Previous Article | Next Article 
Journal of Virology, December 2001, p. 12220-12227, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12220-12227.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Recovery of Infectious Pariacoto Virus from cDNA
Clones and Identification of Susceptible Cell Lines
Karyn N.
Johnson and
L. Andrew
Ball*
Department of Microbiology, University of
Alabama at Birmingham, Birmingham, Alabama 35294
Received 18 June 2001/Accepted 24 September 2001
 |
ABSTRACT |
Pariacoto virus (PaV) is a nodavirus that was recently isolated in
Peru from the Southern armyworm, Spodoptera eridania. Virus particles are non enveloped and about 30 nm in diameter and have T=3 icosahedral symmetry. The 3.0-Å crystal structure
shows that about 35% of the genomic RNA is icosahedrally ordered, with
the RNA forming a dodecahedral cage of 25-nucleotide (nt) duplexes that
underlie the inner surface of the capsid. The PaV genome comprises two
single-stranded, positive-sense RNAs: RNA1 (3,011 nt), which encodes
the 108-kDa catalytic subunit of the RNA-dependent RNA polymerase, and
RNA2 (1,311 nt), which encodes the 43-kDa capsid protein precursor 
.
In order to apply molecular genetics to the structure and assembly of
PaV, we identified susceptible cell lines and developed a reverse
genetic system for this virus. Cell lines that were susceptible to
infection by PaV included those from Spodoptera exigua,
Helicoverpa zea and Aedes albopictus, whereas cells
from Drosophila melanogaster and Spodoptera
frugiperda were refractory to infection. To recover virus from
molecular clones, full-length cDNAs of PaV RNAs 1 and 2 were
cotranscribed by T7 RNA polymerase in baby hamster kidney cells that
expressed T7 RNA polymerase. Lysates of these cells were infectious
both for cultured cells from Helicoverpa zea (corn earworm)
and for larvae of Galleria mellonella (greater wax moth).
The combination of infectious cDNA clones, cell culture infectivity,
and the ability to produce milligram amounts of virus allows the
application of DNA-based genetic methods to the study of PaV structure
and assembly.
| |
INTRODUCTION |
Members of the
Nodaviridae family are small positive-sense RNA viruses with
T=3 icosahedral symmetry (for a review, see references 4, 5, and 37). Each 30-nm particle is assembled from 180 copies of the capsid protein precursor and one copy of each of the
two unique segments of the viral RNA genome (24, 30, 41).
Viruses from the alphanodavirus genus infect primarily insects. In
alphanodaviruses, the 44-kDa capsid protein precursor is
autocatalytically cleaved following virion assembly to yield the two
mature capsid proteins 
(40 kDa) and 
(4 kDa) (20). The maturation cleavage is required for infectivity (38).
The larger genome segment (RNA1) contains 3.0 to 3.2 kb and encodes protein A, the catalytic subunit of the RNA-dependent RNA polymerase (26). The smaller genome segment (RNA2) contains 1.3 to
1.4 kb and encodes the capsid protein precursor, . Both genomic RNAs are capped but not polyadenylated (9, 10, 22, 28). During replication, a subgenomic RNA (RNA3) which is 3' coterminal with RNA1
is transcribed. RNA3 contains about 400 nucleotides (nt) and encodes
one or two small proteins (B1 and B2) of unknown function (5, 16,
22).
The best studied alphanodavirus is Flock House virus (FHV),
and reverse genetic systems that allow the infectious cycle of this
virus to be reconstructed from cDNA clones have been developed. FHV
replication can be initiated and infectious virus can be recovered from
in vitro transcripts of RNA1 and RNA2 transfected into Drosophila melanogaster cells (11) or from specialized cDNA
transcription plasmids transfected into mammalian cells
(2). These approaches have advanced our understanding of
both RNA replication and capsid assembly of this model virus (5,
6, 25, 37). For example, two regions in the FHV capsid protein
precursor that are involved in RNA encapsidation have been identified:
(i) protein that lacks amino acid residues 1 to 31 assembles into
virus-like particles (VLPs) that encapsidate RNA1 but fail to
encapsidate RNA2 (13); (ii) deleting the C-terminal 26 amino acids (aa) results in the assembly of VLPs that encapsidate
cellular RNAs in place of the viral genome segments (36).
However, most of the capsid protein regions involved in the specificity
of FHV RNA encapsidation are not visible in the crystal structure
(8, 14).
The structure of the alphanodavirus Pariacoto virus (PaV) was recently
determined, and it significantly extended our understanding of
RNA-protein interactions in nodavirus virions (43). PaV
was isolated in Peru in 1996 from moribund larvae of the Southern armyworm, Spodoptera eridania (45), and is the
most recent member of the alphanodavirus genus to be characterized. PaV
is the most distantly related of the insect nodaviruses, with its
RNA-dependent RNA polymerase and capsid protein sharing less than 29 and 41% sequence identity, respectively, with those of the other
alphanodaviruses (26, 27).
The three-dimensional structure of PaV is generally similar to that of
the other nodaviruses, but with several novel features (43). First, along each twofold axis of the capsid lies a
25-nt A-form RNA duplex that is visible at high resolution in the
crystal structure and accounts for 1,500 nt, or approximately 35% of
the single-stranded genomic RNA. Thirty such RNA duplexes are arranged as a dodecahedral cage, with discontinuous vertices where the RNA is
less ordered and presumably loops into the interior of the virion.
Second, for the first time in any nodavirus structure, the basic N
terminus of one of the three quasiequivalent capsid protein monomers in
the asymmetric unit is clearly visible from aa 7 to 51, making numerous
contacts with the RNA duplex. Third, the C-terminal 8 aa of the same
subunit are also visible for the first time, lying in a protein channel
at the quasi-threefold axis. Thus, regions of the capsid protein that
likely influence the specificity of RNA encapsidation and particle
assembly are uniquely visible in the PaV structure, making it an
attractive system for analysis by molecular genetics.
Such studies require a method by which infectious PaV particles and
VLPs could be recovered from cDNA clones in quantities large enough for
structural analysis. In our previous work we showed that transfection
of PaV virion RNAs into BHK-21 cells resulted in RNA replication and
the synthesis of both protein A and the capsid protein precursor .
