JGV Papers in Press. Published March 6, 2013 as doi:10.1099/vir.0.050104-0
1 Short communications 2 3 An insect cell line derived from the small brown planthopper supports replication of 4 Rice stripe virus, a tenuivirus 5
6 Yuanyuan Ma, Wei Wu, Hongyan Chen, Qifei Liu, Dongsheng Jia, Qianzhuo Mao, 7 Qian Chen, Zujian Wu and Taiyun Wei* 8 9 Fujian Province Key Laboratory of Plant Virology, Institute of Plant Virology, Fujian 10 Agriculture and Forestry University, Fuzhou, Fujian 350002, PR China 11 12 Running title: Replication of a tenuivirus in a planthopper cell line 13 Keywords: tenuivirus, planthopper cell line, viral replication 14 15 16 The number of words in Abstract: 146 words 17 The number of words in text: 1999 words 18 The number of Figures: 3 19 20 21 22 23 24 25 26 Dr. Taiyun Wei, Telephone number: +86-591-83789270; Fax number: 27 +86-591-83789439; 28 E-mail address: [email protected] 29 30
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1 1 Summary
2 A cell line of the small brown planthopper (SBPH; Laodelphax striatellus) was
3 established to study replication of Rice stripe virus (RSV), a tenuivirus. The SBPH cell
4 line, which had been subcultured through 30 passages, formed monolayers of
5 epithelial-like cells. Inoculation of cultured vector cells with RSV resulted in a
6 persistent infection. During viral infection in the SBPH cell line, the viral nonstructural
7 protein NS3 colocalized with the filamentous ribonucleoprotein particles of RSV, as
8 revealed by electron and confocal microscopy. The knockdown of NS3 expression
9 due to RNA interference induced by synthesized double-stranded RNAs from the NS3
10 gene significantly inhibited viral infection in the SBPH cell line. These results
11 demonstrated that NS3 of RSV might be involved in viral replication or assembly. The
12 persistent infection of the SBPH cell line by RSV will enable a better understanding of
13 the complex relationship between RSV and its insect vector.
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2 1 Plant viruses in the genera Tospovirus, Tenuivirus, Rhabdovirus, Phytoreovirus
2 and Fijivirus are transmitted by their respective insect vectors in a persistent
3 propagative manner (Ammar et al., 2009; Hogenhout et al., 2008). Insights into
4 replication of persistent propagative viruses in their insect vectors will help in
5 developing new strategies to control the transmission of the viruses by the vectors.
6 Continuous cell cultures of insect vectors are strong tools to study the replication cycle
7 for persistent propagative plant viruses in their vector cells because they are more
8 uniform than primary cultures and thus provide more consistent data (Creamer, 1993).
9 Continuous cultures of leafhoppers provide a good system to study the replication
10 cycle of phytoreoviruses in their insect vectors (Kimura & Omura, 1988; Omura &
11 Kimura, 1994; Wei et al., 2006, 2007).
12 Rice stripe virus (RSV), the type species of the genus Tenuivirus, has
13 filamentous ribonucleoprotein particles (RNPs) that contain nucleoprotein,
14 RNA-dependent RNA polymerase and four negative-sense single-stranded RNA
15 segments that encode seven proteins (Toriyama, 1986; Falk & Tsai, 1998; Wei et al.,
16 2009; Xiao et al., 2010). RSV is transmitted by the small brown planthopper (SBPH;
17 Laodelphax striatellus Fallén) in a persistent propagative manner (Toriyama, 1986;
18 Wei et al., 2009). Currently, the mechanism that enables efficient multiplication of
19 RSV in its vector is unknown due to the lack of SBPH cell lines. In previous attempts
20 to establish primary cell cultures, only a small number of cells migrated occasionally
21 from the embryonic explants dissected from SBPH eggs, and eventually the
22 subculture died off (Mitsuhashi, 1969, 2001; Yamada et al., 1970). The present report
23 describes the first successful establishment of continuous cultures of SBPHs for the
24 study of RSV replication.
