Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro
from a full-length cDNA clone
Sandhya Boyapallea,c,1, Randy J. Beckettb, Narinder Pala,
W. Allen Millerb,c,⁎, Bryony C. Bonninga,c
aDepartment of Entomology, Iowa State University, Ames, IA, USA
bDepartment of Plant Pathology, Iowa State University, Ames, IA, USA
cInterdepartmental Graduate Microbiology Program, Iowa State University, Ames, IA, USA
Received 22 October 2007; returned to author for revision 4 December 2007; accepted 5 February 2008
Available online 12 March 2008
no such clones have been reported for the Dicistroviridae, an emerging family of invertebrate viruses. Previously we demonstrated baculovirus-driven
expression of a clonedRhopalosiphum padi virus (RhPV;Dicistroviridae) genome that was infectious to aphids,and we identified a cell line (GWSS-Z10)
from the glassy-winged sharpshooter, that supports RhPV replication. Here we report that RNA transcribed from a full-length cDNA clone is infectious.
Transfection of GWSS-Z10 cells with the RhPV transcript resulted in cytopathic effects, ultrastructural changes, and accumulation of progeny virions,
valuabletool,anda more tractablesystemwithoutinterference frombaculovirusinfection,for investigating replication and pathogenesisofdicistroviruses.
© 2008 Elsevier Inc. All rights reserved.
Keywords: Rhopalosiphum padi virus; Dicistroviridae; Positive strand RNA virus; Infectious clone; Aphid virus; Insect cell culture
Rhopalosiphum padi virus (RhPV, Dicistroviridae family)
(ICTV, 2004) infects at least seven economically important
aphid species (D'Arcy et al., 1981; Gildow and D'Arcy, 1988;
von Wechmar and Rybicki, 1981; Williamson et al., 1989).
These species include R. padi (the bird cherry-oat aphid), a
ubiquitous vector of the major cereal pathogens, Barley yellow
dwarf virus and Cereal yellow dwarf virus. The RhPV genome
is composed of a positive polarity RNA of 10011 nucleotides
(nt), containing two open reading frames (ORFs) with the
structural proteins encoded by the 3′-proximal ORF (Fig. 1)
(Moon et al., 1998). Proteins from both ORFs are translated via
internal initiation of ribosomes directly on the genomic RNA at
internal ribosome entry sites (IRES) (Domier and McCoppin,
2003; Groppelli et al., 2007). Viruses with similar genome
organization have not been isolated from vertebrates.
The Dicistroviridae is an emerging family of invertebrate
viruses. Dicistroviruses infect hosts of economic value such as
bees and shrimp (Mari et al., 2002), as well as pests such as
aphids, the glassy-winged sharpshooter — a major grapevine
pest (Honnicutt et al., 2006), and Drosophila. Aphids cause
over one billion dollars in losses annually to crops and orna-
mental plants in the United States alone. The presence of a
dicistrovirus correlates strongly with occurrence of colony col-
lapse disorder which has reached crisis levels in American
honeybee colonies (Cox-Foster et al., 2007; Yang and Cox-
Foster, 2007). The dicistronic genomes of the Dicistroviridae
have revealed an entirely new mechanism of translation initi-
translation in the absence of initiation factors by mimicking a
tRNA (Schüler et al., 2006). The 5′ UTR IRES works by a
Available online at www.sciencedirect.com
Virology 375 (2008) 401–411
⁎Corresponding author. Plant Pathology Department, Iowa State University,
351 Bessey Hall, Ames, IA 50011 USA. Fax: +1 515 294 9420.
E-mail addresses: email@example.com (S. Boyapalle),
firstname.lastname@example.org (R.J. Beckett), email@example.com (N. Pal),
firstname.lastname@example.org (W.A. Miller), email@example.com (B.C. Bonning).
1Current address: H. Lee Moffitt Cancer Center and Research Institute,
University of South Florida, Tampa, FL 33612, USA.
0042-6822/$ - see front matter © 2008 Elsevier Inc. All rights reserved.
different mechanism (Groppelli et al., 2007). Given the eco-
nomic importance of dicistroviruses, and their value as tools to
investigate fundamental mechanisms of molecular biology, an
infectious viral genomic clone would be extremely useful to
investigate the biology of dicistroviruses. Such knowledge may
as honey bees (Cox-Foster et al., 2007; Yang and Cox-Foster,
2007), (ii) ways to exploit the virus as an agent to control insect
pests such as aphids, and (iii) understanding of the roles of the
novel IRESes in the context of a replicating viral genome.
