Journal of Invertebrate Pathology 94 (2007) 130–139
www.elsevier.com/locate/yjipa
A glassy-winged sharpshooter cell line supports replication of
Rhopalosiphum padi virus (Dicistroviridae)
Sandhya Boyapalle a,1, Narinder Pal a, W. Allen Miller b, Bryony C. Bonning a,¤
a
Department of Entomology, Iowa State University, 418 Science II, Ames, IA 50011-3222, USA
b
Department of Plant Pathology, Iowa State University, Ames, IA 50011, USA
Received 27 July 2006; accepted 26 September 2006
Available online 17 November 2006
Abstract
Rhopalosiphum padi virus (RhPV) (family Dicistroviridae; genus Cripavirus) is an icosahedral aphid virus with a 10 kb positive-sense
RNA genome. To study the molecular biology of RhPV, identiWcation of a cell line that supports replication of the virus is essential. We
screened nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera for susceptibility to RhPV following RNA
transfection. We observed cytopathic eVects (CPE) only in cell lines derived from hemipterans, speciWcally GWSS-Z10 cells derived from
the glassy winged sharp shooter, Homalodisca coagulata and DMII-AM cells derived from the corn leaf hopper, Dalbulus maidis. Translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles in the cytoplasm of GWSS-Z10
cells were examined by Western blot analysis, Northern blot hybridization and electron microscopy. Infectivity of the GWSS-Z10 cell
derived-virus particles to the bird cherry-oat aphid, R. padi, was conWrmed by RT-PCR and Western blot. The GWSS-Z10 cell line provides a valuable tool to investigate replication, structure and assembly of RhPV.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Insect cell line; Dicistrovirus; Aphid virus
1. Introduction
Rhopalosiphum padi virus (RhPV), which was Wrst isolated from the bird cherry-oat aphid, R. padi, belongs to the
family Dicistroviridae (Fauquet et al., 2005). The dicistroviruses are restricted to invertebrate hosts and possess a positive-sense RNA genome with a characteristic dicistronic
arrangement (Christian and Scotti, 1998). Other members
of the family include Drosophila C virus (DCV) (Johnson
and Christian, 1998), and Cricket paralysis virus
(CrPV)(Reinganum et al., 1970). The dicistrovirus genome
has two open reading frames (ORF); the 5⬘ ORF encodes a
polyprotein that includes the non-structural proteins
*
Corresponding author. Fax: +1 515 294 5957.
E-mail address: bbonning@iastate.edu (B.C. Bonning).
1
Present address: Interdisciplinary Oncology Department, H. Lee
MoYtt Cancer Center & Research Institute, University of South Florida,
Tampa, FL 33612, USA.
0022-2011/$ - see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.jip.2006.09.010
including RdRp, while the 3⬘ ORF encodes the structural
polyprotein. Proteins from both ORFs are translated via
internal initiation of ribosomes directly on the genomic
RNA at internal ribosome entry sites (IRES) (Domier
et al., 2000; Wilson et al., 2000; Royall et al., 2004). Activity
of the RhPV 5⬘ IRES has been demonstrated in mammalian, Drosophila, and wheat germ in vitro translation systems, and also in Spodoptera frugiperda (Sf21) cells, which
are commonly used for baculovirus expression of recombinant proteins (Royall et al., 2004). Structural proteins accumulate in vast excess of non-structural proteins in infected
cells (Wilson et al., 2000).
RhPV is known to infect seven species of aphid: R. padi,
Schizaphis graminum, R. ruWabdominalis (D’Arcy et al.,
1981; Gildow and D’Arcy, 1988), R. maidis, Metopolophium
dirhodum, Diuraphis noxia and Sitobion avenae (Wechmar
and Rybicki, 1981; Williamson et al., 1989). In contrast to
RhPV which has been isolated only from hemipteran species, and DCV which has been isolated only from dipteran
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
species, CrPV has been isolated from members of the
Orthoptera, Hymenoptera, Lepidoptera, Hemiptera, and
Diptera (Christian and Scotti, 1998). CrPV and DCV replicate readily in several Drosophila cell lines, and CrPV replicates in a number of other insect cell lines (Scotti et al.,
1996; Christian and Scotti, 1998). Cell lines that support
replication of dicistroviruses other than CrPV and DCV
are unknown. Unfortunately, with respect to Wnding cell
lines that support replication of RhPV, it has not been possible to establish continuous cell lines derived from aphids
(Peters and Black, 1970; Hirumi and Maramorosch, 1971;
Christian and Scotti, 1998). Primary cell lines cultured from
Acrythosiphon pisum (Hirumi and Maramorosch,
1971)(Boyapalle, unpublished observation) and Hyperomyzus lactucae (Peters and Black, 1970; Hirumi and Maramorosch, 1971) were viable only for a few weeks.
