Virus Research 210 (2015) 54–61
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Virus Research
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Nonstructural protein Pns12 of rice dwarf virus is a principal
regulator for viral replication and infection in its insect vector
∗ ∗
Qian Chen, Hongyan Chen, Dongsheng Jia, Qianzhuo Mao, Lianhui Xei , Taiyun Wei
Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, PR China
a r t i c l e i n f o a b s t r a c t
Article history: Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms.
Received 15 May 2015
The molecular mechanisms underling the formation of the viroplasm during infection of rice dwarf
Received in revised form 9 July 2015
virus (RDV), a plant reovirus, in its leafhopper vector cells remain poorly understood. Viral nonstruc-
Accepted 11 July 2015
tural protein Pns12 forms viroplasm-like inclusions in the absence of viral infection, suggesting that the
Available online 19 July 2015
viroplasm matrix is basically composed of Pns12. Here, we demonstrated that core capsid protein P3 and
nonstructural protein Pns11 were recruited in the viroplasm by direct interaction with Pns12, whereas
Keywords:
nonstructural protein Pns6 was recruited through interaction with Pns11. The introduction of dsRNA
Rice dwarf virus
from Pns12 gene into cultured insect vector cells or intact insect strongly inhibited such viroplasm for-
Nonstructural protein Pns12
Viroplasm mation, preventing efficient viral spread in the leafhopper in vitro and in vivo. Thus, nonstructural protein
Viral replication Pns12 of RDV is a principal regulator for viral replication and infection in its insect vector.
RNA interference © 2015 Elsevier B.V. All rights reserved.
1. Introduction ons (Zhong et al., 2003; Miyazaki et al., 2010; Wei et al., 2006a,b).
The precise timing and molecular mechanisms of these processes
Plant viruses in the Reoviridae family (Plant reoviruses) are largely unknown. However, the development of the cultured
comprise the genera Phytoreovirus, Fijivirus and Oryzavirus and insect vector cell in monolayers (VCMs) derived from the rice green
are transmitted by cicadellid leafhoppers or planthoppers in leafhopper (N. cincticeps) is starting to shed light on investigating
a persistent-propagative manner (Attoui et al., 2012). Plant RDV replication cycle in depth (Miyazaki et al., 2010; Wei et al.,
reoviruses are icosahedral, double-layered particles with 10 or 12 2006a,b, 2007; Zhou et al., 2007; Chen et al., 2011, 2012).
double-stranded RNA (dsRNA) segments (Attoui et al., 2012). Rice In our previous work using VCMs, we have demonstrated that
dwarf virus (RDV), a phytoreovirus in the family Reoviridae, is core particles assembly takes place exclusively within the dense
transmitted mainly by the leafhopper species Nephotettix cincti- globular inclusions termed viroplasms in the cytoplasm of insect
ceps in a persistent-propagative manner (Zhong et al., 2003). The vector cells, while intact double-layered RDV virions are assembled
genome of RDV encodes at least seven structural proteins (P1, P2, at the periphery of the viroplasm (Wei et al., 2006b). Viroplasm
P3, P5, P7, P8 and P9) and five nonstructural proteins (Pns4, Pns6, formation is orchestrated by complex networks of interactions
Pns10, Pns11 and Pns12) (Omura and Yan, 1999; Nakagawa et al., involving nonstructural proteins and structural proteins. The non-
2003). Replication of the RDV genome begins with the assortment structural proteins Pns6, Pns11 and Pns12 are all present within the
and packaging of the 12 viral mRNA and is followed by minus- viroplasm matrix induced by RDV infection, but only Pns12 forms
strand synthesis to produce new dsRNA genome segments (Zhong viroplasm-like inclusions when expressed in non-host insect Sf9
et al., 2003; Miyazaki et al., 2010; Wei et al., 2006a,b). In a cou- cells, suggesting that Pns12 is a minimal viral factor required for
pled process, core proteins P1, P3, P5 and P7 are first assembled to viroplasm formation (Wei et al., 2006b). Currently, the molecular
form core particles for the transcription of the bulk of viral mRNAs mechanisms that govern the genesis and maturation of viroplasm,
(Zhong et al., 2003; Miyazaki et al., 2010; Wei et al., 2006a,b). the intermolecular interactions among viroplasm components, and
Subsequently, progeny core particles are thought to be coated by the specific roles that viroplasms play in viral persistent infection
outer capsid proteins P2, P8 and P9 to generate mature RDV viri- in its insect vector all remain poorly understood.
