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Journal of Virology logoLink to Journal of Virology
. 2017 Mar 29;91(8):e02261-16. doi: 10.1128/JVI.02261-16

Two White Spot Syndrome Virus MicroRNAs Target the Dorsal Gene To Promote Virus Infection in Marsupenaeus japonicus Shrimp

Qian Ren a,b, Xin Huang b, Yalei Cui a, Jiejie Sun c, Wen Wang b, Xiaobo Zhang a,✉
Editor: Grant McFaddend
PMCID: PMC5375696  PMID: 28179524

ABSTRACT

In eukaryotes, microRNAs (miRNAs) serve as regulators of many biological processes, including virus infection. An miRNA can generally target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs has not yet been extensively explored during virus infection. This study found that the Spaztle (Spz)-Toll-Dorsal-antilipopolysaccharide factor (ALF) signaling pathway plays a very important role in antiviral immunity against invasion of white spot syndrome virus (WSSV) in shrimp (Marsupenaeus japonicus). Dorsal, the central gene in the Toll pathway, was targeted by two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) during WSSV infection. The regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study contributes novel insights into the viral miRNA-mediated Toll signaling pathway during the virus-host interaction.

IMPORTANCE An miRNA can target diverse genes during virus-host interactions. However, the regulation of gene expression by multiple miRNAs during virus infection has not yet been extensively explored. The results of this study indicated that the shrimp Dorsal gene, the central gene in the Toll pathway, was targeted by two viral miRNAs during infection with white spot syndrome virus. Regulation of Dorsal expression by viral miRNAs suppressed the Spz-Toll-Dorsal-ALF signaling pathway in shrimp in vivo, leading to virus infection. Our study provides new insight into the viral miRNA-mediated Toll signaling pathway in virus-host interactions.

KEYWORDS: virus, microRNAs, Dorsal, Toll signaling pathway, shrimp

INTRODUCTION

MicroRNAs (miRNAs) are small noncoding RNAs that serve as regulators in many cellular processes, including virus infection (1). miRNAs originating from the virus are expressed during virus infection, and the expression levels of host miRNAs are altered (2–7). In shrimp, white spot syndrome virus (WSSV) can express 89 viral miRNAs (8). The miRNAs of the host and/or virus are involved in virus infection processes in various ways. For instance, dengue virus can upregulate the expression of a host miRNA to enhance viral replication (9). In contrast, host miRNA can inhibit the replication of hepatitis B virus by regulating the expression of a host gene that is beneficial in virus infection (10). WSSV genome-encoded miRNAs promote virus replication by regulating the genes of the host or virus (11, 12). WSSV genome-encoded miRNA can undergo RNA editing to facilitate entry to viral latency during virus infection (8). In this context, host miRNAs and virus genome-encoded miRNAs play essential roles in virus-host interactions.

During virus infection processes, miRNAs can target the key genes for virus infection and/or target the genes participating in key biological processes to facilitate virus infection. The entry of viruses into host cells is speculated to be dependent on receptor-mediated endocytosis. The transferrin receptor (TfR) is a target of miRNA during mink enteritis virus infection (13). Moreover, the viral replicase complex is important in virus replication, and eukaryotic translation elongation factor 1A1 (EEF1A1) can stabilize the components of the viral replicase complex. The downregulation of host miR-33a-5p during Japanese encephalitis virus infection enhances virus replication resulting from the upregulation of the EEF1A1 gene, the target gene of miR-33a-5p (14). Furthermore, during virus infection, apoptosis of host cells can be triggered to inhibit virus invasion. Thus, the key genes for apoptosis are possibly the targets of miRNAs. In shrimp, miR-1000 targets the host p53 gene, thereby inhibiting apoptosis, leading to WSSV replication in shrimp (15). Human miR-942 can suppress hepatitis C virus-induced apoptosis by inhibiting the expression of interferon (IFN)-stimulated gene 12a (16). WSSV-miR-N24, one of the viral miRNAs identified during WSSV infection, can suppress apoptosis in shrimp by targeting the host caspase 8 gene (5).

Generally, one miRNA can target diverse genes during virus infection. Host miR-532-5p targets SEC14 and spectrin domains 1 and transforming growth factor β-activated kinase 1 (TAK1)-binding protein 3, thereby inhibiting West Nile virus replication (17). During influenza virus infection, host miR-485 not only regulates the expression of host genes but also targets the influenza virus gene to inhibit virus replication (18). During WSSV infection in shrimp, viral WSSV-miR-66 targets two virus genes (wsv094 and wsv177), and WSSV-miR-68 targets two other virus genes (wsv248 and wsv309) (11). These findings revealed that one miRNA can target multiple genes. Conversely, a gene may be regulated by multiple miRNAs. Both miR-320a and miR-140 can target the feline transferrin receptor gene to inhibit mink enteritis virus infection (13). Host gga-miR-221 and gga-miR-222 target chicken BCL-2-modifying factor, inducing apoptosis to promote tumorigenesis during avian leukosis virus infection (19). However, regulation of the expression of one gene by several miRNAs during virus infection has not yet been extensively investigated.

To fill this knowledge gap, this study characterized the WSSV miRNAs. WSSV, a virus with a 305-kb circular double-stranded genomic DNA, is a new member of the genus Whispovirus belonging to the family Nimaviridae (20). WSSV is a major pathogen in shrimp and causes great economic losses in crustacean aquaculture industries worldwide (21). The results showed that WSSV genome-encoded miRNAs (WSSV-miR-N13 and WSSV-miR-N23) could simultaneously target the Dorsal gene of its host shrimp. The involvement of viral miRNAs in the Spaztle (Spz)-Toll-Dorsal-antilipopolysaccharide factor (ALF) signaling pathway promoted virus infection in vivo.