To develop a reverse genetic system, cDNA transcription plasmids
encoding PaV RNA1 and RNA2 were transfected into BHK-21 cells infected
with a recombinant vaccinia virus that expressed T7 RNA polymerase to
drive primary transcription (27). The cDNA clones
initiated RNA replication and viral protein synthesis, but since BHK-21
cells are not susceptible to infection with PaV no evidence of
infectivity was obtained. In the present study, we surveyed several
insect cell lines for their susceptibility to infection with PaV and
then used the infectious cell culture system to validate the recovery
of clonally derived virus.
| |
MATERIALS AND METHODS |
Cells and virus.
Except where indicated, all insect cell
lines were maintained at 28°C in medium supplemented with 10%
heat-inactivated fetal bovine serum and antibiotics.
Drosophila line 1 and line 2 (DL-1 and DL-2)
(39) cells were maintained in Schneider's medium
(Gibco/BRL), and Spodoptera frugiperda (Sf9) cells
(44) were maintained in Grace's medium (Gibco/BRL). Cell
lines derived from Aedes aegypti whole larvae (Ae-59)
(34), Aedes albopictus whole larvae (ATC-15) (42), Spodoptera exigua larvae (Se-1)
(21), and Helicoverpa zea fat body cell lines
(BCIRL-HZ-FB27 and BCIRL-HZ-FB33, referred to here as FB27 and FB33,
respectively) and a midgut cell line (BCIRL-HZ-MG8, referred to here as
MG8) (29; A. H. McIntosh, personal communication)
were maintained in ExCell-401 medium (JRH Biologicals) with 10%
heat-inactivated fetal bovine serum, except for the MG8 line, which was
maintained in serum-free medium.
Baby kidney hamster-derived cell line BSR-T7/5 cells (7)
were grown at 37°C as monolayer cultures in modified Eagle's medium (Gibco/BRL catalogue number 41200-015) supplemented with 5% fetal calf
serum and 5% newborn calf serum in an atmosphere containing 5%
CO2. BSR-T7/5 cells were subcultured in the continuous
presence of the antibiotics penicillin and streptomycin, and in
alternate passages G418 was added to 1 mg/ml to ensure maintenance of
the T7 polymerase gene. Wild-type PaV was purified as described
previously (27) from Galleria mellonella larvae
inoculated with the original field material collected in 1996 (45).
cDNA clones.
The PaV cDNA clones PaV1(0,0) and PaV2(0,0)
were described previously (27). Briefly, reverse
transcription (RT)-PCR copies of the two PaV genome segments were
ligated into the transcription vector TVT7R(0,0) between the T7
promoter and cDNA sequences that encode the hepatitis delta virus
antigenomic ribozyme, followed by the T7 terminator. Following
autocatalytic cleavage by the hepatitis delta virus ribozyme, the
resulting T7 transcripts had precise 5' and 3' termini with no
additional nucleotides at either end, hence the designation (0,0). When
these plasmids were transfected into BHK-21 cells previously infected
with a recombinant vaccinia virus that expressed T7 RNA polymerase, we
observed a low level of replication of PaV RNA1 and RNA2. However, for
use in BSR-T7/5 cells which express T7 RNA polymerase constitutively
(7) and thus eliminate the need for vaccinia virus
infection, it proved necessary to optimize the PaV RNA1 clone by adding
a single guanylate residue at the transcription initiation site (see
Results and Discussion). The additional nucleotide was introduced into
plasmid PaV1(0,0) using Quik Change as described by the supplier
(Stratagene). A small DNA fragment spanning the mutation was
substituted into PaV1(0,0) to give PaV1(1,0), and the nucleotide
sequence of the substituted fragment was verified.
Screening for cells susceptible to infection with PaV.
To
screen cells for susceptibility to infection with PaV, insect cell
lines were plated in 35-mm-diameter wells of six-well plates at an
appropriate cell density to achieve 50 to 90% confluence. Cells were
allowed to attach for at least 1 h at 28°C, washed once with
serum-free medium, and overlayed with 1 ml of serum-free medium
containing 5 × 1010 PaV or FHV particles. After
2 h of adsorption, 1 ml of serum-containing medium was added and
incubation was continued at 28°C. RNA replication was assayed 24 h
postinfection by metabolic labeling as described below.
Transfection of BSR-T7/5 cells.
For transfection with virion
RNA (vRNA) or with plasmids, BSR-T7/5 cells were plated in
35-mm-diameter wells of six-well tissue culture plates and grown
overnight at 37°C to reach 80 to 90% confluency. The cells were
washed twice with Dulbecco's minimal essential medium (DMEM)
(catalogue number 12100-103; Gibco/BRL) and then overlayed with 1 ml of
DMEM containing 20 µl of Lipofectamine 2000 (Gibco/BRL) and 0.1 to
0.5 µg of PaV vRNA or 2.5 µg of PaV1(1,0) with or without 2.5 µg
of PaV2(0,0). After incubation for 24 h at 28°C, the
transfection mix was removed and replaced by MEM containing serum.
Incubation was continued at 28°C until 48 h posttransfection, at
which time the cells were either harvested or radiolabeled as described below.
RNA and protein labeling, extraction, and analysis.
The
products of RNA replication were radiolabeled by metabolic
incorporation of [3H]uridine for 2 or 4 h in the
presence of actinomycin D as previously described (2). For
BSR-T7/5 cells actinomycin D was used at a working concentration of 5 µg/ml, whereas for insect cell lines it was necessary to increase the
concentration of actinomycin D to 20 µg/ml to satisfactorily inhibit
DNA-dependent RNA synthesis. Total RNA was extracted from cells or
virions using an RNAgents kit (Promega) as suggested by the
manufacturer. RNAs were resolved by electrophoresis on 1%
agarose-formaldehyde gels and visualized by fluorography
(32).
The products of protein synthesis were metabolically labeled by
incorporation of either [
35S]methionine-cysteine or
[
3H]leucine for 1 h in DMEM lacking either
methionine and cysteine
or leucine, respectively. Total cell proteins
were analyzed by
sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE)
using standard techniques (
31).
For fluorography, SDS-PAGE gels
were treated with 1 M sodium salicylate
for 10 min before drying
and exposure to X-ray
film.
Western blot analysis.
Proteins were transferred from
SDS-polyacrylamide gels to polyvinylidene difluoride membranes in
Towbin buffer (10 mM Tris base, 96 mM glycine in 10% methanol) by
using a semidry graphite electroblotter (Millipore) for 2 h at a
constant current of 80 mA. PaV antiserum raised in rabbits against the
original isolate of PaV (45) was a gift from Jean-Louis
Zeddam. PaV antiserum was diluted 3,000-fold for use as the primary
antibody in the Immun-Star chemiluminescent protein detection system
(Bio-Rad) according to the manufacturer's instructions.
Virus neutralization.
For neutralization assays, PaV
antiserum was diluted 20-fold in phosphate-buffered saline before
addition of 1 µl to each 20 µl of virus or BSR-T7/5 lysate sample.
The samples were then incubated at room temperature for 30 min.
Propagation and purification of clonally derived PaV.
Larvae
of the greater wax moth (G. mellonella) were reared at
31°C as previously described (27). Late-instar larvae
(average weight, 150 mg) were injected with lysates derived from either mock-transfected BSR-T7/5 cells (12 larvae) or cells transfected with
plasmids PaV1(1,0) + PaV2(0,0) (58 larvae). Larvae were incubated at 31°C for 8 days before being frozen at 
20°C. Larval
homogenates were clarified by low-speed centrifugation, and the virus
was pelleted through a 30% sucrose cushion. The redissolved virus pellet was injected into 92 larvae (average weight, 220 mg), with 12 larvae left uninfected. The larvae were collected after 8 days of
incubation and frozen at 20°C until virus purification. Clonally derived PaV was purified from frozen infected larvae as previously described (27) except that 50 mM Tris (pH 7.4) buffer was
used throughout purification. Virus was resuspended in 50 mM Tris (pH 7.4) buffer and stored in aliquots at 80°C. Virus concentration was
determined using an extinction coefficient at 260 nm of 4.15/mg and a
particle mass of 9.2 × 106 g/mol, which was
calculated on the basis of the protein and RNA content of a PaV
particle (27, 43).
| |
RESULTS AND DISCUSSION |
Cell lines susceptible to infection with PaV.
Nine insect cell
lines were assayed for susceptibility to infection with PaV (Table
1). We determined infectivity based on replication of PaV RNA in cells following exposure to 5 × 1010 PaV particles, which corresponds to a multiplicity of
infection (MOI) of about 104 particles per cell. RNA
replication was assayed by metabolic labeling of RNAs with
[3H]uridine in the presence of actinomycin D, followed by
separation of RNAs on denaturing agarose-formaldehyde gels and
detection of the RNAs by fluorography. Input RNA was not detected by
this method because only the products of RNA replication are
actinomycin D resistant and became labeled. PaV infection led to
detection of RNA replication in five of the cell lines: those from
A. albopictus (mosquito; ATC-15), S. exigua (beet
armyworm; Se-1) and H. zea (corn earworm; FB33, FB27 and
MG8). The level of RNA labeled varied among these cell lines but was
highest in FB33 cells. However, even in FB33 cells the level of RNA
labeled was at least 100-fold less than during FHV infection of
Drosophila DL-1 or DL-2 cells. FHV infection of FB33 cells
led to a similarly low level of RNA labeling. Under the conditions used
for the infectivity assay, there was no evidence of PaV infection in
the mosquito cell line Ae-59, a fall armyworm line (Sf9), or the two
Drosophila cell lines (DL-1 and DL-2) that are permissive
for other alphanodaviruses such as FHV, Black beetle virus
(BBV), and Boolarra virus (12, 15, 35). The
lack of detection of RNA replication in Sf9 cells following exposure to
PaV is consistent with the previous finding by Zeddam et al.
(45) that S. frugiperda larvae were not
susceptible to infection by PaV. Based on the results of the
infectivity assay, the H. zea fat body cell line FB33 was
chosen for use in further experiments with PaV.
The results of the infectivity assays suggested that PaV and FHV may
have cell specificities that only partially overlap.
Of the nine cell
lines tested, only FB33 cells were susceptible
to both viruses. This is
understandable in view of the different
isolation sites of the two
viruses, their highly diverged genome
sequences (
26,
27),
and the distinct virion surface structures
in the putative
receptor-binding region (
43).
Titration of infectivity of PaV in FB33 cells.
The initial
infectivity assays used arbitrary cell densities and a high MOI. To
optimize the infectivity assay for FB33 cells, we examined the effect
of cell density on RNA replication. Cells were plated at a range of
densities (5 × 105 to 4 × 106 cells per
35-mm-diameter well), and replicating RNAs were labeled 24 h
postinfection. The optimal cell density for infection with PaV was
found to be 2 × 106 cells in a 35-mm-diameter well,
equivalent to 70 to 80% confluency (data not shown). Using this
optimum cell density, we tested the effects of various MOIs ranging
from 4 × 104 to 4 × 10
2 PaV
particles per cell (Fig. 1). RNA1 and
RNA2 were clearly labeled at MOIs down to 4 × 102,
and a weak signal was detected at 4 × 101 particles
per cell. No signal was detected at lower MOIs. Thus, PaV infection of
FB33 cells could be readily detected at or above a MOI of 400 particles
per cell, a level similar to the previously reported optimal MOIs for
synchronous infection of DL-1 cells with BBV (17) and
consistent with the high particle-to-PFU ratio for FHV (38,
41). In addition to the intense signals seen for the PaV genomic
RNAs, some weaker bands that were also present in the mock-infected
cells were visible. We attribute these to residual labeling of rRNAs
(Fig. 1).
View larger version (63K):
[in this window]
[in a new window]
|
FIG. 1.
Infectivity of PaV in FB33 cells. Helicoverpa
zea FB33 cells were infected with 10-fold dilutions of purified
PaV (MOIs of 4 × 104 to 4 × 100) or
mock infected and incubated at 28°C. After 22 h of incubation,
actinomycin D was added at 20 µg/ml, and 30 min later replicating
RNAs were metabolically labeled by incorporation of
[3H]uridine (20 µCi/ml) for a period of 2 h before
total cellular RNAs were harvested. RNAs were resolved by
electrophoresis on a 1% agarose-formaldehyde gel and visualized by
fluorography. PaV genomic RNA1 and RNA2 are identified on the left. The
faint bands below RNA2 are cellular RNAs that are present in all
samples including that from mock-infected cells.
|
|
Despite the evidence of PaV RNA replication, infected FB33 cells showed
no cytopathic effects even 5 days after infection
at an MOI of
10
4 particles per cell, and only low levels of PaV capsid
proteins
were detected in cell lysates (data not shown). In view of
these
results, it was not surprising that attempts to develop a plaque
assay for PaV in FB33 cells, as was achieved for FHV and BBV in
DL-2
cells (
41), were unsuccessful. Nevertheless, PaV infection
could be passed repeatedly on FB33 cells, although infectivity
increased by only 3 to 4 logs in a single passage. This contrasts
with
the infection of DL-2 cells with FHV, which is highly productive
(
40). Despite the low yields, FB33 cells provided a simple
infectivity
assay for wild-type PaV and recombinant viruses recovered
from
cDNA
clones.
PaV RNA and protein synthesis in BSR-T7/5 cells.
We showed
previously that PaV RNA replication can be initiated in BHK-21 cells
from the full-length cDNA clones PaV1(0,0) and PaV2(0,0)
(27). These plasmids were constructed such that the major
RNA transcripts made by T7 RNA polymerase initiated on the first
nucleotide of the PaV RNA (adenosine) and, after cleavage by the HDV
ribozyme, had terminal nucleotides that corresponded exactly to those
of the PaV genomic RNAs. In these original experiments, T7 RNA
polymerase was provided by infection of BHK-21 cells with a recombinant
vaccinia virus (27). However, since the presence of
vaccinia virus would complicate experiments designed to produce infectious PaV, for the present study we used the BHK-derived cell line
BSR-T7/5, which stably expresses T7 RNA polymerase under the control of
a cytomegalovirus pol II promoter (7).
The replication of FHV RNAs transcribed from cDNA clones in these cells
has been characterized in detail (1).
BSR-T7/5 cells were transfected with vRNA, or with plasmid PaV1(0,0)
with or without PaV2(0,0), and 48 h posttransfection
RNA
replication products were labeled with [
3H]uridine.
Despite strong replication of PaV vRNA, no RNAs were
labeled in the
cells transfected with PaV1(0,0) with or without
PaV2(0,0) (data not
shown). Therefore, while previous experiments
had shown that PaV cDNA
transcripts could initiate RNA replication,
and BSR-T7/5 cells could
support authentic vRNA replication (Fig.
2, lane 4), the original PaV
transcription plasmids failed to
initiate RNA replication in BSR-T7/5
cells.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 2.
Replication of RNAs in BSR-T7/5 cells transfected with
PaV cDNA clones. BSR-T7/5 cells were mock transfected (lane 3),
transfected with 100 ng of vRNA (lane 4), or transfected with 2.5 µg
of the following plasmids: PaV1(1,0) (lane 1); PaV1(1,0) + PaV2(0,0) (lane 2); PaV1(2,0) (lane 5); or PaV1(2,0) + PaV2(0,0)
(lane 6). Following transfection, cells were incubated at 28°C for
48 h, at which time 5 µg of actinomycin D per ml was added, and
incubation was continued for 30 min before the RNAs were metabolically
labeled by incorporation of [3H]uridine (20 µCi/ml) for
4 h. Total cellular RNAs were harvested, resolved by
electrophoresis on a 1% agarose-formaldehyde gel, and visualized by
fluorography. PaV RNA1, RNA2, and RNA3 are identified on the left.
|
|
To overcome this problem, we inserted either one or two additional G
residues at the transcription initiation site in the
PaV1 plasmid to
improve the sequence context of the T7 promoter
and thereby enhance the
level of DNA-templated primary transcription
(
33). An
additional G residue at the initiation site for T7
transcription was
also found necessary for replication initiated
by plasmids containing
FHV1 cDNA (
3). BSR-T7/5 cells were transfected
with PaV
vRNA, PaV1(1,0), or PaV1(2,0) alone or in combination
with PaV2(0,0),
and 48 h posttransfection RNA was metabolically
labeled with
[
3H]uridine as before. Total cellular RNA was
extracted and resolved
on denaturing agarose-formaldehyde gels, and
replication products
were visualized by fluorography (Fig.
2). In cells
transfected
with PaV1(1,0) (Fig.
2, lane 1) or PaV1(2,0) alone (Fig.
2,
lane
5), RNA replication was detected by the presence of labeled RNAs
corresponding to authentic PaV RNA1 and RNA3 (Fig.
2, lane 4).
Additionally, a small amount of an RNA1 dimer (
1,
27),
seen
most clearly in the vRNA sample (Fig.
2, lane 4), was noted in
cells transfected with plasmids for RNA1. In cells transfected
with
PaV1(1,0) + PaV2(0,0) (Fig.
2, lane 2) or PaV1(2,0) + PaV2(0,0)
(Fig.
2, lane 6), the RNA replication products comigrated with
authentic RNA1 and RNA2. These results indicate that PaV RNA
replication
can be initiated in BSR-T7/5 cells from the cDNA
transcription
plasmids PaV1(1,0) or PaV1(2,0) in conjunction with
PaV2(0,0).
PaV replication initiated from these plasmids
establishes an authentic
pattern of RNA products, including the
RNA2-mediated suppression
of RNA3 synthesis (
19,
46).
Plasmid PaV1(1,0) was chosen for
further
experiments.
At least one additional G nucleotide following the resected T7 promoter
was required to initiate PaV RNA replication in cells
that expressed T7
RNA polymerase constitutively, but not in those
that expressed the
enzyme from a recombinant vaccinia virus (
27).
In the
latter system, T7 transcripts are capped and methylated
in the
cytoplasm by the vaccinia virus guanylyl- and methyltransferases
(
18), which will enhance their translation efficiency. In
contrast,
primary transcripts made in the cytoplasm of BSR-T7/5 cells
are
presumably not capped and thus poorly translated. Consistent with
this interpretation, infection of BSR-T7/5 cells with wild-type,
nonrecombinant vaccinia virus rescued the replication of RNA1
from
PaV1(0,0), confirming that vaccinia virus contributed to
the recovery
of RNA replication from transcripts of this plasmid
(data not shown).
We speculate that adding a G residue to the
T7 promoter increased the
level of primary transcripts made from
PaV1(1,0), thus compensating for
their lack of cap structures.
Unlike RNA1, which encodes the
RNA-dependent RNA polymerase, RNA2
need provide no translation product
before it can replicate, and
the transcripts made from PaV2(0,0)
evidently sufficed to initiate
RNA2 replication. In both expression
systems, once RNA replication
was established presumably all products
would be capped and methylated
by PaV-specific
enzymes.
To examine PaV protein synthesis, BSR-T7/5 cells were transfected
with authentic vRNA or with plasmids PaV1(1,0) + PaV2(0,0)
and proteins were labeled with [
35S]methionine-cysteine
or [
3H]leucine 48 h posttransfection. Cytoplasmic
extracts were resolved
by SDS-PAGE, and labeled proteins were
visualized by fluorography
(Fig.
3A). A
protein of the size expected for protein
was labeled
both in cells
transfected with PaV vRNA and in cells transfected
with PaV1(1,0) + PaV2(0,0) plasmids (Fig.
3A, lanes 2, 4, 6, and
8), but not in
mock-transfected cells (Fig.
3A, lanes 3 and 7)
or in cells that
received PaV1(1,0) alone (Fig.
3A, lanes 1 and
5). As expected given
the short period of labeling, little or
no cleavage of protein
into
the mature capsid proteins
and
was observed. Extensive shutoff
of host protein synthesis was
seen in cells transfected with vRNA. In
cells transfected with
PaV1(1,0) alone or together with PaV2(0,0),
[
3H]leucine labeling revealed an additional protein with
an apparent
Mr of about 14 kDa (Fig.
3A, lanes 5 and 6) that was not present
in the untransfected cell sample (Fig.
3A,
lane 7). The identification
of this as the B2 protein is described
below.
View larger version (35K):
[in this window]
[in a new window]
|
FIG. 3.
Viral proteins synthesized in BSR-T7/5 cells transfected
with PaV cDNA clones. (A) In vivo labeling. Cells were mock transfected
(lanes 3 and 7) or transfected with 100 ng of vRNA (lanes 4 and 8), or
2.5 µg of PaV1(1,0) (lanes 1 and 5), or 2.5 µg each of
PaV1(1,0) + PaV2(0,0) (lanes 2 and 6). After 48 h of
incubation at 28°C, cells were preincubated for 30 min with medium
lacking either methionine and cysteine (lanes 1 to 4) or leucine (lanes
5 to 8), and then proteins were metabolically labeled for 1 h with
[35S]methionine-cysteine (lanes 1 to 4) or
[3H]leucine (lanes 5 to 8). Cytoplasmic extracts were
harvested, resolved by SDS-PAGE on 12.5% gels, and visualized by
fluorography. Proteins and B2 are indicated on the left, and the
migration positions of molecular mass markers are shown on the right.
(B) Western blot analysis. Samples 5 to 8 from panel A were also
resolved by SDS-PAGE on a 12.5% mini-gel (lanes 2 to 5) along with 250 ng of purified wild-type PaV virus particles (lane 1). Proteins were
transferred to a polyvinylidene difluoride membrane, probed with a
rabbit antiserum raised against purified PaV particles, and visualized
by chemiluminescence.
|
|
To confirm the identity of the major labeled protein, the samples were
examined by Western blot analysis using a polyclonal
rabbit antiserum
raised against authentic PaV. Cytoplasmic extracts
were resolved on
SDS-12.5% polyacrylamide gels, transferred to
membranes, and
probed with antiserum against purified PaV (Fig.
3B). In purified virus
particles the major protein was the mature
capsid protein
, although
small amounts of the capsid precursor
protein
and some putative
breakdown products were also present
(Fig.
3B, lane 1). BSR-T7/5 cells
transfected with PaV vRNA contained
proteins that comigrated with both
and
(Fig.
3B, lane 5),
suggesting synthesis and some cleavage
of the capsid precursor
protein. In comparison, cells
transfected with the cDNA plasmids
PaV1(1,0) + PaV2(0,0)
(Fig.
3B, lane 3) contained less capsid
protein precursor
and no
detectable mature capsid protein
.
The smaller amount of capsid
proteins in this sample was consistent
with both the analysis of
labeled proteins (Fig.
3A) and the lower
level of RNA replication
compared to vRNA-transfected cells (Fig.
2).
In addition to the capsid proteins, two minor bands were detected in
BSR-T7/5 cell extracts by Western blot analysis. One
of these was
present in all samples, including the mock-infected
control, and
therefore must represent a cellular protein. The
other immunoreactive
protein was present in all the lysates except
the mock-transfected
control (Fig.
3B, lanes 2, 3, and 5) and
was similar in size to the
14-kDa protein detected by [
3H]leucine labeling (Fig.
3A). This protein was RNA1 related and
corresponded in size to either
of the two overlapping open reading
frames (ORFs) in PaV RNA3
(
26) that are analogous to the ORFs
that encode the B1 and
B2 proteins in FHV RNA3 (
16,
22,
23).
Additionally, the
14-kDa protein was more abundant in the absence
of PaV2(0,0),
suggesting that it was translated from RNA3, which
is inhibited by RNA2
(Fig.
2). To determine whether the 14-kDa
protein was the product of
one of these ORFs, point mutations
were introduced into PaV1(1,0) to
disrupt the initiation codons
of either the B1 or the B2 ORF. The first
AUG of the B1 ORF was
mutated to GUG, which changed the initiating
methionine of B1
to a valine without altering the amino acid
sequence of the B2
ORF. Because B1 is in the same reading frame as
protein A, the
latter was also mutated at this position (M880V). The
first AUG
of the B2 ORF was mutated to ACG, which changed the
B2-initiating
methionine to threonine without altering the protein A or
B1 amino
acid sequences. The proteins synthesized in BSR-T7/5 cells
transfected
with the mutant plasmids were analyzed by
[
3H]leucine labeling and Western blot analysis, and the
results
identified the 14-kDa protein as a product of the B2 ORF (data
not
shown).
Reactivity of the PaV antiserum with the B2 protein was unexpected, as
B2 is a nonstructural protein that is not normally
present in purified
virus preparations (Fig.
3B, lane 1). We prepared
a second antiserum by
inoculation of a rabbit with gradient-purified,
clonally derived PaV
(see Fig.
5, lane 5). This antiserum also
detected B2 protein in the
extracts of vRNA-transfected BSR-T7/5
cells shown in Fig.
3B. Since
both antisera were polyclonal, we
cannot exclude the possibility that
both of the purified virus
samples used as antigens contained
undetectable levels of B2 protein.
However, it seems more likely that
some virus replication and
concomitant B2 protein synthesis occurred in
each of the rabbits
in which the antisera were raised. The host-range
of PaV has not
been examined, and another alphanodavirus,
Nodamura virus, is
able to replicate in some mammals
(
5); thus, it is possible
that PaV replicated in the
immunized rabbit. In these experiments
we found no evidence for
expression of the B1 ORF, which in PaV
RNA3, unlike the situation in
FHV, begins downstream of the B2
ORF. However, the predicted amino acid
sequence of the B1 ORF
contains only one methionine, no cysteines, and
one leucine, so
it is possible that synthesis of B1 protein would not
have been
detected by using the methods described
here.
Recovery of infectious virus from BSR-T7/5 cells.
To test
whether the cDNA clones yielded infectious virus, lysates were prepared
from BSR-T7/5 cells that had been transfected with PaV1(1,0) + PaV2(0,0). An aliquot of the lysate and a control sample containing
109 purified PaV particles were treated with anti-PaV
antiserum, with RNase A, or both. FB33 cells were infected with the
treated and mock-treated samples, and 24 h postinfection RNAs were
metabolically labeled with [3H]uridine, resolved on
denaturing agarose gels, and visualized by fluorography (Fig.
4). RNA replication was evident in cells infected with untreated PaV particles and with the untreated lysate from BSR-T7/5 cells that had been transfected with PaV1(1,0) + PaV2(0,0) (Fig. 4, lanes 1 and 5). RNase A treatment did not affect this infectivity (Fig. 4, lanes 2 and 6). In contrast, treatment with
anti-PaV antiserum decreased the infectivity of both authentic and
cDNA-derived PaV to below the limits of detection (Fig. 4, lanes 3 and
7). These results demonstrate that infectious PaV was generated in
BSR-T7/5 cells following transfection with plasmids that contained cDNA
copies of PaV RNA1 and RNA2.
View larger version (52K):
[in this window]
[in a new window]
|
FIG. 4.
Neutralization of authentic and clonally derived PaV.
BSR-T7/5 cells were transfected with 2.5 µg of PaV1(1,0) + PaV2(0,0) and incubated at 28°C for 48 h before the cells were
washed and lysed by freezing and thawing. These lysates (lanes 5 to 8)
and purified wild-type PaV particles (lanes 1 to 4) were incubated with
RNase A (lanes 2 and 6), PaV antiserum (lanes 3 and 7), or both RNase A
and PaV antiserum (lanes 4 and 8), or mock-treated with PBSM (lanes 1 and 5). The treated samples were used to infect FB33 cells. After
22 h of incubation at 28°C, actinomycin D was added at 20 µg/ml, and 30 min later replicating RNAs were metabolically labeled
by incorporation of [3H]uridine (20 µCi/ml) for a
period of 2 h before total cellular RNAs were harvested. RNAs were
resolved by electrophoresis on a 1% agarose-formaldehyde gel and
visualized by fluorography. PaV RNA1 and RNA2 are indicated
on the left.
|
|
Growth of PaV in G. mellonella larvae.
In order to
prepare a large stock of clonally derived virus, a lysate of BSR-T7/5
cells transfected with PaV1(1,0) + PaV2(0,0) was injected into
G. mellonella larvae. Following 8 days of incubation at
31°C, the nonpupated larvae (approximately 70%) were collected and
frozen at 20°C. Cadavers were homogenized in 50 mM Tris (pH 7.4),
and the clarified homogenates were pelleted through a 30% sucrose
cushion and resuspended in 50 mM Tris (pH 7.4). The resuspended pellets
were subjected to SDS-PAGE, and the proteins were stained with
Coomassie brilliant blue (Fig. 5). Two
major proteins were visible in extracts from infected larvae (Fig. 5,
lane 4) that were not present in uninfected samples (Fig. 5, lane 3).
One of these bands comigrated with the major capsid protein from
purified wild-type PaV (Fig. 5, lane 2), and the other was about 4 kDa larger, which is the size expected for the PaV capsid protein precursor
. As described previously (27), the small capsid protein was not visualized by Coomassie blue staining. These results indicated that BSR-T7/5 cells transfected with PaV1(1,0) + PaV2(0,0) generated PaV particles that were infectious for G. mellonella larvae.
View larger version (57K):
[in this window]
[in a new window]
|
FIG. 5.
Protein composition of clonally derived virus purified
from G. mellonella larvae. Larvae were injected with lysates
of BSR-T7/5 cells transfected with plasmids PaV1(1,0) + PaV2(0,0) or
mock transfected. After 8 days of incubation at 31°C, partially
purified virus was amplified by a second passage in larvae and clonally
derived PaV was purified from these larvae by sedimentation through a
sucrose gradient. Samples were resolved on an SDS-12.5%
polyacrylamide gel and visualized by staining with Coomassie blue.
Molecular mass markers are shown on the left (lane 1). Shown are
authentic PaV (lane 2; 1.5 µg); partially purified samples from
larvae injected with lysates of mock-transfected BSR-T7/5 cells (lane
3) or PaV1(1,0) + PaV2(0,0)-transfected BSR-T7/5 cells (lane 4);
clonally-derived, sucrose gradient-purified PaV (lanes 5 and 6; 5 and 1 µg respectively).
|
|
To amplify the clonally derived virus, the larval extracts described
above were repassaged in
G. mellonella larvae, and after
8 days of incubation at 31°C the virus was purified as described
in
Materials and Methods. Seven milligrams of recombinant virus
was
purified from 92
G. mellonella larvae. The purified clonally
derived virus was examined by SDS-PAGE and found to contain a
single
major protein that comigrated with the major capsid protein
of
authentic wild-type PaV (Fig.
5, lanes 5 and 6). Furthermore,
at
equivalent MOIs (similar to those shown in Fig.
1), the recombinant
and
wild-type viruses produced similar patterns of labeled RNAs
in infected
FB33 cells (data not
shown).
In summary, we have described a cell-based infectivity assay for PaV
and used it to recover infectious virus from cDNA clones.
Although
infected FB33 cells produced only modest amounts of virus,
amplification of the clonally derived virus in
G. mellonella
larvae
provided quantities of virus which have previously been
sufficient
for structural examination. Previous studies of nodavirus
assembly
have focused on FHV partly because of the availability of
susceptible
cell lines and infectious cDNA clones (
25,
37). The work described
above makes PaV only the second
nodavirus for which these experimental
tools are available and provides
methods to investigate the mechanisms
of RNA encapsidation and virion
assembly. These studies will be
guided by the high-resolution structure
of PaV, which revealed
many contacts between the capsid proteins and
the genomic RNAs
of this
virus.
| |
ACKNOWLEDGMENTS |
We thank Jean-Louis Zeddam (Station de recherches de pathologie
comparée, INRA-CNRS, 30380 Saint-Christol-les-Alès) for the
gift of PaV antiserum, K. Conzelmann and M. Schnell (Max von Pettenkofer Institute & Gene Center, Ludwig-Maximilians-University Munich, D-81377 Munich, Germany) for BSR-T7/5 cells, and Art McIntosh and Cindy Goodman (USDA, Agricultural Research Service, Biological Control of Insects Research Laboratory, Columbia, Mo.) for providing the following cell lines: Se-1, Ae-59, ATC-15, FB33, FB27 and MG8. We
thank members of the A. Ball and Gail Wertz laboratories for
discussions and critical reading of the manuscript.
