Journal of General Virology (1993), 74, 2731-2735. Printed in Great Britain
2731
Expression of potyvirus proteins in insect cells infected with a recombinant
baculovirus
D. W . Thornbury, 1. J. F. J. M . van den H e u v e l , t J. A. L e s n a w 2 and T. P. Pirone 1
Departments o f a Plant Pathology and 2 Biological Sciences, University o f Kentucky, Lexington, Kentucky 40546,
U.S.A.
The N-terminal portion (P1-HC-Pro-P3) of the tobacco
vein mottling virus (TVMV) polyprotein was expressed
in insect cells and larvae by a recombinant baculovirus.
The proteases necessary to process this TVMV polyprotein fragment were active in insect cells, since mature
P1, HC-Pro and P3 proteins were detected by specific
antisera in Western blots. Antisera to P1, HC-Pro and
P3 also recognized polypeptides with apparent M r values
predicted for the intermediate processing products of
the polyprotein fragment. The results of this study
indicate that the autocatalytic processing of TVMV
HC-Pro from the polyprotein is supported by insect cells.
Helper component activity in extracts of cells infected
with recombinant baculovirus was not detected by aphid
transmission assay.
The potyviruses are filamentous, positive-strand RNA
plant viruses which have an approximately 10 kb genome
encoding a polyprotein which is proteolytically processed
into at least eight products (Dougherty & Carrington,
1988). The N-terminal third of the genome codes for
three proteins, P1, HC-Pro and P3, each of which have
been detected in tobacco vein mottling virus (TVMV)infected plants (Thornbury et al., 1985; RodrfguezCerezo & Shaw, 1991). In tobacco etch virus (TEV) two
of these proteins, HC-Pro and P1 (35K), contain protease
domains which are responsible for their release from the
polyprotein (Carrington et al., 1989; Verchot et aI.,
1991). The N-terminal portion of the HC-Pro protein
contains the helper component domain necessary for
transmission of potyviruses by aphids (Thornbury et al.,
1985; Atreya et al., 1992) and approximately 20K of the
C-terminal portion contains the proteolytic domain
which cleaves between a G I ~ G l y dipeptide located
between HC-Pro and P3 (Carrington et al., 1989).
Verchot et al. (1991) showed in wheat germ cell-free
translations that the N terminus of TEV HC-Pro is
cleaved by a protease domain at the C terminus of the
TEV P1 protein.
Potyvirus proteins have been expressed in cell-free
translation systems, Escherichia coli, and transgenic
plants. Cell-free translation systems have been used to
identify and map the potyvirus genes and gene products
(Dougherty & Hiebert, 1980; Hellmann et al., 1980,
1983; Hiebert et al., 1984), and to characterize the
proteases involved in processing the polyprotein (Carrington & Dougherty, 1987; Carrington et al., 1988,
1989; Hellmann et al., 1988; Verchot et al., 1991). E. coli
expression systems have been used to produce polypeptides for generation of antisera to coat protein and
NIb (Nagel & Hiebert, 1985), TEV HC-Pro (Carrington
et al., 1990), TVMV P1 (34K) and P3 (42K) (RodriguezCerezo & Shaw, 1991), and to study processing by the
NIa protease (Garcia et al., 1989; LaLibert6 et al., 1992).
Transgenic plant systems support proper processing of
the polyprotein and produce biologically active HC-Pro
(Berger et al., 1989; Carrington et al., 1990).
Expression of potyvirus proteins in the baculovirus
expression vector system (BEVS) is an attractive alternative to bacterial and cell-free systems because insect cells
support post-translational modifications in a manner
similar to other eukaryotic systems and are thus capable
of producing authentic proteins (Maeda, 1989; Luckow,
1991). A major advantage of the BEVS over the use of
expression in transgenic plants is that less time is required
to reach the stage of purification of the recombinant
protein. In this study we explore the suitability of the
BEVS for expression of potyvirus genes. We report
proper processing of a potyvirus polypeptide in insect
cells into mature-sized proteins and describe problems
with recovery of helper component activity.
Production of recombinant baculovirus expressing
potyvirus proteins was mediated using the transfer vector
pJVP10Z, kindly provided by Dr C. Richardson (Biotechnology Research Institute, Montreal, Quebec,
Canada). This vector contains two promoters derived
t Present address: DLO Research Institute for Plant Protection,
P.O. Box 9060, 6700 GW Wageningen, The Netherlands.