25 The SBPH cell line was established by adapting the protocol described by
3 1 Kimura and Omura (1988). Nonviruliferous SBPHs eggs that had been oviposited 8
2 days earlier in leaf sheaths of rice plants were surface-sterilized with 70% ethanol and
3 then rinsed in sterile Tyrode's solution. Embryonic fragments were dissected from the
4 eggs in Tyrode's solution, treated with 0.25% trypsin in Tyrode′s solution for 15 min
5 and then incubated with Kimura’s insect medium at 25°C. The medium was changed
6 every 7-10 days. New cells started to migrate from the SBPH embryonic explants
7 within 48 h after the cultures were set up. Figure 1A showed the epithelial-like cells
8 that migrated from the embryonic explants to form monolayers by 12 days after
9 preparation. When the cells grew almost confluently in the dish about 1 month after
10 initiation, they were transferred to new dishes and again cultured to confluence (about
11 2 wk after subculture). Such cells were then transferred to culture flasks for further
12 subculturing. After 30 passages of subculturing at 10-day intervals, the dominant cell
13 type in the established SBPH cell line was epithelial-like, approximately 25-50 μm in
14 diameter, with dense cytoplasm around the nuclei, which were approximately 7.5-20
15 μm in diameter (Fig. 1B). This is the first report of the establishment of a continuous
16 planthopper cell line.
17 Such SBPH vector cell monolayers (VCMs) were used for RSV infection. RSV
18 inoculum was prepared from infected plants, essentially as described previously
19 (Kimura & Omura, 1988; Omura & Kimura, 1994). Briefly, infected leaves were
20 surface-sterilized with 70% ethanol and then rinsed sterile distilled water. The
21 sterilized leaves were ground in a solution of 0.1 M histidine that contained 0.01 M
22 MgCl2, pH 6.2 (His-Mg) and centrifuged. The supernatant was then used as the
23 inoculum. VCMs were washed twice with His-Mg and then inoculated with RSV. After
24 2 h, VCMs were washed twice with His-Mg and covered with Kimura’s insect medium.
25 VCMs were cultured to confluence in the culture flasks and then subcultured at
4 1 intervals of 10 days through 10 passages. We then analyzed the accumulation of RNP
2 and nonstructural protein NS3 of RSV in virus-infected VCMs by SDS-polyacrylamide
3 gel electrophoresis (SDS-PAGE) and immunoblotting with RNP- and NS3-specific
4 antibodies, respectively, as reported previously (Zhang et al., 2008; Takahashi et al.,
5 1991). Our results showed that RNP and NS3 of RSV accumulated in virus-infected
6 VCMs even after 10 passages (Fig. 2A, B).
7 To further observe the subcellular localization of RNP and NS3, virus-infected
8 VCMs on coverslips with 4% paraformaldehyde, probed with RNP-specific antibodies
9 that had been conjugated to FITC (Invitrogen; RNP-FITC Abs) and NS3-specific
10 antibodies that had been conjugated to rhodamine (Invitrogen; NS3-rhodamine Abs)
11 and then examined by confocal microscopy, as described previously (Wei et al., 2006,
12 2007). No specific fluorescence was detected in uninfected cells after incubation with
13 either RNP-FITC or NS3-rhodamine Abs (Fig. 2C, D). In RSV-infected VCMs, RNP
14 and NS3 were detected as discrete punctate inclusions throughout the cytoplasm (Fig.
