Chang-Jun Guo

The JAK and STAT family members of the mandarin fish Siniperca chuatsi: Molecular cloning, tissues distribution and immunobiological activity

The JAK/STAT signal transduction pathway plays a critical role in host defence against viral and bacterial infections. In the present study, we report cDNA cloning and characterization of the JAK family (mJAK1-3 and mTYK2) and STAT family members (mSTAT1, mSTAT3-6) from the mandarin fish Siniperca chuatsi. To our knowledge, JAK2, TYK2 and STAT6 genes were cloned from fish for the first time. The mJAK family proteins consist of 1112-1177 residues with a FERM domain, an SH2 domain, a pseudokinase domain, and a tyrosine kinase domain. The mSTAT family members contain 716-786 residues with similar architecture, including an N-terminal domain, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a transcription activation domain. Multiple sequence alignments of mJAKs/mSTATs and phylogenetic analysis showed that mJAK1 was closed to mTYK2, and mJAK2 was closed to mJAK3. Quantitative real-time PCR results revealed that mJAK/mSTAT family members were expressed in most tissues examined except muscle. In mandarin fish fry cells, the expressions of IRF-1, Mx, SOCS1 and SOCS3 genes were significantly induced by poly(I:C) stimulation, indicating that the mJAK/mSTAT signal pathway is activated by poly(I:C). Furthermore, expressions of all four mJAKs and four mSTATs were all up-regulated after poly(I:C) stimulation, but expression of mSTAT5 was inhibited by poly(I:C). These results suggest that mandarin fish has the JAK/STAT signal transduction pathways similar to those in mammals, and these signalling pathways may play an important role in regulation of antiviral responses in fish.

Entry of Tiger Frog Virus (an Iridovirus) into HepG2 Cells via a pH-Dependent, Atypical, Caveola-Mediated Endocytosis Pathway

ABSTRACT Tiger frog virus (TFV), in the genus Ranavirus of the family Iridoviridae, causes high mortality of cultured tiger frog tadpoles in China. To explore the cellular entry mechanism of TFV, HepG2 cells were treated with drugs that inhibit the main endocytic pathways. We observed that TFV entry was inhibited by NH4Cl, chloroquine, and bafilomycin, which can all elevate the pH of acidic organelles. In contrast, TFV entry was not influenced by chlorpromazine or overexpression of a dominant-negative form of Esp15, which inhibit the assembly of clathrin-coated pits. These results suggested that TFV entry was not associated with clathrin-mediated endocytosis, but was related to the pH of acidic organelles. Subsequently, we found that endocytosis of TFV was dependent on membrane cholesterol and was inhibited by the caveolin-1 scaffolding domain peptide. Dynamin and actin were also required for TFV entry. In addition, TFV virions colocalized with the cholera toxin subunit B, indicating that TFV enters as caveola-internalized cargo into the Golgi complex. Taken together, our results demonstrated that TFV entry occurs by caveola-mediated endocytosis with a pH-dependent step. This atypical caveola-mediated endocytosis is different from the clathrin-mediated endocytosis of frog virus 3 (FV3) by BHK cells, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection in lower vertebrates.

Infectious Spleen and Kidney Necrosis Virus (a Fish Iridovirus) Enters Mandarin Fish Fry Cells via Caveola-Dependent Endocytosis

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. Megalocytiviruses have been implicated in more than 50 fish species infections and currently threaten the aquaculture industry, causing great economic losses in China, Japan, and Southeast Asia. However, the cellular entry mechanisms of megalocytiviruses remain largely uncharacterized. In this study, the main internalization mechanism of ISKNV was investigated by using mandarin fish fry (MFF-1) cells. The progression of ISKNV infection is slow, and infection is not inhibited when the cells are treated with ammonium chloride (NH4Cl), chloroquine, sucrose, and chlorpromazine, which are inhibitors of clathrin-dependent endocytosis. The depletion of cellular cholesterol by methyl-β-cyclodextrin results in the significant inhibition of ISKNV infection; however, the infection is resumed with cholesterol replenishment. Inhibitors of caveolin-1-involved signaling events, including phorbol 12-myristate 13-acetate (PMA), genistein, and wortmannin, impair ISKNV entry into MFF-1 cells. Moreover, ISKNV entry is dependent on dynamin and the microtubule cytoskeleton. Cofraction analysis of ISKNV and caveolin-1 showed that ISKNV colocates with caveolin-1 during virus infection. These results indicate that ISKNV entry into MFF-1 cells proceeds via classical caveola-mediated endocytosis and is dependent on the microtubules that serve as tracks along which motile cavicles may move via a caveola-caveosome-endoplasmic reticulum (ER) pathway. As a fish iridovirus, ISKNV entry into MFF-1 cells is different from the clathrin-mediated endocytosis of frog virus 3 entry into mammalian cells (BHK-21) at 28°C, which has been recognized as a model for iridoviruses. Thus, our work may help further the understanding of the initial steps of iridovirus infection.

Involvement of caveolin-1 in the Jak–Stat signaling pathway and infectious spleen and kidney necrosis virus infection in mandarin fish (Siniperca chuatsi)

Abstract Caveolae, the major source of caveolin-1 protein, are specialized invaginated microdomains of the plasma membrane that act as organizing centers for signaling molecules in the immune system. In the present study, we report the cloning and characterization of caveolin-1 (mCav-1) from mandarin fish (Siniperca chuatsi) and study on the roles of mCav-1 in the fish Jak–Stat signaling pathway and in virus infection. The cDNA sequence of mCav-1 was 707bp in size, encoding a protein of 181 amino acids, which was different from the mammalian protein (178 amino acids). The deduced amino acid sequence of mCav-1 shared similar architecture with vertebrate caveolin-1 proteins, but mCav-1 lacked a phosphorylation site (y14). The major subcellular location of mCav-1 was in the caveolae, where the protein appeared to have major functions. Real-time PCR revealed that the expression of the mandarin fish Mx, IRF-1, SOCS1, and SOCS3 genes involved in the poly(I:C)-induced Jak–Stat signaling pathway was impaired by the mCav-1 scaffolding domain peptide (mSDP). In mandarin fish fry (MFF-1) cells, the protein levels of mCav-1 were markedly up-regulated at 12 and 24h post-infection with ISKNV (infectious spleen and kidney necrosis virus). In addition, ISKNV entry into MFF-1 cells was significantly inhibited by mSDP, and the inhibition was dose-dependent. Thus, ISKNV infection was apparently associated with mCav-1 protein and may utilize the caveolae-related endocytosis pathway. The findings reported here further our understanding of the function of caveolin-1 in the complex signal transduction network in fish immune systems and in the cellular entry mechanism of iridoviruses.

A novel viral SOCS from infectious spleen and kidney necrosis virus: interacts with Jak1 and inhibits IFN-α induced Stat1/3 activation.

Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-α-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations (F18D, S66A, S85A, and R64K) of ISKNV-vSOCS significantly impaired the inhibition of IFN-α-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-α-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.

Mandarin fish caveolin-1 interaction with major capsid protein of infectious spleen and kidney necrosis virus and its role in early stages of infection

ABSTRACT Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus from the family Iridoviridae. ISKNV is one of the major agents that cause mortality and economic losses to the freshwater fish culture industry in Asian countries, particularly for mandarin fish (Siniperca chuatsi). In the present study, we report that the interaction of mandarin fish caveolin 1 (mCav-1) with the ISKNV major capsid protein (MCP) was detected by using a virus overlay assay and confirmed by pulldown assay and coimmunoprecipitation. This interaction was independent of the classic caveolin 1 scaffolding domain (CSD), which is responsible for interacting with several signaling proteins and receptors. Confocal immunofluorescence microscopy showed that ISKNV MCP colocalized with mCav-1 in the perinuclear region of virus-infected mandarin fish fry (MFF-1) cells, which appeared as soon as 4 h postinfection. Subcellular fractionation analysis showed that ISKNV MCP was associated with caveolae in the early stages of viral infection. RNA interference silencing of mCav-1 did not change virus-cell binding but efficiently inhibited the entry of virions into the cell. Taken together, these results suggested that mCav-1 plays an important role in the early stages of ISKNV infection.

The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase β

The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase β protein (scIKKβ), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKβ. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

The potential role of microfilaments in host cells for infection with infectious spleen and kidney necrosis virus infection

BackgroundInfectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus from the family Iridoviridae. Megalocytivirus causes severe economic losses to tropical freshwater and marine culture industry in Asian countries and is devastating to the mandarin fish farm industry in China particularly.MethodsWe investigated the involvement of microfilaments in the early and late stages of ISKNV infection in MFF-1 cells by selectively perturbing their architecture using well-characterized inhibitors of actin dynamics. The effect of disruption of actin cytoskeleton on ISKNV infection was evaluated by indirect immunofluorescence analysis or real-time quantitative PCR.ResultsThe depolymerization of the actin filaments with cytochalasin D, cytochalasin B, or latrunculin A reduced ISKNV infection. Furthermore, depolymerization of filamentous actin by inhibitors did not inhibit binding of the virus but affected virus internalization in the early stages of infection. In addition, the depolymerization of actin filaments reduced total ISKNV production in the late stages of ISKNV.ConclusionsThis study demonstrated that ISKNV required an intact actin network during infection. The findings will help us to better understand how iridoviruses exploit the cytoskeleton to facilitate their infection and subsequent disease.

Molecular cloning of IKKβ from the mandarin fish Siniperca chuatsi and its up-regulation in cells by ISKNV infection.

The IκB kinase β (IKKβ) plays crucial roles in regulating activation of nuclear factor-kappa B (NF-κB) in response to proinflammatory factors and microbial and viral infections. Here, we report the cloning of an IKKβ cDNA (named SicIKKβ) from the mandarin fish Siniperca chuatsi. The full-length cDNA is 4052bp and contains an ORF that encodes a predicted protein of 743-amino acid residues. The deduced amino acid sequence of SicIKKβ has the same domain organization as human IKKβ, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. Quantitative RT-PCR showed that SicIKKβ was ubiquitously expressed in tissues of mandarin fish, and its expression in mandarin fish fry (MFF-1) cells was up-regulated during the course of ISKNV infection.

Budding of Tiger Frog Virus (an Iridovirus) from HepG2 Cells via Three Ways Recruits the ESCRT Pathway.

The cellular endosomal sorting complex required for transport (ESCRT) pathway is a multifunctional pathway involved in cell physiological activities. While the majority of RNA viruses bearing L-domains are known to hijack the ESCRT pathway to complete the budding process, the budding of large and complex enveloped DNA viruses, especially iridoviruses, has been rarely investigated. In the present study, we use the tiger frog virus (TFV) as a model to investigate whether iridoviruses are released from host cells through the ESCRT pathway. Inhibition of class E proteins and auxiliary proteins (VPS4A, VPS4B, Tsg101, Alix, and Nedd4.1) reduces extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in TFV release. The respective interactions of TFV VP031L, VP065L, VP093L with Alix, Tsg101, Nedd4 suggest the underlying molecular mechanism by which TFV gets access to the ESCRT pathway. Co-depletion of Alix, Tsg101, and Nedd4.1 induces a significant reduction in extracellular virion production, which implies the functional redundancy of host factors in TFV budding. Those results are first observation that iridovirus gains access to ESCRT pathway through three ways of interactions between viral proteins and host proteins. Our study provides a better understanding of the budding mechanism of enveloped DNA viruses.