RESULTS

Effects of host Spz-Toll-Dorsal-ALF pathway on virus infection.

The Toll signaling pathway is important in the activation of antimicrobial peptides (AMPs) in the innate immune system. The ligand Spatzle (Spz) can trigger the Toll pathway by binding Toll proteins on the cell membrane, activating the transcription factor Dorsal (22). To elucidate the role of the Toll pathway in virus infection, this work investigated the expression levels of the Spz, Toll, and Dorsal genes in shrimp in response to WSSV infection. As shown in Fig. 1A, Spz, Toll1, Toll4, Toll6, Toll7, and Dorsal were significantly upregulated in WSSV-infected shrimp compared with their levels of regulation in control shrimp; the expression of Toll2 or Toll3 in infected shrimp was similar to that in control shrimp, whereas Toll5 was significantly downregulated in response to WSSV infection. These data suggest that Spz, Toll1, Toll4, Toll6, Toll7, and Dorsal play important roles in virus infection.

FIG 1.

FIG 1

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Effects of the host Spaztle-Toll-Dorsal-ALF pathway on virus infection. (A) Expression profiles of Spz, Toll genes, and Dorsal in shrimp in response to virus infection. Shrimp were challenged with WSSV. At different time points postinfection, gene expression levels were evaluated through quantitative real-time PCR. PBS was used as a control. (B) Silencing of gene expression in shrimp. siRNA specific for the Spz, Toll1, Toll4, Toll6, Toll7, or Dorsal sequence and WSSV were coinjected into the shrimp for gene knockdown. The scrambled siRNA and WSSV alone were used as controls. At different time points postinfection, gene expression levels were examined using quantitative real-time PCR. (C) Effects of silencing of gene expression on virus replication in shrimp. The WSSV copies in shrimp treated with Spz-, Toll1-, Toll4-, Toll6-, Toll7-, or Dorsal-siRNA were quantified by real-time PCR. WSSV only and Spz-, Toll1-, Toll4-, Toll6-, Toll7-, and Dorsal-siRNA-scrambled were used as controls. (D) Influence of Spz, Toll1, Toll4, Toll6, Toll7, and Dorsal gene silencing on expression of shrimp ALF in vivo. Sequence-specific siRNA and WSSV were coinjected into the shrimp, followed by detection of shrimp ALF using quantitative real-time PCR. WSSV only and Spz-, Toll1-, Toll4-, Toll6-, Toll7-, and Dorsal-siRNA-scrambled were used as controls. (E) Silencing of ALF expression in shrimp. siRNA specific for the ALF sequence was coinjected with WSSV into shrimp to knock down ALF. Scrambled siRNA was included in the injection mixtures as a negative control. WSSV only was used as a positive control. ALF expression in shrimp was quantified using real-time PCR. (F) Effects of shrimp ALF silencing on WSSV infection. The WSSV copies of ALF-siRNA-treated shrimp were quantified using real-time PCR. WSSV only and ALF-siRNA-scrambled were used as controls. In all panels, asterisks indicated significant differences (*, P < 0.05; **, P < 0.01) between treatments.

To explore the influence of Spz, Toll1, Toll4, Toll6, Toll7, and Dorsal on virus infection, we knocked down the expression levels of these genes by injecting sequence-specific small interfering RNA (siRNA) into shrimp in vivo (Fig. 1B). The results indicated that silencing of Spz, Toll1, Toll4, Toll6, Toll7, or Dorsal significantly increased the number of WSSV copies in shrimp compared with the number in the controls (the controls consisted of shrimp treated with scrambled sequences of siRNA specific for the different genes [Spz-, Toll1-, Toll4-, Toll6-, Toll7-, or Dorsal-siRNA-scrambled] and infected with WSSV only) (Fig. 1C). These findings show that Spz, Toll1, Toll4, Toll6, Toll7, and Dorsal exert negative effects on WSSV replication.

The transcription factor Dorsal is required for the expression of AMPs, including ALFs (23). The effects of Dorsal, Spz, Toll1, Toll4, Toll6, and Toll7 on the expression of shrimp ALF were determined by silencing these genes in shrimp by using sequence-specific siRNA followed by ALF detection at the mRNA level. The results revealed that shrimp ALF was obviously downregulated in shrimp treated with siRNA specific for Spz, Toll1, Toll4, Toll6, Toll7, or Dorsal compared with its levels of regulation in the controls (shrimp treated with Spz-, Toll1-, Toll4-, Toll6-, Toll7-, or Dorsal-siRNA-scrambled and infected with WSSV only) (Fig. 1D). The data show that the Spz-Toll-Dorsal pathway plays a crucial role in ALF expression in shrimp.

ALF is an AMP and is involved in the antiviral immunity of shrimp (24). This work further evaluated the influence of ALF on virus infection by knocking down ALF expression in shrimp using sequence-specific siRNA in vivo (Fig. 1E). The results according to the number of virus copies detected indicated that silencing of ALF significantly increased the number of WSSV copies in shrimp compared with those in the controls (shrimp treated with ALF-siRNA-scrambled and infected with WSSV only) (Fig. 1F), demonstrating that ALF exerts a negative effect on WSSV infection in shrimp in vivo.

