JOURNAL OF VIROLOGY, Dec. 1984, p. 1024-1027 Vol. 52, No.

3
0022-538X/84/121024-04$02.00/0
Copyright C) 1984, American Society for Microbiology

Protein Synthesis in a Lymantria dispar Cell Line Infected by

Cytoplasmic Polyhedrosis Virus
M. ARELLA," S. BELLONCIK,2* DEVAUCHELLE'

Laboratoire Associe 203, Equipe de Virologie du Centre National de la Recherche Scientifique, Universite de Rouen,
Rouen, France,' and Centre de Recherche en Virologie, Institut Armand-Frappier, C.P. 100, Laval, Quebec,
Canada H7N 4Z32
Received 2 December 1983/Accepted 27 August 1984

The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order

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Lepidoptera, Euxoa scandens, was studied in eight different lepidopterean cell lines. Lymantria dispar cells,
which were found to support viral replication, more efficiently, were used to follow the kinetics of appearance
of viral-specific polypeptides by a 2-h pulse with [35S]methionine. Five polypeptides (ca. 120,000 molecular
weight [120K], 105K, 66K, 46K, and 28K) were identified as components of the polyhedral inclusion bodies,
and two polypeptides (112K and 39K) were assigned as viral-particle polypeptides. All these polypeptides were
present after 24 h and were still being produced 96 h after infection. The rate of synthesis of the major
polyhedral polypeptide (28K) increased in the time course of infection, whereas the background of cellular
polypeptides seemed to be unaffected. An indirect immunoperoxidase technique, after sodium dodecyl sulfate-
polyacrylamide gel electrophoresis was blotted to a nitrocellulose membrane, showed that traces of the major
polyhedral polypeptide were found from 8 h postinfection.

Cytoplasmic polyhedrosis viruses (CPVs) are one of the
six genuses in the Reoviridae family and are found mainly in
insects (14). Thus, they possess double-stranded RNA
genome in the form of 10 segments that are not linked
covalently, and their coding assignment for one type of CPV
has been established (15; P. P. C. Mertens, Ph.D. thesis,
Oxford University, England, 1979). Differences in the elec-
trophoretic mobilities of genome segments between different
isolates of CPV provides a basis for a provisional classifica-
tion of 13 distincts virus types of CPV (20, 21).
Apart from host range and morphology, CPVs are distin-
guished among the Reoviridae by the production of large
polyhedral inclusion bodies (polyhedra), containing four to
six polypeptides (21), in the cytoplasm of virus-infected cells
(for a review, see references 18 and 20).
Although several reports have described the polypeptide
composition of CPV purified polyhedra and viral particles (7,
12, 19, 21, 22), there are no descriptions of intracellular
polypeptide synthesis of viral origin after CPV infection in a
tissue culture system. Recently it was reported by McCrae
(15) that the absence of a suitable tissue culture system for
CPV growth in an impediment to the study of the molecular
details of viral replication and gene expression. However,
this assertion must be denied since successful infection of
invertebrate cell lines with different types of CPV has been
reported (4, 6, 7, 13, 24). In our efforts to understand the
replication strategies of CPVs in cell culture we report here
the infection of eight lepidopterean cell lines and the poly-
peptide patterns of one of these cell lines (Lymantria dispar)
after infection with a CPV isolation from the lepidopterean
Euxoa scandens (EsCPV) (24). Finally, we adapted an
indirect immunoperoxidase technique to identify EsCPV
proteins after sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was blotted to a nitrocellulose
membrane.
The seed virus (EsCPV) was obtained from diseased
* Corresponding author.
1024
larvae (24), and the procedure proposed by Payne and
Tinsley (22) was adopted for purification. Established cell
lines from Bombyx mori (strain SPC Bm 36) (9), Choriston-
eura fumiferana (IPRICF 124) (27), Choristoneura fumifer-
ana Ti (IPRICF-124) (2), Lymantria dispar (SCLd 135) (23),
Malacosoma disstria (IPRIMd 66) (9), Papilio xuthius
(strains Px 58 and Px 64) (16); and Trichoplusia ni (8) were
propagated in plastic flasks (25 cm2) (Bioblock, Strasbourg,
France) at 28°C using the insect medium of Grace (4)
supplemented with 10% fetal calf serum and antibiotics (100
U of penicillin and 100 pLg of streptomycin per ml of culture
medium). Before infection, exponential growth cultures of
cells were seeded in multiwell plates (1.5 cm in diameter), set
at 4 x 105 cells per ml, and incubated at 28°C. After 24 h, the
medium was discarded, and 0.2 ml of viral suspension was
added to each well for an adsorption period of 1 h. The cell
sheet was then washed three times with culture medium, and
0.5 ml of medium was added to each well. The viral
suspension used was titrated at 106.23 50% tissue culture
infective dose per ml with a technique described by Bellon-
cik and Chagnon (1). For radiolabeling of cells, at the
indicated times postinfection the insect medium of Grace
without methionine and supplemented with 2% dialyzed fetal
calf serum was added to the cultures for 1 h. Then it was
replaced by the same medium containing 20 pCi of L-
[35S] methionine (specific activity, 1,465 Ci/mmol; Radio-
chemical Centre, Amersham, England) per ml for 2 h. Cell
lysates for SDS-PAGE analysis were prepared by discarding
the supernatant of cultures and resuspending the cells in the
following buffer: 50 mM Tris-hydrochloride pH (6.8) con-
taining 2% SDS, 2% 3-mercaptoethanol, and 15% glycerol.
After the trichloracetic acid-precipitable material was boiled
for 2 min, the radioactivity incorporated in it was evaluated
in duplicate 5-,u portions from all samples dried onto What-
man 3 filter paper disks (2 by 2 cm). The filters were
immersed into ice-cold 5% trichloroacetic acid, washed
twice with absolute ethanol, and dried in benzidine and air.
The radioactivity was then counted in 5 ml of a toluene-
based scintillation fluid with a liquid spectrometer (Inter-
 ;~ ~ .
VOL. 52, 1984 NOTES 1025