The nucleotide sequences of many dicistrovirus genomes have
been determined (Czibener et al., 2000; de Miranda et al., 2004;
1998; Munster et al., 2002; Wilson et al., 2000) and production of
infectious transcripts of an RT-PCR product of Black queen cell
virus (BQCV) has been reported (Benjeddou et al., 2002), but the
the construction of a full-length clone of the RhPV genome, and
the glassy-winged sharp shooter Homalodisca coagulata (GWSS-
Z10) (Boyapalle et al., 2007; Kamita et al., 2005) and corn leaf-
hopper, Dalbulus maidis (DMII-AM) (McIntosh, unpublished
data). The virus-like particles derived from RNA transfections of
the insect cells, are also infectious to aphids.
Production of RhPV transcripts
To allowsynthesis of a full-length, infectioustranscript, cDNA
encompassing the entire RhPV genome (nts 1 to 10,011, along
with non-viral bases atthe ends) was transferred from pAcRhPV6
(Pal et al., 2007) into pGEM3ZF (Promega) to create pRhPV6-1.
that in vitro transcription of Acc65 I-linearized pRhPV6-1 should
yield a full-length transcript with 15 non-viral bases at the 5′ end
and 5 non-viral bases at the 3′ end (Fig. 1). We call this transcript
RhPV6-1. Sequencing of the genome cloned into pRhPV6-1
(GenBank accession number EU282007) revealed a total of 119
differences compared to the previously published RhPV sequence
(Moon et al., 1998), with 10 changes in the 5′ UTR, 65 in ORF 1,
of which 52 were silent, 9 in the IGR, 32 in ORF 2 of which 22
were silent, and 3 in the 3′ UTR (Fig. 1; Table 1).
Translation in vitro
Totestifviralproteinscould besynthesizedasexpected from
a wheat germ translation extract. We expect polyproteins of
Fig. 1. Map of the full-length cDNA clone, pRhPV6-1. Genome organization of RhPV is shown with the known functions of the two ORFs (hel, helicase; prot,
protease; pol, RNA-dependent RNA polymerase; VP, virion protein). Molecular weights of VPs 1, 2 and 3 are indicated above each. Approximate positions of amino
acids encoded by RhPV6-1 that differ from those encoded by the previously published RhPV sequence (Moon et al., 1998), are indicated by black (conservative
changes) and white (nonconservative changes) arrowheads. See also Table 1. Sequences at the ends of the viral insert are shown below, with viral bases in bold text.
Arrows indicate the beginning and end of the in vitro transcript obtained with T7 polymerase from Acc65 I-linearized pRhPV6-1. An SP6 promoter (not shown)
downstream of the Acc65 I site was used to transcribe negative strand RNA.
Nucleotide (NT) and amino acid (AA) sequence differences between RhPV6-1
and RhPV (Moon et al., 1998)
5′ UTRORF1 IGRORF23′ UTR
NT NTAA NTAA
For amino acid sequences, the first letter and number represent the amino acid
sequence in the published sequence. The second letter represents the amino acid
encoded by RhPV6-1 at that position.
aConservative amino acid changes. There are no changes within the con-
served motifs of the non-structural proteins.
402 S. Boyapalle et al. / Virology 375 (2008) 401–411
~229 kDa and 90 kDa from ORFs 1 and 2, respectively. These
may then be proteolytically processed if conditions in wheat
germ extract permit. Translation of the RhPV6-1 transcript was
compared with that of viral RNA. In both cases, the35S-met
of ~90 kDa was always observed, which corresponds to the
expected mass of the uncleaved polyprotein translated from
ORF 2 (Fig. 1). Thus wheat germ extract appears to lack activity
necessarytoprocessviralcoatproteins. Aproduct ofORF1was
not detected, probably because the expected 229 kDa poly-
protein is too large to be detected in this gel system.
Cytopathic effects in transfected cells
GWSS-Z10 cells transfected with RhPV6-1 transcript showed
cytopathic effects (CPE) by 4 days post transfection (dpt). In
contrast, healthy cells in the control wells had characteristic
fibroblast-like extensions (Fig. 3A). The transfected cells lost their
fibroblast-like extensions, became rounded and were of uniform
size (Fig. 3B). Approximately 90% of the cells detached from the
with RNA from RhPV virions (Boyapalle et al., 2007). There was
evidence of cell lysis and examination with a phase contrast
microscope revealed that the cells had a granular appearance.
RhPV6-1 transcripts caused similar CPE at 8–10 dpt when trans-
fected in DMII-AM cells derived from the corn leafhopper,
permissive for RhPVreplication (Boyapalle et al., 2007).