These findings collectively show that the Spaztle-Toll-Dorsal signaling pathway exerts considerable negative effects on WSSV infection in shrimp in vivo by activating the expression of an antiviral peptide.

Host Dorsal as the target of viral miRNAs.

The findings presented above revealed that the transcription factor Dorsal plays a central role in virus infection. The WSSV genome can encode 89 viral miRNAs (8). The interactions between Dorsal and WSSV miRNAs were investigated to further explore the viral miRNA-mediated regulation of Dorsal gene expression in shrimp. The target prediction, obtained using the TargetScan, MiRanda, and Pictar algorithms, showed that both WSSV-miR-N13 and WSSV-miR-N23 possibly target Dorsal in shrimp (Fig. 2A), suggesting that viral miRNAs exert considerable effects on WSSV infection.

FIG 2.

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Dorsal in the host as a target of viral miRNA. (A) Viral miRNAs predicted to target shrimp Dorsal. As predicted, the 3′ UTR of Dorsal was targeted by WSSV-miR-N13 and WSSV-miR-N23. (B) Plasmid construction. The 3′ UTR of Dorsal and the EGFP gene were cloned into the pIZ/EGFP V5-His vector using the XbaI and SacII restriction sites to generate the EGFP-Dorsal plasmid. For the controls, the 3′ UTR sequences of Dorsal complementary to the seed sequences of WSSV-miR-N13 and WSSV-miR-N23 were mutated, generating mutant EGFP-ΔDorsal-N13 and mutant EGFP-ΔDorsal-N23. (C) Direct interaction between viral WSSV-miR-N13 and host Dorsal in insect cells. The synthesized WSSV-miR-N13-mimic and the EGFP-Dorsal plasmid, consisting of EGFP and the Dorsal 3′ UTR, were cotransfected into insect High Five cells. EGFP-ΔDorsal-N13 and control miRNA were included in the cotransfection as controls. At 48 h after cotransfection, the fluorescence of the cells was examined (top), and the relative fluorescence intensity of the cells was determined (bottom). (D) Direct interaction between viral WSSV-miR-N23 and Dorsal in insect cells. Insect cells were cotransfected with WSSV-miR-N23-mimic and the EGFP-Dorsal plasmid. EGFP-ΔDorsal-N23 and control miRNA were used as controls. After cotransfection for 48 h, the fluorescence of the cells was examined (top), and the relative fluorescence intensity of the cells was determined (bottom).

To characterize the interactions between the viral miRNAs and the Dorsal gene in the host, we constructed the EGFP-Dorsal plasmid, which expressed the enhanced green fluorescent protein (EGFP) and the Dorsal 3′ untranslated region (UTR) of shrimp (Fig. 2B). The synthesized WSSV-miR-N13-mimic or WSSV-miR-N23-mimic and the EGFP-Dorsal plasmid were cotransfected into insect High Five cells. The results showed that the fluorescence intensity in the cotransfected cells was significantly reduced compared with that in the control cells (Fig. 2C and D), indicating that WSSV-miR-N13 and WSSV-miR-N23 can directly target Dorsal in the host.

Effects of viral miRNAs on virus infection in shrimp.

Two viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) were significantly upregulated in shrimp in response to WSSV infection (Fig. 3A), indicating that these viral miRNAs are involved in virus-host interactions. To further evaluate the effects of miRNA-Dorsal interactions on virus infection in vivo, we overexpressed or silenced WSSV-miR-N13 or WSSV-miR-N23 in WSSV-infected shrimp, and then we examined the level of Dorsal mRNA or Dorsal protein expression. Quantitative real-time PCR data revealed that overexpression of viral miRNA (WSSV-miR-N13 or WSSV-miR-N23) significantly reduced the level of Dorsal expression compared with that in the controls (Fig. 3B). The Western blot analyses showed results similar to those of the quantitative real-time PCR analysis (Fig. 3C). In contrast, the Dorsal mRNA or protein level was significantly increased in the viral miRNA-silenced (AMO-WSSV-miR-N13 or AMO-WSSV-miR-N23, where AMO is an anti-miRNA oligonucleotide) WSSV-infected shrimp compared with that in the controls (Fig. 3D and E). These findings reveal that both WSSV-miR-N13 and WSSV-miR-N23 can target Dorsal in the host in vivo.

FIG 3.