A cell culture system that allows replication of RhPV
would be an asset for further study of the biology of this
virus. Nine cell lines derived from species within the Lepidoptera, Diptera and Hemiptera were tested for susceptibility to infection by RhPV. Here, we report
identiWcation of one hemipteran cell line that supports
the replication and packaging of infectious RhPV, and a
second hemipteran cell line that is likely to support replication of RhPV.
2. Materials and methods
2.1. Viruses and cells
RhPV was puriWed from infected colonies of R. padi
maintained at Iowa State University as described previously (D’Arcy et al., 1981). CrPV and DCV were propagated in cultured Drosophila melanogaster cells (Schneider’s
line 2: S2)(Schneider, 1972). The viruses were puriWed from
infected S2 cells as described previously (Krishna et al.,
2003).
Sf21 cells derived from the fall armyworm, Spodoptera
frugiperda (Vaughn et al., 1977) were maintained in TC-100
insect cell medium (Sigma Chemicals, St. Louis, MO, USA)
supplemented with fetal bovine serum (FBS; Gibco-BRL)
to a Wnal concentration of 10%. The Sf9 cell line, which was
derived from the Sf21 cell line, was maintained in Hink’s
TNM (JRH Biosciences, Lenexa, KA, USA) containing 3%
FBS. Tn5B1-4 “High-Five” cells derived from the cabbage
looper Trichoplusia ni (Wickham et al., 1992) were maintained in Excel 405 (JRH Biosciences) serum free medium.
OnE cells derived from the European corn borer, Ostrinia
nubialis were maintained in Excel 405 supplemented with
FBS to a Wnal concentration of 3%. S2 cells derived from
vinegar Xy D. melanogaster were maintained in Schneider’s
medium (Sigma Chemicals, St. Louis, MO, USA) with 10%
FBS. Cell lines derived from the leaf hopper Agallia constricta (AC-20) (McIntosh et al., 1973), the velvetbean caterpillar
Anticarsia
gemmatalis
(BCIRL-AG-AM1)
(McIntosh and IgonoV, 1989), corn leaf hopper Dalbulus
maidis (DMII-AM; A. McIntosh, University of Missouri,
131
CO), glassy-winged sharpshooter Homalodisca coagulata
(GWSS-Z10) (Kamita et al., 2005) were maintained in
Excel 405 with 10% FBS. All cell culture media were supplemented with penicillin–streptomycin (Sigma) to a Wnal
concentration of 1% with the exception of Schneider’s
medium which was supplemented with penicillin–streptomycin–amphotericin B (Wnal concentration 1%: Invitrogen).
2.2. Isolation and transfection of viral RNA
Viral RNA was extracted using the Absolutely RNA®
RT-PCR MiniPrep kit (Stratagene) according to the manufacturer’s speciWcations. The RNA concentration was determined by measurement of absorbance at 260 nm. To test the
ability of the cell lines to support replication of RhPV, DCV
and CrPV, the following procedure was employed (Masoumi et al., 2003). Cells were seeded into four wells of a 6-well
cell culture plate (Fisher ScientiWc) at a density of 2 £ 105
cells per well for Sf9 and AC-20 cells; 1 £ 105 for Sf21, HighFive, OnE and AG-AM1 cells; 6 £ 105 for S2 cells. GWSSZ10 and DMII-AM cells were seeded such that they were
near conXuent. The cells were allowed to attach to the surface for 4 h at room temperature. Media and Xoating cells
were then removed and the cells washed with serumfree medium. One well was treated with 0.5 g DOTAP
(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium
methyl-sulfate (Alexis) in 1 ml of serum-free medium. The
second, third and fourth wells were treated with 0.5 g
DOTAP in 1 ml serum-free medium along with 4 g of
RhPV RNA, 2 g DCV RNA and 2 g CrPV RNA, respectively. All treatments were maintained at 28 °C for 6 h, cells
were gently washed twice with serum-containing media
except High-Five cells which were washed in serum-free
medium. The cells were then incubated in 1.5 ml of their
respective media at 28 °C. Cells were examined daily for
cytopathic eVects (CPE) and harvested after 4–8 days.
For production of virus for aphid infectivity experiments, GWSS-Z10 cells in two 60 mm dishes (2.5 £ 106 cells
per dish) were transfected with 30 g of RhPV RNA as
described above. Infected cells were harvested 4 days posttransfection (dpt).
