1 Kamiura Station, Japan Fisheries Research Agency, Oita 879-2602, Japan
2 Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
3 Graduate School of Biosphere Science, Hiroshima University, Higashihiroshima, 739-8528, Japan
Correspondence
Kazuyuki Mise
kmise{at}kais.kyoto-u.ac.jp
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ABSTRACT |
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Present address: University of Alabama at Birmingham, Birmingham, AL 35294, USA.
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INTRODUCTION |
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In Flock house virus (FHV), the most extensively studied virus among the alphanodaviruses, which naturally infect insects, a single subgenomic RNA3 is synthesized from RNA1 during RNA replication. This subgenomic RNA3 encodes two non-structural proteins, B1 and B2 (Friesen & Rueckert, 1982; Guarino et al., 1984
). The crucial roles of FHV proteins B1 and B2 in virus multiplication were originally unclear, since both proteins could be eliminated without inhibiting viral RNA replication in some mammalian cells (Ball, 1995
) and in yeast cells (Price et al., 2000
). Interestingly, however, a recent report demonstrated that FHV protein B2 inhibits RNA silencing in cultured Drosophila cells and in transgenic plants (Li et al., 2002
). More recently, Li et al. (2004)
demonstrated that protein B2 of Nodamura virus (NoV), the type species of the genus Alphanodavirus, also suppresses RNA silencing in cultured Drosophila and Anopheles cells. For betanodaviruses, there have been preliminary reports on subgenomic RNA3 and its encoded protein (protein B) for Dicentrarchus labrax encephalitis virus (Delsert et al., 1997
) and SJNNV (Nagai & Nishizawa, 1999
). RNA3 of Atlantic halibut nodavirus (AHNV) was recently identified in infected fish cells (Sommerset & Nerland, 2004
). Some previous reports for betanodaviruses have designated the RNA3-encoded protein as protein B because RNA3s of betanodaviruses have only one open reading frame (ORF), whereas those of most alphanodaviruses contain two ORFs. However, to avoid future confusion in the literature, protein B2 in this report is used for SJNNV protein B, following Johnson et al. (2001)
. We previously reported that a subgenomic 0·4 kb RNA was observed in SJNNV-infected fish cells. This RNA hybridized with both positive and negative strand-specific riboprobes for the 3'-proximal region of SJNNV RNA1, but not for that of RNA2 (Iwamoto et al., 2001
). These results prompted an investigation into the production of SJNNV RNA3 and protein B2 during viral multiplication, as described for alphanodaviruses. In this study, we have determined the primary structure of SJNNV RNA3 and detected its encoded protein B2 in infected fish cells using an antiserum raised against recombinant protein B2. Moreover, we have shown that protein B2 can suppress RNA silencing in plants, using an Agrobacterium-mediated transient system.
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METHODS |
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Determination of the 5' end of the SJNNV subgenomic RNA.
E-11 cells were inoculated with SJNNV and cultured at 25 °C for 16 h. Total cellular RNA was extracted from the infected cells using Isogen (Nippon Gene) according to the manufacturer's instructions, and then separated by electrophoresis on 2 % agarose gels (NuSieve 3 : 1 agarose; Cambrex), and stained with ethidium bromide. The subgenomic RNA (ca. 0·4 kb) was isolated from the gels using Gene-Capsule (Geno Technology) according to the manufacturer's instructions, and was used as a template for the rapid amplification of cDNA ends (RACE) method (Frohman et al., 1988) to determine the 5'-terminal sequences. Prior to 5' RACE, the recovered RNA was treated with tobacco acid pyrophosphatase (TAP) (Nippon Gene), according to the supplier's instructions, to remove the 5' cap structure or left untreated. 5' RACE was carried out as previously described (Iwamoto et al., 2001
) by using the synthetic oligonucleotide primers SJ1-3Ec, ANCH, SJ1R11 and AUAP (Table 1
).
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Preparation of His-tagged protein B2 (SJpB2) and anti-SJpB2 antiserum.
A putative SJNNV ORF B2 was amplified by PCR by using pSJ1TK19 as a template and with forward primer SJ3B2-Nde and reverse primer SJ3B2-Bam (Table 1), which contain NdeI and BamHI recognition sites, respectively, to facilitate cloning. Amplified products were digested with NdeI and BamHI and the ORF B2 fragment was isolated from a 2 % agarose gel. This fragment was cloned into the NdeI and BamHI sites of the pET16b vector (Novagen) according to standard protocols (Sambrook & Russell, 2001
), to obtain pETSJpB2. pETSJpB2 was transformed into the Escherichia coli strain BL21(DE3) (Novagen). Recombinant protein B2 (SJpB2) was expressed as an N-terminal-polyhistidine-tagged protein and was purified using Ni-NTA resin (Qiagen) according to the manufacturer's procedure. Anti-SJpB2 antiserum was raised in a New Zealand White rabbit, immunized with a mixture of the SJpB2 and complete Freund's adjuvant (Difco) as previously described (Nguyen et al., 1996
).
