Yi-Bing Zhang

Molecular regulation of interferon antiviral response in fish.

Interferon (IFN) response is the first line of host defense against virus infection. The recent years have witnessed tremendous progress in understanding of fish IFN antiviral response. Varied number of IFN genes has been identified in different fish species but obviously, they do not show a one-to-one orthologous relationship with mammalian IFN homologs. These genes are divided into two groups with different abilities to induce downstream gene expression through binding to different receptor complexes. Consistently, some fish IFN-stimulated genes such as Mx and PKR have been confirmed for their antiviral effects. In this review, we focus on how fish cells respond to IFNs and how fish IFNs are triggered through TLR pathway and RLR pathway. We highlight the roles of IRF3 and IRF7 in activation of fish IFN response. In addition, the unique mechanisms underlying IRF3/7-dependent fish IFN response and auto-regulation of fish IFN gene expression are discussed.

Fish MITA Serves as a Mediator for Distinct Fish IFN Gene Activation Dependent on IRF3 or IRF7

In mammals, cytosolic sensors retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) activate multiple signaling cascades initiating IFN-α/β expression. IFN regulatory factor 3 (IRF3) is required for the activation of IFN-β, which, in turn, primes the expression of most IFN-α genes by IFN-induced IRF7 through the STAT1 pathway. In fish, RIG-I overexpression inhibits virus infection by induction of IFN response; however, the subtle signaling cascade mechanism remains to be identified. In this study, we clone an ortholog of MITA, a recently identified adaptor responsible for RLR pathway, from crucian carp (Carassius auratus L.), and demonstrate its ability to suppress viral replication through IRF3/7-dependent IFN response. The pivotal signaling molecules of RLR pathway, including RIG-I, melanoma differentiation-associated gene 5, laboratory of genetics and physiology 2, and TANK-binding kinase 1, are also cloned and characterized, confirming that the RLR-mediated IFN activation is conserved from fish to mammals. Further characterization of distinct IFN gene activation reveals that zebrafish IFN1 and IFN3 are induced by the MITA pathway but are dependent on distinct transcription factors. Whereas fish IFN genes cannot be classified into IFN-α or IFN-β, zebrafish IFN1 is primarily regulated by IRF3, thereby resembling that of IFN-β, and zebrafish IFN3 is regulated by IRF7, thereby resembling of those of IFN-αs. In contrast with mammalian IFN-α/β, zebrafish IFN1 and IFN3 are induced by the basally expressed IRF3 or IRF7, both of which are upregulated by IFN and virus infection. Collectively, these data suggest that IFN genes in fish and mammals have evolved independently to acquire a similar mechanism triggering their expression.

Characterization of Fish IRF3 as an IFN-Inducible Protein Reveals Evolving Regulation of IFN Response in Vertebrates

In mammals, IFN regulatory factor (IRF) 3 is a critical player in modulating transcription of type I IFN and IFN-stimulated genes (ISGs). In this study, we describe the roles of crucian carp (Carassius auratus L.) IRF3 in activating fish IFN and ISGs. Fish IRF3 exhibits a large sequence divergence from mammalian orthologs. Whereas mammalian IRF3 is constitutively expressed, fish IRF3 protein is significantly upregulated by IFN, poly-IC, and other stimuli known as IFN inducers in mammals. The IFN-inducible property of fish IRF3 is consistent with the comparative analysis of 5′ flanking regulatory region of vertebrate IRF3 genes, which reveals the presence of typical IFN-stimulated response elements in fish and amphibians, but an absence in tetrapods. Furthermore, either IFN or poly-IC induces phosphorylation and cytoplasmic-to-nuclear translocation of IRF3, which seems essential for its function in that phosphomimic active IRF3 exhibits stronger transactivation than wild type IRF3. Finally, overexpression of fish IRF3 activates production of IFN that in turn triggers ISG transcription through Stat1 pathway, whereas transfection of dominant negative mutant IRF3-DN abrogates poly-IC induction of ISGs, probably owing to blockade of IFN production. Therefore, regulation of IFN response by vertebrate IRF3 is another ancient trait. These data provide evidence of the evolving function of vertebrate IRF3 on regulating IFN response.