2.3. Western blot analysis of proteins
Harvested cells were washed twice in phosphate-buVered
saline (PBS) and resuspended in 100 l of PBS. Aliquots were
boiled for 5 min in 100 l of 2£ protein dissociation buVer
(2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mM
Tris–HCl and 0.01% bromophenol blue, pH 6.8) and proteins
resolved by SDS–PAGE. Approximately 150 ng of puriWed
virus alone was run as a positive control in each gel. Ten
microliter samples were resolved by electrophoresis on 4–
12% SDS–PAGE gels (Bio-Rad Laboratories), after which
each gel was treated for 30 min with Towbin buVer (10 mM
Tris base, 96 mM glycine in 10% methanol). Proteins were
transferred to Hybond-P membrane (Amersham Pharmacia)
132
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
in Towbin buVer using an electro blotter apparatus (BioRad) for 1 h at 100 V. The membrane was placed for 2 h at
room temperature in a blocking solution consisting of 5%
skimmed milk and 0.1% Tween 20 in Tris-buVered saline
(TBS). Rabbit polyclonal anti-RhPV antiserum was puriWed
as described previously (Bassham and Raikhel, 1998), diluted
100-fold in blocking solution, and incubated with the membrane for 1 h at room temperature. Polyclonal anti-DCV and
anti-CrPV antisera (Christian and Scotti, 1994) were diluted
2000-fold in blocking solution, and incubated with the membrane for 1 h at room temperature. Membranes were washed
twice with 0.1% Tween 20 in TBS before reaction with goat
anti-rabbit immunoglobulin conjugated with horseradish
peroxidase (Sigma). After washing for 1 h, the bound enzyme
was detected using the ECL detection system (Amersham
Pharmacia).
2.4. Plasmids
Full length double stranded cDNA of RhPV was cloned
between the EcoRI–KpnI sites of pGEM-3ZF (Promega) to
produce the plasmid pGEM-3ZF.RhPV (Boyapalle and R.
Beckett, unpublished data). The T7 promoter was positioned upstream of the 5⬘ end of the RhPV sequence and
the SP6 promoter downstream of the 3⬘ end of the RhPV
sequence.
2.5. Preparation of in vitro transcripts
The plasmid pGEM-3ZF.RhPV was linearized with
Acc651, and used as template for in vitro transcription of
full length positive-strand RNA from the T7 promoter at
the 5⬘ end using the T7 MegaScript kit (Ambion). Full
length negative-sense RNA was transcribed by linearizing
the plasmid with EcoRI, and using it as template for in vitro
transcription from the SP6 promoter using the SP6 Megascript kit (Ambion). Strand-speciWc 32P-labeled RNA
probes were prepared from the T7 and SP6 promoters in
the vector using T7 or SP6 RNA polymerase, respectively,
following digestion of the plasmid with HpaI.
2.6. Northern blot hybridization
To analyze the production of viral RNA over the course
of infection, GWSS-Z10 cells were transfected with RhPV
RNA or full-length negative-sense RhPV transcript with
0.5 g DOTAP as described above. Transfected cells were
harvested at various hours post-transfection (hpt) and total
cellular RNA was extracted using Trizol (Invitrogen)
according to the manufacturer’s speciWcations. RNA was
analyzed by Northern blot as described previously (Koev
et al., 1999). Approximately 100 ng of viral RNA spiked
with total RNA from mock infected cells, or full-length
negative-sense RhPV transcript were run as positive controls. A 32P-labeled probe complementary to the 1.9 kb 3⬘terminal sequence of RhPV was used to detect production
of positive-strand RNA in the cells. A 32P-labeled probe
complementary to the 1.2 kb 5⬘ end sequence of RhPV was
used to detect negative-sense RhPV RNA in the cells.
2.7. PuriWcation of virus particles from infected GWSS-Z10
cells
GWSS-Z10 cells were harvested when extensive CPE
were observed at 4 dpt and the virus particles were puriWed
as described previously (Krishna et al., 2003). PuriWed virus
particles were negatively stained with 2% uranyl acetate
and examined by TEM, or used to test for infectivity to
aphids (Section 2.9).
2.8. Immunogold electron microscopy
GWSS-Z10 cells and DMII cells were grown to near
conXuence in T-25 Xasks (Fisher ScientiWc), two Xasks for
each cell line. The cells were transfected with 1 g of
DOTAP and 10 g of RhPV RNA. GWSS-Z10 and DM11
cells were harvested at 4 or 10 dpt, respectively. The cells
were pelleted and Wxed in 1% paraformaldehyde–0.5% gluteraldehyde–0.05% sodium cacodylate, pH 7.1, for 10 min at
4 °C, then in 2% paraformaldehyde–2.5% gluteraldehyde–
0.05% sodium cacodylate, pH 7.1, for 30 min at 4 °C. After
washing the cells three times (10 min each time) with 0.05 M
sodium cacodylate, the cells were dehydrated with a series
of ethanol concentrations (50%, 70%, 85%, 95%, 3£ 100%)
for 30 min for each step at 4 °C. Cells were then inWltrated
with ethanol: LR White Resin using ratios of 1:1 (2 h at
4 °C), and 1:3 (overnight at 4 °C) followed by pure LR
White (24 h at 4°C, with a resin change after 8 h). The cells
were then embedded in gelatin capsules, and resin was polymerized at 4 °C for 48 h under UV light.