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Effects of viral miRNAs on virus infection in shrimp. (A) Time course expression patterns of viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) in shrimp. The shrimp were infected with WSSV. The expression levels of viral miRNAs in shrimp were monitored at different time points postinfection by using quantitative real-time PCR. (B) Influence of overexpression of viral miRNA on Dorsal mRNA level in WSSV-infected shrimp. WSSV-miR-N13-mimic or WSSV-miR-N23-mimic and WSSV were coinjected into shrimp to overexpress the viral miRNA (WSSV-miR-N13 or WSSV-miR-N23). WSSV alone and WSSV plus control miRNA were included in the injection mixtures as controls. The expression level of Dorsal mRNA in shrimp was examined at 24 h postinfection by using quantitative real-time PCR. (C) Western blot analysis of the Dorsal protein in WSSV-infected shrimp after the overexpression of WSSV-miR-N13 or WSSV-miR-N23. The viral miRNA was overexpressed in the WSSV-infected shrimp. The Dorsal protein in shrimp was detected at 24 h postinfection through Western blot analysis. Shrimp β-actin was used as a control. (D) Impact of viral miRNA silencing on transcription of Dorsal in WSSV-infected shrimp. AMO-WSSV-miR-N13 or AMO-WSSV-miR-N23 and WSSV were coinjected into the shrimp to knock down the viral miRNA. The expression of Dorsal mRNA in shrimp was examined at 24 h postinfection by using quantitative real-time PCR. WSSV only and control AMO were used as controls. (E) Effects of viral miRNA knockdown on expression of the Dorsal protein in shrimp. Shrimp were coinjected with AMO and WSSV. The dorsal protein in shrimp was evaluated at 24 h postinfection through Western blot analysis. (F) Effects of viral miRNA overexpression on virus infection in vivo. WSSV-miR-N13 or WSSV-miR-N23 was overexpressed in shrimp, and then the number of WSSV copies was determined using quantitative real-time PCR. WSSV-miR-N13-scrambled, WSSV-miR-N23-scrambled, and WSSV only were used as controls. (G) Influence of WSSV-miR-N13 or WSSV-miR-N23 silencing on virus infection in vivo. AMO-WSSV-miR-N13 or AMO-WSSV-miR-N23 and WSSV were coinjected into the shrimp. The number of WSSV copies was determined at 24 h postinfection. WSSV alone was used as a control. AMO-WSSV-miR-N13-scrambled and AMO-WSSV-miR-N23-scrambled were also used as controls. Asterisks indicate significant differences (**, P < 0.01) between treatments.

The results showed that WSSV-miR-N13 overexpression significantly increases the number of WSSV copies in shrimp compared with the number in the controls (shrimp treated with WSSV-miR-N13-scrambled and infected with WSSV only) (Fig. 3F). WSSV-miR-N23 overexpression yielded similar results (Fig. 3F). When viral miRNAs (WSSV-miR-N13 or WSSV-miR-N23) were silenced, the number of WSSV copies in shrimp significantly decreased compared with the number in the controls (shrimp treated with AMO-WSSV-miR-N13-scrambled or AMO-WSSV-miR-N23-scrambled and infected with WSSV only) (Fig. 3G). These findings indicate that these two viral miRNAs play essential roles in WSSV infection in vivo.

Viral miRNA-mediated pathway in virus infection.

To further explore the involvement of viral miRNAs in the Spaztle-Toll-Dorsal-ALF signaling pathway, we overexpressed or knocked down WSSV-miR-N13 or WSSV-miR-N23 and then we monitored ALF expression. The results indicated that the level of ALF mRNA was significantly downregulated in shrimp in which WSSV-miR-N13 or WSSV-miR-N23 was overexpressed compared with the level of regulation in the controls (Fig. 4A), demonstrating that the viral miRNAs regulated the Spaztle-Toll-Dorsal-ALF signaling pathway. Western blot analysis showed similar results (Fig. 4B). Moreover, ALF mRNA expression was obviously upregulated when viral miRNA (WSSV-miR-N13 or WSSV-miR-N23) was silenced compared with the level of regulation in the controls (Fig. 4C). Western blot analyses showed that silencing of WSSV-miR-N13 or WSSV-miR-N23 significantly increased the ALF protein level in virus-infected shrimp (Fig. 4D).

FIG 4.

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Viral miRNAs mediated the pathway for virus infection. (A) Effects of viral miRNA overexpression on expression levels of ALF mRNA in shrimp in vivo. WSSV-miR-N13-mimic or WSSV-miR-N23-mimic and WSSV were coinjected into shrimp. At 24 h after infection with WSSV, the level of ALF mRNA was quantified using quantitative real-time PCR. WSSV only, WSSV-miR-N13-scrambled, and WSSV-miR-N23-scrambled were used as controls. (B) Impact of viral miRNA overexpression on expression of the ALF protein in virus-infected shrimp. The shrimp were coinjected with miRNA and WSSV. The ALF protein in shrimp was detected at 24 h postinfection through Western blot analysis. Shrimp β-actin was used as a control. (C) Influence of viral miRNA silencing on ALF expression. AMO-WSSV-miR-N13 or AMO-WSSV-miR-N23 and WSSV were coinjected into the shrimp to knock down viral miRNA expression. After 24 h, the transcription level of ALF was detected using quantitative real-time PCR. AMO-WSSV-miR-N13-scrambled, AMO-WSSV-miR-N23-scrambled, and WSSV only were used as controls. (D) Western blot analysis of ALF protein in WSSV-infected shrimp after silencing of WSSV-miR-N13 or WSSV-miR-N23. The viral miRNA was silenced in the WSSV-infected shrimp. At 24 h postinfection, the ALF protein in shrimp was examined through Western blot analysis. (E) Mode for the viral miRNA-mediated signaling pathway in virus infection. WSSV-miR-N13 and WSSV-miR-N23 targeted the Dorsal gene only in shrimp.

These findings collectively reveal that viral miRNAs (WSSV-miR-N13 and WSSV-miR-N23) negatively regulate the Spaztle-Toll-Dorsal-ALF signaling pathway by targeting the host Dorsal gene only, resulting in virus infection in shrimp in vivo (Fig. 4E).

