAg
coding sequence was isolated as a 1.3-kb BamHI fragment
from pHBs4 (20) and inserted into the BamHI site of pTF7-3
(see Materials and Methods). A 2.5-kb EcoRV fragment
containing the gp160 coding sequence was cleaved from
mpPE-env and inserted into the BamHI site of pTF7-3 made
blunt with Klenow polymerase. Recombinant plasmids containing HBsAg and HIV gpl60 coding sequences juxtaposed
by T7 promoter (410) and termination (TO) sequences and
flanked by vaccinia virus tk DNA sequences were isolated
and recombined into the vaccinia virus genome by homologous recombination. TK- recombinant viruses vTF7HB-1
and vPE-5, which contained the HBsAg and gpl60 coding
sequences, respectively, were isolated and purified.
Under conditions established for the synthesis of 3-gal,
cells were coinfected with recombinant viruses vTF7-3 and
vTF7HB-1 and harvested at various times after infection,
and the levels of HBsAg in cell extracts and culture medium
were quantitated by radioimmunoassay (AUSRIA II; Abbott
Laboratories). Approximately 4 ,ug of HBsAg per 3 x 106
cells was detected by 48 h after infection (Table 5). HBsAg
synthesis was not detected in either cell extract or culture
medium until 6 h after infection. This delay coincided with
the kinetics of 3-gal synthesis shown previously and the
requirement for replication of the template.
HBsAg consists of a major protein of apparent molecular
weight 22,000 to 25,000 (P1) and a higher-molecular-weight
glycoprotein (P2) (14). As seen in Fig. 5A, two polypeptides
with estimated molecular weights of 22,000 and 27,000,
corresponding to P1 and P2, respectively, were present in
lysates of cells coinfected with vTF7-3 and vTF7HB-1. The
time course of synthesis of the HBsAg polypeptides by
immunoblot analysis parralleled the radioimmunoassay results for the detection of the 22-nm particles (Table 5).
Northern blot analysis of steady-state bulk RNA extracted
from cells at various times after coinfection with vTF7-3 and
vTF7HB-1 is shown in Fig. SB. A discrete 1.4-kb HBsAgspecific transcript was detected 6 h after infection and
1
3
6
12
24
TABLE 5. Production of HBsAg in the vaccinia virus-T7 systema
HBsAg (ng/3 x 106 cells)
Time postinfection (h)
Cell extract
Culture medium
<1
<1
13
288
1
3
6
12
24
48
<1
<1
<1
50
625
1,188
1,720
2,250
CV-1 cell monolayers were coinfected with vaccinia virus recombinants
vTF7-3 and vTF7HB-1 at 10 PFU/cell per virus. At 2 h, the virus inoculum
was replaced with 2.5 ml of Eagle medium containing 2.5% FBS. At the
indicated times after infection, cells were collected and separated from culture
medium by centrifugation. Cell pellets were suspended in 2.5 ml of medium
with 2.5% FBS, freeze-thawed three times, and sonicated. Equal portions of
cell extracts and culture medium were tested for HBsAg by
a
radioimmunoassay.
accumulated over a 24-h period. The onset of stable accumulation of HBsAg RNA and protein synthesis paralleled
each other, as found with ,3-gal.
We also compared HIV envelope protein synthesis by the
hybrid vaccinia virus-T7 system with that of a conventional
vaccinia virus recombinant. The vaccinia virus recombinant,
vPE-7, contained the same gp160 coding sequence described
above placed downstream of the vaccinia virus P7.5 promoter. Synthesis of HIV env proteins was demonstrated by
polyacrylamide gel electrophoresis of lysates of cells infected with the straight vaccinia virus recombinant vPE-7 or
coinfected with vTF7-3 and vPE-5, blotting to nitrocellulose,
and immunoprobing with serum from a patient with acquired
immunodeficiency syndrome (AIDS). Several cell types
were used in this analysis, BSC41, CV-1, and H9 lymphocytes. Immunoblot analyses of extracts from infected H9
and CV-1 cells are shown in Fig. 6. A major band comigrating with authentic gpl60 precursor and additional ones
corresponding to gpl20 and gp4l cleavage polypeptides were
observed. Densitometry of the bands corresponding to gpl60
indicated a relative intensity ratio of 7.5:1 for the hybrid
vaccinia virus-T7 and straight vaccinia virus expression
systems, respectively. The amount of gpl6O-cross-reactive
A 1
3
6
12
24
-
4
B 1
3
6
9
12
24
_~~~~~
-3 3
~
..
