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Lysyl oxidases (LOXs) are copper-dependent monoamine oxidases, and they play critical roles in extracellular matrix (ECM) remodeling. The LOX and LOX-like (LOXL) proteins also have a variety of biological functions, such as development and growth regulation, tumor suppression, and cellular senescence. However, the functions of LOXLs containing repeated scavenger receptor cysteine-rich (SRCR) domains in immunity are rarely reported. In this study, we characterized the antiviral and antibacterial functions of a lysyl oxidase-like (LOXL) protein containing tandem SRCR domains in Marsupenaeus japonicus. The mRNA level of LoxL was significantly upregulated in the hemocytes and intestines of shrimp challenged using white spot syndrome virus (WSSV) or bacteria. After the knockdown of LoxL via RNA interference, WSSV replication and bacterial loads were apparently increased, and the survival rate of the shrimp decreased significantly, suggesting that LOXL functions against pathogen infection in shrimp. Mechanistically, LOXL interacted with the envelope proteins of WSSV or with lipopolysaccharide and peptidoglycan from bacteria in shrimp challenged using WSSV or bacteria, and it promoted the expression of a battery of antimicrobial peptides (AMPs) via the induction of Dorsal nuclear translocation against viral and bacterial infection. Moreover, LOXL expression was also positively regulated by Dorsal in the shrimp challenged by pathogens. These results indicate that, by acting as a pattern recognition receptor, LOXL plays vital roles in antiviral and antibacterial innate immunity by enhancing the expression of AMPs in shrimp.

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of

AMPK plays significant roles in the modulation of metabolic reprogramming and viral infection. However, the detailed mechanism by which AMPK affects viral infection is unclear. The present study aims to determine how AMPK influences white spot syndrome virus (WSSV) infection in shrimp (Marsupenaeus japonicus). Here, we find that AMPK expression and phosphorylation are significantly upregulated in WSSV-infected shrimp. WSSV replication decreases remarkably after knockdown of Ampkα and the shrimp survival rate of AMPK-inhibitor injection shrimp increases significantly, suggesting that AMPK is beneficial for WSSV proliferation. Mechanistically, WSSV infection increases intracellular Ca2+ level, and activates CaMKK, which result in AMPK phosphorylation and partial nuclear translocation. AMPK directly activates mTORC2-AKT signaling pathway to phosphorylate key enzymes of glycolysis in the cytosol and promotes expression of Hif1α to mediate transcription of key glycolytic enzyme genes, both of which lead to increased glycolysis to provide energy for WSSV proliferation. Our findings reveal a novel mechanism by which WSSV exploits the host CaMKK-AMPK-mTORC2 pathway for its proliferation, and suggest that AMPK might be a target for WSSV control in shrimp aquaculture. AMPK and its downstream signaling pathways are implicated in the white spot syndrome virus (WSSV) response of the shrimp Marsupenaeus japonicus, with WSSV using this pathway to promote its proliferation. [ABSTRACT FROM AUTHOR]

Previous studies have shown that the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway has antiviral functions or is beneficial for viral replication, however, the detail mechanisms by which mTORC1 enhances viral infection remain unclear. Here, we found that proliferation of white spot syndrome virus (WSSV) was decreased after knockdown of mTor (mechanistic target of rapamycin) or injection inhibitor of mTORC1, rapamycin, in Marsupenaeus japonicus, which suggests that mTORC1 is utilized by WSSV for its replication in shrimp. Mechanistically, WSSV infects shrimp by binding to its receptor, polymeric immunoglobulin receptor (pIgR), and induces the interaction of its intracellular domain with Calmodulin. Calmodulin then promotes the activation of protein kinase B (AKT) by interaction with the pleckstrin homology (PH) domain of AKT. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, ribosomal protein S6 kinase (S6Ks), for viral protein translation. Moreover, mTORC1 also phosphorylates eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), which will result in the separation of 4EBP1 from eukaryotic translation initiation factor 4E (eIF4E) for the translation of viral proteins in shrimp. Our data revealed a novel pathway for WSSV proliferation in shrimp and indicated that mTORC1 may represent a potential clinical target for WSSV control in shrimp aquaculture. Author summary: White spot syndrome virus (WSSV) is the causative pathogen of white spot disease (WSD) and represents the most destructive viral disease of shrimp. The virus has evolved various strategies to escape from host defenses or exploit host biological pathways for its reproduction. Studies on viral immune-escape mechanisms can provide new strategies for disease prevention and control in shrimp aquaculture. Mechanistic target of rapamycin (mTOR) plays a central role in the regulation of cell growth and metabolism, which nucleates two distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) with diverse functions at different levels of the signaling pathway. mTORC1 is reported to be exploited by viruses in their reproduction. However, the detail mechanism remains unclear. In this study, we identified a new mechanism of mTOR being hijacked by WSSV in shrimp (Marsupenaeus japonicus). WSSV infects shrimp by its receptor, pIgR and induces the interaction of the intracellular domain of pIgR with Calmodulin. Calmodulin subsequently promotes the activation of AKT by interaction with the pleckstrin homology domain of the kinase. Activated AKT phosphorylates mTOR and results in the activation of the mTORC1 signaling pathway to promote its downstream effectors, S6Ks, for viral protein synthesis. Moreover, mTORC1 also phosphorylates 4EBP1, which results in the separation of 4EBP1 from eIF4E for the translation of viral proteins in shrimp. Our study reveals a novel strategy for WSSV proliferation in shrimp and indicates that the components of mTORC1 may represent potential clinical targets for WSSV control in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