Synthesis of positive sense RNA following transfection of
GWSS-Z10 cells with RhPV6-1 transcripts
Northern blot hybridization was used to detect viral RNA
following transfection of GWSS-Z10 cells with the RhPV6-1
transcript. Large amounts of input positive sense viral RNA and
negative sense in vitro transcript were detected at early time points
(12 and 24 h post transfection, hpt) (Fig. 4). Most of this RNAwas
low molecular weight, indicating rapid degradation. However a
significant proportion of the positive sense RNA detected at 72, 96
contrast, in cells transfected with non-replicatable (negative sense)
and very little or no full-length negative strand was detected at 72
clear, negative sense transcript always migrated as a doublet.
Detection of replicative negative sense RNA
While the northern blot hybridization data suggest virus repli-
negative sense viral RNA in cells inoculated with positive sense
RhPV transcript. Negative strands accumulate only if the viral
GWSS-Z10 cells, at 48, 72, and 96 hpt (Fig. 5). No PCR product
Fig. 2. In vitro translation of RhPV6-1 RNA in wheat germ extract. RhPV6-1 and
uncloned viral RNAwere translated at the indicated concentrations of potassium
Fig. 3. GWSS-Z10 cells transfected with RhPV6-1 transcript. (A) GWSS-Z10 cells
transcript in DOTAP. Cells are shown 4 days post transfection (dpt). In panel B, note
the increase in rounded cells (arrows) and loss of the fibroblast-like extensions that
of Boyapalle et al. (2007) for comparison. Reprinted from Journal of Invertebrate
Pathology, 94, Boyapalle, S., Pal, N., Miller., W.A., Bonning, B.C., A glassy-
winged sharpshooter cell line supports replication of Rhopalosiphum padi
virus (Dicistroviridae), 130–137, © 2007, with permission from Elsevier.
403S. Boyapalle et al. / Virology 375 (2008) 401–411
was detected at 0 and 24 hpt, indicating that detectable negative
sense RNA was not synthesized in the cells until some point
between 24 and 48 hpt. This slow replication may explain the
dominance of inoculum RNA detected by northern blot
hybridization (Fig. 4). As controls, positive sense RNA was not
amplified by the primers designed to detect negative strand, nor
was DNA amplified as indicated by lack of amplification in the
absence of reverse transcriptase (Fig. 5).
Detection of virions in infected cells
To determine whether virions could be observed in the in-
fected cells, GWSS-Z10 cells were transfected as described
above with the RhPV6-1 positive sense transcript, and the cells
harvested at 4 dpt for purification of virus particles (Krishna
et al., 2003) when CPE were pronounced. In preparations from
were detected by negative staining in transmission electron
micrographs (TEM). The particles were indistinguishable from
those purified from RhPV-infected aphids (Fig. 6A, B). No
particles were detected in preparations from uninfected cells
(Fig. 6C). Thus transfection of cells with RhPV6-1transcript led
to accumulation of RhPV-sized particles.
Ultrastructural analysis of cells transfected with the RhPV6-1
RhPV6-1-transfected cells showed striking alterations in ultra-
small 200 nm membrane-bound vesicles described previously in
RhPV-infected aphid gut cells (Gildow and D'Arcy, 1990). Pre-
sence of RhPV antigen in RhPV6-1-transfected GWSS-Z10 and
usingantiserumagainst RhPVvirions (Fig.7,E–H).Occasionally,
gold label associated with the inclusion bodies and with cell mem-
brane, which may indicate a site of viral endocytosis or release
(Fig. 7H). Mock infected GWSS-Z10 or DMII-AM cells exhibited
neither cytoplasmic inclusions nor labeling (Fig. 7A, C, D).
Infection of aphids with virions generated from transfected
We next determined whether virus produced in RhPV6-1-
colonies were established from a single apterous female and the
by western blot analysis and RT-PCR (Pal et al., 2007). Aphids
were inoculated by membrane feeding on a sucrose solution
containing virions from RhPV6-1-transfected GWSS-Z10 cells.
Aphids were virus-positive by western blot analysis two weeks
after this acquisition feeding, in three out of three independent
trials. Awestern blot from one of the trials is shown in Fig. 8.
To visualize the site of virus infection in the aphid, infected
aphids were fixed, sectioned and treated with purified anti-RhPV
silver-enhanced immunogold labeling was visible in the midgut
RhPV6-1-derived virions (Fig. 9E, F), six weeks post acquisition
feeding. No labeling was detected in virus-free aphids (Fig. 9C).