FIG. 4. Blot analysis of lacZ transcripts. Monolayers of CV-1
cells were coinfected with recombinant viruses vTF7-3 and
vTF7LZ-1 at 10 PFU/cell for each virus. At 1, 3, 6, 12, and 24 h after
infection, total RNA was extracted, fractionated on formaldehydeagarose gels, blotted, and hybridized with 32P-labeled pTF7LZ-1
DNA as described in Materials and Methods. The size of the major
band (in kilobases) is indicated on the right.
FIG. 5. Synthesis of HBsAg. (A) Immunoblot analysis of HBsAg
synthesis. CV-1 cells were coinfected with vTF7-3 and vTF7HB-1 at
10 PFU/cell for each virus. At 1, 3, 6, 12, 24, and 48 h after infection,
cell lysates were prepared, separated by SDS-polyacrylamide gel
electrophoresis, and transferred to nitrocellulose, and HBsAg was
detected immunochemically with anti-HBsAg monoclonal antibody,
CN324 (19). (B) Blot analysis of HBsAg transcripts. CV-1 cell
monolayers were coinfected as stated above. Total RNA was
extracted at 1, 3, 6, 9, 12, and 24 h after infection, separated on
formaldehyde-agarose gels, blotted, and probed with 32P-labeled
pTF7HB-1 DNA as described in Materials and Methods. The size of
the major band (in kilobases) is indicated on the right.
_
VOL. 7, 1987
CV-1
H9
-
_
~~~9pl 63
_
qgp120
a~~~ p4 l
Vac
VACCINIA VIRUS-T7 HYBRID EXPRESSION SYSTEM
Vac T7
HIV
Vac T7
Vac
@
if
^,
X
HIV
FIG. 6. Synthesis of HIV envelope proteiins. Duplicate cultures
of H9 suspension cells or CV-1 cell monolay,ers were infected with
vPE-7 (Vac) or coinfected with vTF7-3 andI vPE-5 (VacMT7) at a
multiplicity of 10 PFU/cell per virus. At 24 h after infection, cell
ellec-
lysates were prepared, separated by SDS-po lyacrylamide gel
trophoresis, blotted, and immunologically prc bed with serum from a
patient with AIDS as described in Materia ls and Methods. Cell
extracts prepared from H9 cells infected wiith HIV were used as
controls.
radioimmunohybrid
vaccinia virus-T7 system produced enve lope glycoprotein at
a consistently higher level than the straight vaccinia virus
system in the three cell types tested, wit! h the highest level of
1.5 ,ug produced per 3 x 106 infected BS' C-1 cells over a 24-h
period.
material
was
also measured by
a compe tition
assay (J. Bess, manuscript in prepar-ation). The
DISCUSSION
In a previous communication, we des,cribed a simple and
rapid way of expressing genes in mamnnialian cells by using
an appropriately engineered recombinanIt vaccinia virus that
synthesizes bacteriophage T7 RNA p olymerase (5). The
target gene, flanked by 17 regulatory seq uences, was present
in a plasmid and transfected into infec ted cells. Since the
level of expression was higher than tlhat of conventional
transient-expression systems, this prociLedure has considerable utility. Nevertheless, the level of e xpression is limited
by the calcium phosphate-mediated trarisfection procedure.
We therefore explored the possibility of introducing the
target gene as well as T7 RNA polymeirase into a recombinant vaccinia virus. Our initial efforts wiere aimed at inserting both genes into the same virus. We were unsuccessful,
however, apparently because of the lo w viability of such
double recombinants. Difficulty in cons tructing E. coli vectors with 17 RNA polymerase and T7 promoters has been
encountered previously (F. W. Studier, personal communication). We therefore adopted an altezrnative coinfection
strategy. This procedure involves the 4construction of two
recombinant vaccinia viruses: one conttaining the T7 RNA
polymerase gene under control of a vacccinia promoter and
the other containing a target gene flani ked by T7 promoter
and terminator regulatory elements. Ce 1ls infected with the
two viruses expressed a-gal at a 14-fold higher rate than was
obtained with the previous virus-plasmiid system. This increase may be attributed to more synchronous infection with
a virus, amplification of the template cluring virus replication, and better tissue culture conditioins with omission of
calcium phosphate.
