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JOURNAL OF VIROLOGY, Feb. 2011, p. 1439–1451 Vol. 85, No. 4
0022-538X/11/$12.00 doi:10.1128/JVI.02220-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Modulation of Stress Granules and P Bodies during
Dicistrovirus Infection
䌤
Anthony Khong and Eric Jan*
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver BC V6T 1Z3, Canada
Received 22 October 2010/Accepted 16 November 2010
Stress granules (SGs) are dynamic cytosolic aggregates composed of ribonucleoproteins that are induced
during cellular stress when protein synthesis is inhibited. The function of SGs is poorly understood, but they
are thought to be sites for reorganizing mRNA and protein. Several viruses can modulate SG formation,
suggesting that SGs have an impact on virus infection. In this study, we have investigated the relationship of
SG formation in Drosophila S2 cells infected by cricket paralysis virus (CrPV), a member of the Dicistroviridae
family. Despite a rapid shutoff of host translation during CrPV infection, several hallmark SG markers such
as the Drosophila TIA-1 and G3BP (RasGAP-SH3-binding protein) homologs, Rox8 and Rin, respectively, do
not aggregate in CrPV-infected cells, even when challenged with potent SG inducers such as heat shock,
oxidative stress, and pateamine A treatment. Furthermore, we demonstrate that a subset of P body markers
become moderately dispersed at late times of infection. In contrast, as shown by fluorescent in situ hybridiza-
tion, poly(A)
ⴙ
RNA granules still form at late times of infection. These poly(A)
ⴙ
RNA granules do not contain
viral RNA nor do they colocalize with P body markers. Finally, our results demonstrate that the CrPV viral 3C
protease is sequestered to SGs under cellular stress but not during virus infection. In summary, we propose
that dicistrovirus infection leads to the selective inhibition of distinct SGs so that viral proteins are available
for viral processing.
In response to environmental stress such as oxidative stress
or heat shock, cells respond by shutting down overall protein
synthesis. This results in the disassembly of polyribosomes,
leading to stalled initiation complexes that are dynamically
recruited to cytoplasmic foci called stress granules (SGs) (re-
viewed in references 2, 4, and 9). SGs are not required for
global translation repression (8, 36, 41, 44, 49) or global
mRNA stability (8, 26). Instead, it has been proposed that SGs
are sites where the increased local concentration of proteins
and mRNAs allows for remodeling and redistribution of
mRNPs (9). Alternatively, it has been shown that specific pro-
teins can be selectively sequestered to or away from SGs, thus
affecting biochemical processes in the cell. For instance, re-
cruitment to SGs of specific proteins such as RACK1, which is
required to activate the apoptosis-inducing MTK1 kinase dur-
ing mild stress, can inhibit apoptosis (3). Interestingly, it has
become apparent that viruses can affect SG formation, sug-
gesting that SGs influence the virus life cycle (reviewed in
reference 4). Because the function of SGs is still poorly under-
stood, further examination into the interplay between SGs and
virus infection may shed light on this process.
The primary trigger for SG assembly is the inhibition of
protein synthesis, where stalled initiation complexes are shut-
tled to and aggregate into SG foci. SG formation can occur in
cells that respond to environmental stresses or through the
addition of chemicals that block the activity of specific trans-
lation initiation factors (reviewed in references 2 and 9). One
of the best-described pathways is through eIF2␣(eukaryotic
initiation translation factor 2␣) phosphorylation (30, 33). In
response to distinct stresses, eIF2␣kinases are activated to
target and phosphorylate eIF2␣, which inhibits a key step in
translation initiation, leading to stalled initiation complexes on
mRNA and subsequent movement to SGs (33). SGs can also
be induced in an eIF2␣-independent manner. For example,
treatment of cells with hippuristanol or pateamine A (PatA),
which alters the activity of the helicase, eIF4A, also induces SG
formation (12, 42). In general, inhibition of translation that
results in the release of translating ribosomes will trigger SG
assembly. However, there are exceptions. In one study, pre-
venting 60S subunit joining with the 40S subunit does not lead
to SG assembly, suggesting that translational repression can be
uncoupled from SG induction (44). Thus, SGs may form only
through the inhibition of specific translation factors or within a
defined window during translation initiation (9).
SGs are nonmembranous dense complexes composed of sev-
eral proteins and RNA. In addition to stalled initiation com-
plexes composed of translation initiation factors such as
eIF4E, eIF2, eIF3, poly(A)-binding protein (PABP), and the
small 40S ribosomal subunit, SGs contain hallmark protein
markers such as T-cell intracellular antigen 1 (TIA-1), TIA-1-
related protein (TIAR), and GTPase (Src homology 3 [SH3]
domain) binding protein G3BP (31, 60). TIA-1 and TIAR are
closely related proteins that contain RNA recognition motifs
and are implicated in RNA metabolism (59). G3BP, a member
of the Ras signaling pathway, was discovered by its ability to
bind to the SH3 domain of RasGAP and has since been im-
plicated in a number of biological processes including RNA
metabolism (28). All three proteins contain domains that are
important for the aggregation and formation of SGs. For ex-
ample, TIA-1 and TIAR lacking the QN-rich prion-like do-
main, which allows for self-aggregation, can no longer form
* Corresponding author. Mailing address: Department of Biochem-
istry and Molecular Biology, 5457-2350 Health Sciences Mall, Univer-
sity of British Columbia, Vancouver, BC V6T 1Z3 Canada. Phone:
(604) 827-4226 Fax: (604) 822-5227. E-mail: ej@interchange.ubc.ca.
䌤
Published ahead of print on 24 November 2010.
1439
SGs (23). There are currently more than 50 proteins that are
associated with SGs (reviewed in references 2 and 9). How-
ever, not all SGs are compositionally homogeneous. For ex-
ample, tristetraprolin (TTP) is recruited to SGs when cells are
treated with the mitochondrial inhibitor FCCP [carbonyl cya-
nide-p-(trifluoromethoxy)phenylhydrazone] but not with ar-
senite (56). Thus, compositionally distinct SGs may form de-
pending on the type of cellular stress. Furthermore, a recent
report using a small interfering RNA (siRNA) screen identi-
fied more than 100 genes involved in the assembly and disas-
sembly of SGs (49). Which of these genes are specifically reg-
ulating the assembly of distinct SGs remains to be determined.
Another key component of SGs is mRNA. Currently, it is not
clear if all mRNAs or a distinct subset of mRNAs are recruited
to SGs. Interestingly, a recent report described the recruitment
of cleaved tRNAs to SGs under angiotensin-induced stress,
suggesting that distinct RNA populations may exist in SGs
(15).
