Identification of Functional Gene Modules Associated With STAT-Mediated Antiviral Responses to White Spot Syndrome Virus in Shrimp.
ABSTRACT: White spot syndrome virus (WSSV) is one of the major threats to shrimp aquaculture. It has been found that the signal transducer and activator of transcription (STAT) protein plays an important role in the antiviral immunity of shrimp with a WSSV infection. However, the mechanism that underlies the STAT-mediated antiviral responses in shrimp, against WSSV infection, remains unclear. In this work, based on the gene expression profiles of shrimp with an injection of WSSV and STAT double strand RNA (dsRNA), we constructed a gene co-expression network for shrimp and identified the gene modules that are possibly responsible for STAT-mediated antiviral responses. These gene modules are found enriched in the regulation of the viral process, JAK-STAT cascade and the regulation of immune effector process pathways. The gene modules identified here provide insights into the molecular mechanism that underlies the STAT-mediated antiviral response of shrimp, against WSSV.
Project description:Although the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is part of the antiviral response in arthropods such as Drosophila, here we show that white spot syndrome virus (WSSV) uses a shrimp STAT as a transcription factor to enhance viral gene expression in host cells. In a series of deletion and mutation assays using the WSSV immediate-early gene ie1 promoter, which is active in shrimp cells and also in insect Sf9 cells, an element containing a STAT binding motif was shown to be important for the overall level of WSSV ie1 promoter activity. In the Sf9 insect cell line, a specific protein-DNA complex was detected by using electrophoresis mobility shift assays (EMSA) with the 32P-labeled STAT binding motif of the WSSV ie1 promoter as the probe. When recombinant Penaeus monodon STAT (rPmSTAT) was overexpressed in Sf9 cells, EMSA with specific antibodies confirmed that the STAT was responsible for the formation of the specific protein-DNA complex. Another EMSA showed that in WSSV-infected P. monodon, levels of activated PmSTAT were higher than in WSSV-free P. monodon. A transactivation assay of the WSSV ie1 promoter demonstrated that increasing the level of rPmSTAT led to dose-dependent increases in ie1 promoter activity. These results show that STAT directly transactivates WSSV ie1 gene expression and contributes to its high promoter activity. We conclude that WSSV successfully annexes a putative shrimp defense mechanism, which it uses to enhance the expression of viral immediate-early genes.
Project description: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.
Project description:MicroRNAs (miRNAs) have important roles in post-transcriptional regulation of gene expression. During viral infection, viruses utilize hosts to enhance their replication by altering cellular miRNAs. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays crucial roles in the antiviral responses. In this study, two miRNAs (miR-9041 and miR-9850) from Macrobrachium rosenbergii were found to promote white spot syndrome virus (WSSV) replication. The up-regulation of miR-9041 or miR-9850 suppresses STAT expression in the gills of M. rosenbergii, which subsequently down-regulates the expression of its downstream dynamin (Dnm) genes: Dnm1, Dnm2, and Dnm3. Knockdown of miR-9041 and miR-9850 restricts WSSV replication by up-regulating STAT and Dnm gene expression. The silencing of STAT, Dnm1, Dnm2, or Dnm3 led to an increase of the number of WSSV copies in shrimp. The injection of recombinant Dnm1, Dnm2, or Dnm3 proteins could inhibit WSSV replication in vivo. Overall, our research indicates the roles of host miRNAs in the enhancement of WSSV replication by regulating the host JAK/STAT pathway.
Project description:White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including ?, ?2, ?2 and ?2. Knockdown of clathrin coat AP17, or ? subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2? and ? subunits of P. monodon are reported. PmAP-2 ? was up-regulated about 4-fold at 6 and 36?h post-WSSV infection. Knockdown of PmAP-2? delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2? co-localized with WSSV particles at plasma membrane. In addition, PmAP-2?-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2? is associated with the JAK/STAT and the Toll pathway.
Project description:MicroRNAs (miRNAs), the small non-coding RNAs, play a pivotal role in post-transcriptional gene regulation in various cellular processes. However, the miRNA function in shrimp antiviral response is not clearly understood. This research aims to uncover the function of pmo-miR-315, a white spot syndrome virus (WSSV)-responsive miRNAs identified from Penaeus monodon hemocytes during WSSV infection. The expression of the predicted pmo-miR-315 target mRNA, a novel PmPPAE gene called PmPPAE3, was negatively correlated with that of the pmo-miR-315. Furthermore, the luciferase assay indicated that the pmo-miR-315 directly interacted with the target site in PmPPAE3 suggesting the regulatory role of pmo-miR-315 on PmPPAE3 gene expression. Introducing the pmo-miR-315 into the WSSV-infected shrimp caused the reduction of the PmPPAE3 transcript level and, hence, the PO activity activated by the PmPPAE3 whereas the WSSV copy number in the shrimp hemocytes was increased. Taken together, our findings state a crucial role of pmo-miR-315 in attenuating proPO activation via PPAE3 gene suppression and facilitating the WSSV propagation in shrimp WSSV infection.
Project description: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.
Project description:Viruses, in particular DNA viruses, generate microRNAs (miRNAs) to control the expression of host and viral genes. Due to their essential roles in virus-host interactions, viral miRNAs have attracted extensive investigations in recent years. To date, however, most studies on viral miRNAs have been conducted in cell lines. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blotting, the expression of viral miRNAs was tissue specific in vivo. The results indicated that the viral miRNA WSSV-miR-N24 could target the shrimp caspase 8 gene, and this miRNA further repressed the apoptosis of shrimp hemocytes in vivo. As a result, the number of WSSV copies in shrimp in vivo was significantly increased compared with the control level (WSSV only). Therefore, our study presents the first report on the in vivo molecular events of viral miRNA in antiviral apoptosis.
Project description:In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis, the expressions of viral miRNAs were tissue-specific in vivo. In this study, the viral miRNAs from white spot syndrome virus (WSSV) were characterized in shrimp in vivo. On the basis of our previous study and small RNA sequencing in this study, a total of 89 putative WSSV miRNAs were identified. As revealed by miRNA microarray analysis and Northern blots, the expressions of viral miRNAs were tissue-specific in vivo. Therefore, our study presented the first report on the in vivo molecular events of viral miRNA in the antiviral apoptosis.
Project description:Recent studies have shown that the ubiquitin (Ub) proteasome pathway (UPP) is closely related to immune defense. We have identified a ubiquitin-conjugating enzyme, E2, from the Chinese white shrimp, Fenneropenaeus chinensis (FcUbc). Injection of recombinant FcUbc protein (rFcUbc) reduced the mortality of shrimp infected with white spot syndrome virus (WSSV) and inhibited replication of WSSV. rFcUbc, but not a mutant FcUbc (mFcUbc), bound to WSSV RING domains (WRDs) from four potential E3 ligase proteins of WSSV in vitro. Importantly, rFcUbc could ubiquitinate the RING domains (named WRD2 and WRD3) of WSSV277 and WSSV304 proteins in vitro and the two proteins in WSSV-infected Drosophila melanogaster Schneider 2 (S2) cells. Furthermore, overexpression of FcUbc increased ubiquitination of WSSV277 and WSSV304 during WSSV infection. In summary, our study demonstrates that FcUbc from Chinese white shrimp inhibited WSSV replication and could ubiquitinate WSSV RING domain-containing proteins. This is the first report about antiviral function of Ubc E2 in shrimp.
Project description:Many types of small GTPases are widely expressed in eukaryotes and have different functions. As a crucial member of the Rho GTPase family, Cdc42 serves a number of functions, such as regulating cell growth, migration, and cell movement. Several RNA viruses employ Cdc42-hijacking tactics in their target cell entry processes. However, the function of Cdc42 in shrimp antiviral immunity is not clear. In this study, we identified a Cdc42 protein in the kuruma shrimp (Marsupenaeus japonicus) and named it MjCdc42. MjCdc42 was upregulated in shrimp challenged by white spot syndrome virus (WSSV). The knockdown of MjCdc42 and injection of Cdc42 inhibitors increased the proliferation of WSSV. Further experiments determined that MjCdc42 interacted with an arginine kinase (MjAK). By analyzing the binding activity and enzyme activity of MjAK and its mutant, ?MjAK, we found that MjAK could enhance the replication of WSSV in shrimp. MjAK interacted with the envelope protein VP26 of WSSV. An inhibitor of AK activity, quercetin, could impair the function of MjAK in WSSV replication. Further study demonstrated that the binding of MjCdc42 and MjAK depends on Cys271 of MjAK and suppresses the WSSV replication-promoting effect of MjAK. By interacting with the active site of MjAK and suppressing its enzyme activity, MjCdc42 inhibits WSSV replication in shrimp. Our results demonstrate a new function of Cdc42 in the cellular defense against viral infection in addition to the regulation of actin and phagocytosis, which has been reported in previous studies. IMPORTANCE The interaction of Cdc42 with arginine kinase plays a crucial role in the host defense against WSSV infection. This study identifies a new mechanism of Cdc42 in innate immunity and enriches the knowledge of the antiviral innate immunity of invertebrates.