TABLE 1. Yield of CPV polyhedra in various lepidoptera cell (heavy plus light chain), and peroxidase labeled (Institut
linesa Pasteur Production, Paris, France). After extensive wash-
No. of cells No. of ings, the peroxidase activity was revealed with the medium
Cell line containing Nol of polyhe- polyhe- of Graham and Karnovsky (5).
polyhedra dra (x 104) dra per In Table 1 we show that the L. dispar cell line is the more
(%) cell
efficient to support EsCPV replication as compared with
Lymantria dispar 57.7 ± 5.6
714 ± 25.8 12.21 seven other tested, established lepidopterean cell lines.
Bombyx mori 32.8 ± 6.8 411.3 ± 59.5 6.98 Results presented in this table were obtained, after an
Choriston eura fumiferana 6.8 ± 2.9 65.6 ± 5.2 0.46 incubation time of 96 h at an optimum temperature of 28°C;
Choristoneura fumiferana ti 5.1 + 2.5 37.0 ± 18.7 0.40 increasing incubation time to 168 h did not significantly alter
Trichoplusia ni 6.1 ± 1.2 21.6 ± 6.6 0.26
Malacosoma disstria 3.3 ± 2.8 5.2 ± 0.9 0.14 the yield of infection. At that time efforts to clone L. dispar
Papilio xuthus strain 58 1.2 ± 0.3 4.1 ± 1.5 0.05 cells with improved susceptibility to EsCPV infection were
Papilio xuthus strain 64 2.2 ± 0.3 6.5 + 1.8 0.07 unsuccessful. It is noteworthy to mention that the EsCPV
a Results of this table are the average of six different experiments. Incuba-
polyhedra obtained in all of these cell cultures are of cuboid
shape, compared with the spherical shape obtained with the