We optimized the new expression sy!stem by varying the
multiplicihigh multiplicimultiplicities of each virus. Except at ^iery high
ties, the best result occurred with equtal amounts of each
virus and reached a maximum at a multiiplicity of 10 PFU of
semy
2543
each. We had anticipated that relatively lower amounts of
the virus containing the T7 RNA polymerase might be
required, but this was not the case.
T7 RNA polymerase was detected in cells within 3 h and
continued to increase during the remainder of the infectious
cycle, consistent with the presence of early and late transcriptional regulatory signals in the vaccinia virus promoter
used for expression. Nevertheless, target gene transcripts
were barely detected at 6 h and were much more abundant at
later times. The most likely explanation is that the template
is not accessible to the T7 RNA polymerase until the DNA in
the virus particles is completely uncoated or replicated. The
inhibition of expression when DNA replication was prevented with araC was consistent with the latter interpretation. Greater expression at late times may also be a consequence of increased template, since 10,000 to 20,000 copies
of vaccinia virus DNA were made.
T7 RNA polymerase seems to recognize its own transcriptional regulatory signals in a normal fashion in mammalian
cells. The target gene RNAs were of the appropriate size,
suggesting correct initiation and termination. A small fraction of the HBsAg transcripts were larger than predicted,
possibly because of readthrough of the T7 termination signal
and stopping at a downstream site in vaccinia virus DNA
that mimicked a T7 signal. Alternatively, some transcripts
may have started upstream at a vaccinia virus promoter. The
accumulation of transcripts, as judged by Northern blotting,
indicated that the RNA was fairly stable. This is an intriguing
result, since previous studies have suggested that in vaccinia
virus-infected cells, both cellular and viral mRNAs have
very short half-lives (13, 17, 18). It is our intention to further
characterize the T7 transcripts, with particular regard to the
structure of their 5' and 3' ends.
The general usefulness of the hybrid vaccinia virus-T7
system for mammalian cell expression was demonstrated by
synthesis of both HBsAg and the HIV gpl60 envelope
protein. Judging from their sizes, both of these proteins
appear to be properly glycosylated, and the latter was also
processed into subunits. Thus far, we have obtained higher
levels of expression with the vaccinia virus-T7 system than
with conventional recombinant vaccinia viruses. For a-gal
and gpl60, the hybrid system was six to seven times better,
while for HBsAg the difference was less. The absolute
amount of protein made was estimated to be about 5.4, 1.8,
and 1.5 ,ug per 3 x 106 over a 24-h period for 3-gal, HBsAg,
and HIV gpl60, respectively. These values increased approximately threefold over a 48-h incubation period, which
compares favorably with amounts reported for other mammalian expression systems (6, 12). Since the vaccinia virusT7 system is so new, we anticipate further increases in
efficiency. The fact that expression only occurred when the
two viruses were mixed may make this system particularly
useful for expression of toxic substances in mammalian
cells.
ACKNOWLEDGMENTS
We thank A. H. Rosenberg and F. W. Studier (Brookhaven
National Laboratory) for providing plasmid pAR2529, P. L. Cote
and J. L. Gerin (Georgetown University Schools of Medicine and
Dentistry)J. for
monoclonal antibody
(CN324)
against
and L. Arthur
Bessproviding
Cancer
for
HBsAg,
(National
Institute)
quantitation of HIV envelope proteins
by radioimmunoassay,
N.
Cooper for tissue cultures cells, and J. Drogin for typing the
manuscript.
2544
MOL. CELL. BIOL.
FUERST ET AL.
LITERATURE CITED
1. Burnette, W. N. 1981. "Western blotting": electrophoretic
transfer of proteins from sodium dodecyl sulfate-polyacryl_.
amide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal. Biochem. 112:195-204.
2. Chakrabarti, S., K. Brechling, and B. Moss. 1985. Vaccinia viruc
expression vector: coexpression of ,B-galactosidase providevisual screening of recombinant virus plaques. Mol. Cell. Biol.
5:3403-3409.
3. Cochran, M. A., C. Puckett, and B. Moss. 1985. In vitro
mutagenesis of the promoter region for a vaccinia virus gene:
evidence for tandem early and late regulatory signals. J. Virol.