Several lines of evidence indicate that transcript RhPV6-1
was infectious in GWSS-Z10 cells. CPE observed in the cells
transfected with the RhPV6-1 transcript were similar to those
seen with the viral RNA transfections (Boyapalle et al., 2007).
Although there was no striking increase in the amount of viral
RNA detected in GWSS-Z10 cells (Fig. 4A), more viral RNA
was present at the longest time points compared to cells
Fig. 4. Northern blot hybridization of GWSS-Z10 cellular RNA following
transfection. GWSS-Z10 monolayer was transfected with 10 µg of RNA using
DOTAP, and incubated at 28 °C until harvesting after the number of hours
indicated above each lane. Following extraction, total cellular RNAs, along with
100 ng RhPV6-1 RNA (lane +), or RNA from mock infected GWSS-Z10 cells
(lane −), were resolved on a formaldehyde agarose gel and transferred to a nylon
membrane. Bottom panels represent ethidium bromide stained ribosomal RNAs
as gel loading indicators. (A) RNA from cells transfected with RhPV6-1 T7
polymerase transcript. Positive sense RNA was detected using an in vitro
synthesized,32P-labeled, 3′ end negative sense RNA probe. (B) RNA from cells
transfected with negative sense full-length RhPV transcript. RNA was detected
using in vitro synthesized, 5′ end positive sense RNA probe.
404 S. Boyapalle et al. / Virology 375 (2008) 401–411
transfected with the non-replicatable negative sense RNA (Fig.
4B). It is likely that the cell can replicate only a small fraction of
the massive amount (10 μg) of input RNA. Hence, a vast excess
of inoculum RNA is degraded, predominating over, but not
preventing detection of, replicated RNA. Detection of the
negative strand RNA by RT-PCR at 48, 72 and 96 hpi (Fig. 5) in
GWSS-Z10 cells inoculated with plus sense RhPV6-1 trnascript
clearly demonstrated that viral replication occurred. In contrast,
negative strand RNA was detected from GWSS-Z10 cells
transfected with uncloned viral RNA by 12 hpt (Boyapalle et
al., 2007). The delayed accumulation of the RNA transcript,
relative to viral RNA is typical for transcripts of a number of
other viruses (Gritsun and Gould, 1995; Iwamoto et al., 2001;
Khromykh and Westaway, 1994; Lai et al., 1991; Sit and
AbouHaidar, 1993). Benjeddou et al. (2002) showed that the
infectivity of RNA transcripts generated for Black queen cell
virus (BQCV) was 350-fold lower than the infection efficiency
of viral RNA, although the recovered virus was as infectious as
wild type (Benjeddou et al., 2002).
Low infectivity of the transcript may be explained by the
presence of 15 non-viral bases at the 5′ end. Even short 5′ non-
viral extensions usually greatly inhibit replication of transcripts
(Bujarski and Miller, 1992). Also, infectivity of RhPV6-1
transcript is probably reduced by the absence of the VPg and
poly(A) tail that are present at the ends of the natural viral
genome. Absence of a poly(A) tail would destabilize the ino-
culum RNA (Boyer and Haenni, 1994; Garneau et al., 2007)
relative to the natural genome which contains a 60–70 nt poly
(A) tail (Moon et al., 1998). Poly(A) tails are also essential for
efficient translation (Gallie, 1991; Preiss and Hentze, 1998;
Tarun and Sachs, 1995), so the absence of a poly(A) tail is likely
to delay virus gene expression. That the RhPV6-1 transcript is
the 3′ end that may sufficiently substitute for the poly(A) tail.
In host specificity studies of RhPVin aphids, RhPV particles
occurred free in the cytoplasm and also packed in crystalline
arrays in large membranous vesicles (Gildow and D'Arcy,
1990). Our EM observations (Fig. 7) revealed formation of
electron dense amorphous cytoplasmic structures that were
labeled sporadically by anti-RhPVantibodies. Similar structures
were observed within the cytoplasm of these cells when
transfected with viral RNA (Boyapalle et al., 2007), indicating
Fig. 5. Detection of the negative sense RhPV6-1 transcript in infected cells. Strand-specific RT-PCR was carried out on total RNA extracted from GWSS-Z10 cells
transfected with the positive sense RhPV6-1 transcript. Cells were harvested at the hpt indicated. Negative strand-specific primer was used in lanes at left giving
significant products at 48, 72 and 96 hpt. Also shown, 1 kb DNA ladder (L); mock infected GWSS-Z10 cells (M); negative control using positive strand virion RNA as
template, (−); negative sense, full-length in vitro transcript as positive control (+). “No RTcontrols”: controls for PCR reactions without reverse transcriptase on RNA
extracted from infected cells show that PCR products are from RNA and not potential contaminating pRhPV6-1 DNA.