Functional Domains and the Antiviral Effect of the Double-Stranded RNA-Dependent Protein Kinase PKR from Paralichthys olivaceus

ABSTRACT The double-stranded RNA (dsRNA)-dependent protein kinase PKR is thought to mediate a conserved antiviral pathway by inhibiting viral protein synthesis via the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). However, little is known about the data related to the lower vertebrates, including fish. Recently, the identification of PKR-like, or PKZ, has addressed the question of whether there is an orthologous PKR in fish. Here, we identify the first fish PKR gene from the Japanese flounder Paralichthys olivaceus (PoPKR). PoPKR encodes a protein that shows a conserved structure that is characteristic of mammalian PKRs, having both the N-terminal region for dsRNA binding and the C-terminal region for the inhibition of protein translation. The catalytic activity of PoPKR is further evidence that it is required for protein translation inhibition in vitro. PoPKR is constitutively transcribed at low levels and is highly induced after virus infection. Strikingly, PoPKR overexpression increases eIF2α phosphorylation and inhibits the replication of Scophthalmus maximus rhabdovirus (SMRV) in flounder embryonic cells, whereas phosphorylation and antiviral effects are impaired in transfected cells expressing the catalytically inactive PKR-K421R variant, indicating that PoPKR inhibits virus replication by phosphorylating substrate eIF2α. The interaction between PoPKR and eIF2α is demonstrated by coimmunoprecipitation assays, and the transfection of PoPKR-specific short interfering RNA further reveals that the enhanced eIF2α phosphorylation is catalyzed by PoPKR during SMRV infection. The current data provide significant evidence for the existence of a PKR-mediated antiviral pathway in fish and reveal considerable conservation in the functional domains and the antiviral effect of PKR proteins between fish and mammals.

Transcription of Il17 and Il17f Is Controlled by Conserved Noncoding Sequence 2

T helper 17 (Th17) cells specifically transcribe the Il17 and Il17f genes, which are localized in the same chromosome region, but the underlying mechanism is unclear. Here, we report a cis element that we previously named conserved noncoding sequence 2 (CNS2) physically interacted with both Il17 and Il17f gene promoters and was sufficient for regulating their selective transcription in Th17 cells. Targeted deletion of CNS2 resulted in impaired retinoic acid-related orphan receptor gammat (RORγt)-driven IL-17 expression in vitro. CNS2-deficient T cells also produced substantially decreased amounts of IL-17F. These cytokine defects were associated with defective chromatin remodeling in the Il17-Il17f gene locus, possibly because of effects on CNS2-mediated recruitment of histone-modifying enzymes p300 and JmjC domain-containing protein 3 (JMJD3). CNS2-deficient animals were also shown to be resistant to experimental autoimmune encephalomyelitis (EAE). Our results thus suggest that CNS2 is sufficient and necessary for Il17 and optimal Il17f gene transcription in Th17 cells.

Molecular characterization and IFN signal pathway analysis of Carassius auratus CaSTAT1 identified from the cultured cells in response to virus infection.

Type I interferon (IFN) exerts its pleiotropic effects mainly through the JAK-STAT signaling pathway, which is presently best described in mammals. By subtractive suppression hybridization, two fish signaling factors, JAK1 and STAT1, had been identified in the IFN-induced crucian carp Carassius auratus L. blastulae embryonic (CAB) cells after treatment with UV-inactivated grass carp hemorrhagic virus (GCHV). Further, the full-length cDNA of STAT1, termed CaSTAT1, was obtained. It contains 2926 bp and encodes a protein of 718 aa. CaSTAT1 is most similar to rat STAT1 with 59% identity overall and displays all highly conserved domains that the STAT family possesses. Like human STAT1beta, it lacks the C-terminus acting as transcriptional activation domain in mammals. By contrast, only a single transcript was detected in virus-induced CAB cells. Expression analysis showed that CaSTAT1 could be activated by stimulation of CAB cells with poly I:C, active GCHV, UV-inactivated GCHV or CAB IFN, and displayed diverse expression patterns similar to that of mammalian STAT1. Additionally, the expression of an antiviral gene CaMx1 was also induced under the same conditions, and expression difference between CaSTAT1 and CaMx1 was revealed by induction of CAB IFN. These results provide molecular evidence supporting the notion that the fish IFN signaling transduction pathway is similar to that in mammals. Fish IFN exerts its multiple functions, at least antiviral action, through a JAK-STAT pathway.