Inoculation of E-11 cells and host fish.
E-11 cells grown in eight-chamber slides (Nunc) and 12-well plates (Iwaki) were inoculated with wild-type SJNNV virions. After incubation for 24 h at 25 °C, the cells in eight-chamber slides were fixed with methanol and those in the 12-well plates were suspended in 50 µl Laemmli sample buffer (Laemmli, 1970). SJNNV-free striped jack larvae hatched at the Japan Fisheries Research Agency (formerly the Japan Sea-farming Association) were inoculated with wild-type SJNNV virions as previously described (Iwamoto et al., 2001
). At 3 days post-inoculation, 50 mg moribund fish (fresh weight) was collected and suspended in 50 µl Laemmli sample buffer. A portion of the inoculated fish was fixed with 10 % formalin, embedded in paraffin and sectioned.
Immunological detection of SJNNV protein B2.
Immunofluorescence staining of fixed samples was performed as previously described (Iwamoto et al., 1999). Five microlitre samples from the E-11 cells and striped jack larvae were resolved by Tricine-SDS-PAGE. The gels were essentially prepared according to the method of Schägger & von Jagow (1987)
and consisted of a stacking gel [4 % acrylamide (acrylamide : bisacrylamide=29 : 1)], a spacer gel (10 % acrylamide) and a separating gel (16·5 % acrylamide). Western blot analysis was performed as described by Damayanti et al. (1999)
, except that electroblotting was done in Towbin buffer (10 mM Tris base, 96 mM glycine in 40 % methanol) and anti-SJpB2 antiserum was diluted 1000-fold for use as the primary antibody.
Assay for silencing-suppression activity.
All primers for the following PCR contained an appropriate restriction endonuclease recognition sequence for cloning (Table 1). PCR products containing a 5' leader (5'-AAGGAGATATAACA-3') and SJNNV ORF B2 were amplified from pSJ1TK19 and pSJ1
B2NC, digested with BamHI and EcoRI and cloned into the BamHI/EcoRI sites of pBICP35 (Mori et al., 1991
) to create pBICSJB2 and pBICSJ
B2NC, respectively. A PCR product containing the same 5' leader sequence and the SJNNV CP gene was amplified from pSJ2TK30, digested with BamHI (within the primer sequence and at nt 1062 of pSJ2TK30) and cloned into the BamHI site of pBICP35 to obtain pBICSJCP. The Agrobacterium-mediated transient assay, established in transgenic plants (Nicotiana benthamiana line 16c; Voinnet et al., 2000
) that express green fluorescent protein (GFP), GFP imaging under UV light and RNA analyses were carried out as previously described (Takeda et al., 2002
).
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RESULTS |
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We used an Agrobacterium strain carrying pBICGFP as an RNA silencing inducer, together with a second Agrobacterium strain bearing another plasmid that expresses one of several proteins to be tested. A mixture of Agrobacterium carrying pBICSJB2 and Agrobacterium carrying pBICGFP was co-infiltrated into line 16c leaves. As controls, Agrobacterium carrying either pBICP35 (empty vector) or pBICNSs was co-infiltrated with that carrying pBICGFP. pBICNSs contains the Tomato spotted wilt virus (TSWV) NSs protein, which has been shown to suppress RNA silencing in a similar assay (Takeda et al., 2002). For simplicity, each Agrobacterium strain will hereafter be referred to by the name of the plasmid it carries. The leaf patches receiving pBICGFP plus pBICP35 did not show strong green fluorescence (Fig. 5
b) at 6 days post-infiltration (p.i.). In contrast, the patches that received pBICGFP plus pBICSJB2 showed bright green fluorescence (Fig. 5a
). Patches that received pBICGFP plus pBICNSs showed even stronger green fluorescence (Fig. 5d
). These results indicate that SJNNV protein B2 suppressed RNA silencing of GFP, although the suppression was not as potent as that mediated by the TSWV NSs protein. Infiltration with pBICGFP plus pBICSJ
B2NC did not demonstrate strong green fluorescence, indicating that RNA silencing had not been suppressed in the patch (Fig. 5c
).