DISCUSSION

miRNAs regulate gene expression at the posttranscription level. This regulation is generally a one-to-one type of regulation (i.e., one miRNA to one mRNA). To avoid the consequences of miRNA off-targeting, viruses and/or hosts needed to develop the strategy of utilizing two or more miRNAs to regulate the expression of one gene, which is important in host biological processes or in virus infection. Regulation of expression of a gene by only one miRNA does not guarantee the success of the regulation of gene expression. Some studies have found that multiple miRNAs regulate the expression of one gene. The programmed cell death protein 4, which is a tumor suppressor protein, is important in developing human cancers (25). Its expression is tightly regulated by miR-21 and miR-499 (25). Three genes of the Wnt signaling pathway can be targeted by multiple miRNAs (26). A number of miRNAs are coexpressed as clusters (27), and human miRNA clusters regulate the activating protein 1, NF-κB, c-Myc, and p53 signaling pathways (28). To successfully inhibit virus infection, a host utilizes two miRNAs (miR-320a and miR-140) to regulate the expression of a virus receptor gene (TfR) (13). Epstein-Barr virus (EBV) miRNAs collectively suppress the release of proinflammatory cytokines, such as interleukin-12 (29). Multiple members of the small ubiquitin-like modifier signaling network can be targeted by one or more EBV miRNAs (30). However, the mechanism of the regulation of expression of one gene by multiple miRNAs has not yet been intensively explored. This study revealed that two viral miRNAs simultaneously target a host transcription factor (Dorsal), which is required in the Toll signaling pathway, leading to virus infection. During the virus-host interaction, successful infection by the virus and successful replication of the virus in its host are important for the continuation of virus life. To facilitate infection, the virus employs a strategy of utilizing its multiple miRNAs to target important genes that function as inhibitors of virus infection in the host. In this context, two or more miRNAs targeting the same gene can ensure the success of important biological processes.

The Toll-Dorsal signaling pathway is necessary in the resistance of a host to virus infection (31–35). Toll receptors can trigger the Toll-Dorsal signaling pathway. Multiple Toll proteins exist in an animal, and these Toll proteins perform diverse functions. In humans, Toll-like receptor 4 (TLR4) can bind lipopolysaccharide on the surface of bacteria (36), whereas TLR3 recognizes double-stranded RNA, leading to the activation of the NF-κB pathway (37). Drosophila Toll7 recognizes viruses and activates antiviral autophagy (38), Drosophila Toll8 negatively regulates the antimicrobial response (39), and Drosophila Toll1 regulates the systemic immune response (40). This study showed that Toll1, Toll4, Toll6, and Toll7 play important roles in antiviral immunity in shrimp. Activation of the Spz-Toll-Dorsal pathway by WSSV infection induced ALF expression. ALF, which is an AMP, demonstrates both antibacterial and antiviral activities in shrimp, preventing pathogen invasion (24, 41). Dorsal plays a central role in the Spz-Toll-Dorsal-ALF signaling pathway, which is required for antiviral immune defense in shrimp, and this study showed that the WSSV genome encodes two viral miRNAs to suppress Dorsal expression in the host. The interaction between viral miRNA and Dorsal in other viruses has not yet been investigated. Bracovirus encodes two proteins (H4 and N5) with homology to inhibitor κB (IκB). N5 can bind to Dorsal to escape from the host immune attack during virus infection (42). Moreover, activation/inactivation of a transcription factor can alter biological processes. Inactivation of the transcription factor can be either direct or indirect. Ank proteins of polydnavirus can directly interact with host NF-κB homodimers and inhibit the activation of relish transcription factor (43). In shrimp, the WSSV genome-encoded miRNA (WSSV-miR-22) targets STAT in the host and enhances virus replication (12). Our study contributes new insights into the viral miRNA-mediated Spz-Toll-Dorsal-ALF signaling pathway in virus-host interactions.

Viruses have developed a number of strategies to escape the host immune attack during the virus-host interaction. Except for miRNAs, viruses can utilize virus genome-encoded proteins to inhibit host antiviral responses. Spring viremia of carp virus P protein can suppress the production of carp interferon (IFN) by reducing the phosphorylation of IFN regulatory factor 3 (44). Additionally, Nipah virus matrix protein inhibits the host IκB kinase epsilon (IKKε) kinase-mediated IFN type I antiviral response by targeting the host E3-ubiquitin ligase TRIM6 (45). A recent study revealed that the WSSV genome can encode protein kinase 1 to defeat the host cell's iron-withholding defense mechanism by interacting with host ferritin (46). In this context, viruses possess complicated mechanisms to escape the host immunity. These mechanisms merit further investigation.

MATERIALS AND METHODS

Shrimp culture and WSSV challenge.

Shrimp (Marsupenaeus japonicus) were obtained from a local shrimp farm in Hangzhou, Zhejiang Province, China, and cultured in groups of 20 individuals in the same tank filled with seawater at room temperature; the shrimp were acclimatized for 3 days prior to the experiments. Three shrimp were randomly selected for PCR detection of WSSV by using WSSV-specific primers (5′-TATTGTCTCTCCTGACGTAC-3′ and 5′-CACATTCTTCACGAGTCTAC-3′) to ensure that the shrimp were WSSV free prior to the conduct of the experiments. The virus-free shrimp were infected with WSSV (105 copies/ml) by injecting 100 μl of WSSV solution into the lateral area of the fourth abdominal segment. The virus copy number used during injection was 104. At different times postinfection (0, 6, 12, 24, and 36 h), the shrimp were collected for later use.

Detection of mRNA or miRNA by quantitative real-time PCR.