This work was supported by NIH grant R01AI18270.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, University of Alabama at Birmingham, BBRB 373/17, 845 19th St. South, Birmingham, AL 35294-2170. Phone: (205) 934-0864. Fax: (205) 934-1636. E-mail: AndyB{at}uab.edu.
| |
REFERENCES |
| 1.
| Albariño, C. G., B. D. Price,
L. D. Eckerle, and L. A. Ball. Characterization and
template properties of RNA dimers generated during Flock House Virus
RNA replication. Virology, in press.
|
| 2.
|
Ball, L. A.
1992.
Cellular expression of a functional nodavirus RNA replicon from vaccinia virus vectors.
J. Virol.
66:2335-2345[Abstract/Free Full Text].
|
| 3.
|
Ball, L. A.
1995.
Requirements for the self-directed replication of flock house virus RNA 1.
J. Virol.
69:720-727[Abstract].
|
| 4.
|
Ball, L. A.,
D. A. Hendry,
J. E. Johnson,
R. R. Rueckert, and P. D. Scotti.
2000.
Nodaviridae, p. 747-755.
In
M. H. V. van Regenmortel, C. M. Fauquet, D. H. L. Bishop, E. B. Carstens, M. K. Estes, S. M. Lemon, D. J. McGeoch, J. Maniloff, M. A. Mayo, C. R. Pringle, and R. B. Wickner (ed.), Virus taxonomy, 7th Report of the International Committee on Taxonomy of Viruses. Academic Press, San Diego, Calif.
|
| 5.
|
Ball, L. A., and K. L. Johnson.
1998.
Nodaviruses of insects, p. 225-267.
In
L. K. Miller, and L. A. Ball (ed.), The insect viruses. Plenum Publishing Corporation, New York, N.Y.
|
| 6.
|
Ball, L. A., and K. L. Johnson.
1999.
Reverse genetics of nodaviruses.
Adv. Virus Res.
53:229-244[Medline].
|
| 7.
|
Buchholz, U. J.,
S. Finke, and K. K. Conzelmann.
1999.
Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.
J. Virol.
73:251-259[Abstract/Free Full Text].
|
| 8.
|
Cheng, R. H.,
V. S. Reddy,
N. H. Olson,
A. J. Fisher,
T. S. Baker, and J. E. Johnson.
1994.
Functional implications of quasi-equivalence in a T=3 icosahedral animal virus established by cryo-electron microscopy and X-ray crystallography.
Structure
2:271-282[Medline].
|
| 9.
|
Dasgupta, R.,
A. Ghosh,
B. Dasmahapatra,
L. A. Guarino, and P. Kaesberg.
1984.
Primary and secondary structure of black beetle virus RNA2, the genomic messenger for BBV coat protein precursor.
Nucleic Acids Res.
12:7215-7223[Abstract/Free Full Text].
|
| 10.
|
Dasmahapatra, B.,
R. Dasgupta,
A. Ghosh, and P. Kaesberg.
1985.
Structure of the black beetle virus genome and its functional implications.
J. Mol. Biol.
182:183-189[CrossRef][Medline].
|
| 11.
|
Dasmahapatra, B.,
R. Dasgupta,
K. Saunders,
B. Selling,
T. Gallagher, and P. Kaesberg.
1986.
Infectious RNA derived from transcription from cloned cDNA copies of the genomic RNA of an insect virus.
Proc. Natl. Acad. Sci. USA
83:63-66[Abstract/Free Full Text].
|
| 12.
|
Dearing, S. C.,
P. D. Scotti,
P. J. Wigley, and S. D. Dhana.
1980.
A small RNA virus isolated from the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae).
N. Z. J. Zool.
7:267-269.
|
| 13.
|
Dong, X. F.,
P. Natarajan,
M. Tihova,
J. E. Johnson, and A. Schneemann.
1998.
Particle polymorphism caused by deletion of a peptide molecular switch in a quasi-equivalent icosahedral virus.
J. Virol.
72:6024-6033[Abstract/Free Full Text].
|
| 14.
|
Fisher, A. J., and J. E. Johnson.
1993.
Ordered duplex RNA controls capsid architecture in an icosahedral animal virus.
Nature (London)
361:176-182[CrossRef][Medline].
|
| 15.
|
Friesen, P.,
P. Scotti,
J. Longworth, and R. Rueckert.
1980.
Black beetle virus: propagation in Drosophila line 1 cells and an infection-resistant subline carrying endogenous black beetle virus-related particles.
J. Virol.
35:741-747[Abstract/Free Full Text].
|
| 16.
|
Friesen, P. D., and R. R. Rueckert.
1982.
Black beetle virus: messenger RNA for protein B is a subgenomic viral RNA.
J. Virol.
42:986-995[Abstract/Free Full Text].
|
| 17.
|
Friesen, P. D., and R. R. Rueckert.
1981.
Synthesis of black beetle virus proteins in cultured Drosophila cells: differential expression of RNAs 1 and 2.
J. Virol.
37:876-886[Abstract/Free Full Text].
|
| 18.
|
Fuerst, T. R.,
P. L. Earl, and B. Moss.
1987.
Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes.
Mol. Cell. Biol.
7:2538-2544[Abstract/Free Full Text].
|
| 19.
|
Gallagher, T. M.,
P. D. Friesen, and R. R. Rueckert.
1983.
Autonomous replication and expression of RNA1 from black beetle virus.
J. Virol.
46:481-489[Abstract/Free Full Text].
|
| 20.
|
Gallagher, T. M., and R. R. Rueckert.
1988.
Assembly-dependent maturation cleavage in provirions of a small icosahedral insect ribovirus.
J. Virol.
62:3399-3406[Abstract/Free Full Text].
|
| 21.
|
Gelernter, W. D., and B. A. Federici.
1986.
Continuous cell line from Spodoptera exigua (Lepidoptera: Noctuidae) that supports replication of nuclear polyhedrosis viruses from Spodoptera exigua and Autographa californica.
J. Invertebr. Pathol.
48:199-207[CrossRef].
|
| 22.
|
Guarino, L. A.,
A. Ghosh,
B. Dasmahapatra,
R. Dasgupta, and P. Kaesberg.
1984.
Sequence of the black beetle virus subgenomic RNA and its location in the viral genome.
Virology
139:199-203[CrossRef][Medline].
|
| 23.
|
Harper, T. A.
1994.
Characterization of the proteins encoded from the nodaviral subgenomic RNA. Ph.D. Thesis.
University of Wisconsin Madison.
|
| 24.
|
Hosur, M. V.,
T. Schmidt,
R. C. Tucker,
J. E. Johnson,
T. M. Gallagher,
B. H. Selling, and R. R. Rueckert.
1987.
Structure of an insect virus at 3.0 angstrom resolution. Proteins: Struct.
Funct. Genet.
2:167-176.
|
| 25.
|
Johnson, J. E., and V. Reddy.
1998.
Structural studies of nodaviruses and tetraviruses, p. 171-223.
In
L. K. Miller, and L. A. Ball (ed.), The insect viruses. Plenum Publishing Corporation, New York, N.Y.
|
| 26.
|
Johnson, K. N.,
K. L. Johnson,
R. Dasgupta,
T. Gratsch, and L. A. Ball.
2001.
Comparisons among the larger genome segments of six nodaviruses and their encoded RNA replicases.
J. Gen. Virol.
82:1855-1866[Abstract/Free Full Text].
|
| 27.
|
Johnson, K. N.,
J. Zeddam, and L. A. Ball.
2000.
Characterization and construction of functional cDNA clones of Pariacoto virus, the first Alphanodavirus isolated outside Australasia.
J. Virol.
74:5123-5132[Abstract/Free Full Text].
|
| 28.
|
Kaesberg, P.,
R. Dasgupta,
J.-Y. Sgro,
J.-P. Wery,
B. H. Selling,
M. V. Hosur, and J. E. Johnson.
1990.
Structural homology among four nodaviruses as deduced by sequencing and X-ray crystallography.
J. Mol. Biol.
214:423-435[CrossRef][Medline].
|
| 29.
|
Kariuki, C. W.,
A. H. McIntosh, and C. L. Goodman.
2000.
In vitro host range studies with a new baculovirus isolate from the diamondback moth Plutella xylostella (L.) (Plutellidae: Lepidoptera).
In Vitro Cell. Dev. Biol.
36:271-276.
|
| 30.
|
Krishna, N. K., and A. Schneemann.
1999.
Formation of an RNA heterodimer upon heating of nodavirus particles.
J. Virol.
73:1699-1703[Abstract/Free Full Text].
|
| 31.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[CrossRef][Medline].
|
| 32.
|
Laskey, R. A., and A. D. Mills.
1975.
Quantitative film detection of 3H and 14C polyacrylamide gels by fluorography.
Eur. J. Biochem.
56:335-341[Medline].
|
| 33.
|
Ling, M. L.,
S. S. Risman,
J. F. Klement,
N. McGraw, and W. T. McAllister.
1989.
Abortive initiation by bacteriophage T3 and T7 RNA polymerases under conditions of limiting substrate.
Nucleic Acids Res.
17:1605-1618[Abstract/Free Full Text].
|
| 34.
|
Peleg, J., and A. Shahar.
1972.
Morphology and behaviour of cultured Aedes aegypti mosquito cells.
Tissue Cell
4:55-62[Medline].
|
| 35.
|
Reinganum, C.,
J. B. Bashiruddin, and G. F. Cross.
1985.
Boolarra virus: a member of the Nodaviridae isolated from Oncopera intricoides (Lepidoptera: Hepialidae).
Intervirology
24:10-17[Medline].
|
| 36.
|
Schneemann, A., and D. Marshall.
1998.
Specific encapsidation of nodavirus RNAs is mediated through the C terminus of capsid precursor protein alpha.
J. Virol.
72:8738-8746[Abstract/Free Full Text].
|
| 37.
|
Schneemann, A.,
V. Reddy, and J. E. Johnson.
1998.
The structure and function of nodavirus particles: a paradigm for understanding chemical biology.
Adv. Virus Res.
50:381-446[Medline].
|
| 38.
|
Schneemann, A.,
W. Zhong,
T. M. Gallagher, and R. R. Rueckert.
1992.
Maturation cleavage required for infectivity of a nodavirus.
J. Virol.
66:6728-6734[Abstract/Free Full Text].
|
| 39.
|
Schneider, I.
1972.
Cell lines derived from late embryonic stages of Drosophila melanogaster.
J. Embryol. Exp. Morph.
27:353-365[Medline].
|
| 40.
|
Scotti, P. D.,
S. Dearing, and D. W. Mossop.
1983.
Flock house virus: a nodavirus isolated from Costelytra zealandica (White) (Coleoptera: Scarabaeidae).
Arch. Virol.
75:181-189[CrossRef][Medline].
|
| 41.
|
Selling, B. H., and R. R. Rueckert.
1984.
Plaque assay for black beetle virus.
J. Virol.
51:251-253[Abstract/Free Full Text].
|
| 42.
|
Singh, K. R.
1971.
Propagation of arboviruses in Singh's Aedes cell lines. I. Growth of arboviruses in Aedes albopictus and A. aegypti cell lines.
Curr. Top. Microbiol. Immunol.
55:127-133[Medline].
|
| 43.
|
Tang, L.,
K. N. Johnson,
L. A. Ball,
T. Lin,
M. Yeager, and J. E. Johnson.
2001.
The structure of Pariacoto virus reveals a dodecahedral cage of duplex RNA.
Nat. Struct. Biol.
8:77-83[CrossRef][Medline].
|
| 44.
|
Vaughn, J. L.,
R. H. Goodwin,
G. J. Tompkins, and P. McCawley.
1977.
The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae).
In Vitro
13:213-217[Medline].
|
| 45.
|
Zeddam, J. L.,
J. L. Rodriguez,
M. Ravallec, and A. Lagnaoui.
1999.
A noda-like virus isolated from the sweetpotato pest Spodoptera eridania (Cramer) (Lep.; Noctuidae).
J. Invertebr. Pathol.
74:267-274[CrossRef][Medline].
|
| 46.
|
Zhong, W. D., and R. R. Rueckert.
1993.
Flock House Virus-Down-regulation of subgenomic RNA3 synthesis does not involve coat protein and is targeted to synthesis of its positive strand.
J. Virol.
67:2716-2722[Abstract/Free Full Text].
|
Journal of Virology, December 2001, p. 12220-12227, Vol. 75, No. 24
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.24.12220-12227.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Johnson, K. N., Tang, L., Johnson, J. E., Ball, L. A.
(2004). Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions. J. Virol.
78: 11371-11378
[Abstract]
[Full Text]
-
Johnson, K. N., Ball, L. A.
(2003). Virions of Pariacoto virus contain a minor protein translated from the second AUG codon of the capsid protein open reading frame. J. Gen. Virol.
84: 2847-2852
[Abstract]
[Full Text]
Top
Abstract
Introduction