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Interestingly, at 12 days p.i., necrosis was observed in the patches infiltrated with pBICGFP plus pBICSJB2 in the N. benthamiana line 16c expressing GFP (data not shown). To further explore this observation, we tested the necrosis-inducing activity of several constructs in non-transgenic wild-type N. benthamiana plants. Although necrosis was observed in the patches infiltrated with pBICSJB2 alone (Fig. 6b), no necrosis was observed in the patches that received pBICP35, pBICSJ
B2NC or pBICNSs (Fig. 6a, c and d
). The patches that received pBICSJCP, and therefore express SJNNV CP, did not show necrosis (Fig. 6e
).
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DISCUSSION |
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The sequence UAA is conserved at the 5' end of SJNNV RNAs 1 and 2 (Iwamoto et al., 2001). This study found that, in SJNNV RNA3, the dinucleotide UA is conserved at the 5' terminus and that this RNA contained a 5' cap structure. Thus, SJNNV RNAs 1, 2 and 3 share the initial dinucleotide UA, a situation similar to that seen for the alphanodavirus FHV whereby RNAs 1, 2 and 3 share GU at the 5' end (Ball, 1995
). According to the report by Nishizawa et al. (1997)
, betanodaviruses can be divided into four genotypes: SJNNV, Redspotted grouper nervous necrosis virus (RGNNV), Tiger puffer nervous necrosis virus and Barfin flounder nervous necrosis virus (BFNNV). The sequence UAA is also present at the 5' ends of RNAs 1 and 2 of Greasy grouper nervous necrosis virus (RGNNV-type), Sevenband grouper nervous necrosis virus (RGNNV-type) and AHNV (BFNNV-type) (Grotmol et al., 2000
; Iwamoto et al., 2004
; Sommerset & Nerland, 2004
; Tan et al., 2001
). Further, these viruses share the 5'-terminal sequence of SJNNV RNA3, 5'-UAGUCAA-3', at nucleotide positions 27302736 in RNA1. These results suggest that this sequence may contain important determinants of subgenomic RNA3 synthesis and that these viruses may synthesize RNA3 by a common mechanism.
For FHV, RNA3 synthesis is greatly reduced in baby hamster kidney BHK21 cells when the first nucleotide of the RNA3 sequence in RNA1 is changed from G to A. In addition, RNA3 synthesis is essentially eliminated by a G-to-T or G-to-C substitution at the same position (Ball, 1995; Eckerle & Ball, 2002
). In a more recent study in yeast cells, the same G-to-T substitution greatly inhibited positive-strand RNA3 accumulation, although negative-strand RNA3 accumulated to wild-type levels (Price et al., 2000
). In our current study, the transfection experiments using three RNA1 mutants that had substitutions at the first nucleotide of RNA3 from U to A, C or G, showed that negative-strand RNA3 accumulation was not significantly reduced compared to the wild-type (Fig. 2b
). In contrast, positive-strand RNA3 accumulation for the mutants was essentially abolished (Fig. 2a
). Altogether, changing the first nucleotide of the SJNNV subgenomic RNA selectively inhibited production of positive- but not of negative-strand RNA3. This result suggests that synthesis of negative-strand subgenomic RNA3 may precede synthesis of positive-strand RNA3. This result also suggests that SJNNV subgenomic RNA3 may be synthesized not by internal initiation but by premature termination as proposed for FHV and some plant positive-strand RNA viruses (Price et al., 2000
; White, 2002
).
There are two ORFs found in RNA3 of FHV, Black beetle virus, Pariacoto virus and NoV (Ball, 1995; Guarino et al., 1984
; Johnson et al., 2000
, 2003
). However, SJNNV RNA3 has a single ORF from nt 27 to 254, encoding a predicted protein (designated protein B2) of 8·3 kDa, like RNA3 of the alphanodavirus Boolarra virus (Harper, 1994
). SJNNV protein B2 (75 aa) is the smallest amongst those identified from known nodaviruses. Moreover, SJNNV protein B2 differs from alphanodavirus B2 proteins in its amino acid sequence (Johnson et al., 2001
). These differences between SJNNV protein B2 and alphanodavirus B2 proteins prevented prediction of SJNNV protein B2 function, which prompted us to further examine SJNNV protein B2. Immunofluorescence staining of SJNNV-infected E-11 cells and striped jack larvae showed that protein B2 was specifically detected in the cytoplasm of E-11 cells, as well as in the central nervous system and retina of striped jack larvae. Similar localization was observed when CP and/or virions were detected with anti-SJNNV antiserum. However, localization of protein B2 and CP were different to some extent in E-11 cells (Fig. 3
). This result suggests that protein B2 was localized at limited sites in infected cells. The mutant SJ1
B2N lacks the N-terminal 20 aa of protein B2 (Fig. 4
). The truncated protein B2 could barely be detected in the transfected cells (T. Iwamoto & K. Mise, unpublished data). However, no fluorescent cells were observed in the transfection with mutant SJ1
B2C or SJ1
B2NC (data not shown), both of which were designed to express a truncated protein B2 lacking the C-terminal 33 aa (Fig. 4
). These results suggest that the anti-SJpB2 antiserum may recognize the C-terminal region of protein B2. Alternatively, the lack of the C-terminal region could affect the stability of protein B2.