53:30-37.
4. Dunn, J. J., and F. W. Studier. 1983. Complete nucleotide
sequence of bacteriophage T7 DNA and the locations of T7
genetic elements. J. Mol. Biol. 166:477-535.
5. Fuerst, T. R., E. G. Niles, F. W. Studier, and B. Moss. 1986.
Eukaryotic transient-expression system based on recombinant
vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc. Natl. Acad. Sci. USA 83:8122-8126.
6. Hsiung, N., R. Fitts, S. Wilson, A. Milne, and D. Hamer. 1984.
Efficient production of hepatitis B surface antigen using a
bovine papilloma virus-metallothionein vector. J. Mol. Appl.
Genet. 2:497-506.
7. Kozak, M. 1984. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs.
Nucleic Acids Res. 12:857-872.
8. Lehrach, D., D. Diamond, J. M. Wozney, and H. Boedtkes. 1977.
RNA molecular weight determinations by gel electrophoresis
under denaturing conditions: a critical re-examination. Biochemistry 16:4743-4751.
9. Mackett, M., G. L. Smith, and B. Moss. 1984. General method
for the production and selection of infectious vaccinia virus
recombinants expressing foreign genes. J. Virol. 49:857-864.
10. Mackett, M., G. L. Smith, and B. Moss. 1985. The construction
and characterization of vaccinia virus recombinants expressing
foreign genes, p. 191-211. In D. Rickwood and B. D. Hames
(ed.), DNA cloning, vol. 2. IRL Press, Washington, D.C.
11. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
cloning: a laboratory manual, p. 90-93. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
Moriarty, A. M., B. H. Hoyer, J. W. Shih, J. L. Gerin, and D. H.
Hamer. 1981. Expression of the hepatitis B virus surface antigen
in cell cultures by using a simian virus 40 vector. Proc. Natl.
Acad. Sci. USA 78:2606-2610.
Oda, K., and W. K. Joklik. 1967. Hybridization and sedimentation studies on "early" and "late" vaccinia messenger RNA.
J. Mol. Biol. 27:395-419.
Peterson, D. L., N. Nath, and F. Gavilanes. 1982. Structure of
hepatitis B surface antigen. J. Biol. Chem. 257:10414-10420.
Ratner, L., W. Haseltine, R. Patarca, K. J. Livak, B. Starcich,
S. F. Josephs, E. R. Doran, J. A. Rafalski, E. A. Whitehorn, K.
Baumeister, L. Ivanoff, S. R. Petteway, Jr., M. L. Pearson, J. A.
Lautenberger, T. S. Papas, J. Ghrayeb, N. T. Chang, R. C.
Gallo, and F. Wong-Staal. 1985. Complete nucleotide sequence
of the AIDS virus, HTLV-III. Nature (London) 313:277-284.
Rhim, J. S., H. Y. Cho, and R. J. Huebner. 1975. Non-producer
human cells induced by murine sarcoma virus. Int. J. Cancer
15:23-29.
Rice, A. P., and B. E. Roberts. 1983. Vaccinia virus induces
cellular mRNA degradation. J. Virol. 47:529-539.
Sebring, E. D., and N. P. Salzman. 1967. Metabolic properties of
early and late vaccinia messenger ribonucleic acid. J. Virol.
1:550-575.
Shih, J. W., P. J. Cote, G. M. Dapolito, and J. L. Gerin. 1980.
Production of monoclonal antibodies against hepatitis B surface
antigen (HBsAg) by somatic cell hybrids. J. Virol. Methods
1:257-273.
Smith, G. L., M. Mackett, and B. Moss. 1983. Expression of
hepatitis B surface antigen by infectious vaccinia virus recombinants. UCLA Symp. Mol. Cell. Biol. New Series 8:543-554.
Thomas, P. S. 1980. Hybridization of denatured RNA and small
DNA fragments transferred to nitrocellulose. Proc. Natl. Acad.
Sci. USA 77:5201-5205.
Towbin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic
transfer of proteins from polyacrylamide gels to nitrocellulose
sheets: procedure and some applications. Proc. Natl. Acad. Sci.
USA 76:4350-4353.
Zoiler, M. J., and M. Smith. 1984. Oligonucleotide primers and
a single-stranded DNA template. DNA 3:479-488.