Gold labeling of the grids was carried out as described
by Erickson (Erickson et al., 1993). Grids were treated with
25 l of TBS-supplemented buVer (0.05 M Tris, 0.85%
NaCl, pH 8.3–8.5, 0.5% normal goat serum, 0.5% normal
pig serum and 0.5% BSA) with 3% non-fat dry milk for 2 h
at room temperature. Grids were then immersed in 50 l
drops of 1:10 puriWed rabbit polyclonal anti-RhPV antiserum diluted in TBS-supplemented buVer with 3% non-fat
dry milk for 5 h at 37 °C. After washing the grids three times
in TBS-supplemented buVer, the grids were treated with
25 l drops of 1:100 dilution of secondary goat anti-rabbit
antibody conjugated with 10 nm gold particles (Ted Pella
Inc.) for 1 h at room temperature. After stream washing and
three washes in drops of distilled water (10 min each wash),
grids were dried and stained with 2% aqueous uranyl acetate for 5 min and examined on a JOEL 1200 EX scanning/
transmission electron microscope at 80 kV.
2.9. Infectivity of virus particles to aphids
Clones of uninfected R. padi were initiated parthenogenetically from single apterous adults. The vertical transmission rate of RhPV in R. padi is approximately 28% (D’Arcy
et al., 1981). Progeny of each family were tested for the
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
presence of RhPV by Western blot and RT-PCR. Virus-free
families were pooled for production of the virus-free stock.
For Western blot analysis, at least six aphids from each
treatment were ground in a microfuge tube in 200 l protein
dissociation buVer (2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mM-Tris–HCl, 0.01% bromophenol blue,
pH 6.8), boiled for 5 min and proteins resolved by SDS–
PAGE. Approximately 150 ng of puriWed virus was run as a
positive control. Ten microliter samples were resolved
by electrophoresis on 4–12% SDS–PAGE gels (Bio-Rad
Laboratories). Western blotting with puriWed polyclonal
anti-RhPV antiserum was as described in Section 2.3. For
RT-PCR, total RNA from at least 10 aphids per replicate
was extracted using Trizol (Invitrogen) according to the
manufacturer’s speciWcations. RT-PCR was carried out
using the primers 5⬘-TTAATTTCGAACCCCGTCAG-3⬘
(from RhPV nt 848) and 5⬘-CTCAGTTTCGGGCTCT
CTTG-3⬘ (from RhPV nt 2387). PCR conditions used were
94 °C for 1 min, 58 °C for 1 min, 72 °C for 2 min for 30 cycles
with a Wnal extension at 72 °C for 10 min. PCR products
were visualized in ethidium bromide-stained 1% agarose
gels. Virus-free aphid clones were maintained in a separate
building from the RhPV-infected colony.
To test for replication and infectivity of GWSS-Z10 cellderived virus particles to aphids, R. padi (40 aphids for each
of two replicate experiments) were allowed to feed for 16 h on
ParaWlm sachets containing puriWed virus particles in 25%
sucrose in 0.01 M phosphate buVer, pH 7 (Mittler, 1988; Rasochova and Miller, 1996). Control aphids were fed on 25%
sucrose alone. After the acquisition period, aphids were
transferred to cages and maintained on oats for 17 days. To
determine whether aphids were infected with RhPV we used
RT-PCR and Western blot analysis for detection of RhPV
RNA and proteins respectively as described above. The
in vitro-transcribed positive-sense transcript (Section 2.5) was
used as a positive control for RT-PCR.
133
and DOTAP as transfection agent. DCV and CrPV RNAs
were used as positive controls for RhPV transfection experiments. A summary of the transfection results is presented
in Table 1. The Drosophila (S2) cell line was permissive to
CrPV and DCV as described previously, and exhibited distinct CPE 2–3 dpt (Plus et al., 1975; Reinganum, 1975; Plus
et al., 1978). By 5 dpt there were no living S2 cells in wells
transfected with CrPV or DCV. The cells treated with
DOTAP alone (negative control) were healthy at 5 dpt and
continued to divide. AG-AM1 cells transfected with CrPV
or DCV showed signs of swelling and hypotrophy, with
some evidence of cell lysis and blebbing, and detached from
the surface (Plus et al., 1975; Plus et al., 1978; Masoumi
et al., 2003). CPE were more pronounced in CrPV RNAtransfected cells (3–4 dpt) compared to those transfected
with DCV RNA (5 dpt).
For the GWSS-Z10 cell line, cells showed signs of CPE
by 3–4 days for both CrPV and DCV. The cells lost their
characteristic Wbroblast-like extensions, became rounded
and were of uniform size. They detached from the substrate
with approximately 90% of the cells Xoating. On examination with a phase contrast microscope, the cells had a granular appearance with some evidence of cell lysis. In
contrast, cells in the control wells were dividing and healthy
with characteristic Wbroblast-like extensions. There were no
granular inclusions in the cytoplasm of the control cells.
There were only a few Xoating cells in the control wells,
which in contrast to the infected cells, varied in size and
shape.