SG assembly is dynamic. Under stress, stalled initiation com-
plexes are shuttled to SGs (30, 33, 34). Upon recovery from the
stress, SGs disassemble, and stalled initiation complexes can
now reenter the translation cycle and assemble onto polyribo-
somes. Inhibiting mRNA release from translating ribosomes
by treating cells with translation elongation inhibitors such as
emetine or cycloheximide can inhibit SG formation (31).
Moreover, fluorescence recovery after photobleaching
(FRAP) studies have shown that proteins shuttle in and out of
SGs rapidly (20, 24, 31, 32, 45). Thus, SG assembly and disas-
sembly are in equilibrium with translating polysomes, and this
process is likely highly regulated. A recent study reported that
dynein and kinesin motors are required for SG assembly and
disassembly, indicating that RNPs are delivered to SGs on
microtubule tracks (41).
P bodies (PBs) are another type of cytoplasmic RNA gran-
ule distinct from SGs (reviewed in references 16 and 50).
Normally present under basal conditions, PBs become larger
and more abundant under cellular stress such as oxidative
stress and glucose deprivation (57). Like SG formation, PB
formation is dependent on the pool of nontranslating mRNAs,
and PBs are composed of proteins involved in the mRNA
degradation pathway including DCP1, DCP2, LSM1 to LSM7,
XRN1, and EDC3 (50). In addition, PBs are sometimes com-
posed of factors involved in the microRNA (miRNA) repres-
sion machinery and nonsense-mediated decay (50). PBs often
associate with and are adjacent to SGs and share a subset of
proteins with SGs such as the decapping enzyme eIF4E and
Rck/p54 (32). These observations have led to the idea that
mRNPs cycle between cytosol, PBs, and SGs (9, 32). Finally,
although inhibition of protein synthesis can lead to an increase
in the number and size of SGs and PBs, the study by Ohn et al.
has shown that SGs and PBs can assemble independently,
suggesting that formation of these RNA granules may be spe-
cifically regulated (49).
The importance of SGs and PBs has been highlighted by
observations that several viruses can modulate SG assembly
(reviewed in reference 4). Viruses including the alphavirus
Semliki Forest virus, rotavirus, dengue virus, and West Nile
virus (WNV) inhibit SG formation (14, 43, 46). Moreover, it
appears that these viruses may inhibit SG formation via distinct
strategies. In poliovirus-infected cells, translation arrest early
in infection leads to induction of G3BP-containing SGs that
disassemble at later times of infection (63). This study showed
that SG disassembly is due to cleavage of G3BP by the polio-
virus 3C protease and that expression of a noncleavable G3BP
in poliovirus-infected cells attenuates virus replication (63).
However, a recent study reported that SGs containing
poly(A)
⫹
RNA and TIA-1 are still induced in poliovirus-in-
fected cells, suggesting that compositionally distinct SGs may
be modulated during infection (51). In WNV-infected cells, the
recruitment of TIA-1 and TIAR to the minus-strand viral
RNA for viral replication may play a part in inhibiting SG
assembly (14, 39). These studies suggest that viruses disassem-
ble or modulate SGs to evade being sequestered to nontrans-
lating cytoplasmic foci and that SG induction may act as a host
antiviral response. Further investigation into the antiviral role
of SGs is needed. Like SGs, PBs also play important roles
during virus infection. For example, depletion of PBs increases
translation of viral RNAs in HIV-infected cells (47). More-
over, it was demonstrated that miR-29A interactions with the
HIV RNA are sequestered to PBs, suggesting that PBs play an
antiviral role (47). Clearly, understanding how viruses modu-
late SGs and PBs may provide insight into the significance of
these mRNPs during the viral life cycle.
Dicistroviruses are positive-sense, single-stranded RNA vi-
ruses that primarily infect insects and arthropods. Members of
the Dicistroviridae family include the cricket paralysis virus
(CrPV), Drosophila C virus (DCV), Taura syndrome virus
(TSV), and Israeli acute paralysis virus (IAPV) (reviewed in
reference 6). Several of these viruses have had a negative
impact economically and agriculturally. TSV infection led to
the collapse of shrimp farms on the American continent in the
1990s (7), whereas IAPV has been recently linked to colony
collapse disorder in honeybees (11). Dicistroviruses encode an
8- to 10-kb-long RNA genome which contains two open read-
ing frames, encoding the nonstructural and structural proteins
(reviewed in reference 6). Both open reading frames are trans-
lated by distinct internal ribosome entry sites (21, 29, 64).
CrPV infection of Drosophila cells leads to a rapid shutoff of
host protein synthesis concomitant with phosphorylation of
eIF2␣(21, 64). Given that these properties are cellular signa-
tures for SG induction, we investigated whether SG assembly is
affected during dicistrovirus infection. We demonstrate that
SGs and also PBs are modulated in both CrPV- and DCV-
infected cells, suggesting that disruption of these RNA gran-
ules is important for virus infection. Our results also show that
the CrPV 3C protease is sequestered in SGs under cellular
stress but becomes dispersed during viral infection. We pro-
pose that CrPV inhibits SG formation to keep viral proteins
and RNA available for viral replication and translation. We
discuss the potential role that SGs and PBs may have in the
Drosophila antiviral response.
MATERIALS AND METHODS
Cell culture and virus. S2 cells (Drosophila Schneider 2) were maintained and
passaged in M3-BPYE (M3 insect medium supplemented with bactopeptone-
yeast extract) medium containing 10% fetal bovine serum. CrPV and DCV
production in Drosophila S2 cells has been previously described (21). A total of
3⫻10
9
cells were infected with 1 ml of virus suspension for 30 min. S2 cells were
subsequently incubated in 60 ml of conditioned medium for 14 h at 25°C. The
cells were pelleted, resuspended in phosphate-buffered saline (PBS), and sub-
jected to four freeze-thaw cycles.
1440 KHONG AND JAN J. VIROL.
Viral titers and yields were determined as described previously (21). UV-
irradiated CrPV was produced as described previously (21). The lack of repli-
cation of the UV-irradiated CrPV was confirmed by infecting S2 cells and
probing for the viral RNA by Northern blot analysis.