Fig. 6. Transmission electron microscopy of RhPV virions. Virions were purified from GWSS-Z10 cells or aphids in 10–40% sucrose gradients and negatively stained
with 2% uranyl acetate and analyzed by TEM. (A) Virus particles purified from R. padi infected with uncloned RhPV. (B) Virus particles purified from GWSS-Z10
cells 4 d after transfection with the RhPV6-1 transcript. (C) Material obtained after performing virus particle purification procedure on control GWSS-Z10 cells,
without transfection with the RhPV6-1 transcript. Arrows indicate examples of virions estimated to be between 26 and 27 nm in diameter. Bars represent 200 nm.
405 S. Boyapalle et al. / Virology 375 (2008) 401–411
that the transcript-initiated infection resembles the natural in-
Infection of aphids with RhPV6-1 revealed accumulation of
viral antigen in the midgut. (Fig. 9E, F). Label detected in the
cells (Fig. 9D) and debris is apparent within the midgut lumen in
Fig. 9F. For natural, uncloned virus, the virus appeared to
accumulate initially in the midgut, and then in the hindgut, and
Fig. 7. Electron microscopy of GWSS-Z10 and DMII-AM cells. (A, B) Transmission electron micrographs of GWSS-Z10 cells that were not transfected (A) or
transfected with RhPV6-1 transcript (B). Black arrows indicate electron dense structures, possibly virus particles, within membranous vesicles that are absent in
untransfected cells. (C–H) Immunoelectron microscopy using RhPVantiserum. (C, D) Mock infected GWSS-Z10 and DMII-AM cells respectively. (E, F) GWSS-Z10
cells 4 d after transfection with 10 µg RhPV6-1 transcript; (G, H), DMII-AM cells, 8 days after transfection with 10 µg RhPV6-1 transcript. Panels E and G have
labeled oval shaped electron dense inclusion bodies in the cytoplasm (white arrowheads); and (F and H) show labeling with gold particles in the cytoplasm and the cell
membrane (black arrows). Scale bars in A, B, D and G indicate 500 nm. Bars in C, E, F, and H indicate 200 nm.
406 S. Boyapalle et al. / Virology 375 (2008) 401–411
was also detected in the hemocoel (Gildow and D'Arcy, 1990). It
is unclear whether dark staining of the hindgut in RhPV-infected
aphids (Fig. 9A, B) resulted from intense, merged silver staining
of virus. Panels showing details from Fig. 9B illustrate a dif-
ference in the midgut and hindgut labeling.
Accumulation of RhPV6-1-derived virus in the living or-
ganism provides important evidence that the RNA and virions
that accumulated in cell culture contain all the biological pro-
perties expected of a fit virus. The replication requirements are
much more stringent in the organism than in cell culture, be-
cause movement of virus between cells and throughout the
organism, as well as ability to evade the robust immune system
of the whole organism, requires virus-encoded functions that
may not be necessary for replication in cell culture.
Previously we reported construction of a full-length clone of
the RhPV genome (pAcRhPV6) that was infectious when
expressed in lepidopteran cells from a baculovirus (Pal et al.,
2007). In contrast to results described here, the baculovirus-
expressed RhPV RNA had a 5′ cap and a poly(A) tail because it
was derived from a pol II promoter. Also, that RNA contained
over 100 non-viral bases in each UTR, and these non-viral bases
were maintained stably, even in aphids. Thus, it is not surprising
that the RhPV6-1 transcript tolerates a much smaller number of
Fig. 8. Detection of RhPV virion proteins in aphids by western blot using
purified anti-RhPV antibody. RhPV-positive aphids from lab colony (+; n=8
aphids); aphids fed GWSS-Z10-derived RhPV6-1 virions (RhPV6-1; n=8);
aphids from RhPV-free colony (−; n=15). The major coat proteins of 28 and
30 kDa are indicated.
Fig. 9. Light microscopy showing immunolocalization of RhPVand RhPV6-1 by silver enhancement of infected aphids. (A) Representative RhPV-positive aphid from
the RhPV-infected colony. (B) Phase contrast image of part of (A) with detail of midgut and hindgut. (C) Aphid from virus-free colony. (D) Phase contrast detail of
midgut of RhPV-positive aphid showing labeling (which appears white) in lumen. (E) Aphid after feeding on virions purified from GWSS-Z10 cells transfected with
RhPV6-1 transcript. (F) Phase contrast image of midgut shown in (E) with detail of labeling. m, midgut h, hindgut, e, epithelial cell, n, nucleus. Scale bars: 20 μm in
panels A, C, 50 μm in panels B, D, and F, 100 μm in panel E.