In this work, we provided the evidence that Pns12 of RDV could
directly bind to Pns11 and core capsid protein P3, whereas Pns11
could directly bind to Pns6, indicating that Pns12 may act as a scaf-
∗
Corresponding authors. Fax: +86 591 83789439. fold for the recruitment of other viral proteins into viroplasm. To
E-mail addresses: [email protected] (L. Xei), [email protected] (T. Wei).
http://dx.doi.org/10.1016/j.virusres.2015.07.012
0168-1702/© 2015 Elsevier B.V. All rights reserved.
Q. Chen et al. / Virus Research 210 (2015) 54–61 55
further investigate the functional roles of viroplasm in the infection as templates for the synthesis of dsRNAs. A T7 RiboMAX Express
cycle of RDV in insect vector, we used RNA interference (RNAi), RNA interference System kit (Promega) was used to synthesize
a conserved sequence-specific gene silencing mechanism that is dsRNAs in vitro for Pns12 or GFP genes according to the manufac-
induced by dsRNAs (Fire et al., 1998), to knock down the expres- turer’s instructions. VCMs were transfected with 0.5 g/l dsRNAs
sion of Pns12 gene in vivo and in vitro. We determined that the in the presence of Cellfectin II reagent (Invitrogen) for 8 h. At
assembly of the viroplasm by viral nonstructural protein Pns12 is 72 h after the treatment of dsRNAs, total RNA from transfected
essential for persistent infection of RDV in its insect vector. cells was extracted with TRIzol Reagent (Invitrogen). DIG-labeled
negative-sense RNA transcripts of Pns12 or GFP genes were gener-
2. Materials and methods ated in vitro with T7 RNA polymerase using a DIG RNA Labeling
kit (Roche). Northern blots were produced using a DIG North-
2.1. Cells, viruses and antibodies ern starter kit (Roche), as described previously (Chen et al.,
2012).
VCMs, maintained in LBM growth medium, were originally For examining the effects of synthesized dsRNAs on viral infec-
developed from embryonic fragments of N. cincticeps collected from tion, after a 8-h treatment with dsRNAs, VCMs were inoculated
Fujian Province, China (Kimura and Omura, 1988). The rice sam- with RDV at a multiplicity of infection (MOI) of 10 in a solu-
ples of RDV were initially collected from Yunnan Province, China, tion of 0.1 M histidine that contained 0.01 M MgCl2 (pH 6.2;
and propagated for several generations via transmission by N. cinc- His-Mg) for 2 h as described previously (Chen et al., 2011, 2012).
◦
ticeps at 25 ± 3 C in laboratory. RDV virions were purified from At 48 h post-inoculation (hpi), VCMs were fixed, immunolabeled
infected rice plants without the usage of CCl4 as described by Zhong with virus-specific IgG conjugated to FITC (virus-FITC) and Pns12-
et al. (2003). Rabbit polyclonal antisera against the Pns6, Pns11, specific IgG conjugated to rhodamine (Pns12-rhodamine) and then
Pns12 and virus were prepared as described previously (Wei et al., processed for immunofluorescence microscopy as described previ-
2006b). IgGs purified from specific polyclonal antisera were con- ously (Chen et al., 2011, 2012). Furthermore, at 48 hpi, total proteins
jugated directly to fluorescein isothiocyanate (FITC) or rhodamine were extracted from infected cells and analyzed by a Western blot
according to the manufacturer’s instructions (Invitrogen). assay using Pns12- and P8-specific IgGs, respectively. Insect actin
was detected with actin-specific antibodies as a control to confirm
2.2. Yeast two-hybrid assay loading of equal amounts of proteins in each lane.
Yeast two-hybrid screening was performed using a Matchmake
2.5. Effect of synthesized dsRNAs from the Pns12 gene of RDV on
Gold Yeast two-hybrid system (Clontech) according to the manu-
viral infection in the leafhopper
facturer’s protocol. Full-length cDNA of RDV P3, Pns6 and Pns12
amplified by PCR were cloned in pGBKT7 and pGADT7 vector as
The dsRNAs were microinjected into leafhoppers using the
bait and prey plasmids, respectively. Each of these constructs was
method reported for the brown planthopper (Liu et al., 2010).
transformed into yeast strain AH109 to confirm that the baits
Briefly, 30 second-instar nymphs of the leafhopper were kept on
and preys were not toxic or self-activating. For protein interaction
RDV-infected rice for 2 days and then microinjected with 64 nl dsR-
test, yeast strain AH109 was co-transformed with the bait and the
NAs (0.5 g/l) into the thorax using a Nanoject II Auto-Nanoliter
prey pairs. The transformants were cultured on SD double-dropout
Injector (Spring). The microinjected leafhoppers were placed on
(DDO) medium (SD/-Leu /-Trp), SD triple-dropout (TDO) medium
healthy rice seedlings. At different days post-first access to diseased
(SD/-His/-Leu/-Trp) and SD quadruple-dropout (QDO) medium
plants (padp), the intestines and the salivary glands of leafhoppers
(SD/-Ade/-His/-Leu/-Trp) to show specific interactions. Then the
were dissected, fixed and immunolabeled with virus-FITC, Pns12-
positive ones were picked and streaked on QDO/X plates (contain-
rhodamine and actin dye phalloidin-Alexa Fluor 647 carboxylicacid
ing 20 g mL-1 X-␣-Gal) to measured ␣-Galactosidase activity. The
(Invitrogen), as described previously (Chen et al., 2011, 2012). At
pGBKT7-53/ pGADT7-T interaction was used as a positive control,
15 days padp, the accumulation of Pns12 and P8 of RDV was ana-
while the vectors pGBKT7-Lam/pGADT7-T were used as a negative
lyzed with a Western blot assay using Pns12- and P8-specific IgGs, control.
respectively.
2.3. GST pull-down assay
3. Results
Vector of pGEX-3X was used to construct plasmids express-
ing GST fusion proteins of P3, Pns11 and Pns12 as probe proteins, 3.1. Interaction among nonstructural proteins Pns6, Pns11 and
and pDEST17 vector was used to construct plasmids expressing Pns12 and core capsid protein P3 of RDV
His fusion proteins of Pns6, Pns11 and Pns12 as target proteins.
The constructs were transformed into Escherichia coli BL21 (DE3) Our previous data demonstrated that the nonstructural pro-
pLysS (Invitrogen) and expressed GST and His fusion proteins after teins Pns6, Pns11 and Pns12 are the constituents of viroplasm
induction with IPTG. GST fusion proteins were immobilized on glu- matrix in virus-infected insect vector cells (Wei et al., 2006b). Fur-
tathione sepharose beads (glutathione-sepharose 4 Fast Flow, GE) thermore, Pns12 has the ability of self-association to form the
and then incubated with lysates from E. coli BL21 expressing His viroplasm-like inclusions in the absence of viral infection (Wei
fusion proteins with gentle shaking. Thereafter, beads were washed et al., 2006b). To explore how Pns6 and Pns11 are recruited in
6 times with washing buffer and proteins were eluted by boiling in the viroplasm, the yeast two-hybrid system was used to determine
loading buffer. Purified proteins and GST pull-down products were whether there were interactions among Pns6, Pns11 and Pns12. Our
then analyzed by Western blotting. results showed that Pns12 interacted with Pns11, but not with Pns6
(Fig. 1A). Pns6 was also found to bind to Pns11 (Fig. 1A). To confirm
2.4. Effect of synthesized dsRNAs from Pns12 gene of RDV on viral these interactions, we fused Pns6, Pns11 and Pns12 with a His or
infection in the continuous cell cultures derived from leafhopper GST and assayed the interaction using GST pull-down methodol-
ogy, as described previously (Kong et al., 2014; Huo et al., 2014).
A 600-bp segment of the RDV Pns12 gene (bases 141–740) As shown in Fig. 1B, positive interactions were confirmed between
and a 585-bp segment of the GFP gene were amplified by PCR Pns6 and Pns11 and between Pns11 and Pns12, but not between
56 Q. Chen et al. / Virus Research 210 (2015) 54–61
Fig. 1. Protein interactions between Pns6, Pns11, Pns12 and P3 of RDV. (A) Yeast two-hybrid assay of protein–protein interactions. Transformants on plate of SD-Trp-Leu-
His-Ade medium were shown. (+) Positive control, i.e., pGBKT7-53/ pGADT7-T; (–) negative control, i.e., pGBKT7-Lam/pGADT7-T; Pns6+Pns12, pGBKT7-Pns6/pGADT7-Pns12;
Pns11+Pns12, pGADT7-Pns11/pGBKT7-Pns12; Pns6+Pns11, pGBKT7-Pns6/pGADT7-Pns11; P3+Pns6, pGBKT7-P3/pGADT7-Pns6; P3+Pns11, pGBKT7-P3/pGADT7-Pns11;
P3+Pns12, pGBKT7-P3/pGADT7-Pns12. (B) GST pull-down assay of protein–protein interactions. Pns6, Pns11 or Pns12 of RDV was fused with His to act as bait proteins;
GFP was fused with His as a control. Pns6, Pns12 or P3 of RDV was fused with GST as prey proteins. GST pull-down products were analyzed by immunoblotting with
antibodies against GST to detect prey proteins and with antibodies against His to detect bound proteins.
Pns6 and Pns12. These data suggested that Pns11 was recruited 3.2. Transfection of dsRNAs from the Pns12 gene inhibited the
in the viroplasm by direct interaction with Pns12, whereas Pns6 assembly of viroplasm and viral infection in insect vector cells
was recruited through interaction with Pns11. Thus, Pns6, Pns11
and Pns12 of RDV could bind to each other to form the viroplasm Since we have shown the central position of Pns12 in the pro-
matrix. tein interaction network during viroplasm formation (Fig. 1) and
Our observations show that core particles are assembled within its ability to self-associate (Wei et al., 2006b), we then explored its
the interior regions of viroplasm induced by RDV infection in insect functional role in viroplasm formation in RDV infected insect vec-
vector cells (Wei et al., 2006b). To explore how the core capsid pro- tor cells using dsRNAs specific for Pns12 and GFP genes (dsPns12
tein P3 is recruited in the viroplasm, the yeast two-hybrid and GST and dsGFP)-mediated RNAi. RNAi is induced by dsRNAs and man-
pull-down assays were used to determine whether proteins Pns6, ifested by the appearance of small interfering RNAs (siRNAs) that
Pns11 or Pns12 could specifically bind to P3. Both protein interac- correspond to the mRNA target sequence (Chen et al., 2011, 2012).
tion methods revealed that P3 specifically interacted with Pns12, Small RNAs approximately 21 nt long were detected from RNAs
but not with Pns6 or Pns11 (Fig. 1). Our results clearly showed that extracted from dsRNA-transfected cells at 72 h after transfection
P3 is recruited and retained in the viroplasm by direct interaction with the synthesized dsRNAs (Fig. 2A). These results showed that
with Pns12. Thus, Pns12 could directly bind to core particles within RNAi was triggered by dsRNAs in VCMs.
the viroplasm. Eight hour after transfection of dsPns12 or dsGFP, we inoculated
VCMs with RDV at a MOI of 10. At 48 hpi, VCMs were fixed, immuno-
Q. Chen et al. / Virus Research 210 (2015) 54–61 57
Fig. 2. The nonstructural protein Pns12 of RDV were essential for viral replication or assembly in VCMs. (A) Detection of small RNAs in VCMs transfected with dsRNAs. Total
RNA was probed with DIG-labeled riboprobes corresponding to negative-sense RNA of the genes of Pns12 or GFP. The 5.8S rRNA was used as a control. (B) Eight hours after
transfection with dsGFP or dsPns12, VCMs were infected with RDV. At 48 hpi, VCMs were immunolabeled with virus-FITC (green) and Pns12-rhodamine (red), and then
examined with confocal microscopy. (C) Inhibition of viral protein expression in VCMs transfected with dsRNAs. Proteins were analyzed by immunoblotting with antibodies
against Pns12 or P8. An actin-specific antibody was used as the control. (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
labeled with virus-FITC and Pns12-rhodamine, and processed for (Fig. 2B). The accumulation of viral proteins in the cell lysates was
immunofluorescence, as described previously (Chen et al., 2011, then analyzed using Western blotting assay with antibodies against
2012). In VCMs transfected with dsGFP, viral infection was observed Pns12 or outer capsid protein P8. Our results showed that the treat-
in 100% of cells, and viroplasms containing Pns12 were abundant ment of dsPns12 significantly inhibited the expression of these viral
(Fig. 2B). However, in VCMs transfected with dsPns12, RDV infec- proteins (Fig. 2C). These results suggested that Pns12 of RDV was
tion was restricted to a limited number of the initially infected involved in viroplasm formation and viral infection. The mRNA of
cells, and the formation of viroplasms was almost entirely blocked genomic segment S12 of RDV
58 Q. Chen et al. / Virus Research 210 (2015) 54–61
Fig. 3. RNAi induced by dsPns12 inhibited the replication and spread of RDV in vivo. (A and B) Second-instar nymphs of leafhoppers were microinjected with dsGFP or dsPns9.
At 8 and 15 days padp (panels A and B, respectively), internal organs of leafhoppers were immunolabeled with virus-FITC (green), Pns12-rhodamine (red) and actin dye
phalloidin-Alexa Fluor 647 carboxylicacid (blue). The images were merged with green fluorescence (viral antigens), red fluorescence (Pns12 antigens), and blue fluorescence
(actin dye). Insets show green fluorescence (viral antigens) and red fluorescence (Pns12 antigens) of the merged images in the boxed areas in each panel. (C) Treatment with
dsPns12 reduced the expression of Pns12 and P8 proteins in leafhoppers as shown by Western blotting. fc, filter chamber; mg, midgut; amg, anterior midgut; pmg, posterior
midgut; sg, salivary glands. Bars, 100 m. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.3. Microinjection of dsRNAs from the Pns12 gene inhibits the of RDV in its leafhopper vector. Second-instar nymphs of leafhop-
spread of RDV from the initially infected site in the filter chamber pers were allowed a 2-day acquisition on virus-infected rice plants,
of its insect vector and then microinjected with dsGFP or dsPns12. At 8 days padp,
viral antigens and viroplasms containing Pns12 were seen in the
After ingested by the leafhopper vector, RDV establishes its pri- entire intestine in 60% of leafhoppers receiving dsGFP (Fig. 3A,
mary infection in the epithelial cells of the filter chamber, then Table 1). However, viral infection was restricted in the epithelial
infects other tissues in the insect (Chen et al., 2011, 2012; Ahlquist, regions of the filter chamber and only observed in 30% of leafhop-
2006). An RNAi strategy was used to investigate the functional role pers receiving dsPns12 (Fig. 3A, Table 1). At 15 days padp, viral
of nonstructural protein Pns12 during the early persistent infection antigens and viroplasms containing Pns12 had accumulated in the
Q. Chen et al. / Virus Research 210 (2015) 54–61 59
Table 1
Inhibition of viral infection and spread in the bodies of leafhoppers treated with dsPns12 via microinjection.
Treatments No. of Pns12- or viral antigens-positive insects detected by immunofluorescence at 8 and 15 days padp (n = 30)
padp (days) Limited regions of filter chamber Entire intestine Salivary glands
dsGFP 8 0 18 0
15 0 25 15
dsPns12 8 9 0 0
15 9 0 0
salivary glands in 50% of leafhoppers that received dsGFP (Fig. 3B, of dsPns12 should also specifically knock down the expression of
Table 1). As expected, the treatment of dsPns12 inhibited the spread Pns12OPa and Pns12OPb in insect vector. Whether these two small
of RDV in the salivary glands of its insect vector (Fig. 3B, Table 1). proteins are involved in the formation of viroplasm during viral
The decrease in the expression of Pns12 and P8 proteins of RDV infection in insect vector will be investigated in future.