Plasmids and transfection. pAc5.1 GFP-PABP (where GFP is green fluores-
cent protein) and pAc5.1 V5-eIF3 S9 were generously provided by Pam Silver
(Harvard). GFP-DCP1a and GFP-GW182 were gifts from Elisa Izaurralde (Max
Planck). Red fluorescent protein (RFP)-AGO2 and GFP-AGO1 were gener-
ously provided by Andrew Simmonds (University of Alberta). Full-length Rox8,
FMR1, and CrPV viral proteins were amplified by reverse transcription-PCR
(RT-PCR) from RNA extracted from S2 cells infected with CrPV. Full-length
Rin (Rasputin) cDNA was PCR amplified from a clone obtained from the
Drosophila Genomics Resource Center. Rox8 and Rin were fused with GFP by
PCR amplification and ligated into pAc5.1 to produce Rox8-GFP and Rin-GFP.
FMR1 and the viral 3C protein were PCR amplified and ligated into pAc5.1
fused in frame with the V5 tag to produce FMR1-V5 and 3C-V5.
For transient transfections, 1 ⫻10
7
cells were transfected with 20 gof
plasmid DNA using Lipofectamine 2000 (Invitrogen) according to the manufac-
turer’s protocol. Cells were incubated for 18 h. S2 cells were infected with CrPV
or DCV at a multiplicity of infection (MOI) of 10. In detail, S2 cells were
centrifuged and washed once with PBS. Cells were infected with virus for 30 min,
and subsequently conditioned medium was added. Cells were treated with 50 nM
pateamine A (generous gift of Jerry Pelletier, McGill University) or 500 M
arsenite or incubated at 42°C to induce SGs. In preparation for microscopy, a
total of 1 ⫻10
6
S2 cells were plated on coverslips precoated with 0.5 mg/ml
concanavalin A (Calbiochem) for 1 h. Cells were subsequently fixed with 3%
paraformaldehyde in PBS and permeabilized in 0.5% Tween in PBS.
In situ hybridization. In preparation for in situ hybridization, S2 cells were
incubated in hybridization buffer (2⫻SSC [1⫻SSC is 0.15 M NaCl plus 0.015 M
sodium citrate], 20% formamide, 0.2% bovine serum albumin [BSA], 1 g/l
Saccharomyces cerevisiae tRNA) for 15 min at 37°C. Subsequently, S2 cells were
hybridized with 1 mg/ml DNA probes, oligo(dT) (40) conjugated to Cy-3 or Cy-5,
antisense CrPV RNA conjugated to Cy-5, or antisense 18S rRNA conjugated to
Cy-3 overnight at 46°C in hybridization buffer (IDT). The next day, S2 cells were
washed with 2⫻SSC with 20% formamide twice for 5 min each at 37°C, 2⫻SSC
twice for 5 min each at 37°C, and 1⫻SSC once for 5 min. If needed, cells were
costained by indirect immunofluorescence. The 18S rRNA probe sequence was
GTCCTAGATACTACCACAAAAGTTGATAG. The antisense CrPV RNA
probe sequence was ACTAAATAAAATATACAATAAAT.
Indirect immunofluorescence assays. After in situ hybridization, S2 cells were
washed with PBS and incubated with antibodies in PBS containing 5% horse
serum for1hat25°C. The antibodies include 1:200 rabbit-anti-deIF4E (gener-
ous gift from Paul Lasko, McGill University), 1:500 rabbit anti-V5 IgG (Sigma),
1:200 anti-double-stranded RNA ([dsRNA] English & Scientific Consulting Bt),
and 1:500 mouse anti-O-GlcNAc IgG (Covance). Cells were subsequently
washed with PBS three times and incubated for1hat25°C with the secondary
antibodies goat anti-rabbit Texas Red IgG (1:300; Invitrogen) and goat anti-
mouse Alexa Fluor 568 IgG (1:500; Invitrogen). Cells were then washed three
times with PBS. Coverslips were mounted on slides with Prolong Gold Antifade
Reagent (Invitrogen). Images were acquired by a confocal microscope (Olympus
FV1000 using Olympus Fluoview, version 2.0a) with a 60⫻oil immersion lens.
Images shown are representative of a single Z-section. Photoshop CS2 software
was used to process the images. Quantitation of the percentage of cells with SGs
or PBs was measured by manually counting the number of cells with ⱖ3orⱖ10
visible foci per cell.
Immunoblotting. S2 cells were lysed in lysis buffer (20 mM HEPES, 150 mM
NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, 10 mM tetrapyrophos-
phate, 100 mM NaF, 17.5 mM -glycerophosphate, and a protease inhibitor
cocktail [Roche]). Equal amounts of lysates were loaded and separated on an
SDS-PAGE gel and then transferred to a polyvinylidene difluoride Immo-
bilon-FL membrane (Millipore). The membrane was blocked for 30 min in 5%
skim milk in TBST (20 mM Tris, 150 mM NaCl, 0.1% Tween) and incubated at
4°C overnight with the following antibodies: 1:1,000 mouse anti-GFP (Roche),
1:5,000 rabbit anti-V5 (Sigma), and 1:1,000 rabbit-anti-deIF4E (generous gift
from Paul Lasko, McGill University). Membranes were washed with TBST three
times and incubated with IRDye 800CW goat-anti-rabbit IgG (Li-Cor Bio-
sciences) or IRDye 680CW goat anti-mouse 1:10,000 for1hatroom tempera-
ture in 5% skim milk in TBST. The membranes were washed three times in
TBST and scanned on an Odyssey imager (Li-Cor Biosciences). In some exper-
iments, a 1:30,000 dilution of donkey anti-rabbit IgG-horseradish peroxidase
(Amersham) or a 1:10,000 dilution of goat anti-mouse IgG-horseradish peroxi-
dase (Santa Cruz Biotechnology) was also used to detect proteins by chemilu-
minescence (Millipore).
RESULTS
Induction of stress granules in Drosophila S2 cells. Previous
reports demonstrated that SGs can be induced in Drosophila
S2 cells under oxidative and heat shock stress, stresses that
inhibit translation initiation in an eIF2␣phosphorylation-de-
pendent and -independent manner (19, 41). To further provide
a comprehensive analysis of SG formation in Drosophila cells,
we monitored the formation of mRNA granules by in situ
hybridization using an oligo(dT)-Cy3 in S2 cells treated with
arsenite (oxidative stress), heat shock (42°C), or pateamine A
(PatA). PatA inhibits translation initiation by sequestering
eIF4A from the cap-binding complex, which results in the
assembly of SGs in mammalian cells (12, 42). In untreated S2
cells, the poly(A)
⫹
RNA was primarily diffuse in the cyto-
plasm. Treatment of cells with arsenite or heat shock for 1 h
induced poly(A)
⫹
RNA granules (⬃100%) (Fig. 1). Similarly,
treatment of cells with PatA also induced poly(A)
⫹
RNA gran-
ules (⬃100%), demonstrating that the induction of RNA gran-
ules when eIF4A activity is inhibited is a conserved process
from Drosophila to mammals (Fig. 1). To determine whether
these poly(A)
⫹
RNA granules are SGs, we monitored the
subcellular localization of a panel of SG protein markers. Be-
cause antibodies to several Drosophila SG markers are not yet
available, we transiently transfected expression vectors con-
taining SG markers fused with either GFP or V5. As observed
previously with some of the markers (19), expression of the
fusion SG markers was cytoplasmically diffuse in untreated
cells (Fig. 1). In contrast, in all stressed cells tested, exog-
enously expressed Rox8-GFP, GFP-PABP, FMR1-V5, and
Rin-GFP were recruited to granules that colocalized with
poly(A)
⫹
RNA granules (Fig. 1A to C and E). Rox8 and Rin
are the Drosophila homologues of the mammalian TIA-1 and
G3BP, respectively, which are classic markers of SGs (31, 60).