407 S. Boyapalle et al. / Virology 375 (2008) 401–411
non-viral bases. However, here we show for the first time that
infection can be initiated from a transcript lacking a 5′ cap (as
does the natural virus) and lacking a poly(A) tail which is new,
because both the baculovirus-expressed construct and the
natural viral RNA (Moon et al., 1998) have a long poly(A) tail.
Importantly, lepidopteran cells are not permissive for RhPV
RNA replication except when the RNA is expressed from a
baculovirus, suggesting baculovirus replication and gene ex-
pression is necessary to suppress host defense pathways to
allow RhPV accumulation (Boyapalle et al., 2007; Pal et al.,
2007). In contrast, the RhPV6-1 transcript infects the same cell
lines (GWSS-Z10 and DMII-AM) that are permissive to
uncloned RhPV RNA (Boyapalle et al., 2007). Finally, bacu-
lovirus expression is unlike natural infection because the former
produces RhPV6-1 virions in the nucleus. In contrast, RNA
inoculation requires replication only in the cytoplasm as in a
normal infection (Gildow and D'Arcy, 1990). For the above
reasons, use of wild type and mutant RhPV6-1 transcripts to
initiate infection should allow rapid analysis of RhPV repli-
cation requirements in cell culture in a somewhat more “natural”
way then via baculovirus expression.
In summary, RhPV is the first dicistrovirus for which a full-
length infectious transcript from a cloned genome has been
demonstrated. Previously, infectious transcripts were obtained
from an uncloned RT-PCR product of BQCV. However, in the
absence of an infectious clone, the genome could not easily be
engineered for research, as pointed out by the authors (Benjed-
dou et al., 2002). This development of a cloned, infectious
genomic RNA of RhPV will provide new opportunities for
investigating the molecular biology, persistence, pathogenesis
and interaction of the virus with the host insect. This method
clones of other members of the Dicistroviridae. The association
of at least four dicistroviruses with the decimation of honey bees
through colony collapse disorder (CCD), including the Israeli
acute paralysis virus that may be a primary contributor to CCD
(Cox-Foster et al., 2007), emphasizes the importance of
understanding fundamental aspects of dicistrovirus biology.
Materials and methods
Cells and viruses
RhPV was purified from infected colonies of R. padi
maintained as described by D'Arcy et al. (1981). Cell lines de-
University of Missouri, Columbia), and glassy-winged sharp
shooter, H. coagulata (GWSS-Z10) (Kamita et al., 2005) were
maintained in Excel 405 (JRH Biosciences), supplemented with
10% FBS and 1% penicillin–streptomycin (Sigma).
Construction of a full-length cDNA clone for RhPV
Construction of the full-length cDNA clone of RhPV in the
baculovirus expression vector (pAcRhPV6) is described in (Pal
from the pGEM3ZF vector, which has T7 and SP6 promoter
sequences flanking the multiple cloning site. The full-length
cDNA was cut from pAcRhPV6 using EcoRI and KpnI and
ligated into pGEM3ZF to create pRhPV6-1. The infectious
In vitro transcription of full-length RhPV RNA
In vitro transcription of the above described cDNA clone
(pRhPV6-1) was carried out using T7 RNA polymerase (MEGA-
script™ High Yield Transcription Kit, Ambion). Briefly, the
plasmid, purified using a plasmid miniprep kit (Bio-Rad), contain-
linear DNA template. RNA was transcribed according to the
synthesize the full-length positive sense RNA transcript.
Full-length negative sense RNA transcribed from EcoRI-cut
pRhPV6-1 starting from the SP6 promoter using the SP6
at37°Cfor 2handthendigestedwithDNase I(providedwiththe
transcription kit) for 30 min at 37 °C. The reaction mixture was
extracted twice with phenol–chloroform with a second round of
Dnase I digestion before the second phenol–chloroform extrac-
tion. The transcripts were ethanol precipitated at −20 °C for 2 h.
The RNA pellet was washed with 70% ethanol, dried and
resuspended in 100 µl of RNA suspension solution (Ambion).
RNA was analyzed after each extraction by electrophoresis in
0.8% agarose gels, followed by ethidium bromide staining. RNA
concentration was determined at A260.
