Long-Feng Lu

IFN Regulatory Factor 10 Is a Negative Regulator of the IFN Responses in Fish

IFN regulatory factor (IRF) 10 belongs to the IRF family and exists exclusively in birds and fish. Most IRFs have been identified as critical regulators in the IFN responses in both fish and mammals; however, the role of IRF10 is unclear. In this study, we identified IRF10 in zebrafish (Danio rerio) and found that it serves as a negative regulator to balance the innate antiviral immune responses. Zebrafish IRF10 (DrIRF10) was induced by intracellular polyinosinic:polycytidylic acid in ZF4 (zebrafish embryo fibroblast-like) cells. DrIRF10 inhibited the activation of zebrafish IFN1 (DrIFN1) and DrIFN3 promoters in epithelioma papulosum cyprinid cells in the presence or absence of polyinosinic:polycytidylic acid stimulation through direct interaction with the IFN promoters, and this inhibition was also shown to block IFN signaling. Overexpression of DrIRF10 was able to abolish the induction of DrIFN1 and DrIFN3 mediated by the retinoic acid–inducible gene I–like receptors. In addition, functional domain analysis of DrIRF10 showed that either the DNA binding domain or the IRF association domain is sufficient for its inhibitory activity for IFN signaling. Lastly, overexpression of DrIRF10 decreased the transcription level of several IFN-stimulated genes, resulting in the susceptibility of host cells to spring viremia of carp virus infection. Collectively, these data suggest that DrIRF10 inhibits the expression of DrIFN1 and DrIFN3 to avoid an excessive immune response, a unique regulation mechanism of the IFN responses in lower vertebrates.

Distinctive Structural Hallmarks and Biological Activities of the Multiple Cathelicidin Antimicrobial Peptides in a Primitive Teleost Fish

Cathelicidin antimicrobial peptides (CAMPs) represent a crucial component of the innate immune system in vertebrates. Although widely studied in mammals, little is known about the structure and function of fish CAMPs. Further to the previous findings, two more cathelicidin genes and multiple transcripts from rainbow trout were identified in the present study. Interestingly, we found that trout have evolved energy-saving forms of cathelicidins with the total deletion of the characteristic cathelin-like domain. Sequence analysis revealed that salmonid CAMPs have formed a special class of antimicrobial peptides in vertebrates with three distinctive hallmarks: the N terminus is intensified by positive charges, the central region consists of repetitive motifs based on RPGGGS, and the C terminus is lowly charged. Immunofluorescence localization of trout CAMPs demonstrated that these peptides expressed mainly at the mucosal layer of gut. Meanwhile, signals around sinusoids were also detected in head kidney. Moreover, the biological activities of trout CAMPs were proved to be mediated by the N terminus. Additionally, the repetitive motifs characteristically existing in Salmonidae increased the structural flexibilities of peptides and further increased the antibacterial and IL-8–stimulating activities. Unlike most α helical and cytotoxic mammalian CAMPs, trout CAMPs, mainly consisting of β-sheet and random coil, exhibited no cytotoxic activities. The distinctive structural features of trout CAMPs provide new insights into the understanding of the evolution of CAMPs in vertebrates. Moreover, the high bacterial membrane selectivity of trout CAMPs will help to design excellent peptide antibiotics.

Spring Viremia of Carp Virus N Protein Suppresses Fish IFNφ1 Production by Targeting the Mitochondrial Antiviral Signaling Protein

For a virus to replicate efficiently, it must try and inhibit host IFN expression because IFN is an important host defense at early stages after viral infection. For aquatic viruses, the mechanisms used to escape the hosts IFN system are still unclear. In this study, we show that the N protein of spring viremia of carp virus (SVCV) inhibits zebrafish IFNφ1 production by degrading the mitochondrial antiviral signaling protein (MAVS). First, the upregulation of IFNφ1 promoter activity stimulated by polyinosinic:polycytidylic acid, retinoic acid–inducible gene I (RIG-I) or MAVS was suppressed by the SVCV infection. However, the upregulation by the downstream factor of the RIG-I–like receptor signaling pathway, TANK-binding kinase 1, was not affected. Notably, at the protein level, MAVS decreased remarkably when cells were infected with SVCV. Second, consistent with the result of the SVCV infection, overexpression of the N protein of SVCV blocked the IFNφ1 transcription activated by MAVS and downregulated MAVS expression at the protein level but not at the mRNA level. Further analysis demonstrated that the N protein targeted MAVS for K48-linked ubiquitination, which promoted the degradation of MAVS. These data indicated that fish MAVS could be degraded by the N protein of SVCV through the ubiquitin-proteasome pathway. To our knowledge, this is the first article of a fish RIG-I–like receptor pathway interfered by an aquatic virus in an ubiquitin-proteasome manner, suggesting that immune evasion of a virus also exists in lower vertebrates.