Other signature SG markers include translation initiation fac-
tors and the small ribosomal subunit (30, 33, 34). We moni-
tored the subcellular localization of a subunit of the translation
factor eIF3 (fusion V5-eIF3 S9) and the endogenous cap-
binding protein, eIF4E, by indirect immunofluorescence. We
also detected the small ribosomal subunit by fluorescent in situ
hybridization using probes that hybridize to the 18S rRNA.
Under all stress conditions, a fraction of V5-eIF3 S9, endoge-
nous eIF4E, and the 18S rRNA partially colocalized with
poly(A)
⫹
RNA granules, consistent with previous reports that
stalled translation complexes are recruited to SGs (Fig. 1D, F,
and G). Recently, it has been shown that a subset of O-
GlcNAc-modified proteins colocalizes with SG markers (49).
In untreated cells, O-GlcNAc staining by immunofluorescence
showed punctate foci that did not colocalize with poly(A)
⫹
granules. In PatA-treated cells, a small fraction of O-GlcNAc
staining colocalized with the poly(A)
⫹
RNA granules, suggest-
ing that, as in the mammalian cells, the GlcNAc modification
may be important for assembly of SGs in Drosophila cells (Fig.
1H). In summary, we have established that SGs can be induced
in Drosophila cells under a number of stresses using a compre-
hensive panel of SG protein markers.
Modulation of SGs during cricket paralysis virus infection.
In CrPV-infected S2 cells, complete shutoff of host translation
occurs by 3 to4hofinfection, concomitant with phosphory-
lation of eIF2␣; thus, we would predict that SGs would form
VOL. 85, 2011 RNA GRANULE REGULATION DURING DICISTROVIRUS INFECTION 1441
during this time (21). For this analysis, cells with ⱖ3 visible foci
per cell were counted as SG-containing cells. We found that
this approach provided a reproducible and stringent assess-
ment of a cell containing SGs. In uninfected cells, only ⬃3to
11% of transfected cells exhibited punctate foci containing
Rox8-GFP, Rin-GFP, or PABP-GFP (Fig. 2A to C). Although
host translation shutoff occurs by 4 h postinfection (hpi), only
⬃15 to 20% of transfected cells at 4 and 6 hpi showed recruit-
ment of Rox8-GFP and Rin-GFP to SGs, compared to ⬃9to
11% of mock-infected cells, indicating that the majority of
infected cells displayed cytoplasmically diffuse Rox8-GFP and
Rin-GFP (Fig. 2A and B). Similarly, staining of V5-eIF3 S9
and FMR1-V5 by indirect immunofluorescence was primarily
diffuse in both mock-infected cells and at 6 hpi (Fig. 2D and F).
Also, no change in O-GlcNAc staining was observed in mock-
and CrPV-infected cells, and there was a lack of colocalization
between O-GlcNAc staining and poly(A)
⫹
granules at 6 hpi
(Fig. 2E). In summary, these results indicate that SG assembly
as defined by these SG protein markers is inhibited during
CrPV infection. However, unlike results for the SG markers
above, the number of transfected cells exhibiting foci that
contain GFP-PABP increased to 21% and 43% by 4 and 6 hpi,
respectively, compared to ⬃3% in mock-infected and at 2 hpi
(Fig. 2C). This suggests that the assembly of SGs composed of
GFP-PABP during CrPV infection is distinct from that of
other SG markers. Interestingly, the percentage of cells con-
taining ⱖ3 poly(A)
⫹
RNA granules increased during CrPV
infection from ⬃20% of mock-infected cells to ⬎90% of cells
by 8 hpi (Fig. 2G). By using a more stringent analysis, the
number of cells exhibiting ⱖ10 foci containing poly(A)
⫹
RNA
granules also increased from ⬃20% in mock-infected cells to
⬎70% in CrPV-infected cells by 8 hpi (Fig. 2G). In general, we
found that the poly(A)
⫹
RNA granules appeared smaller and
more numerous than those observed in cells treated with PatA,
heat shock, or arsenite. In every case, all SGs markers (Rox8-
GFP, Rin-GFP, V5-eIF3, FMR1-V5, and O-GlcNAc staining)
except for GFP-PABP failed to colocalize with poly(A)
⫹
RNA
granules (Fig. 2A, B, and D to F). Although primarily diffuse,
a fraction of GFP-PABP colocalized with the poly(A)
⫹
RNA
granules during infection (Fig. 2C). Thus, CrPV infection in-
hibits SG formation but not GFP-PABP-containing SGs or
poly(A)
⫹
RNA granules in S2 cells.
FIG. 1. SG markers colocalize with poly(A)
⫹
RNA granules in stressed Drosophila S2 cells. S2 cells were transiently transfected with expression
vectors containing Rox8-GFP (A), Rin-GFP (B), GFP-PABP (C), V5-eIF3 S9 (D), or FMR1-V5 (E). A day later, S2 cells were either left untreated
(control) or were treated with 50 nM PatA or 500 M arsenite or heat-shocked at 42°C for 1 h. Poly(A)
⫹
RNA was detected by fluorescence in
situ hybridization using Cy3- or Cy5-oligo(dT) (40) (red fluorescence). S2 cells were subsequently costained with antibodies to V5 (D and E), eIF4E
(F), or O-GlcNAc (H) or cohybridized with Cy3-antisense 18S rRNA (G). All SG markers except for poly(A)[supb]⫹[r] RNA are shown in the
green channel. Enlarged views of the boxed areas highlight different channels and merged views.