Fish virus-induced interferon exerts antiviral function through Stat1 pathway

Virus-induced interferons (IFNs) have been identified in various fish species and display antiviral activities similar to mammalian type I IFNs. However, apart from the mammalian IFN system, the IFN signaling pathway remains largely unknown. Using transient transfection and recombinant protein, we are reporting in this study that a crucian carp (Carassius auratus L.) IFN exhibits strong antiviral activity against grass carp hemorrhagic virus (GCHV) infection and also mediates Poly I:C-induced antiviral response, which correlates with its ability to induce a set of IFN-stimulated genes (ISGs). Strikingly, overexpression of wild-type Stat1 increases the effects of IFN on both the expression of ISGs and the inhibition of virus infection, whereas a dominant negative mutant of Stat1 (Stat1-Delta C), which lacks of the C-terminal transcriptional activation domain (TAD), inhibits the antiviral activity of IFN and reduces the expression of ISGs, demonstrating that fish IFN induces the expression of ISGs and host antiviral response through Stat1 pathway reminiscent that of mammalian IFNs. Significantly, unlike mammalian type I IFNs, recombinant fish IFN is able to upregulate IFN itself, which is enhanced by overexpression of Stat1 but impaired by knockdown of Stat1, indicating a positive feedback loop in regulation of IFN itself. These results provide strong evidence for the existence of an evolutionary conserved Stat1 pathway between fish and mammals, which is indispensable for fish virus-induced IFN antiviral response.

Zebrafish IRF1 Regulates IFN Antiviral Response through Binding to IFN phi 1 and IFN phi 3 Promoters Downstream of MyD88 Signaling

In mammals, type I IFNs (mainly IFN-α/β) are primarily regulated by transcription factors of the IFN regulatory factor (IRF) family. Fish IFNs do not show a one-to-one orthologous relationship with mammalian type I IFN homologues. Using a bacterial one-hybrid reporter screening system and an overexpression approach to explore the molecular mechanism underlying fish IFN induction, we identified zebrafish Danio rerio IRF (DrIRF)1 as a positive regulator of the fish IFN antiviral response. Among 12 zebrafish IRF family genes, DrIRF1 is most abundant in zebrafish immune tissues, including head kidney and spleen; upon virus infection, it is one of most significantly induced genes. Overexpression of DrIRF1 induces the expression of IFN and IFN-stimulated genes, hence protecting epithelioma papulosum cyprini cells against spring viremia of carp virus infection. As a transcription factor with constitutively nuclear retention, DrIRF1 directly binds to the IFN-stimulated regulatory element/IRF-binding element sites of zebrafish IFN promoters, which are dependent on four conserved amino acids of the N-terminal DNA-binding domain helix α3 motif. Mutation of either residue reveals a differential requirement for DrIRF1-mediated activation of zebrafish IFNϕ1 and IFNϕ3 promoters. Notably, C-terminal phosphorylation of DrIRF1 is observed and is not required for in vitro binding of DrIRF1 to fish IFN promoters. Unlike DrIRF3 and DrIRF7, which are responsible for differential expression of zebrafish IFNϕ1 and IFNϕ3 through the retinoic acid–inducible gene I–like receptor pathway, DrIRF1 works in concert with MyD88 to activate zebrafish IFNϕ3 but not IFNϕ1. These results provide insights into the evolving function of IRF1 as a positive IFN regulator.

Cooperative Roles of Fish Protein Kinase Containing Z-DNA Binding Domains and Double-Stranded RNA-Dependent Protein Kinase in Interferon-Mediated Antiviral Response

ABSTRACT The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) inhibits protein synthesis by phosphorylating eukaryotic translation initiation factor 2α (eIF2α). In fish species, in addition to PKR, there exists a PKR-like protein kinase containing Z-DNA binding domains (PKZ). However, the antiviral role of fish PKZ and the functional relationship between fish PKZ and PKR remain unknown. Here we confirmed the coexpression of fish PKZ and PKR proteins in Carassius auratus blastula embryonic (CAB) cells and identified them as two typical interferon (IFN)-inducible eIF2α kinases, both of which displayed an ability to inhibit virus replication. Strikingly, fish IFN or all kinds of IFN stimuli activated PKZ and PKR to phosphorylated eIF2α. Overexpression of both fish kinases together conferred much more significant inhibition of virus replication than overexpression of either protein, whereas morpholino knockdown of both made fish cells more vulnerable to virus infection than knockdown of either. The antiviral ability of fish PKZ was weaker than fish PKR, which correlated with its lower ability to phosphorylate eIF2α than PKR. Moreover, the independent association of fish PKZ or PKR reveals that each of them formed homodimers and that fish PKZ phosphorylated eIF2α independently on fish PKR and vice versa. These results suggest that fish PKZ and PKR play a nonredundant but cooperative role in IFN antiviral response.

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. In this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value<or=5e-3; Identity>45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGFbeta1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XlBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia.