A white spot syndrome virus microRNA promotes the virus infection by targeting the host STAT

JAK/STAT pathway plays an important role in invertebrates during virus infection. However the microRNA (miRNA)-mediated regulation of JAK/STAT is not intensively investigated. Viral miRNAs, encoded by virus genome, have emerged as important regulators in the virus-host interactions. In this study, a WSSV (white spot syndrome virus)-encoded miRNA (WSSV-miR-22) was characterized in shrimp during virus infection. The results showed that the viral miRNA could promote WSSV infection in shrimp by targeting the host STAT gene. When the expression of JAK or STAT was knocked down by sequence-specific siRNA, the WSSV copies in shrimp were significantly increased, indicating that the JAK/STAT played positive roles in the antiviral immunity of shrimp. The further findings revealed that TEP1 and TEP2 were the effectors of JAK-STAT signaling pathway. The silencing of TEP1 or TEP2 led to an increase of WSSV copies in shrimp, showing TEP1 and TEP2 were involved in the shrimp immune response against virus infection. Therefore our study presented a novel viral miRNA-mediated JAK/STAT-TEP1/TEP2 signaling pathway in virus infection.

Scientific RepoRts | 5:18384 | DOI: 10.1038/srep18384 polymerase gene 13 . In the marine animal virus white spot syndrome virus (WSSV), the viral miRNAs WSSV-miR-66 and WSSV-miR-68 could target the WSSV genes and further promote WSSV infection 14 . All the viral target genes play negative roles in the WSSV infection in shrimp, indicating that the virus could encode some viral proteins to precisely balance the virus invasion and virus latency in animals 14 .
WSSV is one of the most devastating shrimp pathogens found in farmed penaeid shrimp and other crustaceans, and it has caused serious damage to the world wide shrimp culture industry 15 Toll, immune deficiency (IMD) and Janus family tyrosine kinase and signal transducer and activator of transcription (JAK/STAT) pathways are regarded as three main signaling pathways regulating humoral immunity of shrimp 16 . Recently, increasing evidences have suggested that JAK/STAT pathway plays an important role in invertebrate organisms during virus infection 17 . In mammals, infectious virus induces the production of interferon and interleukin, which is recognized by cytokine receptors, and then leads to the activation of JAK, which in turn phosphorylates the cytoplasmic domain of the receptor to allow recruitment and phosphorylation of STAT. Activated STAT dimers translocate to the cell nucleus and bind to specific DNA sites, where they act as activators of transcription mechanism 18 . In Drosophila, this pathway activates at least two gene families, thioester-containing protein (TEP) and TOT 19 . TEP families of proteins, which are characterized by a unique intrachain β -cysteinyl-γ -glutamyl thioester bond, are classified into two subfamilies: the alpha-2-macroglobulin (A2M) subfamily and the C3 subfamily 20 . Pl-A2M2 from Pseudomonas libanensis/gessardii may be important for the immune defense in crayfish intestine and function as a pattern recognition protein in crayfish cuticular tissues 21 .
As reported, WSSV can target host STAT. However, instead of inhibiting or disrupting its activity, WSSV exploits the host STAT by using it to bind to the promoter region of the WSSV immediate early gene ie1 and thus enhance ie1 transcription 17 . To better understand the miRNA-mediated regulation of JAK/STAT pathway in shrimp during virus infection, a WSSV-encoded miRNA (WSSV-miR-22) was characterized in the present study. The results indicated that the viral miRNA could promote WSSV infection in shrimp by targeting the host STAT gene.

Effects of WSSV-miR22 on virus infection in shrimp.
In an attempt to reveal the roles of viral miRNA WSSV-miR-22 in virus infection, the expression profile of WSSV-miR-22 was characterized in shrimp in vivo. Northern blotting data indicated that WSSV-miR-22 could be detected in WSSV-challenged shrimp at 24, 36 and 48 h post-infection (Fig. 1A). Then WSSV-miR-22 was overexpressed in shrimp. Under the condition that WSSV-miR-22 was overexpressed in shrimp, the number of WSSV copies was examined. As shown in Fig. 1B, the overexpression of WSSV-miR-22 led to significant increases of WSSV copies from 24 h to 48 h post-infection compared with the controls (WSSV-miR-22-scrambled and WSSV only). On the contrary, when the WSSV-miR-22 expression was knocked down by AMO-WSSV-miR-22, the WSSV copies were significantly decreased by comparison with the controls (Fig. 1C). These data indicated that WSSV-miR-22 played a positive role in the virus replication.
To evaluate the effects of WSSV-miR-22 on the expressions of immediate early (ie) genes of WSSV, the ie1 expression in shrimp treated with WSSV and WSSV-miR-22-mimic or AMO-WSSV-miR-22 was examined. The results showed that the WSSV-miR-22 overexpression led to a significant increase of the ie1 expression compared with the positive control WSSV only, while the control miRNA had no effect on the expression of ie1 (Fig. 1D). The WSSV-miR-22 silencing significantly decreased the ie1 mRNA level (Fig. 1E). The results indicated that WSSV-miR-22 could promote the expression of WSSV immediate early gene.
Taken together, these findings presented that the viral miRNA took a positive effect on the virus infection.
The interaction between viral miRNA and host STAT gene. To reveal the pathways mediated by the viral miRNA, the target genes of WSSV-miR-22 were analyzed. Based on the target prediction using the TargetScan, miRanda, and Pictar algorithms, it was found that WSSV-miR22 could target the shrimp STAT gene ( Fig. 2A), which is reported to be involved in the antiviral immunity of shrimp 17  To characterize the interaction between the viral miRNA and the host STAT gene, the synthesized WSSV-miR-22 and the plasmid EGFP-STAT consisting of EGFP and the STAT 3′ UTR were co-transfected into insect High Five cells (Fig. 2B). The results showed that the fluorescence intensity in the co-transfected cells was significantly decreased compared with the intensity in the control cells (Fig. 2B,C). These data presented that WSSV-miR-22 was directly interacted with the STAT gene.
In order to explore the interaction between WSSV-miR-22 and STAT in vivo, WSSV-miR-22 was overexpressed or silenced in shrimp, followed by the detection of STAT mRNA level. The results indicated that the WSSV-miR-22 overexpression led to significantly decreases of the STAT expression compared with the positive control WSSV only, while the control miRNA had no effect on the expression of STAT, showing that WSSV-miR-22 inhibited the expression of STAT gene in shrimp (Fig. 2D). When the expression of WSSV-miR-22 was knocked down by AMO-WSSV-miR-22, the expression of STAT was significantly upregulated (Fig. 2E). These findings showed that WSSV-miR-22 could interact with the host STAT gene in vivo by targeting its 3′ UTR.
The role of host STAT in virus infection. The data showed that the host STAT gene was the target of WSSV-miR-22. Therefore the role of STAT in virus infection was explored in shrimp. Quantitative real-time PCR indicated that the STAT mRNA was detected in all the shrimp tissues examined (Fig. 3A). The results revealed that the shrimp STAT was significantly upregulated in response to the WSSV infection (Fig. 3B) To evaluate the influence of STAT on virus infection, the STAT gene expression was knocked down by sequence-specific siRNA (STAT-siRNA) in shrimp in vivo, followed by the detection of virus copy. It was revealed that the expression of STAT gene was silenced compared with the control WSSV only, while STAT-siRNA-scrambled had no effect on the STAT gene expression (Fig. 3C), showing that the siRNA was highly specific. Under In all panels, asterisks indicated significant differences (*p < 0.05; **p < 0.01) between treatments.
the condition that the expression of STAT gene was knocked down, the number of WSSV copies in shrimp was significantly increased compared with the control (WSSV only) (Fig. 3D). The data indicated that the host STAT gene played a negative role in the WSSV infection in vivo. The influence of host JAK gene on virus infection. JAK/STAT signaling pathway has been proved to be very important in antiviral process of vertebrate 22 and invertebrate 17,23 . JAK, as one of key components of JAK/ STAT pathway, is upstream gene of STAT. Therefore the effect of host JAK on virus infection was investigated. As shown in Fig. 4A, the expression of JAK was detected in all the examined tissues and it was mainly expressed in the stomach and intestine tissues of shrimp. In response to the WSSV infection, JAK was significantly upregulated in shrimp (Fig. 4B). The results suggested that JAK was involved in the virus infection in shrimp.