Lysyl oxidases (LOXs) are copper-dependent monoamine oxidases, and they play critical roles in extracellular matrix (ECM) remodeling. The LOX and LOX-like (LOXL) proteins also have a variety of biological functions, such as development and growth regulation, tumor suppression, and cellular senescence. However, the functions of LOXLs containing repeated scavenger receptor cysteine-rich (SRCR) domains in immunity are rarely reported. In this study, we characterized the antiviral and antibacterial functions of a lysyl oxidase-like (LOXL) protein containing tandem SRCR domains in Marsupenaeus japonicus. The mRNA level of LoxL was significantly upregulated in the hemocytes and intestines of shrimp challenged using white spot syndrome virus (WSSV) or bacteria. After the knockdown of LoxL via RNA interference, WSSV replication and bacterial loads were apparently increased, and the survival rate of the shrimp decreased significantly, suggesting that LOXL functions against pathogen infection in shrimp. Mechanistically, LOXL interacted with the envelope proteins of WSSV or with lipopolysaccharide and peptidoglycan from bacteria in shrimp challenged using WSSV or bacteria, and it promoted the expression of a battery of antimicrobial peptides (AMPs) via the induction of Dorsal nuclear translocation against viral and bacterial infection. Moreover, LOXL expression was also positively regulated by Dorsal in the shrimp challenged by pathogens. These results indicate that, by acting as a pattern recognition receptor, LOXL plays vital roles in antiviral and antibacterial innate immunity by enhancing the expression of AMPs in shrimp. [ABSTRACT FROM AUTHOR]

Trained immunity is driven by metabolism and epigenetics in innate immune cells in mammals. The phenomenon of trained immunity has been identified in invertebrates, including shrimp, but the underlying mechanisms remain unclear. To elucidate mechanisms of trained immunity in shrimp, the metabolomic changes in hemolymph of Marsupenaeus japonicus trained by the UV-inactivated white spot syndrome virus (UV-WSSV) were analyzed using tandem gas chromatography–mass/mass spectrometry. The metabolomic profiles of shrimp trained with UV-WSSV followed WSSV infection showed significant differences comparison with the control groups, PBS injection followed WSSV infection. 16 differential metabolites in total of 154 metabolites were identified, including D-fructose-6-phosphate, D-glucose-6-phosphate, and D-fructose-6-phosphate, and metabolic pathways, glycolysis, pentose phosphate pathway, and AMPK signaling pathway were enriched in the UV-WSSV trained groups. Further study found that histone monomethylation and trimethylation at H3K4 (H3K4me1 and H3K4me3) were involved in the trained immunity. Our data suggest that the UV-WSSV induced trained immunity leads to metabolism reprogramming in the shrimp and provide insights for WSSV control in shrimp aquaculture. [ABSTRACT FROM AUTHOR]