In vitro translation
In vitro translation reactions with35S-met labeling (25 μl)
using wheat germ extract were conducted according to the
manufacturer's protocols (Promega). Translation reactions with
full-length RhPV transcripts using 10 µg RhPV6-1 transcript or
viral RNA contained 128 or 153 mM potassium acetate. Five
microliters of the translation product was examined by 10%
polyacrylamide SDS-PAGE (Wang and Miller, 1995). The gels
were dried and imaged by using a STORM 840 Phosphoimager
GWSS-Z10 and DMII-AM cells at ~70% confluency were
used for RNA transfection. Ten micrograms of in vitro-produced
positive sense transcript, negative sense transcript or viral RNA
et al., 2003). CPE were observed in GWSS-Z10 cells at 4 dpt and
in DMII-AM cells at 8–10 dpt. The cells were harvested at 0, 12,
24, 48, 72, 96, or 120 hpt for extraction of total RNA.
Northern blot hybridization
GWSS-Z10 cells transfected with positive or negative sense
RhPV6-1 RNA were harvested at 0, 0.2, 12, 24, 48, 72, 96, or
120 hpt and total cellular RNA was extracted using Trizol
408S. Boyapalle et al. / Virology 375 (2008) 401–411
(Invitrogen) according to the manufacturer's specifications. RNA
1999). Approximately 100 ng of the viral RNA or full-length
negative sense RhPV transcript were run alongside as positive
controls. A32P-labeled probe complementary to the 1.9-kb 3′-
terminal sequence of RhPV (plasmid digested with Hpa I and
transcribed using SP6 RNA polymerase) was used to detect
complementary to the 1.2-kb 5′ end sequence of RhPV (plasmid
digested with Hpa I and transcribed using T7 RNA polymerase)
was used to detect negative sense RNA inoculum in the cells.
Detection of negative sense RhPV RNA by RT-PCR
The cells transfected with RhPV6-1 positive sense transcript
were analyzed at 0, 24, 48, 72, and 96 hpt for the presence of the
antisense RNA replicative intermediate using strand-specific RT-
PCR using the primers at positions 7476 and 8780 of the RhPV
sequence (Moon et al., 1998). Negative sense, full-length in vitro
transcript served as a positive control and viral RNA served as a
was carried out using Superscript RT (Gibco-BRL) according to
RNAwas degraded by digestion with RNase A (1 µg, Promega).
The reaction mixture was then extracted once with phenol–
chloroform and precipitated with ethanol. The cDNA was then
PCR amplified with sense and antisense primers using ExTaq
polymerase (Invitrogen). “No RTcontrols”, i.e. controls for PCR
reactions without the RTreaction were alsorun toshowthat PCR
products resulted from RNA and not from the pRhPV6-1 DNA
plasmid, which was a potential contaminant from transfections
with the RhPV6-1 positive sense transcript.
Purification of RhPV6-1 virions
GWSS-Z10 cells transfected with RhPV6-1 transcript were
harvested when extensive CPE were observed (typically at
4 dpt) and the virions were purified as described (Krishna et al.,
2003). Purified virions were negatively stained with 2% uranyl
acetate and examined by TEM.
Transfection of GWSS-Z10 and DMII-AM cells
GWSS-Z10 cells and DMII-AM cells were grown to ~70%
confluency in T-25 flasks (Fisher Scientific). The cells were
transfected with 20 µg of RhPV6-1 RNA in the presence of 1 µg
DOTAP as described previously for viral RNA (Boyapalle et al.,
2007). The cells were harvested 4 dpt (GWSS-Z10) cells and
10 dpt (DMII-AM cells). The cells were harvested, fixed and
treated for immunoelectron microscopy as described previously
(Boyapalle et al., 2007).
Experimental infection of R. padi
Colonies of uninfected R. padi were generated as described
(D'Arcy et al., 1981). Acquisition of the virus upon aphid
feeding was conducted by membrane feeding as described pre-
viously (Rasochova et al., 1997). Briefly, for membrane feeding
acquisition of virions, aphids were allowed to feed for 16 h on
purified virus preparations derived from one 25 cm2flask of
GWSS-Z10 cells transfected with 20 µg of RhPV6-1 positive
sense transcript as described in (Pal et al., 2007). Three replic-
ates were carried out with at least 30 aphids per replicate. The
infectivity of the virions was tested as follows: Aphids were
tested weekly for RhPV infection beginning at two weeks after
virus acquisition, using RT-PCR and western blot analyses as
described previously (Boyapalle et al., 2007).
Analysis of RhPV infection of aphids by light microscopy
For immunolocalization studies by light microscopy, aphids
fed with virions generated from RhPV6-1 transfection in GWSS-
into head and abdomen with a razor blade while submerged in
fixative (1% paraformaldehyde, 0.5% glutaraldehyde in 0.05 M
sodium cacodylate buffer pH 7.1). The abdomens were fixed in
2% paraformaldehyde-2.5% glutaraldehyde-0.05 M sodium
cacodylate, pH 7.1, for 30 min at 4 °C. After washing three
times (10 min each time) with 0.05 M sodium cacodylate, the
(50%,70%,85%,95%,3×100%) for 30minateachstepat4 °C.
Abdomens were then infiltrated with ethanol: LR White Resin
(London ResinCompany Ltd, England) using ratiosof1:1 (2h at
4 °C), and 1:3 (overnight at 4 °C) followed by pure LR White
embedded in gelatin capsules, and resin was polymerized at 4 °C
for 48 h under UV light (Vandenbosch, 1991).
LR White embedded sections were placed on Probe-On Plus
slides (Fisher Scientific) withadropof distilledwateranddriedfor
of TBS-Tween supplemented buffer (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, 0.05% Tween-20) with 3% non-fat dry milk for 2 h at
of 1:10 purified rabbit polyclonal anti-RhPVantiserum (Boyapalle
et al., 2007) diluted in TBS-Tween supplemented buffer with 3%
non-fat dry milk for 4 h at 37 °C. The slides were stream washed
with TBS-Tween buffer, and then washed 3×15 min in TBS-
Tween supplemented buffer. The sections were then treated with
temperature. After stream washing and three washes in drops of
immunolabeled slides was carried out using silver enhancement
Goldmark kit according to the manufacturer's specifications (Elec-
briefly in Xylene, and coverslipped with Permount. The slide
without immunolabeled sections was used as a control for non-
specific silver precipitations.
Transmission electron microscopy (TEM)
The cells were pelleted and fixed in 1% paraformaldehyde-
0.5% gluteraldehyde-0.05% sodium cacodylate, pH 7.1, for
409 S. Boyapalle et al. / Virology 375 (2008) 401–411
10 min at 4 °C, then in 2% paraformaldehyde-2.5% gluter-
aldehyde-0.05% sodium cacodylate, pH 7.1, for 30 min at 4 °C.
After three 10 min washes with 0.05 M sodium cacodylate, cells
were subjected to secondary fixation in 1% osmium tetroxide in
the same buffer for 1 h at room temperature. Samples were
dehydrated in an ethanol series (25–100% ethanol), and trans-
ferred to acetone and infiltrated with Embed 812 epoxy resin
(Electron Microscopy Science, Fort Washington, PA) in a
stepwise manner with ratios of acetone to Embed of 3:1, 1:1, 1:3
and finally transferred into pure resin. Each infiltration step was
allowed to proceed from 30 min to 1 day. Specimens were cast
in pure resin and allowed to harden at 50 °C for 1 day followed
by 60 °C for 2 days. Ultrathin sections were cut with a Leica
Ultramicrotome and positively stained using 5% uranyl acetate
and Reynolds lead citrate. Sections were observed with a JEOL
1200EX scanning and transmission electron microscope at
Immunoelectron microscopy (IEM)
The cells were pelleted and fixed 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% gluter-
aldehyde-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. The cells were
infiltrated with LR White resin (Electron Microscopy Science,
Fort Washington, PA), in a stepwise manner with ratios of
Ethanol to LRWhite of 1:1 (2 h at 4 °C), 1:3 (overnight at 4 °C)
and finally transferred to pure resin. Specimens were cast in
pure resin and allowed to harden at 4° under UV light for 1 day.
Ultrathin sections were cut with a Leica Ultramicrotome. Grids
were treated with 25 μl of TBS-supplemented buffer (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 μl
drops of 1:10 purified rabbit polyclonal anti-RhPV antiserum
diluted in TBS-supplemented buffer with 3% non-fat dry milk
for 5 h at 37 °C. After washing the grids three times in TBS-
supplemented buffer, the grids were treated with 25 μl drops of
1:100 dilution of secondary goat anti-rabbit antibody con-
jugated 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 JEOL 1200EX scanning/transmission electron
microscope at 80 kV.
The authors thank Drs. Art McIntosh, University of Missouri,
of DMII-AM and GWSS-Z10 cells respectively; and Tracey
Pepper and Harry Horner of the ISU Microscopy and Nanoima-
ging Facility for technical guidance. This study was supported by
an Iowa State University Carver Trust grant, the Iowa State
of Iowa funds.
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