1442 KHONG AND JAN J. VIROL.
FIG. 2. Granules containing PABP and poly(A)
⫹
RNA but not Rox8-GFP or Rin-GFP are formed in CrPV-infected cells. S2 cells transiently
transfected with expression vectors containing Rox8-GFP (A), Rin-GFP (B), GFP-PABP (C), FMR1-V5 (D), or V5-eIF3 S9 (F) were mock
infected or infected for 2, 4, or 6 h with CrPV at an MOI of 10. The V5 fusion proteins and O-GlcNAc-modified proteins (E) were detected by
staining using anti-V5 and anti-GlcNAc antibodies, respectively. (G) Quantitation of the percentage of cells containing poly(A)
⫹
granules.
(H) Western blot analysis of the indicated SG protein markers in mock- and CrPV-infected cells at 6 hpi. (I) Inhibition of SGs in Drosophila C
virus-infected cells. S2 cells transiently transfected with the expression vector containing Rox8-GFP were mock-infected or infected for 12 h with
DCV at an MOI of 10. Poly(A)
⫹
RNA was detected by in situ hybridization using Cy3-oligo(dT) (40) (red fluorescence). Percentages represent
the proportion of transfected cells containing at least three granules expressing GFP or staining with V5 or O-GlcNAc. Enlarged views of the boxed
areas highlight different channels and merged views. More than 100 cells were analyzed in multiple fields for each experiment. The average of three
independent experiments is shown.
VOL. 85, 2011 RNA GRANULE REGULATION DURING DICISTROVIRUS INFECTION 1443
1444 KHONG AND JAN J. VIROL.
In poliovirus-infected cells, the inhibition of G3BP-contain-
ing SGs late in infection is due to cleavage of G3BP by the viral
3C protease (63). To rule out the possibility that the diffuse
localization of SG proteins in CrPV-infected cells may be due
to degradation or cleavage, we monitored the status of these
proteins by Western blot analysis. The SG markers, Rin-GFP,
eIF4E, Rox8-GFP, FMR1-V5 and V5-eIF3 S9, remained in-
tact and stable in mock- and CrPV-infected cells at 6 hpi (Fig.
2H). Similarly, it has been shown that PABP is not cleaved or
degraded during CrPV infection (21). Thus, the lack of SG
induction in CrPV-infected cells is not due to loss of these SG
markers.
To determine whether SG assembly is actively inhibited dur-
ing CrPV infection, cells were challenged with PatA treatment
for 1 h prior to fixation at different times of infection. PatA
treatment of mock-infected cells or cells infected for2hin-
duced SGs, as demonstrated by colocalization of poly(A)
⫹
RNA granules with Rox8-GFP, Rin-GFP, and GFP-PABP
(Fig. 3A to C), indicating that the pathway leading to SG
assembly is intact during the first2hofinfection (83 to 100%
of cells contained SGs). By 4 and 6 hpi, only 9 to 15% of
PatA-treated cells showed foci containing Rox8-GFP and Rin-
GFP while the rest of the cells exhibited diffuse GFP expres-
sion (Fig. 3A and B). In contrast, close to ⬃100% of infected
cells at 4 and 6 hpi exhibited ⱖ3 poly(A)
⫹
RNA granules per
cell (Fig. 3). Moreover, similar to observations made during
CrPV infection without PatA treatment, these poly(A)
⫹
RNA
granules did not colocalize with Rox8-GFP and Rin-GFP (Fig.
3A and B). In the case of GFP-PABP, only a fraction of
GFP-PABP colocalized with poly(A)
⫹
RNA granules in PatA-
treated cells at 4 and 6 hpi, which is also similar to observations
in CrPV-infected cells (Fig. 2C and 3C). To determine whether
CrPV infection can inhibit other SG assembly pathways, we
challenged infected cells with arsenite and heat shock. Similar to
results in PatA-treated cells, Rox8-GFP remained diffuse and did
not colocalize with poly(A)
⫹
RNA granules late in infection (Fig.
3D and E). In summary, these results demonstrate that the as-
sembly of Rox8-GFP and Rin-GFP SGs but not poly(A)
⫹
RNA
granules is inhibited in CrPV-infected cells.
DCV infection inhibits SG formation. We next asked
whether the related dicistrovirus, Drosophila C virus (DCV),
also inhibits SG formation. Like CrPV infection, DCV infec-
tion also leads to host translation shutoff but at a later time
point than that in CrPV-infected cells (10). At 12 hpi in DCV-
infected cells, poly(A)
⫹
RNA granules accumulated visible
foci which did not contain Rox8-GFP (Fig. 2I). The Rox8-GFP
remained cytoplasmically diffuse. When challenged with PatA
for 1 h at 12 hpi, DCV-infected cells still displayed diffuse
Rox8-GFP which did not colocalize with poly(A)
⫹
RNA gran-
ules (Fig. 2I). Given that both CrPV and DCV infection leads
to inhibition of Rox8-containing SGs and induction of
poly(A)
⫹
RNA granules, this may suggest a general property
of dicistrovirus infections in general.
CrPV replication is required for modulation of SGs. To
determine whether CrPV replication is required for the inhi-
bition of SGs, S2 cells were infected with UV-inactivated
CrPV. UV-inactivated CrPV was replication defective and did
not produce viral proteins in S2 cells (data not shown). UV-
inactivated CrPV failed to induce foci containing Rox8-GFP or
poly(A)
⫹
granules at 4 hpi, a time when poly(A)
⫹
granules are
normally detected in CrPV-infected cells (Fig. 4). When chal-
lenged with PatA for1hat4hpi, cells infected with UV-
inactivated CrPV exhibited foci that contained both Rox8-GFP
and poly(A)
⫹
granules (Fig. 4), thus demonstrating that viral
replication is necessary for inhibition of SG formation during
CrPV infection.
SGs are not maintained in CrPV-infected cells. Our exper-
iments indicate that the formation of SGs containing markers
such as Rox8 and Rin is inhibited after 4 hpi in CrPV-infected
cells (Fig. 2 and 3). It is possible that CrPV infection may not
only inhibit the assembly of but also promote the disassembly
FIG. 3. CrPV infection inhibits SG formation in response to PatA treatment. S2 cells transiently transfected with expression vectors containing
Rox8-GFP (A), Rin-GFP (B), or GFP-PABP (C) were either mock infected or CrPV infected (MOI of 10) for 2, 4, or 6 h and treated with 50
nM PatA (A to C) or 500 M arsenite (D) or heat shocked at 42°C (E) for 1 h prior to fixation. Poly(A)
⫹
RNA was detected by in situ hybridization
using Cy3-oligo(dT) (40) (red fluorescence). Enlarged views of boxed areas highlight different channels and merged views. Percentages of
transfected cells containing at least three GFP-containing granules are shown. More than 100 cells were analyzed in multiple fields for each
experiment. The average of three independent experiments is shown.