For mRNA detection, total RNAs were extracted from shrimp gills by using an RNAprep Pure tissue kit (Tiangen, Beijing, China) according to the manufacturer's instructions. RNA quality was assessed by electrophoresis on a 1.0% agarose gel, and the total RNA concentration was determined by measuring the absorbance at 260 nm on a spectrophotometer. The first strand of cDNA was synthesized using a PrimeScript first-strand cDNA synthesis kit (TaKaRa, Japan) with oligo(dT) as the primer. Actin was used as a control. The primers for PCR were gene specific (Spz, 5′-CTGGTGGCTGGAAGACAAAGA-3′ and 5′-CTCAGATGGGCAGAGATAGGT-3′; Toll1, 5′-ACGATGATGTATGCCTTTTC-3′ and 5′-GCTTGATGTCACTGTTTGCT-3′; Toll2, 5′-TCCTCATTCTCTCTGGTGTT-3′ and 5′-TCATACTTCTTGTTGTTGTCGT-3′; Toll3, 5′-GAGTGTGGGGAAGTCAGG-3′ and 5′-TTAGGAGGTGGGTGGAAG-3′; Toll4, 5′-AGTGAACATACGACCGATTT-3′ and 5′-GTGTCCAGGTTGTAGAGGC-3′; Toll5, 5′-TTAGGAAGGTGGAAGACGA-3′ and 5′-GGGATGGAGAGGATGTTG-3′; Toll6, 5′-CCTCACCATTACCCTCATC-3′ and 5′-CATACTTCGCATACACCCA-3′; Toll7, 5′-CAGATGCCTGTTCCTGCTT-3′ and 5′-TCCATTGGCACTCGGTCT-3′; Dorsal, 5′-AGACTGGGTTTTCTCATCGTAATC-3′ and 5′-TAAATGGGATCTGACACTTGTGG-3′; ALF, 5′-AGCCTCCTTTTCCTTTCCCCT-3′ and 5′-CACAATCCTGTCAGTTTTTCCGC-3′; β-actin gene, 5′-CAGCCTTCCTTCCTGGGTATGG-3′ and 5′-GAGGGAGCGAGGGCAGTGATT-3′). The reactions were performed using a 2× SYBR Premix Ex Taq kit (TaKaRa, Japan) on a real-time thermal cycler (Bio-Rad, Hercules, CA, USA) according to the manufacturers' instructions. PCR was performed at 95°C for 3 min, followed by 40 cycles at 95°C for 15 s and 60°C for 30 s. Data were quantified by the 2−ΔΔCT threshold cycle (CT) method (47).

For miRNA detection, viral miRNAs were extracted from the gills of WSSV-challenged shrimp by using a mirVana miRNA isolation kit according to the manufacturer's instructions (Ambion, USA). For WSSV-miR-N13 detection, the miRNA first-strand cDNA was synthesized using an all-in-one miRNA first-strand cDNA synthesis kit (GeneCopoeia, USA) following the manufacturer's protocol. A reverse transcriptase reaction was performed at 37°C for 60 min and at 85°C for 5 min. Real-time PCR was conducted using SYBR Premix Ex Taq (RNase H Plus; TaKaRa, Japan) with an WSSV-miR-N13-specific forward primer (5′-GGAATACAACTAGCAAGCACTG-3′) and a universal adaptor PCR primer (GeneCopoeia, USA). U6 was amplified (by the use of U6-specific forward primer 5′-GAATTTGCGTGTCATCCTTGC-3′ and the universal adaptor PCR primer) as an internal control. Real-time PCR was performed at 95°C for 10 min, followed by 40 cycles at 95°C for 5 s and 60°C for 30 s. For WSSV-miR-N23 detection, the first-strand cDNA of WSSV-miR-N23 was synthesized using a TaqMan microRNA reverse transcription kit (Applied Biosystems, USA) according to the instructions in the manufacturer's manual. Reverse transcription was conducted at 16°C for 30 min, 42°C for 30 min, and 85°C for 5 min by using a WSSV-miR-N23-specific reverse transcription primer (5′-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCATTCG-3′) (11). U6 was used as a control with the reverse transcription primer Random6 (Applied Biosystems). Real-time PCR was performed using SYBR Premix Ex Taq (RNase H Plus; TaKaRa, Japan) with WSSV-miR-N23-specific primers (5′-CATTCGTTAGGCACTGGGAAAT-3′ and 5′-GTGCAGGGTCCGAGGTATTC-3′) or U6-specific primers (5′-GTCATCCTTGCGCAGGGGCCA-3′ and 5′-CTCGCTTCGGCAGCACATATA-3′). The real-time PCR was performed at 95°C for 5 min, followed by 40 cycles at 95°C for 5 s and at 60°C for 30 s.

RNA interference (RNAi) assay in shrimp in vivo.

On the basis of the sequences of shrimp Spz, Toll1, Toll4, Toll6, Toll7, Dorsal, and ALF, the siRNAs specifically targeting these genes were separately synthesized according to the design rule for siRNA by using a commercial kit according to the manufacturer's instructions (TaKaRa, Japan). The siRNAs used were Spz-specific siRNA (Spz-siRNA; 5′-CCTGAAGGAGGAGACCTAT-3′), Toll1-siRNA (5′-GCGGCGACAGAGACTCATT-3′), Toll4-siRNA (5′-GGACCATCGGAAACGACTT-3′), Toll6-siRNA (5′-GCGTGATCCCAACACGAAA-3′), Toll7-siRNA (5′-GCCCTTCACGTTACCAGAT-3′), Dorsal-siRNA (5′-GGCACCAGTTGGTGCTGTT-3′), and ALF-siRNA (5′-CCGGAGAACTGGAGCTGTT-3′). For the controls, the siRNA sequences were scrambled, generating scrambled Spz-specific siRNA (Spz-siRNA-scrambled; 5′-GGCGGTACGCACGAATTAA-3′), Toll1-siRNA-scrambled (5′-GCGCAGACGCGACGTATA-3′), Toll4-siRNA-scrambled (5′-GGCCAAGAATCCGCAGTTA-3′), Toll6-siRNA-scrambled (5′-GCACAAGCACGGCTAATCA-3′), Toll7-siRNA-scrambled (5′-GCCTGCACGATCTTTACCA-3′), Dorsal-siRNA-scrambled (5′-GGCTGAGTCCGCGTGTTAT-3′), and ALF-siRNA-scrambled (5′-GCGCTGTCGCGAGATAAGT-3′). The formation of double-stranded siRNAs was monitored by determining their sizes during agarose gel electrophoresis.

The RNAi assay in shrimp was conducted by injecting siRNA (30 μg/shrimp) into the lateral area of the fourth abdominal segment by using a 1-ml sterile syringe. The siRNA (15 μg) and WSSV (105 copies/ml) were coinjected into virus-free shrimp at 100 μl/shrimp. At 12 h after coinjection, the siRNA (15 μg) was injected into the same shrimp (100 μl/shrimp). WSSV alone (105 copies/ml) and control siRNA were included in the injection mixtures as controls. For each treatment, 20 shrimp were used. At different time points after injection, the shrimp were collected for later use. The assays were biologically repeated three times.

Prediction of target genes.

In predicting the target genes of viral miRNAs, three independent computational algorithms, namely, the TargetScan (version 5.1; http://www.targetscan.org), MiRanda (http://www.microrna.org/), and Pictar (http://pictar.mdc-berlin.de/) algorithms, were used to predict the target sites of miRNAs in the 3′ untranslated region (UTR) of the shrimp gene.

Plasmid construction.

To examine whether the viral miRNA (WSSV-miR-N13 or WSSV-miR-N23) can target the Dorsal gene of M. japonicus, we cloned the 3′ UTR of the Dorsal and enhanced green fluorescent protein (EGFP) genes into the pIZ/EGFP V5-His vector (Invitrogen, USA). The Dorsal 3′ UTR was cloned into the pIZ/V5-His vector downstream of EGFP using the primers 5′-GCTCTAGACTTGTTGTTTGACACTTTATTC-3′ and 5′-TCCCCGCGGTATAGACTTAGTGAACAAAAGC-3′, generating an EGFP-Dorsal construct. For the controls, the 3′ UTR sequences of Dorsal complementary to the WSSV-miR-N13 or WSSV-miR-N23 seed sequence were randomly mutated using sequence-specific primers (for the WSSV-miR-N13 mutant, 5′-AAGGAATTCACGCAGCGACAATGTAGGTCTTGCT-3′ and 5′-TACATTGTCGCTGCGTGAATTCCTTATGCAGTATTC-3′; for the WSSV-miR-N23 mutant, 5′-AGTAGAAATAGCGGTGCTGCAGAGATATTATTTAG-3′ and 5′-GCAGCACCGCTATTTCTACTTCAGCTTTGTTGTAAC-3′), yielding the EGFP-ΔDorsal-N13 and EGFP-ΔDorsal-N23 constructs, respectively. All of the recombinant plasmids were confirmed by sequencing.

Cell culture, transfection, and fluorescence assay.

Insect High Five cells (Invitrogen, USA) were cultured at 28°C in Express Five serum-free medium (Invitrogen) supplemented with l-glutamine (Invitrogen). When the cells were at approximately 70% confluence, they were cotransfected with 6 μg/ml EGFP, EGFP-Dorsal, or EGFP-ΔDorsal and 300 nM either synthesized viral miRNA or a synthesized control miRNA. All miRNA mimics were synthesized by Shanghai Gene Pharma Co., Ltd. (Shanghai, China). The transfection was conducted in triplicate by using the Cellfectin transfection reagent (Invitrogen) according to the manufacturer's protocol. At 48 h after cotransfection, cell fluorescence was examined using a Flex Station II microplate reader (Molecular Devices, USA) at an excitation wavelength of 490 and an emission wavelength of 510 nm. The fluorescence values were corrected by subtracting the autofluorescence of cells not expressing EGFP. All experiments were biologically repeated three times.

Silencing or overexpression of WSSV-miR-N13 and WSSV-miR-N23 in shrimp.

Anti-miRNA oligonucleotide (AMO) was injected into WSSV-infected shrimp to silence the viral miRNA. AMO-WSSV-miR-N13 (5′-CAGTGCTTGCTAGTTGTATT-3′) was synthesized (Sangon Biotech, Shanghai, China) with a phosphorothioate backbone and a 2′-O-methyl modification at the 6th, 15th, and 17th nucleotides. The sequence of AMO-WSSV-miR-N23 (5′-CATTCGTTAGGCACTGGGAAAT-3′) was modified with a phosphorothioate backbone and a 2′-O-methyl modification at the 6th, 17th, and 19th nucleotides. For the controls, the sequences of AMO-WSSV-miR-N13 and AMO-WSSV-miR-N23 were scrambled, generating AMO-WSSV-miR-N13-scrambled (5′-AGCTCGTGTCTATGTGTTAT-3′) and AMO-WSSV-miR-N23-scrambled (5′-CATCTGTATCGGACGTGGATAA-3′), respectively. AMO (10 nM) and WSSV (105 copies/ml) were coinjected into virus-free shrimp (100 μl/shrimp). At 12 h after coinjection, AMO (10 nM; 100 μl/shrimp) was injected into the same shrimp. WSSV (105 copies/ml; 100 μl/shrimp) alone and control AMO were included in the injection mixtures as controls. For each treatment, 20 shrimp were used. At various time points after infection with WSSV, shrimp gills were collected for later use.