The P Protein of Spring Viremia of Carp Virus Negatively Regulates the Fish Interferon Response by Inhibiting the Kinase Activity of TANK-Binding Kinase 1

ABSTRACT Spring viremia of carp virus (SVCV) is an efficient pathogen causing high mortality in the common carp. Fish interferon (IFN) is a powerful cytokine enabling host cells to establish an antiviral response; therefore, the strategies that SVCV uses to avoid the cellular IFN response were investigated. Here, we report that the SVCV P protein is phosphorylated by cellular TANK-binding kinase 1 (TBK1), which decreases IFN regulatory factor 3 (IRF3) phosphorylation and suppresses IFN production. First, overexpression of P protein inhibited the IFN promoter activation induced by SVCV and the IFN activity activated by the mitochondrial antiviral signaling protein (MAVS) although TBK1 activity was not blocked by P protein. Second, P protein colocalized and interacted with TBK1. Dominant negative experiments suggested that the TBK1 N-terminal kinase domain interacted with P protein and was essential for P protein and IRF3 phosphorylation. Finally, P protein overexpression reduced the IRF3 phosphorylation activated by TBK1 and reduced host cellular ifn transcription. Collectively, our data demonstrated that the SVCV P protein is a decoy substrate for the host phosphokinase TBK1, preventing IFN production and facilitating SVCV replication. IMPORTANCE TBK1 is a pivotal phosphokinase that activates host IFN production to defend against viral infection; thus, it is a potential target for viruses to negatively regulate IFN response and facilitate viral evasion. We report that the SVCV P protein functions as a decoy substrate for cellular TBK1, leading to the reduction of IRF3 phosphorylation and suppression of IFN expression. These findings reveal a novel immune evasion mechanism of SVCV.

Functions of the two zebrafish MAVS variants are opposite in the induction of IFN1 by targeting IRF7.

IFNs create the first line of host cells to defense viral infection, however, unrestricted expression of IFN can be hazardous to the host. IRF7 is the master regulator of type I IFN expression. To our knowledge, non research about the inhibition of IFN expression by targeting IRF7 has been reported in fish. In this study, we reported that the splicing variant of wildtype MAVS (MAVS_tv1), MAVS_tv2, negatively regulated IRF7-mediated IFN production. Firstly, in vivo, the transcriptional levels of MAVS_tv2 in trunk kidney and spleen from the zebrafish infected with SVCV were monitored. Then, in vitro, the protein expression pattern of MAVS_tv2 in zebrafish cell lines was detected using anti-MAVS_tv2 antibody. Furthermore, overexpression of MAVS_tv2 decreased the activation of IFN1 promoter that induced by IRF7 in a dose-dependent manner, whereas it had little effect on IRF3, a close relative of IRF7. In addition, such inhibition was also observed in IRF7-mediated epcIFN promoter and ISRE activities, but not in the activation of the promoters of type II IFNs and NF-ĸB, due to IRF7 not regulating their expression. Lastly, overexpression of MAVS_tv2 decreased the transcriptional levels of several IFN-stimulated genes activated by IRF7. These findings suggest that MAVS_tv2 is a negative regulator of IFN1 by targeting IRF7.

Regulation pattern of fish irf4 (the gene encoding IFN regulatory factor 4) by STAT6, c-Rel and IRF4

Interferon regulatory factor 4 (IRF4) plays pivotal roles in both innate and adaptive immune responses in mammals. In fish, there are two homologues of irf4, irf4a and irf4b. In this study, we examined the regulatory patterns of zebrafish irf4a and irf4b by STAT6 and c-Rel. Firstly, expression of irf4a and irf4b was monitored in several tissues at mRNA level. By infection with SVCV, irf4a and irf4b were upregulated in both kidney and spleen, and were immediately induced by treatment with poly I:C in ZF4 cells. Moreover, the activation of irf4a promoter was regulated by overexpression of stat6 and c-rel in a cooperation manner, which could be inhibited by mutation of the putative binding sites of STAT6 and c-Rel in irf4a promoter region. However, irf4b promoter was activated slightly only by STAT6 but not c-Rel. Furthermore, overexpression of irf4a inhibited the activation of its own promoter under induction of STAT6 and c-Rel, which was the result of that IRF4a bound to STAT6 and c-Rel directly. In addition, cellular location analysis showed that IRF4a was located only in nuclear region. These data indicate that fish irf4a can also be upregulated by STAT6 and c-Rel.

Fish IRF6 is a positive regulator of IFN expression and involved in both of the MyD88 and TBK1 pathways

Interferon (IFN) regulatory factors (IRF) are the crucial transcription factors for IFN expression, leading host cell response to viral infection. In mammals, only IRF6 is unaffected by IFN expression in the IRF family; however, in fish, a lower vertebrate, whether IRF6 is related to IFN regulation is unclear. In this study, we identified that zebrafish IRF6 was a positive regulator of IFN transcription and could be phosphorylated by both MyD88 and TBK1. First, the transcript level of cellular irf6 was upregulated by treatment with poly I:C (a mimic of viral RNAs), indicating IRF6 might be involved in the process of host cell response to viruses. Overexpression of IRF6 could upregulate IFN promoter activity significantly, meaning IRF6 is a positive regulator of IFN transcription. Subsequently, at the protein regulation level and in the interaction relationship, IRF6 was phosphorylated by and associated with both MyD88 and TBK1. In addition, overexpression of IRF6 activated the transcription of isg15, rig-i and mavs of host cells; meanwhile, the transcripts of p, m and n genes of SVCV were significantly declined in IRF6-overexpressing cells. Taken together, our data demonstrate that fish IRF6 is distinguished from the homolog of mammals by being a positive regulator of IFN transcription and phosphorylated by MyD88 and TBK1, suggesting that differences in the IRF6 regulation pattern exist between lower and higher vertebrates.