Invertebrates rely on innate immunity, including humoral and cellular immunity, to resist pathogenic infection. Previous studies showed that forkhead box transcription factor O (FOXO) participates in mucosal immune responses of mammals and the gut humoral immune regulation of invertebrates. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from shrimp (Marsupenaeus japonicus) and found that it was expressed at relatively basal levels in normal shrimp, but was upregulated significantly in shrimp challenged by Vibrio anguillarum. FOXO played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of the immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity. In the nucleus, activated FOXO directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. Taken together, our results indicated that FOXO exerts its effects on homeostasis of hemolymph and the enteric microbiota by activating the IMD pathway in normal shrimp, and directly and indirectly promoting AMP expression and enhancing phagocytosis of hemocytes against pathogens in bacteria-infected shrimp. This study revealed the different functions of FOXO in the mucosal (local) and systemic antibacterial immunity of invertebrates. Author summary: Shrimp aquaculture is one of the world's fastest growing industries for producing animal proteins and has made a significant contribution to meeting the worldwide increased demand for animal proteins. However, disease outbreaks in aquaculture result in large economic losses to the industry. Studies of shrimp immune mechanisms could provide new strategies for disease prevention and control. The forkhead box transcription factor O family proteins (FOXOs) are involved in various critical biological process of organisms. However, whether FOXO is involved in systemic and cellular immunity regulation in invertebrates remains unknown. In the present study, we identified a FOXO from kuruma shrimp (Marsupenaeus japonicus) and found that it played a critical role in maintaining hemolymph and intestinal microbiota homeostasis by promoting the expression of Relish, the transcription factor of immune deficiency (IMD) pathway for expression of antimicrobial peptides (AMPs) in shrimp. We also found that pathogen infection activated FOXO and induced its nuclear translocation by reducing serine/threonine kinase AKT activity and directly regulated the expression of its target Amp and Relish genes against bacterial infection. Furthermore, FOXO was identified as being involved in cellular immunity by promoting the phagocytosis of hemocytes through upregulating the expression of the phagocytotic receptor scavenger receptor C (Src), and two small GTPases, Rab5 and Rab7, which are related to phagosome trafficking to the lysosome in the cytoplasm. This study revealed the different functions of FOXO in the innate antibacterial immunity of invertebrates. [ABSTRACT FROM AUTHOR]

A small open reading frame (smORF) or short open reading frame (sORF) encodes a polypeptide of <100 amino acids in eukaryotes (50 amino acids in prokaryotes). Studies have shown that several sORF-encoded peptides (SEPs) have important physiological functions in different organisms. Many ribosomal proteins belonging to SEPs play important roles in several cellular processes, such as DNA damage repair and apoptosis. Several studies have implicated SEPs in response to infection and innate immunity, but the mechanisms have been unclear for most of them. In this study, we identified a sORF-encoded ribosomal protein S27 (RPS27) in Marsupenaeus japonicus. The expression of MjRPS27 was significantly upregulated in shrimp infected with white spot syndrome virus (WSSV). After knockdown of MjRPS27 by RNA interference, WSSV replication increased significantly. Conversely, after Mj RPS27 overexpression, WSSV replication decreased in shrimp and the survival rate of the shrimp increased significantly. These results suggested that Mj RPS27 inhibited viral replication. Further study showed that, after Mj RPS27 knockdown, the mRNA expression level of Mj Dorsal, Mj Relish, and antimicrobial peptides (AMPs) decreased, and the nuclear translocation of Mj Dorsal and Mj Relish into the nucleus also decreased. These findings indicated that Mj RPS27 might activate the NF-κB pathway and regulate the expression of AMPs in shrimp after WSSV challenge, thereby inhibiting viral replication. We also found that Mj RPS27 interacted with WSSV's envelope proteins, including VP19, VP24, and VP28, suggesting that Mj RPS27 may inhibit WSSV proliferation by preventing virion assembly in shrimp. This study was the first to elucidate the function of the ribosomal protein Mj RPS27 in the antiviral immunity of shrimp. [ABSTRACT FROM AUTHOR]

The myeloid differentiation factor 2 (MD-2)-related lipid-recognition (ML) domain is found in multiple proteins, including MD-2, MD-1, Niemann-Pick disease type C2, and mite major allergen proteins. The significance of ML proteins in antibacterial signal transduction and in lipid metabolism has been well studied. However, their function in host-virus interaction remains poorly understood. In the current study, we found that the ML protein family is involved in resistance against white spot syndrome virus in kuruma shrimp, Marsupenaeus japonicus. One member, which showed a high similarity to mammalian MD-2/MD-1 and was designated as ML1, participated in the antiviral response by recognizing cholesta-3,5-diene (CD), a lipid component of the white spot syndrome virus envelope. After recognizing CD, ML1 induced the translocation of Rel family NF-κB transcription factor Dorsal into the nucleus, resulting in the expression of Vago, an IFN-like antiviral cytokine in arthropods. Overall, this study revealed the significance of an MD-2 homologue as an immune recognition protein for virus lipids. The identification and characterization of CD-ML1-Dorsal-Vago signaling provided new insights into invertebrate antiviral immunity. [ABSTRACT FROM AUTHOR]