0001-1813 © 1993SGM
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2732
Short communication
from the P10 and polyhedrin genes of Autographa
californica nuclear polyhedrosis virus (AcMNPV)
(Vialard et al., 1990). The P10 promoter directs synthesis
offl-galactosidase as a reporter gene to identify recombinants. Expression of potyvirus genes under the control of
the polyhedrin promoter is achieved by insertion of
constructs into an NheI site 3' to the promoter.
A construct containing bases 206 to 3541 of the
TVMV genome was prepared from cDNA clones using
PCR and specific oligonucleotides which introduced
XbaI sites at the 5' and 3' ends and an in-flame
termination codon after base 3541. The construct
included the initiation codon of the TVMV polyprotein
and the first three cistrons (P1-HC-Pro-P3) of the viral
genome. After digestion with XbaI the 3348 bp fragment
was cloned into the compatible NheI site of pJVP10Z to
generate pTHC 1OZ. The plasmid was CsCl-purified from
E. coli strain DH5~ and the fidelity of the PCR was
confirmed by sequencing.
Spodoptera frugiperda (sfg) cells (Invitrogen), were
cultured in Grace's Antherea Medium (GAM) plus
supplements and 10% fetal bovine serum (FBS)
according to the manufacturer's recommendations. Cells
were cotransfected with the DNA from the plasmid and
wild-type AcMNPV DNA (Invitrogen) by the calcium
phosphate precipitation method (Carstens et al., 1980)
followed by a glycerol shock, using 20% glycerol in
GAM for 1 min at room temperature. Recombinant
plaques were identified by overlay of plaque assay plates
with 0"5 % X-Gal in agarose, as a/~-galactosidase activity
indicator (Vialard et al., 1990). Recombinant plaques
were found to be free of polyhedra after two or three
rounds of plaque purification. Proteins from recombinant plaques were compared on SDS-polyacrylamide
gels with those from wild-type AcMNPV plaques and
were found to be free of the polyhedrin protein.
Baculovirus from plaques positive for fl-galactosidase
was used to infect 2.5 x 106 Sf9 cells following the
procedure described by Summers & Smith (1987). Cells
were harvested 2 to 4 days post-infection (p.i.) and
assayed for expression of potyvirus HC-Pro by SDSPAGE in 10% acrylamide gels (Laemmli, 1970) and
Western blot analysis (Towbin et al., 1979) using antisera
to TVMV HC-Pro (Thornbury et al., 1985). Enrichment
stocks of recombinant baculovirus were made and the
virus titre was determined in plaque assays as described
by Summers & Smith (1987).
The expression of potyvirus peptides in insect cells was
followed after inoculation of Sf9 or Trichoplusia ni cells
(High Five cells, Invitrogen) with the recombinant
baculovirus at an m.o.i, of 10. Cultures of both cell lines
(3 x 106 cells) were inoculated with the recombinant
baculovirus at an m.o.i, of 10. Two days p.i., cells were
collected, washed to remove FBS and resuspended in
1
2
3
4
5
HC-Pro-- ~
Fig. 1. Westernblot detectionof TVMVHC-Pro in supernatant(lanes
1 and 3) and pellet (lanes 2 and 4) fractionsof High Fivecells(lanes 1
and 2) or Sf9 cells (lanes 3 and 4) infected with a recombinant
baculovirus compared with HC-Pro from TVMV-infected plants
(lane 5).
3 ml of 100 mM-Tris-H~SO~ pH 7.2, 20 mM-MgSOa
(TSM). Cells were then lysed by sonication and centrifuged at 16000g. Aliquots of pellet and supernatant
fractions from equal numbers of cells were compared by
Western blot analysis using antiserum to TVMV HC-Pro
(Fig. 1). Four major products with Mr of 126K, 97K,
81K and 52K were detected in supernatant and pellet
fractions (Fig. 1, lanes 1 and 2) of High Five cells; the
latter product comigrated with TVMV HC-Pro extracted
from plants (Fig. 1, lane 5). The only product detected in
Sf9 cells at 2 days p.i. by antiserum to HC-Pro was a
single high M r band in the pellet fraction (Fig. 1, lane 4).