in leafhoppers that received dsPns12 was confirmed using west- Despite the critical role of RDV Pns12 in mediating viroplasm
ern blot (Fig. 3C). The above results indicated that the treatment formation, the molecular mechanism underlying this process is still
of dsPns12 specifically knocked down the expression of Pns12 in unclear. A group of nonstructural proteins encoded by other mem-
the initially infected regions of the filter chamber, which led to the bers in the family Reoviridae may provide some hints. For example,
restriction of viral infection in the filter chamber and blocked viral NSP2 of reoviruses, NS2 of orbiviruses, NSP2/NSP5 of rotaviruses,
spread to the salivary glands. Pns9 of the phytoreovirus rice gall dwarf virus, P9-1 of the fijivirus
rice black streaked dwarf virus, and Pns10 of the oryzavirus rice
4. Discussion ragged stunt virus all possess similar functions as RDV Pns12 in
inducing viroplasm matrix during virus replication cycles (Jia et al.,
During RDV infection of the cultured leafhopper cells, first 2012; Mao et al., 2013; Chen et al., 2014; Fabbretti et al., 1999;
the mRNAs of RDV were transcribed from the viral core parti- Miller et al., 2010; Theron et al., 1996; Thomas et al., 1990; Broering
cles, then the nonstructural proteins Pns6, Pns11 and Pns12 were et al., 2004; Contin et al., 2010; Kar et al., 2007). Among them,
expressed by the host’s cellular translational machinery and asso- Rotavirus NSP2, RBSDV P9-1 and RGDV Pns9 can form octameric
ciated together to form the initial viroplasm matrix, which is the structures that can act as a scaffold for the recruitment of viral pro-
site for viral replication and assembly (Ahlquist, 2006; Akita et al., teins to initiate the viroplasm matrix (Akita et al., 2011, 2012; Jiang
2012; Wei et al., 2006b). In this study, we demonstrated that et al., 2006). The cryo-EM analysis of the native structure of the
knockdown of Pns12 expression by dsPns12 efficiently abolished Pns12, in solution, also forms octameric ring-like structures (Akita
viroplasm formation and prevented viral replication in cultured et al., unpublished data). Such an octameric structure of Pns12 may
leafhopper cells (Fig. 2). Moreover, microinjection of dsPns12 sup- be required to provide a suitable scaffold for the recruitment of
pressed viroplasm formation and restricted viral infection in the viral proteins to initiate the viroplasm matrix for viral replication
initially infected epithelium of the filter chamber, thus, inhibit- and assembly. Our finding of the interactions between Pns6, Pns11
ing the spread of RDV in the body of leafhopper vector (Fig. 3, and Pns12 (Fig. 1) supports this hypothesis and provides a potential
Table 1). These data were consistent with the previous observa- explanation on how Pns6 and Pns11 are recruited and retained in
tion that Pns12 alone was able to form inclusions that are similar the viraplasm matrix.
in morphology to viroplasm in the absence of viral infection (Wei How Pns6, Pns11 and Pns12 are involved in viral replication and
et al., 2006b), and suggested that the formation of the viroplasm assembly in virus-infected insect vector cells? The nonstructural
for progeny virions assembly was a prerequisite for the persistent protein Pns6 is a viral movement protein and Pns11 is a viral RNA-
infection of RDV in its insect vector. In our study, the segment of silencing suppressor in plant hosts (Li et al., 2004; Xu et al., 1998).
Pns12 gene for the synthesis of dsPns12 also contained two small Moreover, both Pns6 and Pns11 have RNA binding activities (Ji et al.,
out-of-phase open reading frames which encoded Pns12OPa and 2011; Li et al., 2004; Xu et al., 1998). Therefore, it is possible that
Pns12OPb, respectively (Suzuki et al., 1996). Thus, the treatment Pns6 or Pns11 acts to recruit or retain viral mRNAs within the viro-
Fig. 4. Proposed model for the assembly of RDV viroplasm in infected cells of its insect vector. Pns12 acts as a scaffold for the recruitment and retaining of core capsid protein
P3 and nonstructural proteins Pns6 and Pns11. RDV RNA binds with Pns6 or Pns11 to form replication and assembly complexes for the production of progeny core particles
within the viroplasm. Pns12 directly binds to core particles. Intact viral particles are assembled at the periphery of the viroplasm.
60 Q. Chen et al. / Virus Research 210 (2015) 54–61
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