On the basis of the sequence of WSSV-miR-N13 (5′-AAUACAACUAGCAAGCACUG-3′) or WSSV-miR-N23 (5′-AUUUCCCAGUGCCUAACGAAUG-3′), WSSV-miR-N13-mimic or WSSV-miR-N23-mimic was synthesized using an in vitro transcription T7 kit for miRNA synthesis (TaKaRa, Japan). The sequence of WSSV-miR-N13 or WSSV-miR-N23 was scrambled to generate the control (WSSV-miR-N13-scrambled, 5′-ACAAACAUACAUGGCAUAGC-3′; WSSV-miR-N23-scrambled, 5′-CAUCCCUAAUCUGGAAUCGUGA-3′). The synthesized miRNAs were dissolved in miRNA solution (50 mM Tris-HCl, 100 mM NaCl, pH 7.5) and quantified spectrophotometrically. Virus-free shrimp were simultaneously injected with miRNA (15 μg) and WSSV (105 copies/ml) at 100 μl/shrimp to overexpress the viral miRNA. At 12 h after coinjection, the miRNA (15 μg) (100 μl/shrimp) was injected into the same shrimp. WSSV only (105 copies/ml) (100 μl/shrimp) and control miRNA were used as controls. For each treatment, 20 shrimp were used. Shrimp gills were collected at different time points postinfection with WSSV. Three shrimp specimens from each treatment were randomly collected for later use.

All experiments were biologically repeated three times.

Detection of WSSV copies through quantitative real-time PCR.

For quantification of WSSV copies in shrimp, the WSSV genome was extracted from virus-infected shrimp tissues using an SQ tissue DNA kit (Omega Bio-Tek, USA), and 150 ng of genomic DNA was used for quantitative real-time PCR analysis. Quantitative real-time PCR was subsequently conducted using WSSV-specific primers (5′-TTGGTTTCAGCCCGAGATT-3′ and 5′-CCTTGGTCAGCCCCTTGA-3′) and a WSSV-specific TaqMan probe (5′-FAM-TGCTGCCGTCTCCAA-TAMRA-3′, where FAM is 6-carboxyfluorescein and TAMRA is 6-carboxytetramethylrhodamine). A linearized plasmid with a 1,400-bp DNA fragment obtained from the WSSV genome was quantified and serially diluted 10-fold for use as an internal standard for quantitative real-time PCR. The real-time PCR mixture (10 μl) contained 5 μl of Premix Ex Taq (TaKaRa, Japan), 0.5 μl of the extracted DNA template or the internal standard plasmid, 0.2 μl of 10 μM (each) primers, and 0.15 μl of 10 μM TaqMan fluorogenic probe. The real-time PCR conditions were as follows: 95°C for 1 min, followed by 45 cycles at 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s.

Western blot analysis.

Shrimp gills were homogenized with a lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, pH 7.8) and then centrifuged at 10,000 × g for 10 min at 4°C. The supernatant proteins were separated by 12.5% SDS-polyacrylamide gel electrophoresis and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% nonfat milk in Tris-buffered saline–Tween 20 (TBST; 10 mM Tris-HCl, 150 mM NaCl, 20% Tween 20, pH 7.5) for 2 h at room temperature, followed by overnight incubation with a primary antibody. The antibody against shrimp Dorsal, ALF, or β-actin was prepared in our laboratory. The Western blot analyses indicated that all antibodies prepared were specific (data not shown). After being washed three times with TBST, the membrane was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (Roche, Switzerland) for 3 h. After rinsing, the membrane was detected with BCIP (5-bromo-4-chloro-3-indolylphosphate)–nitroblue tetrazolium substrate (Sangon Biotech, Shanghai, China).

Statistical analysis.

Data collected from three independent experiments were processed using one-way analysis of variance to calculate the means and standard deviations (SD) from the triplicate assays. Significant differences between treatments were determined using Student's t test, and a P value of <0.05 or <0.01 indicated statistical significance.

Accession number(s).

The sequences of the following genes have been submitted to GenBank: Spz (GenBank accession no. KX424932), Toll1 (GenBank accession no. KX424933), Toll2 (GenBank accession no. KX424934), Toll3 (GenBank accession no. KX424935), Toll4 (GenBank accession no. KX424936), Toll5 (GenBank accession no. KX424937), Toll6 (GenBank accession no. KX424938), Toll7 (GenBank accession no. KX424939), Dorsal (GenBank accession no. KX424930), and ALF (GenBank accession no. KX424931).

ACKNOWLEDGMENTS

This work was financially supported by the National Natural Science Foundation of China (31430089, 31572647), the Natural Science Fund of Colleges and Universities in Jiangsu Province (14KJA240002), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Q.R., X.H., Y.C., and J.S. carried out the experiments. X.Z., Q.R., and W.W. designed the experiments and analyzed the data. X.H., Q.R., and X.Z. wrote the manuscript.

We declare no conflict of interest.

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