This band was also detected in the pellet fraction from
High Five cells (Fig. 1, lane 2). Since the Sf9 cells gave a
lower level of protein expression than High Five cells the
latter were used in the remainder of this study.
Identification of the 126K, 97K and 81K bands was
achieved using antisera to TVMV P 1 and P3 polypeptides
produced in bacteria (Rodriguez-Cerezo & Shaw, 1991)
in addition to HC-Pro antiserum. Four days p.i. 3 x 106
cells infected with the recombinant baculovirus were
collected and analysed by Western blot. Cell pellets,
washed as before, were resuspended in 500 lal of TSM,
diluted with an equal volume of 4 x Laemmli loading
buffer and heated for 5 min at 100 °C. Antisera to
TVMV P1 and P3 detected polypeptides of the expected
size (Fig. 2, lane 1, 34K; lane 2, 42K). Antisera to PI also
recognized the 81K and 126K polypeptides (lane 1)
detected by antisera to TVMV HC-Pro, and antisera to
P3 protein recognized the 97K and 126K products (lane
2). The observation that an 81K product is recognized by
both anti-P1 and anti-HC-Pro sera and that a 97K
protein reacts with antisera to both P3 and HC-Pro
suggests that these polypeptides are intermediate products of polyprotein processing by the protease domains
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1
HC-P
P3
2
3
4
Pl+
-- HC-Pro +
P3
P1 +
HC-P
Table 1. The effect of chaotropic agents and the
zwitterionic detergent C H A P S on activity of helper
component purified from TVMV-infected plants
Treatment of HC-Pro*
-- HC-Pro
P~
P]
Fig. 2. Western blot of T V M V polypeptides expressed by recombinant
baculovirus in High Five cells. Detection of proteins from total cell
extract by antiserum to P1 (lane 1), P3 (lane 2) and HC-Pro (lane 3).
Proteins with M r of 81K and 126K were recognized by P1 and HC-Pro
antisera (lanes 1 and 3) and P3 and HC-Pro antisera detected 97K and
126K proteins (lanes 2 and 3). Lane 4, HC-Pro from TVMV-infected
tobacco plants.
of the TVMV P1 and HC-Pro proteins. The fact that
more of the 81K product than the 97K product is
detected in lanes treated with either HC-Pro, P1 or P3
antiserum suggests that the HC-Pro/P3 junction is more
efficiently cleaved than the P1/HC-Pro junction. This is
also supported by the fact that more P3 is present in lane
2 than P1 in lane 1. Verchot et al. (1992) suggested that
the efficiency of these protease domains may provide
temporal regulation of the release of proteins from the
polyprotein; the BEVS would provide a useful system to
study the order of release of potyvirus proteins. Since the
processing of the P1/HC-Pro junction appears to be
relatively slow, it is possible that the 81K protein may
also have a biological function as well as being a
precursor of P1 and HC-Pro.
HC-Pro must be in a soluble form in order to have
helper component activity in the aphid bioassay (D. W.
Thornbury, unpublished data). Most of the HC-Pro and
other potyvirus polypeptides expressed by recombinant
baculovirus were consistently found in insoluble fractions of cell extracts, and attempts to prepare extracts
having helper component activity from insect cells using
procedures used for plant-derived HC-Pro (Thornbury
et al., 1985) were unsuccessful. Espinoza et al. (1992) and
Blanc et al. (1993) reported similar problems with
extraction of cauliflower mosaic virus helper component
(CaMV aphid transmission factor, ATF) from insect
cells infected with recombinant baculovirus. However,
Blanc et al. (1993) were able to recover active CaMV
ATF expressed by recombinant baculovirus by an
extraction procedure which was least destructive of the
native structure of the expressed protein. Their procedure, which is similar to that used for purification of
2733
Aphid
transmission
of TEV ( % ) t
Control HC-Pro
0-5 M-urea
1 M-urea
2 M-urea
4 M-urea
6 M-urea
0-5 M-guanidin~HC1
1 M-guanidin~HCl
2 M-guanidin~HCl
5 raM-CHAPS
20 mM-CHAPS
80
80
50
20
0
0
0
0
0
40
20
5 raM-CHAPS+ 0-5 M-urea
20 raM-CHAPS+ 05 ~-urea
50
30
* Treatments consisted of chaotropic agents or C H A P S dissolved in
2 x TSM. T V M V HC-Pro purified from plants was diluted 1/10 in each
treatment, incubated 1-5 h at 4 °C, dialysed 18 h against 2 x TSM,
concentrated by precipitation with 8 % P E G and resuspended to its
original volume in 2 x TSM.
t Percentage of infected plants; 10 aphids were placed on each of 10
test plants for each treatment.