15 2C, D). When the images for RNP and for NS3 were merged, RNP and NS3
16 colocalized with the viral inclusions (Fig. 2D), indicating that NS3 was associated with
17 RSV RNPs during viral infection in VCMs.
18 To confirm our observations, we examined infected VCMs on coverslips using
19 immunoelectron microscopy. VCMs were fixed, dehydrated and embedded as
20 described previously (Jia et al., 2012; Wei et al., 2006). Cell sections were then
21 incubated with RNP- and NS3-specific antibodies, respectively, as primary antibodies,
22 followed by treatment with goat anti-rabbit IgG as secondary antibodies that had been
23 conjugated to 15-nm gold particles, as described previously (Jia et al., 2012; Wei et al.,
24 2006). Our results showed that both RNP and NS3 antibodies specifically reacted with
25 the amorphous inclusion bodies in the cytoplasm of virus-infected VCMs (Fig. 2E, F),
5 1 indicating that RNP and NS3 had the same subcellular distribution, as indicated by
2 confocal microscopy (Fig. 2D). Our results confirmed previous data that RNP
3 antibodies of RSV are specific for the amorphous inclusion bodies in the bodies of
4 viruliferous SBPHs (Suzuki et al., 1992). Because RNPs are the infectious unit of RSV
5 (Toriyama, 1986), the association of NS3 with RNPs suggested that NS3 may play a
6 crucial role in viral replication or assembly in cultured vector cells. Furthermore, our
7 observations using transmission electron and confocal microscopy showed that RSV
8 infection of the SBPH cell line did not affect cellular structures and morphology (Fig. 2),
9 suggesting that the SBPH cell line supported a noncytopathic, persistent infection of
10 RSV. Evidently, mechanisms have evolved in the vector cells to allow efficient
11 replication of RSV without any significant pathology.
12 To confirm whether NS3 plays a crucial role in viral replication or assembly, we
13 used an RNA interference (RNAi) strategy to knock down the expression of NS3 in
14 virus-infected VCMs and then analyzed its effect on the formation of viral inclusions
15 and viral infection. The PCR products of a 633-bp segment of RSV NS3 gene (Xiong
16 et al., 2009) and a 717-bp segment of green fluorescence protein (GFP)-encoding
17 gene as controls were used for dsRNA synthesis according to the protocol for the T7
18 RiboMAX Express RNAi System kit (Promega). VCMs were transfected with 0.5 μg/μl
19 dsRNAs in the presence of Cellfectin (Invitrogen) for 24 h. VCMs were then inoculated
20 with RSV, fixed with 4% paraformaldehyde 5 days later and immunostained with
21 RNP-FITC and NS3-rhodamine Abs. Immunofluorescence showed that the treatment
22 of dsRNAs from the GFP gene (dsGFP) did not prevent viral infection or the formation
23 of viral inclusions containing RNPs and NS3 (Fig. 3A). In contrast, the treatment with
24 dsRNAs from the NS3 gene (dsNS3) significantly inhibited viral infection and the
25 accumulation of viral inclusions containing RNPs and NS3 (Fig. 3B). The effects of
6 1 dsNS3 on the accumulation of RNPs and NS3 of RSV were further analyzed by
2 SDS-PAGE and immunoblotting with RNP- and NS3-specific antibodies, respectively,
3 as reported previously (Zhang et al., 2008; Takahashi et al., 1991). As shown in
4 Figure 3C, transfection with dsNS3 resulted in a marked reduction of the
5 accumulation of RNPs and NS3 in viral-infected VCMs. Because RNAi induced by
6 dsRNA is a sequence-specific gene silencing mechanism (Fire et al., 1998; Chen et
7 al., 2012; Jia et al., 2012) and the NS3 gene has no sequence homology with other
8 RSV genes (data not shown), we determined that the knockdown of NS3 expression
9 in VCMs was specifically caused by RNAi induced by dsNS3. Thus, our results
10 indicated that the knockdown of NS3 expression led to the inhibition of the formation
11 of viral inclusions containing NS3, with a significant simultaneous inhibition of the
12 accumulation of RNPs in the VCMs. Due to the association of NS3 with RNPs, these
13 results indicate that NS3 of RSV is essential for RSV infection in its insect vector and
14 may be directly involved in viral replication or assembly. Since RSV NS3 was
15 demonstrated to act as a suppressor of gene silencing in plant host (Xiong et al.,
16 2009), it may have a similar function in insect vector cells.
17 In conclusion, the establishment of a SBPH cell line that supports a
18 noncytopathic, persistent infection of RSV opens new opportunities to elucidate the
19 complex relationship between RSV and its insect vector.