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tion was at 28°C for 96 h.
same inocula in larvae.
The time course of synthesis of viral polypeptides in L.
technique, Paris, France). A total amount of radioactivity of dispar cells infected by EsCPV shows that seven viral-
105 cpm per well of each sample was used for analysis by specific polypeptides appeared starting from 24 h postinfec-
SDS-PAGE after slight modifications of the Laemmli (11) tion (Fig. 1). The remaining viral polypeptides were probably
procedure and in a 14-cm-length gel (apparatus GE 2/4 L, not seen because of comigration with other viral or cellular
Pharmacia, Uppsala, Sweden). The electrophoresis buffer polypeptides or because of their low rate of synthesis. We
contained 25 mM Tris, 192 mM glycine, and 0.1% SDS (pH also show in Fig. la the assumed correspondence of these
8.3). Electrophoresis was conducted at room temperature at polypeptides to viral or polyhedral polypeptides based on
150 V for 6 h. At this time, we performed electroblotting at the Coomassie blue staining of purified viral particles and
250 mA for 6 h in a Bio-Rad Trans-Blot system following the polyhedra (Fig. 2). Five polypeptides (ca. 120,000 molcular
procedure of Towbin et al. (27). Polypeptides were trans- weight [120K], 105K, 66K, 46K, and 28K) were identified as
ferred to a 0.45-,um nitrocellulose membrane Schleicher & components of the polyhedral inclusion bodies, and two
Schull Co., Danel, Federal Republic of Germany) in a buffer polypeptides (112K and 39K) were assigned as viral-particle
of 25 mM Tris-192 mM glycine (pH 8.3) containing 20% (vol/ polypeptides. After blotting SDS-PAGE to a nitrocellulose
vol) methanol. After blotting, the nitrocellulose membrane membrane and using the indirect immunoperoxidase tech-
was saturated for 1 h at 40°C in Tris-hydrochloride (pH 7.4) nique with antibodies directed against whole polyhedra, we
containing 0.3% bovine serum albumin, 0.9% NaCI, 0.2% observed specific staining of the 28K major polyhedral
polyvinylpyrrolidone (Sigma Chemical Co., St. Louis, Mo.), polypeptide from 8 h postinfection. These results are in
and 0.02% Ficoll 400 (Sigma). The membrane was then accordance with those recently published by Payment et al.
incubated for 2 h with antibodies obtained from rabbits and (17) using an enzyme-linked immunosorbent assay. Early
directed against EsCPV polyhedra and viral particles, detection of the 39K polypeptide by staining with the im-
washed several times in buffer, incubated for 1 h with munoperoxidase technique (1 h postinfection; Fig. 3) may be
commercially available sheep anti-rabbit immunoglobulin G due to the parental viral particles entering the cells, since the

X .112V

92

66- -66 p

15- - 4 p

t
I t 1 0 .1
I
# 0, "o $.
.1
4, '. ."i
I
ilim"

t .
O.-

10
..,*
49 39
- v
31-
28 p

T 1 2 4 8 12 18 24 36 48 72 g9
FIG. 1. Appearance of [35S]methionine-labeled EsCPV polypeptides in L. dispar-infected cells. Uninfected cellular polypeptides (T) were
pulse-labeled for 2 h, and infected cell polypeptides were pulse-labeled for 2 h at the indicated times after infection. The migration of the
standard polypeptides used is indicated in the left margin in kilodaltons: carbonic anhydrase (31), ovalbumin (45), bovine serum albumin (66),
and phosphorylase B (92). p, Polyhedral polypeptide; v, viral-particle polypeptide.
1026 NOTES J. VIROL.

shown). Also, it should be noted that no viral polypeptides

were produced in the culture medium precipitated with
trichloroacetic acid and analyzed by SDS-PAGE with infect-
112V
2V ---
ed L. dispar cells (data not shown). An analysis by densitom-
-----
82 V _10 - 92 etry of the autoradiograms presented in Fig. 1 and trichloro-
66 P-
acetic acid precipitation experiments with labeled uninfected
I_III -66 and infected cells proved that cellular polypeptides are
produced in similar amounts (as can be seen in Fig. 1)
49 V 45 throughout the infection cycle, indicating that the cellular
46 P- SF macromolecular synthesis is not shut off by the virus. We
were unable to reduce the cellular background with actino-
39 V I mycin D at concentrations up to 5 Fig per ml of culture
31 medium. Higher concentrations of actinomycin D were
28 p ztS - rapidly toxic to the L. dispar cell line.
In this paper we show that EsCPV may be replicated with

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20
_P.
__14.4
1 2 3 4
FIG. 2. Coomassie blue staining of purified viral particles (lane
2) and polyhedra (lanes 1, 3, and 4). Migration of mo lecular weight
standards (x 103) is shown on the right. Lane 1, puri fied polyhedra
from larvae; lane 2, purified viral particles from larvate; lanes 3 and
4, purified polyhedra from cell culture. p, Polyhedral F:)olypeptide;
viral-particle polypeptide. v'
multiplicities of infection used in this particula .r experiment
are significantly high (50 ,ug of virus proteins p'er ml).
Repeated molecular weight determinations show differ-
ences with the previously published results forr EsCPV (7);
however, these differences can be attributed to the accuracy
of the technique of SDS-PAGE for determiniation of the
molecular weight of polypeptides (29) and Imay also be
attributed to the use of different polypeptide sttandards.
Similar results were obtained when [35S]me thionine was
replaced by a mixture of 14C-protein hydrolyssate, and the
seven polypeptides due to viral infection m igrated in a
manner similar to that with [35S]methionin e (data not
very different yields of polyhedra in all the lepidopterean cell
lines tested. All these cell lines produce cuboid-shaped
polyhedra, which is different from the shape of the polyhedra
produced by the same virus isolated in larvae. This latter
feature seems to be related to cellular conditions for larvae
that are different from those for cell cultures, as discussed in
depth in the review by Payne and Mertens (20).
It is clear that a study of the regulation of protein synthesis
by CPV is facilitated by the use of cell culture rather than
experiments dealing with translation of mRNA or denatured
double-stranded RNA in in vitro systems (15, 26). Hence,
with separation of CPV-specific mRNA at different times
after infection and using in vitro systems for translation, we
are able to account for and compare with CPVs specific
polypeptides produced in infected cell lines.
To our present knowledge this is the first published work
concerning the appearance of CPV polypeptides in infected
cells, and therefore it constitutes a first approach to compar-
ing the molecular biology of CPV replication in cell culture
with the results obtained by using in vitro systems (15). The
pattern of polypeptide synthesis reported here for EsCPV-
infected cells shows that the virus infection does not cause a
decrease in cellular polypeptide synthesis. The situation is
different from the cellular shut-off observed during orbivirus
infection (10) or rotavirus infection, where cellular protein
synthesis may be effectively suppressed with both high
multiplicities of infection and actinomycin D (3).

- 120 p
'- 112 V
- 110 p

92 -

66 - - 66 p

- 46 p
45-

31 - _ms w m 6i.u~hEMS- 28 P
.~.4*

T 1 2 4 112 16 24 304 72 98 cpv

FIG. 3. Electrophoretic blotting of SDS-PAGE of the polypeptide profiles of infected cells to a nitrocellulose membrane. Staining of the
blot by the indirect immunoperoxidase technique is as described in the text. The far right lane contains purified CPV polyhedra and viral
particles. p, Polyhedral polypeptides; v, viral-particle polypeptide.
VOL. 52, 1984
The fact that the rate of synthesis of the major polyhedral
polypeptide (28K) increases during pulse periods at 36, 48,
72, and 96 h after infection suggests the regulation of
translation or transcription of viral mRNA for the polyhedrin
gene. Further studies on CPV replication in L. dispar cells
should serve as a model to help understand the regulation of
CPV transcription in vivo either by host cellular or nascent
viral proteins, as previously suggested by Smith and Furui-
chi (25).
We express our appreciation to S. Barray for helpful discussions,
J. Lecomte and M. Henrichon for the densitometric analysis of the
autoradiograms, and D. Rouleau for her excellent technical assist-
ance.
We acknowledge the financial assistance of the Centre National
de la Recherche Scientifique (France), the F.C.A.C. (Quebec), and
the National Sciences and Engineering Research Council (Canada)
(A9726). The research was a part of a collaboration project of
France-Quebec (FQ 07-06-36).
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