FIG. 4. UV-inactivated CrPV does not inhibit SGs. Transiently
transfected cells expressing Rox8-GFP were mock infected or infected
with UV-inactivated CrPV. Where indicated, cells were treated with
PatA for 1 h prior to fixation. Poly(A)
⫹
RNA was detected by in situ
hybridization using a Cy3-oligo(dT) (40) (red fluorescence). Enlarged
views of boxed areas highlight different channels and merged views.
VOL. 85, 2011 RNA GRANULE REGULATION DURING DICISTROVIRUS INFECTION 1445
of SGs. To address this, after SGs were induced by the addition
of PatA to infected cells at 1 hpi, Rox8-GFP-containing SGs
were monitored at 2 or 4 hpi. The 4-hpi time point was chosen
because this is when Rox8-containing SGs are inhibited during
CrPV infection (Fig. 3). Whereas PatA treatment for1hat2
hpi led to punctate Rox8-GFP granules that colocalized with
poly(A)
⫹
RNA, PatA treatment for3hat4hpiresulted in
diffuse Rox8-GFP and in poly(A)
⫹
RNA granules (Fig. 5). The
lack of Rox8-GFP-containing SGs is not due to the dynamic
property of SGs as cells treated with PatA for 1 or 3 h still
displayed foci containing Rox8-GFP that colocalized with
poly(A)
⫹
RNA granules (Fig. 5). These results indicate that
Rox8-GFP-containing SGs cannot be maintained during the
course of infection.
Characterization of poly(A)
ⴙ
RNA granules in CrPV-in-
fected cells. The lack of colocalization of several SG markers with
poly(A)
⫹
RNA granules in CrPV-infected cells suggests that the
poly(A)
⫹
RNA granules may represent distinct RNA granules.
One possibility is that these poly(A)
⫹
RNA granules are PBs. To
monitor PBs, cells were transfected with plasmids expressing
GFP-GW182, GFP-DCP1, GFP-AGO1, or RFP-AGO2. Expres-
sion of these reporter proteins resulted in distinct punctate GFP
or RFP expression, indicative of basal PB formation (Fig. 6A).
PBs containing GFP-DCP1 or GFP-GW182 were present in
100% of transfected cells, whereas those containing GFP-AGO1
or RFP-AGO2 were present in ⬃50% of cells (cells with ⱖ3 foci
per cell were counted as PB-containing cells). As shown in this
study, at 4 and 6 hpi, poly(A)
⫹
RNA granules were induced in
CrPV-infected cells. However, these granules did not colocalize
with the GFP-GW182, GFP-DCP1, GFP-AGO1, or RFP-AGO2
foci, indicating that these poly(A)
⫹
RNA granules are not PBs.
Interestingly, the number of cells that contained GFP-GW182
and GFP-DCP1 PBs moderately decreased at late times of infec-
tion (Fig. 6A). Compared to mock-infected cells (100% of trans-
fected cells), only ⬃60% of CrPV-infected cells at 6 hpi displayed
ⱖ3 foci containing GFP-GW182 or GFP-DCP1 (Fig. 6A). This
decrease was not due to degradation or cleavage of the PB mark-
ers (Fig. 6B). In contrast, the percentage of cells with PBs con-
taining GFP-AGO1 or RFP-AGO2 remained constant through-
out infection (Fig. 6A). Thus, these results demonstrate that
formation of PBs containing GFP-GW182 or GFP-DCP1 but not
GFP-AGO1 or RFP-AGO2 is inhibited in CrPV-infected cells.
Since the CrPV viral RNA genome has a poly(A) tail (13), we
next investigated the possibility that the poly(A)
⫹
RNA granules
contain CrPV RNA or viral replicative RNA intermediates. By in
situ hybridization using a probe specific to the sense strand of
CrPV RNA, fluorescence increased during the course of CrPV
infection, strongly indicating that the increase in viral RNAs was
specifically detected (Fig. 7). As a control, a probe containing a
randomized sequence did not result in fluorescence signal over
background (data not shown). Despite the increase in viral RNA,
the detectable fluorescence signal did not colocalize with the
poly(A)
⫹
RNA granules (Fig. 7). To determine whether the
poly(A)
⫹
RNA granules may be sites of replicative intermediates,
we used an antibody that specifically recognizes dsRNA (62).
Whereas mock-infected cells displayed basal immunofluores-
cence, the dsRNA staining increased over time in CrPV-infected
cells, consistent with an increase in replicative viral intermediates
(Fig. 7). The dsRNA staining was punctate, indicating that CrPV
replication is localized. However, the dsRNA staining did not
colocalize with the poly(A)
⫹
RNA granules in CrPV-infected
cells (Fig. 7, compare green and red channels). From these re-
sults, we conclude that the virally induced poly(A)
⫹
RNA gran-
ules are not PBs and do not contain viral RNA.
The CrPV 3C protease is sequestered to SGs. Our results
suggest that CrPV replication is required for the inhibition of SG
formation (Fig. 4). One possibility is that a CrPV protein may be
mediating this effect. To begin testing this hypothesis, we sub-
cloned individual CrPV nonstructural and structural proteins. Of
the CrPV proteins tested so far, none can inhibit the formation of
Rox8-GFP-containing SGs in cells treated with PatA. However,
through our analysis, we noticed that in cells expressing the V5-
tagged 3C protease, the staining of V5 by indirect immunofluo-
rescence colocalized with SGs containing Rox8-GFP and
FIG. 5. CrPV infection leads to disassembly of SGs. Transiently transfected cells expressing Rox8-GFP were infected with CrPV for2hor4h.
At 1 hpi, 50 nM Pat A was added. As controls, uninfected cells were treated with 50 nM PatA for1hor3h.Cells were subsequently fixed, and
poly(A)
⫹
RNA was detected by in situ hybridization using a Cy3-oligo(dT) (40) (red fluorescence). Enlarged views of boxed areas highlight
different channels and merged views.
1446 KHONG AND JAN J. VIROL.
poly(A)
⫹
RNA under PatA treatment (Fig. 8, middle panel). We
next tested whether the tagged 3C protease may be colocalizing
with the poly(A)
⫹
RNA granules that are induced in CrPV-
infected cells. As shown previously in CrPV-infected cells, the
Rox8-GFP remained diffuse, and poly(A)
⫹
RNA granules assem-
bled in CrPV-infected cells (Fig. 8, right panel). However, the
tagged 3C protease was not recruited to the poly(A)
⫹
granules
during CrPV infection and remained cytoplasmically diffuse. Be-
cause the viral 3C protease may be susceptible to being seques-
tered to SGs, one possible outcome of inhibiting SG assembly
during CrPV infection is that the 3C protease remains available
for viral processing.
FIG. 6. Virus-induced poly(A)
⫹
RNA granules are not P bodies. (A) S2 cells transiently transfected with expression vectors containing
GFP-AGO1, RFP-AGO2, GFP-DCP1, or GFP-GW182 were either mock or CrPV infected for 2, 4, or 6 h with CrPV at an MOI of 10. PB markers
are shown in green. Poly(A)
⫹
RNA was detected by in situ hybridization using Cy3-oligo(dT) (40) (red fluorescence). Percentages represent the
proportion of transfected cells containing at least three GFP- or RFP-containing granules. Enlarged views of boxed areas highlight different
channels and merged views. (B) Western blot analysis of the indicated PB protein markers in mock- and CrPV-infected cells at 6 hpi.
VOL. 85, 2011 RNA GRANULE REGULATION DURING DICISTROVIRUS INFECTION 1447
DISCUSSION
Viruses have evolved unique mechanisms to evade the
host antiviral response and to usurp host factors for viral
translation and replication. There is increasing evidence
that viruses can modulate SG and PB formation, suggesting
that they can impact virus infection and yield (1, 14, 25, 35,
43, 46, 51, 52, 63). Here, we have demonstrated that infec-
tion by the dicistroviruses CrPV and DCV inhibits the as-
sembly of SGs, as defined by a set of SG protein markers,
but not of granules containing poly(A)
⫹
RNA. Our results
suggest that the inhibition of SG formation may keep viral
proteins available for CrPV processing, translation, and rep-
lication.
SGs are defined as aggregations of nontranslating mRNAs
that colocalize with SG protein markers such as TIA-1, G3BP,
and several translation initiation factors (reviewed in refer-
ences 2 and 9). This study expands on previous reports on SGs
in Drosophila cells, and we have now demonstrated several SG
markers that aggregate and colocalize with poly(A)
⫹
RNA
under a number of cellular stress conditions (19, 41). SGs are
induced in cells treated with heat shock, arsenite, and PatA,
indicating that SG assembly is conserved in Drosophila and
that these SGs are compositionally similar to those in mam-
malian cells (Fig. 1). Using this panel of SG markers, we
explored the relationship of SG formation during dicistrovirus
infection. In CrPV- and DCV-infected cells, despite a rapid
shutoff of host protein synthesis, the majority of the classic SG
markers do not aggregate and remain dispersed (Fig. 2). This
was surprising as we monitored signature SG markers such as
Rox8, Rin, and eIF3. Only the SG marker PABP partially
aggregated and colocalized with poly(A)
⫹
RNA granules. Our
results showed that infected cells challenged with potent SG
inducers such as heat shock, arsenite, or PatA could not induce
aggregation of SG markers after 4 hpi but could at earlier
times of infection (Fig. 3). This suggests that the inhibition of
SG formation in CrPV-infected cells likely targets a general
pathway of SG formation at late times of infection. Our results
also indicate that Rox8-containing SGs are not maintained
during CrPV infection when SGs are induced early in infection
(Fig. 5). Taken together, these results indicate that CrPV in-
fection may lead to the assembly and/or promote the disassem-
bly of SGs at late times of infection.
Despite the lack of aggregation of most SG markers,
poly(A)
⫹
RNA granules were still induced in CrPV-infected
cells (Fig. 2). This observation highlights the importance of
monitoring both SG protein markers and poly(A)
⫹
RNA and
serves as an example that distinct SGs and RNA granules may
be differentially regulated under specific cellular conditions.
Moreover, given that there is more evidence that composition-
ally distinct SGs exist and may be differentially regulated, as we
have observed in our studies, it is imperative to monitor as
many SG markers as possible during a given cellular stress (51,
56). In our studies, the percentage of cells containing poly(A)
⫹
FIG. 7. Virus-induced poly(A)
⫹
RNA granules do not contain
CrPV RNA. S2 cells were mock or CrPV infected (MOI of 10) for 2,
4, or 6 h and then processed for in situ hybridization. Poly(A)
⫹
RNA
was detected by in situ hybridization using a Cy5- or Cy3-oligo(dT) (40)
(red). Viral dsRNA intermediates were detected by indirect immuno-
fluorescence using an anti-dsRNA antibody (green). CrPV RNA was
detected by using a Cy5-conjugated antisense probe that hybridized to
the sense strand of CrPV RNA (green). Enlarged views of boxed areas
highlight different channels and merged views.
FIG. 8. The CrPV 3C-like protease is sequestered to SGs under
cellular stress. S2 cells transiently cotransfected with expression vectors
containing Rox8-GFP and CrPV 3C protease-V5 were left untreated
(control), treated with 50 nM PatA for 1 h, or infected with CrPV for
6 h. Fusion V5 viral proteins were detected by costaining with anti-V5
antibody (gray), and poly(A)
⫹
RNA was detected by in situ hybridiza-
tion using Cy5-oligo(dT) (40) (red). Expression of Rox8-GFP is shown
in green. Enlarged views of boxed areas highlight different channels
and merged views.
1448 KHONG AND JAN J. VIROL.
RNA granules increased during infection, which correlated
with viral replication (Fig. 2). To our surprise, using two ap-
proaches to detect viral RNA by in situ hybridization and by
indirect immunofluorescence using an anti-dsRNA antibody,
we found that these poly(A)
⫹
RNA granules do not contain
viral RNA (Fig. 7). We also showed that these poly(A)
⫹
RNA
granules do not colocalize with PB markers (Fig. 6). Currently,
we do not know if these poly(A)
⫹
RNA granules represent
novel RNA granules or are distinct SGs that do not colocalize
with known classic SG markers but, rather, with a yet to be
discovered aggregating protein complex. A recent report
showed that in poliovirus-infected cells, the induction of
poly(A)
⫹
RNA granules requires ongoing host transcription
(51). In CrPV-infected cells, addition of actinomycin D did not
affect the induction of poly(A)
⫹
RNA granules or lead to the
inhibition of SG marker aggregation (our unpublished results).
In CrPV-infected cells, overall translation is shut off by 4 hpi,
which is approximately the same time when poly(A)
⫹
RNA
granules are induced (21). It is likely that these poly(A)
⫹
RNA
granules represent nontranslating mRNAs that are released
from polysomes during infection. An outstanding question is
whether a subset of mRNAs or all nontranslating mRNAs are
localized to these poly(A)
⫹
RNA granules in infected cells.
What is inhibiting SGs during CrPV infection? UV-inacti-
vated CrPV does not mediate the inhibition of SG assembly,
indicating that CrPV replication is required (Fig. 4). Further-
more, the inhibition of SG formation occurred after ⬃4 hpi,
suggesting that a viral protein may be mediating this effect
(Fig. 2 and 3). We are currently investigating whether specific
CrPV structural and nonstructural proteins inhibit SG forma-
tion. SGs can assemble through the action of aggregating pro-
teins such as TIA-1 and G3BP (31, 60). However, during CrPV
and DCV infection, these and other SG proteins tested in this
study do not aggregate and are not cleaved or degraded (Fig.
2). Besides protein aggregation, posttranslational modifica-
tions of specific proteins appear to be important for SG as-
sembly. It has been reported that the deacetylase activity of
histone deacetylase (HDAC) is required for SG assembly (36).
Moreover, a recent siRNA screen identified several genes
(⬎100) involved in the assembly and disassembly of SGs, sug-
gesting that multiple factors may mediate SG assembly (49).
One interesting outcome from this previous study was the
discovery that the O-GlcNAc modification of proteins may be
important for the assembly of SGs (49). In our studies, the
staining of O-GlcNAc by immunofluorescence was localized to
SGs under cellular stress but not during CrPV infection, sug-
gesting that CrPV infection does not affect this pathway of SG
assembly. Our data suggest that SGs may be actively disrupted
during CrPV infection (Fig. 5). The factors Staufen 1 and
eIF5A have been shown to disrupt SGs by maintaining ri-
bosomes bound to mRNAs, thus preventing initiation com-
plexes from shuttling to SGs (38, 58). It is unlikely that these
factors are responsible for the inhibition of SGs during
CrPV infection since host translation is shut off, and the
majority of polysomes are disassembled (21). Clearly, more
studies are required to elucidate the pathways leading to SG
assembly and disassembly.
Our results demonstrated that PBs containing GW182
and DCP1 disassemble late during CrPV infection (Fig. 6).
It has been proposed that PBs are sites where translationally
stalled mRNPs are shuttled to and destined for degradation
or for reorganization of mRNPs. Moreover, PBs are nor-
mally found adjacent to SGs during cellular stress, and pro-
teins and mRNAs may be exchanged between PBs and SGs
(32). In general, GW182, DCP1, AGO1, and AGO2 interact
with each other to form cytoplasmic PB foci containing
mRNA and miRNAs (5, 18, 27, 55). However, not all PBs
contain the full complement of PB markers, and they can be
compositionally distinct. For instance, PBs containing DCP1
and AGO2 do not colocalize under certain cellular stress
conditions (37), and a recent study revealed that GW182-
and AGO2-containing bodies are distinct from DCP1-con-
taining P bodies (22). Supporting this, CrPV infection af-
fects only granules containing DCP1 and GW182 but not
AGO1 and AGO2 (Fig. 6). During CrPV infection, the virus
may inhibit DCP1- and GW182-containing PBs specifically
in order to keep viral RNAs from degrading. Alternatively,
given that GW182 is a key component in miRNA-dependent
translational control, it is possible that a miRNA that is
normally shuttled to PBs may be required for CrPV infec-
tion (17, 40, 54). It has been shown that Ago2
⫺/⫺
flies are
hypersensitive to CrPV and DCV infection, indicating that
Ago2 is an antiviral factor in Drosophila (61). Moreover, the
CrPV silencing suppressor 1A protein targets and inhibits
AGO2 activity, thereby antagonizing RNA interference
(48). Because AGO2 remains punctate during CrPV infec-
tion, it remains to be seen whether this process may con-
tribute to the suppression of AGO2 activity and affect viral
yield.
We observed that the V5-tagged 3C protease colocalized
with Rox8-GFP and poly(A)
⫹
RNA granules under PatA-
induced stress, suggesting that this viral protein has a ten-
dency to be sequestered to SGs (Fig. 7). Under viral infec-
tion, the 3C protease remained disperse and did not
colocalize with the poly(A)
⫹
RNA granules. Thus, we pro-
pose that one purpose of inhibiting SG formation during
infection is to keep the CrPV 3C protease available and free
for viral protein processing. Alternatively, given that SGs
may sequester specific proteins to regulate apoptosis (3), it
is possible that the inhibition of SGs in CrPV-infected cells
affects other cellular processes.
The significance and function of SGs are not well under-
stood. However, modulation of SGs appears to be a common
host response during viral infection, as several viruses have
been shown to modulate SGs and PBs. Infection with viruses
such as alphaviruses (43, 63) leads to a transient induction of
SGs at early times postinfection, but at later times SGs become
dispersed. Alternatively, the flaviviruses West Nile virus and
dengue virus completely inhibit SG induction even when chal-
lenged with strong SG inducers, similar to results in this study
with dicistrovirus infections (14). There is some indication that
SGs act to limit viral infections. For instance, mouse embryonic
fibroblasts lacking TIA-1 show increased coronavirus produc-
tion (53). In poliovirus-infected cells, expression of a cleavage-
resistant G3BP attenuates virus replication (63). Finally, vi-
ruses may not just modulate SG assembly but may also utilize
key SG components. For instance, the SG marker TIAR binds
to the 3⬘untranslated region (UTR) of the WNV RNA and
augments viral replication (14, 39). In this present study, we
have demonstrated that CrPV infection may limit SG assembly
VOL. 85, 2011 RNA GRANULE REGULATION DURING DICISTROVIRUS INFECTION 1449
to maintain viral proteins available for virus infection. Whether
certain SG proteins, as in the case of TIAR and WNV (14, 39),
are utilized for dicistrovirus infection will be an interesting
aspect to examine in future studies. In summary, our studies
have expanded on the number of viruses that modulate SGs
and further highlight the significance of this process during
virus infection.
ACKNOWLEDGMENTS
We thank Kurt Gustin for critical reading of the manuscript and for
insightful discussions. We are grateful to Natalie Farny, Andrew Sim-
monds, Pamela Silver, and Elisa Izaurralde for providing SG and PB
expression vectors. We also thank Paul Lasko for providing the eIF4E
antibody and Jerry Pelletier for pateamine A. A special thanks goes to
the laboratory of Doug Allan for assistance on the confocal micro-
scope.
This study was supported by a Canadian Institute of Health Re-
search Grant (CIHR) (MOP-81244). E.J. is supported by career de-
velopment awards by Michael Smith Foundation for Health Research
and CIHR.
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