Viral entry into the host cell is the first step towards successful infection. Viral entry starts with virion attachment, and binding to receptors. Receptor binding viruses either directly release their genome into the cell, or enter cells through endocytosis. For DNA viruses and a few RNA viruses, the endocytosed viruses will transport from cytoplasm into the nucleus followed by gene expression. Receptors on the cell membrane play a crucial role in viral infection. Although several attachment factors, or candidate receptors, for the infection of white spot syndrome virus (WSSV) were identified in shrimp, the authentic entry receptors for WSSV infection and the intracellular signaling triggering by interaction of WSSV with receptors remain unclear. In the present study, a receptor for WSSV infection in kuruma shrimp, Marsupenaeus japonicus, was identified. It is a member of the immunoglobulin superfamily (IgSF) with a transmembrane region, and is similar to the vertebrate polymeric immunoglobulin receptor (pIgR); therefore, it was designated as a pIgR-like protein (MjpIgR for short). MjpIgR was detected in all tissues tested, and its expression was significantly induced by WSSV infection at the mRNA and protein level. Knockdown of MjpIgR, and bloking MjpIgR with its antibody inhibited WSSV infection in shrimp and overexpression of MjpIgR facilitated the invasion of WSSV. Further analyses indicated that MjpIgR could independently render non-permissive cells susceptible to WSSV infection. The extracellular domain of MjpIgR interacts with envelope protein VP24 of WSSV and the intracellular domain interacts with calmodulin (MjCaM). MjpIgR was oligomerized and internalized following WSSV infection and the internalization was associated with endocytosis of WSSV. The viral internalization facilitating ability of MjpIgR could be blocked using chlorpromazine, an inhibitor of clathrin dependent endocytosis. Knockdown of Mjclathrin and its adaptor protein AP-2 also inhibited WSSV internalization. All the results indicated that MjpIgR-mediated WSSV endocytosis was clathrin dependent. The results suggested that MjpIgR is a WSSV receptor, and that WSSV enters shrimp cells via the pIgR-CaM-Clathrin endocytosis pathway. [ABSTRACT FROM AUTHOR]

Scavenger receptors are an important class of pattern recognition receptors that play several important roles in host defense against pathogens. The class C scavenger receptors (SRCs) have only been identified in a few invertebrates, and their role in the immune response against viruses is seldom studied. In this study, we firstly identified an SRC from kuruma shrimp, Marsupenaeus japonicus, designated MjSRC, which was significantly upregulated after white spot syndrome virus (WSSV) challenge at the mRNA and protein levels in hemocytes. The quantity of WSSV increased in shrimp after knockdown of MjSRC, compared with the controls. Furthermore, overexpression of MjSRC led to enhanced WSSV elimination via phagocytosis by hemocytes. Pull-down and co-immunoprecipitation assays demonstrated the interaction between MjSRC and the WSSV envelope protein. Electron microscopy observation indicated that the colloidal gold-labeled extracellular domain of MjSRC was located on the outer surface of WSSV. MjSRC formed a trimer and was internalized into the cytoplasm after WSSV challenge, and the internalization was strongly inhibited after knockdown of Mjβ-arrestin2. Further studies found that Mjβ-arrestin2 interacted with the intracellular domain of MjSRC and induced the internalization of WSSV in a clathrin-dependent manner. WSSV were co-localized with lysosomes in hemocytes and the WSSV quantity in shrimp increased after injection of lysosome inhibitor, chloroquine. Collectively, this study demonstrated that MjSRC recognized WSSV via its extracellular domain and invoked hemocyte phagocytosis to restrict WSSV systemic infection. This is the first study to report an SRC as a pattern recognition receptor promoting phagocytosis of a virus. [ABSTRACT FROM AUTHOR]

Plants and invertebrates can suppress viral infection through RNA silencing, mediated by RNA-induced silencing complex (RISC). Trans-activation response RNA-binding protein (TRBP), consisting of three double-stranded RNA-binding domains, is a component of the RISC. In our previous paper, a TRBP homologue in Fenneropenaeus chinensis (Fc-TRBP) was reported to directly bind to eukaryotic initiation factor 6 (Fc-eIF6). In this study, we further characterized the function of TRBP and the involvement of TRBP and eIF6 in antiviral RNA interference (RNAi) pathway of shrimp. The double-stranded RNA binding domains (dsRBDs) B and C of the TRBP from Marsupenaeus japonicus (Mj-TRBP) were found to mediate the interaction of TRBP and eIF6. Gel-shift assays revealed that the N-terminal of Mj-TRBP dsRBD strongly binds to double-stranded RNA (dsRNA) and that the homodimer of the TRBP mediated by the C-terminal dsRBD increases the affinity to dsRNA. RNAi against either Mj-TRBP or Mj-eIF6 impairs the dsRNA-induced sequence-specific RNAi pathway and facilitates the proliferation of white spot syndrome virus (WSSV). These results further proved the important roles of TRBP and eIF6 in the antiviral response of shrimp. [ABSTRACT FROM AUTHOR]