For the DMII cell line, the cells transfected with CrPV
and DCV RNA exhibited CPE in 4–6 dpt. The cells were
rounded and looked strikingly diVerent from cells in the
control wells. Infected cells lost their surface extensions, but
did not Xoat. The cells were clumped and the cytoplasm
appeared granular. None of the other Wve cell lines tested
were permissive for CrPV or DCV replication following
transfection with viral RNA.
3. Results
3.2. RhPV replication in cultured cell lines
3.1. CrPV and DCV infection of cultured cell lines
The ability of RhPV to replicate in various insect cell
lines was tested by transfecting the cells with viral RNA
Of the nine cell lines tested, only the GWSS-Z10 and
DMII cell lines appeared to be permissive to infection by
transfection with RhPV RNA. CPE were observed in
Table 1
Susceptibility of insect cell lines to infection by RhPV, DCV and CrPV by transfection with viral RNA
Order
Lepidoptera
Diptera
Hemiptera
Cell line
Spodoptera frugiperda Sf9
S. frugiperda Sf21
Trichoplusia ni Tn5B1-4 (High-Five)
Ostrinia nubilalis OnE
Anticarsia gemmatalis AG-AM1
Drosophila melanogaster S2
Agallia constricta AC-20
Homalodisca coagulata GWSS-Z10
Dalbulus maidis DMII-AM
RhPV
DCV
CrPV
CPE
Western
CPE
Western
CPE
Western
¡
¡
¡
¡
¡
¡
¡
+
+
¡
¡
¡
¡
¡
¡
¡
¡
+
+
¡
+
+
¡
¡
¡
¡
+
+
¡
+
+
¡
¡
¡
¡
+
+
¡
+
+
¡
¡
¡
¡
+
+
¡
+
+
¡
¡
+
+
CPE, cytopathic eVects; Western, Western blot analyses with the appropriate antiserum; DCV, Drosophila C virus; CrPV, Cricket paralysis virus.
134
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
GWSS-Z10 cells at 4 dpt (Fig. 1B), while the control cells
appeared healthy, with Wbroblast-like extensions (Fig. 1A).
DMII cells showed CPE 8–10 dpt that were similar to those
described above (Fig. 1E). To determine whether CPE
could be caused by non-infectious viral RNA, GWSS-Z10
cells were transfected with the in vitro synthesized fulllength negative-sense RhPV RNA transcript. There was no
diVerence between these cells and control cells (treated with
DOTAP only) at 4 dpt (Fig. 1A and C). This result shows
that the CPE observed resulted from infectious viral RNA.
3.3. Detection of coat proteins following viral RNA
transfections
To determine whether transfection of cells with viral RNA
resulted in synthesis of viral coat proteins, we performed
immunoblots using antiserum against puriWed virus preparations of RhPV, DCV and CrPV (Table 1). RhPV has three
major virion proteins of 30, 29 and 28 kDa (Williamson et al.,
1988). The fourth coat protein (VP4) has not been detected by
Western blot, although it can be detected in protein gels (Gildow and D’Arcy, 1990). The capsid proteins for DCV are
37.7, 33.3 and 29.2 kDa (Jousset et al., 1972), and for CrPV are
35, 34 and 30 kDa (Moore et al., 1985). Viral structural proteins were detected in all cell lines that exhibited CPE following transfection with viral RNA (Fig. 2; Table 1).
3.4. Synthesis of viral RNA following viral RNA transfection
We next examined viral RNA accumulation in GWSSZ10 cells transfected with RhPV RNA. Northern blot
Fig. 2. Detection of dicistrovirus coat proteins. (A) Sucrose gradient-puriWed virus particles were run on an SDS–polyacrylamide gel and coat proteins visualized by Coomassie blue staining. RhPV was puriWed from R.
padi; DCV and CrPV were puriWed from D. melanogaster S2 cells. Molecular mass markers are indicated. (B) Western blot analysis of RhPV
derived from R. padi and puriWed by sucrose gradient centrifugation (R.
padi; positive control), of GWSS-Z10 cells (Z10; negative control), and of
GWSS-Z10 cells transfected with RhPV RNA (Z10-RhPV) at 4 dpt.
hybridization was used to detect accumulation of positive
and negative-sense genomic RNA molecules (Fig. 3A and
C). Input positive-sense viral RNA and negative-sense
in vitro transcript were detected at 12 and 24 hpt (Fig. 3A
and B). Blots revealed that substantial amounts of positivesense viral RNA were present at least up to 96 hpt
(Fig. 3A). While a striking increase is not apparent, the levels must be compared against the large amount of inoculum
Fig. 1. Transfection of GWSS-Z10 cells (A–C) and DMII cells (D–E) with RhPV RNA. (A) GWSS-Z10 cells treated with DOTAP alone (negative control). (B) GWSS-Z10 cells transfected with 10 g of RhPV RNA showing CPE at 4 days post-transfection (dpt). Note the rounding of the cells, and
reduction in Wbroblast-like extensions and cell number. Ninety percent of the cells detached from the Xask. (C) GWSS-Z10 cells transfected with 10 g negative-sense full length in vitro transcript of RhPV (negative control). CPE were not apparent 4 days post-transfection. The characteristic Wbroblast-like
extensions (arrows) were clearly visible in control treatments (A and C). (D) DMII cells treated with DOTAP alone (negative control). Note the Wbroblastlike extensions. (E) DMII cells transfected with 10 g of RhPV RNA showing CPE at 8 days post-transfection. Infected cells (E) became rounded (arrows),
were of uniform size, and detached from the Xask in clumps. Cell division was reduced.
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
135
Fig. 3. Northern blot hybridization of RhPV RNA from transfected GWSS-Z10 cells. (A) Detection of positive-sense genomic RNA. Total RNA
extracted at indicated hours after transfection of GWSS-Z10 cells with viral RNA. Hybridization was carried out using a 32P-labeled 3⬘ end-speciWc, negative-strand riboprobe. Controls are virion RNA extracted from virus particles spiked with total RNA from mock infected cell (+); mock infected GWSSZ10 cells (¡). (B) Control treatments for detection of positive-sense genomic RNA. Total RNA from GWSS-Z10 cells transfected with full-length, in vitro
transcribed negative-strand RhPV RNA. Hybridization was carried out using a 32P-labeled 5⬘ end-speciWc, positive-stranded riboprobe. Controls are
in vitro synthesized full-length negative-sense RhPV6 RNA (¡), in vitro synthesized full-length positive-sense RhPV6 RNA (+; 100 ng). (C) Detection of
negative-sense RhPV RNA in GWSS-Z10 cells. Total RNA was extracted at 12, 48, 96, 120 and 144 after transfection with virion RNA. Hybridization was
carried out with a 5⬘ end positive-strand RNA-speciWc riboprobe. In vitro synthesized full-length negative-sense RhPV6 RNA (¡; 200 ng) was used as positive control. Lower panels show ethidium bromide-stained ribosomal RNA that indicate total amount of RNA loaded.
present at early time points, much of which degraded by
12 hpt (low molecular weight smear). For comparison, nonreplicatable RNA (negative-sense inoculum) levels
decreased rapidly after 24 h, (Fig. 3B).
To conWrm replication of the RhPV RNA, we tried to
detect negative-strand RhPV RNA in cells inoculated with
infectious viral RNA. Negative-strand RNA was indeed
detected in cells inoculated with RhPV RNA as in Fig. 3A,
beyond 96 hpt, (Fig. 3C). The negative-sense RNA was not
detected until 48 hpt. Between 48 and 96 hpt these negativesense RNAs appeared to have reached peak levels. Bands
are not well deWned because the negative-strand accumulates at a many fold lower level than the corresponding positive-sense species (Fig. 3C).
3.5. Detection of virus particles in infected cells
To determine whether virions could be observed in the
infected cells, GWSS-Z10 cells were transfected as described
above and the cells harvested at 4 dpt, when CPE were pronounced, for puriWcation of virus particles (Krishna et al.,
2003). PuriWed virus particles were negatively stained with 2%
uranyl acetate and examined by TEM (Fig. 4B). RhPV particles puriWed from aphids (Fig. 4A) were examined for comparison. In both cases, the size of the particles was 26–27 nm
as observed by Rybicki (Rybicki and Wechmar, 1984). The
apparent size of virus particles puriWed from GWSS-Z10 cells
4 dpt with DCV RNA was 29–30 nm as previously described
(Jousset et al., 1972; Reinganum, 1975.
3.6. Immunolocalization of RhPV in GWSS-Z10 and DMII
cells
To determine the intracellular localization of the virus
particles, GWSS-Z10 and DMII cells were transfected as
described above, harvested 4 dpt and sectioned for examination by TEM. AYnity puriWed RhPV antiserum was used
for immunolocalization of virus proteins. Infected cells
Fig. 4. Virus particles derived from R. padi and GWSS-Z10 cells. (A) R. padi-derived virus particles of RhPV. Virus particles were puriWed from R. padi by
10–40% sucrose gradient centrifugation and negatively stained with 2% uranyl acetate for analysis by TEM. (B) GWSS-Z10 cell-derived virus particles of
RhPV. Virus particles were puriWed from GWSS-Z10 cells 4 days post-transfection with RhPV RNA. The particles in A and B are 26–27 nm in diameter.
(C) GWSS-Z10 cell-derived virus particles of DCV. Virus particles (27–29 nm) were puriWed from GWSS-Z10 cells 4 days post-transfection with DCV
RNA. Virus particles were negatively stained and analyzed by TEM. Bars D 200 nm.