Numerical simulation on the marine landslide due to gas hydrate dissociation

Marine landslide due to gas hydrate dissociation is a kind of potential heavy hazards. Numerical simulation was processed to investigate the deformation, landslide and effects of main factors on the critical scale of dissociation zone to induce marine landslides during/after dissociation of gas hydrate. A simple method for analyzing the critical scale was presented based on the limit equilibrium method. It is shown that the maximum settlement is located near the upper side of the dissociation zone. The soil near the lower side of the dissociation zone uplifts. There is a critical scale of dissociation zone over which landslide will occur under given conditions. Expansion modes of the dissociation zone have obvious effects on the critical scale.

Zebrafish STAT6 negatively regulates IFNφ1 production by attenuating the kinase activity of TANK-binding kinase 1

ABSTRACT The aquatic spring viremia of carp virus (SVCV) causes significant mortality in common carp (Cyprinus carpio), and TBK1 plays a crucial role in the retinoic acid‐inducible gene I (RIG‐I)‐like receptor (RLR) system by phosphorylating its substrates to induce type I interferons (IFNs) and cellular antiviral responses. In this study, we report that zebrafish STAT6 is induced during SVCV infection and reduces IFN&phgr;1 expression by suppressing TBK1 phosphorylation. A typical IFN stimulatory response element (ISRE) motif was found in the promoter region of zebrafish STAT6, and zebrafish STAT6 transcription was significantly upregulated in the early stages of virus infection. Overexpression of STAT6 interfered with IFN&phgr;1 promoter activity in response to SVCV infection. Additionally, TBK1‐, but not MITA‐mediated activation of the IFN&phgr;1 promoter was impaired by STAT6. Co‐immunoprecipitation and Western blot experiments indicated that MITA and IRF3 were significantly phosphorylated by TBK1, and that the N‐terminal kinase domain of TBK1 was critical in this process. In the final step, STAT6 interacted with the N‐terminal kinase domain of TBK1 causing dephosphorylation, which resulted in reductions in the phosphorylation of IRF3 and the production of IFN&phgr;1. These results indicate that fish STAT6 can attenuate the kinase activity of TBK1, leading to suppression of IFN&phgr;1 expression which may in turn facilitate virus replication. HighlightsZebrafish STAT6 interacts with MITA and TBK1.Zebrafish STAT6 decreases the kinase activity of TBK1.Zebrafish STAT6 negatively regulates TBK1‐mediated activation of IFN&phgr;1.Zebrafish STAT6 facilitates virus replication by suppression of IFN&phgr;1.

Grass Carp Reovirus VP41 Targets Fish MITA To Abrogate the Interferon Response

ABSTRACT Although fish possess an efficient interferon (IFN) system to defend against aquatic virus infection, grass carp reovirus (GCRV) still causes hemorrhagic disease in grass carp. To date, GCRVs strategy for evading the fish IFN response is still unknown. Here, we report that GCRV VP41 inhibits fish IFN production by suppressing the phosphorylation of mediator of IFN regulatory factor 3 (IRF3) activation (MITA). First, the activation of the IFN promoter (IFNpro) stimulated by mitochondrial antiviral signaling protein (MAVS) and MITA was decreased by the overexpression of VP41, whereas such activation induced by TANK-binding kinase 1 (TBK1) was not affected. Second, VP41 was colocalized in the cellular endoplasmic reticulum (ER) and associated with MITA. Furthermore, as a phosphorylation substrate of TBK1, VP41 significantly decreased the phosphorylation of MITA. Truncation assays indicated that the transmembrane (TM) region of VP41 was indispensable for the suppression of IFNpro activity. Finally, after infection with GCRV, VP41 blunted the transcription of host IFN and facilitated viral RNA synthesis. Taken together, our findings suggest that GCRV VP41 prevents the fish IFN response by attenuating the phosphorylation of MITA for viral evasion. IMPORTANCE MITA is thought to act as an adaptor protein to facilitate the phosphorylation of IRF3 by TBK1 upon viral infection, and it plays a critical role in innate antiviral responses. Here, we report that GCRV VP41 colocalizes with MITA at the ER and reduces MITA phosphorylation by acting as a decoy substrate of TBK1, thus inhibiting IFN production. These findings reveal GCRVs strategy for evading the host IFN response for the first time.