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21 Acknowledgments
22 The authors are indebted to Yijun Zhou (Jiangsu Academy of Agricultural
23 Sciences, China) for nonviruliferous populations of Laodelphax striatellus and to
24 Toshihiro Omura (National Agricultural Research Center, Japan) for antibodies
25 against purified RNPs of RSV. This work was supported by grants from the programs
7 1 for the National Natural Science Foundation of China (No. 31130044, No. 31171821
2 and No. 31070130), the Natural Science Foundation of Fujian Province ((No.
3 2010J01075 and No. 2011J06008), the Doctoral Fund of Ministry of Education of
4 China (No. 20113515130002) and the New Century Excellent Talent in University (No.
5 NCET-09-0011).
6
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16 Figure Legends:
17 Fig. 1. Light micrographs of primary and continuous cultures of SBPH cells in vitro. (A)
18 Epithelial-like cells that migrated from the embryonic explants (Ex) of SBPH to form a
19 monolayer by 12 days after preparation. (B) Monolayer culture of SBPH cell line after
20 30 passages showing the predominantly epithelial-like cell population. Bars, 20 μm.
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22 Fig. 2. The association of viral nonstructural protein NS3 with RNPs of RSV in
23 virus-infected VCMs. (A, B) Western blot analyses for RNP and NS3 of RSV. Protein
24 extracts were separated by SDS-PAGE and detected with RNP-specific (A) or
25 NS3-specific (B) antibodies, respectively. Lanes M, protein marker; lanes 1 and 5,
10 1 extracts from rice plants infected with RSV; lanes 2 and 6, extracts from viruliferous
2 SBPHs; lanes 3 and 7, extracts from VCMs infected with RSV; lanes 4 and 8, extracts
3 from uninfected VCMs. (C, D) Confocal micrographs of VCMs which were treated with
4 RNP-FITC Abs (green) or with NS3-rhodamine Abs (red). (C) Virus-infected (panel I)
5 and uninfected (panel II) VCMs stained for RNP. The images were merged under the
6 background of transmitted light. Bars, 20 μm. (D) Images with green fluorescence
7 (RNP ABs, panel I), red fluorescence (NS3 ABs, panel II) and the merged green and
8 red fluorescence (merged, panel III) from virus-infected VCMs. No specific
9 fluorescence was detected in uninfected VCMs after incubation with NS3-rhodamine
10 Abs (panel IV). Bars, 5 μm. (E, F) Immunogold labeling of RNP (E) and NS3 (F) in the
11 amorphous inclusion bodies in the cytoplasm of virus-infected VCMs. Bars, 100 nm.
12 Cells were immunolabeled for RNP and NS3 with RNP- and NS3-specific Abs in (E)
13 and (F), respectively, as primary antibodies, followed by treatment with goat
14 anti-rabbit IgG conjugated with 15 nm gold particles as secondary antibodies.
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16 Fig. 3. RNAi induced by dsNS3 inhibited the formation of viral inclusions and RSV
17 infection of VCMs. (A, B) RSV infection was strongly inhibited by RNAi induced by
18 dsNS3. Twenty-four hours after transfection with dsGFP (A) or dsNS3 (B), the VCMs
19 were inoculated with RSV. Five days after viral inoculation, VCMs were stained with
20 RNP-FITC Abs (green) or NS3-rhodamine Abs (red) and examined with confocal
21 microscopy. Images are representative of the results of multiple experiments with
22 multiple preparations. Bars, 5 μm. (C) RNAi induced by dsNS3 reduced the
23 accumulation of RNPs and NS3 of RSV in virus-infected VCMs as shown in Western
24 blot analyses. Protein extracts from cells transfected with dsGFP or dsNS3 were
25 separated by SDS-PAGE and detected with NS3-specific or RNP-specific antibodies,
11 1 respectively. Lower panel: detection of insect actin with actin-specific antibodies
2 (Sigma) as a control to confirm loading of equal amounts of proteins in each lane.
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