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S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
were compared to mock-infected GWSS-Z10 (Fig. 5A and
B) and DMII cells (Fig. 5C and D). In the infected cells,
large electron-dense amorphous aggregates (up to 2 m in
diameter) and oval-shaped inclusions were labeled by antiRhPV antiserum in the cytoplasm (Fig. 5A and C). These
cytoplasmic structures appeared to be enclosed by a membrane. Almost every DMII cell (8–10 dpt), had at least one
virus-induced cytoplasmic structure (Fig. 5C), and most
cells had numerous structures.
Moreover, viral antigen was concentrated in electrondense patches in the cytoplasm, probably consisting of
small vesicles (Fig. 5F, arrow). Mock infected GWSS-Z10
or DMII cells did not exhibit any of these cytoplasmic
inclusions or labeling (Fig. 5B and D).
Fig. 5. Immunogold labeling of RhPV proteins in GWSS-Z10 and DMII cells. (A) GWSS-Z10 cells 4 dpt with 10 g RhPV RNA. (B) Mock-infected
GWSS-Z10 cells. (C) DMII cells, 8 dpt with 10 g RhPV RNA. (D) Mock-infected DMII cells. Note the oval, electron dense inclusion bodies in the cytoplasm in A and C. (E) and (F) detail of infected GWSS-Z10 cells 4 dpt showing labeling of cell membrane (E) and electron dense regions in the cytoplasm
(F). Size bars, 200 nm.
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
137
Fig. 6. Reinfection of GWSS-Z10 cells with supernatant from RhPV RNA transfected GWSS-Z10 cells. (A) Mock-infected GWSS-Z10 cells. (B) Reinfection of GWSS-Z10 monolayer with the supernatant from the GWSS-Z10 cells 6 days after transfection with RhPV RNA. CPE were observed 6–7 days
post-infection with rounding of the cells (arrows), loss of Wbroblast-like extensions and detachment of the cells from the Xask.
3.7. Infectivity of GWSS-Z10 cell-derived virus particles to
GWSS-Z10 cells and aphids
4. Discussion
Fig. 6B shows infection of GWSS-Z10 cells following
treatment with supernatant harvested at 6 dpt from cells
transfected with RhPV RNA. Characteristic CPE were
observed, with rounding of the cells and detachment from
the surface of the Xask. Importantly, supernatant from
transfected GWSS-Z10 cells contained infectious virus at
6 dpt.
To test for production of infectious RhPV in GWSS-Z10
cells, aphids were fed on virus particles puriWed from RhPV
RNA-transfected GWSS-Z10 cells. Aphids were then maintained on oats for 17 days, during which time their numbers
increased at least 5-fold. RT-PCR and Western blot analysis of aphids in two separate replicate experiments, showed
the presence of RhPV RNA and immunoreactive structural
proteins (Fig. 7).
A
We have identiWed one insect cell line that supports replication of RhPV, and a second cell line that is likely to support replication of RhPV. We also identiWed new cell lines
capable of being infected by CrPV and DCV. Although
RhPV, CrPV and DCV are in the same family, cells
responded diVerently to infection. CrPV and DCV caused
CPE in GWSS-Z10 and DMII cells in almost half the time
required for RhPV to do so. Extensive cell lysis was
observed with CrPV and DCV infection while RhPV
infected cells became rounded and detached from the surface with limited cell lysis at 4 dpt. Twice the amount of
RhPV RNA inoculum was required to elicit CPE comparable to those of DCV and CrPV infection. Scotti et al.
observed that DL1 (D. melanogaster) cells appeared to support growth of DCV but without the distinctive CPE
observed for CrPV infections, although the infected cells
B
C
Fig. 7. Detection of RhPV in aphids fed on GWSS-Z10-derived virus particles. (A) Detection of positive-strand RhPV RNA. RT-PCR of in vitro-transcribed positive-sense transcript was used as a positive control (+; 1539 nt product). RT-PCR of RNA extracted from aphids fed on sucrose solution (¡;
negative control) or GWSS-Z10 cell-derived virus particles (two replicates; 1, 2). L, 1 kb ladder. (B) Detection of RhPV structural proteins by Western blot
in infected and RhPV-free aphid colonies. RhPV virions (W, 0.1 g); RhPV-positive R. padi colony (+); virus-free R. padi colony (¡); Acyrthosiphon pisum
negative control. Protein extracts from six aphids were examined by Western blot for each colony. (C) Western blot analysis of aphids fed on GWSS-Z10
cell-derived virus particles. RhPV structural proteins were detected in all four replicates (lanes 1–4; six aphids per lane). Control aphids were fed on
sucrose alone (¡, negative control).
138
S. Boyapalle et al. / Journal of Invertebrate Pathology 94 (2007) 130–139
did clump and detached from the surface of the culture
Xask several days after inoculation (Scotti et al., 1981).
Western blot analysis (Table 1 and Fig. 2) revealed that
viral RNA transfected into GWSS-Z10 and DMII cells was
translated and that the ORF2 polyprotein was eYciently
processed into structural proteins. These data demonstrate
that the two IRES (5⬘ IRES and IGR-IRES) of RhPV are
active in these cell lines. This result is not surprising given
that the 5⬘ IRES functions in wheat and HeLa cell lines.
Detection of structural proteins is not in itself indicative of
a cell line being permissive for virus replication however.
Masoumi et al. (2003) observed that CrPV was unable to
infect and/or replicate in either T. ni or A. aegypti cells even
though both IRES elements were functional in these cell
lines. These Wndings were in general agreement with the
Wndings of Shaw-Jackson and Michiels (1999) for Theiler’s
virus, that IRES elements may be active in cell types that
are not derived from natural hosts of the virus.
While there was no striking increase in the amount of
viral RNA detected in GWSS-Z10 cells (Fig. 3A), consideration of the ratio of Northern blot signal to the amount
loaded on the gel and comparison with cells transfected
with the non-replicatable negative-sense RNA (Fig. 3B)
suggests that viral RNA replication occurred in these cells.
The large amounts of RNA detected at early time points
represent inoculum. It is unlikely that inoculum RNA
would not decrease by 48 or 72 hpt. The low molecular
weight smear at 12 h shows degradation of the inoculum
RNA. Thus, the RNA at the later time points is likely to
result from replication. Consistent with this, low amounts
of negative strand were detected (Fig. 3C). The apparent
detection of negative-strand RNA suggests that RhPV replicates in GWSS-Z10 cells. The virus particles puriWed from
the infected cells were morphologically indistinguishable
from the virus particles puriWed from aphids. These results
indicate that cellular factors required for the processing of
viral gene products and assembly of virus particles were
available in the GWSS-Z10 cell line.
RhPV immunoreactive proteins appear to localize in the
cytoplasm of the infected cell (Fig. 5A and C). In host speciWcity studies of RhPV in aphids, RhPV particles occurred
free in the cytoplasm and also packed in crystalline arrays
in large membrane vesicles (Gildow and D’Arcy, 1990).
Virions were never observed in the nucleus, and the nuclear
envelope and mitochondria of the gut cells remained intact
following virus replication and development of severe cytopathic symptoms. The authors also observed that clusters
of single membrane vesicles were associated with the virions during early stages of infection. Occasionally, a clump
of label was observed associated with the cell membrane
(Fig. 5E), which may indicate a site of viral endocytosis or
release.
DCV and CrPV virions are also found in crystalline
arrays in the cytoplasm of infected cells, as for vertebrate
picornaviruses (Reinganum et al., 1970; Jousset et al., 1972;
Jousset and Plus, 1975. Our EM observations (Fig. 5A and
C) also revealed formation of electron dense amorphous
cytoplasmic structures that were strongly labeled by antiRhPV antibodies. These observations are consistent with
production of similar structures by other viruses: Similar
structures were observed within the cytoplasm of the gut
cells of the pea aphid infected with A. pisum virus (van den
Heuvel et al., 1997) and abundant cytoplasmic structures
loaded with virions were seen when a midgut cell line
derived from Helicoverpa zea was infected with Providence
virus (family Tetraviridae) (Pringle et al., 2003).
The apparent demonstration that RhPV virions produced in GWSS-Z10 cells following transfection with
RhPV RNA, are infectious to GWSS-Z10 cells (Fig. 6) suggests that RNA was packaged within virus particles and
that any receptor required for infection by virus particles is
present in these cells. Western blot and PCR analyses are
required to conWrm this result. However, infection of virusfree aphids with the GWSS-Z10 cell-derived virions
conWrms that infectious virus is produced following transfection of GWSS-Z10 cells with RhPV RNA and that this
virus was transmitted between aphids. The infectivity of
GWSS-Z10 cell-derived virus particles to aphids conWrms
that RhPV does indeed replicate in GWSS-Z10 cells.
We have shown that one hemipteran cell line (GWSSZ10) is permissive to RhPV infection, and that a second cell
line (DMII) is likely to be permissive to RhPV infection. The
GWSS-Z10 cells allow for translation and appropriate processing of viral gene products, RNA replication and packaging of virus particles, thereby producing infectious RhPV.
The GWSS-Z10 cell line provides a valuable tool to investigate the replication, assembly and structure of RhPV.
Acknowledgments
The authors thank Dr. Art McIntosh, USDA-ARS,
Columbia, MO, for providing the DMII, AC-20 and AGAM1 cell lines, and Dr. Shizuo G. Kamita, University of
California, Davis for providing GWSS-Z10 cells. The
authors thank Dr. Peter Christian, National Institute for
Biological Standards and Control, UK for providing CrPV,
DCV and the respective antisera, and Randy Beckett for
assistance with constructing the full-length clone of the
RhPV genome. This material is based upon work supported
by an Iowa State University Carver Trust grant as well as
Hatch Act and State of Iowa funds.
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