CaMV ATF from plants, was not effective for recovery
of TVMV helper component activity expressed in insect
cells (data not shown).
An attempt was made to develop procedures for
solubilization of HC-Pro having helper component
activity. A number of procedures based on solubilization
by chaotropic agents followed by gradual renaturation
were first tested on active helper component which had
been partially purified from infected tobacco plants.
Urea, guanidine-HC1 or the zwitterionic detergent
CHAPS each in 2 x TSM were tested (Table 1). Concentrated TVMV HC-Pro was diluted 10-fold in each
solution and incubated for 1 hour at 4 °C following
which HC-Pro was further diluted 7.5-fold and placed in
dialysis tubing. In the case of urea or guanidine-HC1 the
concentration was then reduced hourly in 0"5 M steps
until reaching 0"5 M, and then in 0-I u per hour steps.
During dialysis, precipitates formed at 0.4 M-urea and
0"1 M-guanidine-HC1. CHAPS treatments were dialysed
against 2 x TSM overnight, samples were then concentrated by precipitation with 8% polyethylene glycol
(Thornbury et al., 1985) and resuspended to their original
volume in 2 x TSM. A control sample was diluted in
2 x TSM and treated in a similar manner. Final concentrates of each treatment and the control were clarified by
centrifugation at 16000 g and the supernatants tested for
helper component activity in the aphid bioassay. No
helper component activity was detected in the soluble
fraction from the renaturation treatments with
guanidine-HC1, whereas treatment with urea at over 1 u
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2734
Short communication
and CHAPS as low as 5 mM resulted in a 25 to 60 %
reduction helper component activity as compared to the
control HC-Pro (Table 1). Extraction procedures used
for plant tissue supplemented with the treatments in
Table 1 that did not destroy helper component activity
were tested for recovery of TVMV HC-Pro expressed in
insect cells. In all cases HC-Pro was found only in pellet
fractions and no fractions contained helper component
activity detectable by aphid bioassay (data not shown).
The active form of potyvirus helper component recovered
from virus-infected plants is probably a dimer of the 52K
HC-Pro protein (Thornbury et al., 1985), and biological
activity may depend upon proper folding during translation and/or proper subunit association during polyprotein processing. Our experiments with helper component from infected plants in the present study indicate
that once the structure necessary for transmission activity
is destroyed it cannot be recovered.
The TVMV gene products expressed by a recombinant
baculovirus appear to have been processed properly and
to have undergone the post-translational modifications
necessary to produce proteins with the same apparent M,,
as those produced in tobacco plants infected with
TVMV. The protease responsible for cleavage of the
P1/HC-Pro junction and the protease domain of HCPro were active in insect cells and produced HC-Pro
protein that comigrated with authentic HC-Pro from
plants. P1 and P3 proteins having the correct M~ were
also produced in the insect cells. These results extend to
TVMV the observation of Verchot et al. (1991) that the
TEV P1 protein contains the protease that cleaves the
P1/HC-Pro junction in the TEV polyprotein.
Although the recovery of helper component activity
from the BEVS has been thus far unsuccessful the results
of these studies suggest that proper post-translational
processing of potyvirus proteins occurs in insect cells.
The expression of potyvirus genes in the BEVS may be
useful for study of the regulation of polyprotein
processing and determining the unknown functions of
proteins such as P1 and P3.
The authors are grateful to M. McGuinness and S. Dutton for
technical assistance. J. F. J. M. van den H. is a recipient of a fellowship
of the Netherlands Organization for Scientific Research (NWO). This
work was supported by NIH Grant 5R01 A113574 to J. A. L., and by
USDA Competitive Grant 91-37303-6505 and USDA Cooperative
Agreement 58-6430-1-126 to T.P.P. and D.W.T. This is Kentucky
Agricultural Experiment Station Journal Series Paper 93-11-54.
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