RNA silencing is a conserved biological response to double-stranded RNA (dsRNA) that regulates gene expression and has evolved in plants as a defence against viruses (Gitlin & Andino, 2003; Hannon, 2002
; Voinnet, 2001
). As a counter-defence, many viruses encode proteins called RNA silencing suppressors' that specifically inhibit the RNA silencing machinery (Li & Ding, 2001
). RNA silencing also appears to contribute to antiviral defence in invertebrates. Importantly, a recent study showed that FHV RNAs can be targeted by RNA silencing in Drosophila cells, and that productive FHV infection requires suppression of RNA silencing by FHV-encoded protein B2 (Li et al., 2002
), a counterpart of SJNNV protein B2. So far, there has been no report of RNA silencing suppressor activity of any fish virus protein. To obtain some insights into the functions of SJNNV protein B2, we used an Agrobacterium-mediated assay to show that SJNNV protein B2 has suppressor activity against RNA silencing, at least in plants. Similar to this result, recent studies have shown that a dsRNA-binding protein from a mammalian reovirus (Lichner et al., 2003
) and the NS1 protein of influenza virus (Bucher et al., 2004
; Delgadillo et al., 2004
) could suppress RNA silencing in plants. In addition, Li et al. (2004)
demonstrated cross-kingdom suppression of RNA silencing in an animal system by the plant viral suppressor, tombusvirus p19. These results and a previous report (Li et al., 2002
) show that the RNA silencing mechanism is conserved between the plant and animal kingdoms and suggest that SJNNV protein B2 may have a role in counteracting RNA silencing in natural infections in fish. The adaptive immune system is a major defence mechanism for vertebrates to protect against viral infection. Generally in fish the adaptive immune system has not fully developed at the larval stage. If SJNNV protein B2 also plays a role in suppression of RNA silencing in fish, RNA silencing may also be an important defence to prevent virus attack in these animals.
There is still a need to investigate whether SJNNV RNAs can be targets of RNA silencing, and whether productive SJNNV infection requires suppression of RNA silencing by an SJNNV-encoded protein B2 in fish cells or in whole fish. Interestingly, the latter possibility could be supported by the observation that SJNNV-related RNAs accumulated to lower levels in fish cells transfected with protein B2-minus mutants (SJ1B2N, SJ1
B2C and SJ1
B2NC) (Fig. 2
). Johnson et al. (2004)
recently reported that B2-minus mutants of NoV accumulate to different levels in transfected cells depending on the cell type, implying that the efficacy of RNA silencing differs between different host cells. Our data suggest that E-11 cells have detectable activity of RNA silencing against viral RNA multiplication. On the other hand, for the SJNNV RNA3-minus mutants (SJ1U2730A, SJ1U2730C and SJ1U2730G), all SJNNV-related RNAs, except for positive-strand RNA3, accumulated to levels similar to those in wild-type-transfected cells. This could be due to the suppressing activity of small amounts of protein B2 barely detected by immunofluorescence.
SJNNV-infected fish exhibit a range of neurological abnormalities, which are characterized by vacuolization and cellular necrosis in the central nervous system and retina (Yoshikoshi & Inoue, 1990). Recently, Guo et al. (2003)
showed that the capsid protein of a betanodavirus, Greasy grouper nervous necrosis virus, induces apoptotic cell death in cultured fish and mammalian cells. In the present study, it was found that SJNNV protein B2, but not its capsid protein, induced necrotic cell death in plants. It is of great interest to test whether protein B2 has necrosis-inducing activity in the nervous system of fish. This may lead to the interesting hypothesis that SJNNV protein B2 could bind to 21 nt siRNAs, and thereby suppress RNA silencing. Then, the function of a putative Drosophila bantam-like microRNA, which suppresses programmed cell death (Brennecke et al., 2003
), could be inhibited, leading to necrosis of the nervous tissue. Detailed analysis of the infectivity of protein B2-minus mutants in fish may shed light on the role of protein B2 in the ability of betanodaviruses to cause nervous necrosis in fish, and on a possible correlation between suppression of RNA silencing and induction of nervous necrosis.
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ACKNOWLEDGEMENTS |
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Received 19 January 2005;
accepted 22 July 2005.
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