Under the condition that JAK gene expression was knocked down by JAK-siRNA in shrimp (Fig. 4C), the number of WSSV copies was evaluated. The data presented that the silencing of JAK gene expression led to significant increases of virus copies compared with the control (WSSV only) (Fig. 4D). However, JAK-siRNA-scrambled took no effect on the WSSV infection (Fig. 4D). These findings indicated that JAK played an important role in the virus infection in vivo.
The effects of host TEP1 and TEP2 on virus infection. In Drosophila melanogaster, it is reported that the expressions of TEP1 and TEP2 are JAK/STAT-dependent and upregulated in response to the bacteria challenge 21 , suggesting the involvement of TEP1 and TEP2 in the innate immunity of invertebrates. To evaluate the roles of TEP1 and TEP2 in the virus infection, the two genes were characterized in shrimp. The results indicated that both mRNAs of TEP1 and TEP2 were detected in all the examined tissues, sharing the similar tissue distributions to those of JAK/STAT (Fig. 5A). In response to the WSSV infection, the expressions of TEP1 and TEP2 were significantly upregulated (Fig. 5B), indicating that the two genes played very important roles in the virus infection. As a control, STAT-siRNA-scrambled was included in the injections. WSSV alone was used as a positive control. (D) The influence of STAT silencing on virus infection. The WSSV copies in gills of STAT-siRNA-treated shrimp were quantified using quantitative real-time PCR. In all panels, statistically significant differences between treatments were indicated by asterisks (**p < 0.01).
Scientific RepoRts | 5:18384 | DOI: 10.1038/srep18384 To explore the effects of TEP1 and TEP2 on the virus infection, the two genes' expressions were silenced, followed by the detection of WSSV copies in shrimp. The data presented that the expressions of TEP1 and TEP2 were knocked down by sequence-specific siRNAs (Fig. 5C). The results showed that the TEP1 silencing and the TEP2 silencing resulted in significant increases of WSSV copies compared with the control (WSSV only), while the TEP1-siRNA-scrambled and TEP2-siRNA-scrambled had no effect on the virus infection (Fig. 5D). These data demonstrated that both TEP1 and TEP2 took great effects on the virus infection in shrimp.

The pathway mediated by viral miRNA in virus infection.
The above data presented that WSSV-miR-22, JAK, STAT, TEP1 and TEP2 were involved in the virus infection in shrimp in vivo. Therefore the pathway mediated by the viral miRNA was further explored. The results indicated that the expressions of TEP1 and TEP2 were significantly downregulated when the expression of WSSV-miR-22 was overexpressed in shrimp (Fig. 6A), suggesting that the viral miRNA, TEP1 and TEP2 shared the same pathway.
To reveal the relationship between STAT and TEPs (TEP1 and TEP2), the expression of STAT was knocked down by STAT-siRNA in shrimp. It was found that the STAT silencing led to significant decreases of both TEP1 and TEP2 mRNA levels compared with the controls (Fig. 6B), showing that TEP1 and TEP2 were the downstream genes of STAT. To evaluate the effect of JAK expression silencing on the expressions of TEP1 and TEP2, the JAK-siRNA-treated shrimp, which were simultaneously infected with WSSV, were subjected to the detections of TEP1 and TEP2 mRNA levels. The quantitative real-time PCR data presented that both the TEP1 and TEP2 were downregulated in the JAK-silenced shrimp by comparison with the controls (Fig. 6C).
Taken together, these findings revealed that the viral miRNA (WSSV-miR-22) could inhibit the JAK/ STAT-TEP1/TEP2 signaling pathway by targeting the host STAT gene, leading to the virus infection in shrimp (Fig. 6D). In all panels, statistically significant differences between treatments were indicated by asterisks (**p < 0.01). The sequence-specific TEP1-siRNA or TEP2-siRNA was injected into shrimp. Then the gene expression was examined. As a negative control, siRNA-scrambled was included in the injections. WSSV alone was used as a positive control. (D) The detection of WSSV copies in shrimp. The gills of siRNA-treated shrimp were subjected to the quantitative real-time PCR analysis to examine the WSSV copies. WSSV alone was used as a positive control. In all panels, asterisks indicated significant differences between treatments (**p < 0.01).

Discussion
The activation/inactivation of transcription factors affect the expressions of a large number of genes, leading to the changes of biological processes. During the virus-host interactions, the regulation of transcription factors' expression becomes the key issues. The host's transcription factors, such as STAT, are often selected by virus as targeted sites. Through the protein-protein interactions, the host's transcription factors can be utilized by virus to enhance its infection. It is evidenced that STAT, an important transcription factor, is involved in the course of virus infection. The measles virus (MV) phosphoprotein (P) can interact with the linker domain of STAT1 and subsequently inhibit the JAK/STAT activation 24 . Hepatitis C virus (HCV) core protein is required for the production of infectious viruses through the interaction with the JAK protein 25 . On the basis of protein-protein interactions, the activity of transcription factor can be turned off by virus. However, this turnoff of transcription factor activity may result in disorders of many biological processes. In recent years, it is found that miRNAs, a kind of regulators participating in the post-transcription regulations of large number of protein-encoding genes, can regulate the genes' expressions by fine tuning 26 . In this context, miRNA-mediated expression regulation of transcription factor may be a better strategy for the virus-host interactions. To reveal the mechanisms of virus-host interactions, the host miRNAs involved in the regulation of transcription factor expression have attracted more and more interests. In human, the host miR-146 and miR-21 are used by human immunodeficiency virus (HIV) and HCV to downregulate the expressions of IRAK1 and TRAF6, leading to the decrease of the activity of NF-κ B 27,28 . The upregulation of human miR-373 by the HCV infection can target the JAK gene and then impair STAT phosphorylation and inhibit the JAK/STAT signal pathway 29 . Up to date, however, the regulation of transcription factor expression mediated by the virus miRNAs has not been explored. In this study, the results indicated that the viral miRNA could promote the WSSV infection by targeting the shrimp STAT. Our study revealed a novel viral miRNA-mediated JAK/STAT-TEP1/ TEP2 signaling pathway in the virus-host interactions.
At present, it is reported that WSSV can encode more than 80 viral miRNAs 30,31 . Among these viral miRNAs, WSSV-miR-N24 targets shrimp caspase 8 gene, leading to the inhibition of apoptotic activity and the promotion of virus infection 32 . Both WSSV-miR-66 and WSSV-miR-68 can enhance virus replication by inhibiting the virus genes' expression 14 . In the present study, the findings contributed a novel aspect of viral miRNA in the virus infection by targeting the host's transcription factor gene. As well known, the JAK/STAT signaling pathway is highly conserved from vertebrates to invertebrates and plays an important role in the antiviral immune response 29,33 . The activation of STAT, the transcription factor of JAK/STAT signaling pathway, triggers expressions of the effect genes. However, the effect genes regulated by the JAK/STAT pathway have not extensively characterized. In the present investigation, it was indicated that the expressions of TEP1 and TEP2 were regulated by JAK/STAT. TEPs have three different families, including alpha-2-macroglobulins (A2Ms), C3/C4/C5 complement factors, and insect TEPs (iTEPs) 34 . Macroglobulin complement-related factors, which belong to the iTEP family, are crucial effectors to defense the flaviviral infection of Aedes aegypti 35 . Our study revealed that the shrimp TEP1 and TEP2, the effectors of JAK-STAT signaling pathway, played important roles in the virus-host interactions. In this context, the regulation of transcription factor's expression mediated by viral miRNA might represent a key issue in the virus-host interactions.

Materials and Methods
Shrimp culture and WSSV challenge. The Marsupenaeus japonicas shrimp (approximately 10 g in body weight and 10 to 12 cm in length) were purchased from an aquaculture market in Hangzhou, Zhejiang Province, China. Before treatments, the shrimp were cultured in groups of 20 individuals in laboratory tanks containing 80 liters of aerated seawater at room temperature. To ensure that the shrimp were WSSV-free before experiments, PCR using WSSV-specific primers (5′ -TATTGTCTC TCCTGACGTAC-3′ and 5′ -CACATTCTTCACGAGTCTAC-3′ ) were conducted. Then the virus-free shrimp were infected with 100 μ l of WSSV virus solution at 10 5 copies/ml by intramuscular injection into the lateral area of the fourth abdominal segment. At different times post-infection (0, 24, 36, and 48 h), five shrimp were randomly collected for each treatment and stored for later use. In the following assays, the shrimp gills were employed. As well known, the shrimp gill is one of the immune organs and is an important target organ of WSSV infection. On the other hand, the shrimp JAK, STAT, Tep1 and Tep2 genes are highly expressed in shrimp gills.
The detection of miRNA by Northern blotting. Total RNAs were extracted from shrimp gills by using the mirVanaP TMP miRNA isolation kit (Ambion, USA) according to the manufacturer's protocol. After separation on a denaturing 15% polyacrylamide gel containing 8 M urea, the small RNAs were transferred to a Hybond-N+ membrane (Amersham Biosciences, Buckinghamshire, United Kingdom). Subsequently, the RNAs were cross-linked under UV light (Ultra-Violet Products Ltd., USA). The membrane was prehybridized in DIG Easy Hyb Granules buffer (Roche, Basel, Switzerland) for 30 min and then hybridized with digoxigenin (DIG)-labeled probes completely complementary to WSSV-miR-22 (5′ -UUUCCUUACGAAUGAAAAGUAA-3′ ) at 42 °C overnight. The DIG-labeled U6 probe (5′ -GGGCCATGCTAATCTTCTCTGTATCGTT-3′ ) was used as a control. Immunological detection was performed using the DIG High Prime DNA labeling and detection starter kit II (Roche, Basel, Switzerland).
For each treatment, 20 shrimp were used. At different times post-infection (0, 24, 36 and 48 h), five shrimp were randomly collected for each treatment and subjected to subsequent analysis. All the experiments were biologically repeated three times.
The quantitative real-time PCR analysis of WSSV copies. Quantitative real-time PCR was performed to examine the WSSV copies in gills of WSSV-infected shrimp. The viral DNA was extracted from shrimp gills using the SQ tissue DNA kit (Omega-Bio-Tek, USA), and then the WSSV copies were detected by real-time PCR with WSSV-specific primers (5′ -TTGGTTTCAGCCCGAGATT-3′ and 5′ -CCTTGGTCAGCCCCTTGA-3′ ) and WSSV-specific TaqMan
Cell culture, transfection, and fluorescence assays. Insect High Five cells (Invitrogen, USA) were cultured at 28 °C in Express Five serum-free medium (SFM) (Invitrogen) containing l-glutamine (Invitrogen). When the cells were at about 70% confluence, they were transfected with 6 μ g of EGFP, EGFP-STAT or EGFP-∆STAT. At the same time, the cells were transfected with 300 nM of either synthesized WSSV-miR-22 or a synthesized control miRNA. All the miRNAs were synthesized by Shanghai Gene Pharma Co., Ltd. (Shanghai, China). The transfections were carried out in triplicate with Cellfectin transfection reagent (Invitrogen) according to the manufacturer's protocol. At 48 h after transfection, the fluorescence of cells was examined with a Flex Station II microplate reader (Molecular Devices, USA) at 490/510 nm for excitation and emission, respectively. The fluorescence values were corrected by subtracting the autofluorescence of cells not expressing EGFP. All the experiments were biologically repeated three times.

Synthesis of siRNAs and
The RNA interference (RNAi) assay was conducted in shrimp by the injection of an siRNA into the lateral area of the fourth abdominal segment at 30 μ g/shrimp using a 1-ml sterile syringe. The siRNA (15 μ g) and WSSV (10 5 copies/ml) were co-injected into virus-free shrimp at a volume of 100 μ l per shrimp. At 16 h after the co-injection, the siRNA (15 μ g) (100 μ l/shrimp) was injected into the same shrimp. At the same time, the siRNAs-scrambled (15 μ g) (100 μ l/shrimp) were co-injected into virus-free shrimp. At 16 h after the co-injection, siRNAs-scrambled (15 μ g) (100 μ l/shrimp) were injected into the same shrimp. WSSV (10 5 copies/ml) (100 μ l/shrimp) alone was included in the injections as a positive control. As a negative control, phosphate-buffered saline (PBS) (0.1M, pH7.4) was used in the injections instead of the siRNAs. For each treatment, 20 shrimps were used. The assays were biologically repeated three times.
Statistical analysis. The data from three independent experiments were analyzed by one-way analysis of variance (ANOVA) to calculate the means and standard deviations (SD) of the triplicate assays 36 .