Vibrio parahaemolyticus is a critical pathogenic bacterium for the kuruma shrimp, Marsupenaeus japonicus. Antibacterial drug application is the main method to control vibriosis in aquaculture; however, drug residues and the emergence of resistant bacteria have restricted their use. Therefore, the present study aimed to identify metabolites that are likely to play roles in the homeostasis of the intestinal microbiota, and to further explore their application in shrimp aquaculture. Metabolomic analysis, using high-performance liquid chromatography tandem mass spectrometry (HPLC/MS) of shrimp intestinal contents, was performed. The results showed that metabolic profiles changed distinctly in shrimp intestines after V. parahaemolyticus challenge compared with that in the control. The levels of 39 metabolites changed significantly after Vibrio challenge. Notably, vitamin D3 metabolism was enhanced in shrimp intestines after Vibrio challenge. Oral administration of 0.1 mg/kg vitamin D3 effectively improved the survival rate of shrimp infected with V. parahaemolyticus. Further studies indicated that vitamin D3 promoted bacterial clearance by regulating the expression of antimicrobial peptides genes in the shrimp intestines. Transcriptomics analysis revealed that vitamin D3 functioned in shrimp intestinal immunity by activating the prophenoloxidase system and Toll signaling pathway to induce the expression of certain antimicrobial peptides to maintain microbial homeostasis. Collectively, we identified vitamin D3 as an immunomodulatory factor that plays an important role in boosting the immune response against pathogenic bacteria in shrimp. • 39 metabolites changed significantly post V. parahaemolyticus challenge. • Vitamin D3 metabolism was enhanced in shrimp intestine post challenge. • Vitamin D3 improved shrimp survival rate. • Vitamin D3 promoted bacterial clearance by regulating the expression of AMPs. • Vitamin D3 functioned in shrimp intestinal immunity. [ABSTRACT FROM AUTHOR]

ATPase Inhibitory Factor 1 (IF1) is a mitochondrial protein that functions as a physiological inhibitor of F 1 F 0 -ATP synthase. In the present study, a mitochondrial ATPase inhibitor factor 1 (Mj ATPIF1) was identified from kuruma shrimp (Marsupenaeus japonicus), which was demonstrated to participate in the viral immune reaction of white spot syndrome virus (WSSV). Mj ATPIF1 contained a mitochondrial ATPase inhibitor (IATP) domain, and was widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine of shrimp. MjATPIF1 transcription was upregulated in hemocytes and intestines by WSSV. WSSV replication decreased after MjATPIF1 knockdown by RNA interference and increased following recombinant Mj ATPIF1 protein injection. Further study found that Mj ATPIF1 promoted the production of superoxide and activated the transcription factor nuclear factor kappa B (NF-κB, Dorsal) to induce the transcription of WSSV RNAs. These results demonstrate that Mj ATPIF1 benefits WSSV replication in kuruma shrimp by inducing superoxide production and NF-κB activation. • A mitochondrial ATPase inhibitor factor 1 was identified from kuruma shrimp. • Mj ATPIF1 was widely distributed in shrimp tissues and upregulated by WSSV challenge. • Mj ATPIF1 promoted the production of mitochondria superoxide. • Mj ATPIF1 triggered the activation of NF-κB by ROS. • Mj ATPIF1 benefits WSSV replication in kuruma shrimp. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) is a threatening pathogenic virus in shrimp culture, and at present, no effective strategy can prevent and control the disease. Intestinal flora and its metabolites are important for the resistance of shrimp to lethal pathogenic viruses. However, the changes of metabolites in the shrimp intestines after WSSV infection remain unclear. We established an artificial oral infection method to infect shrimp with WSSV and analyzed the metabolites in intestinal content of shrimp by HPLC and tandem mass spectrometry. A total of 78 different metabolites and five different metabolic pathways were identified. Among them, we found that the content of linoleic acid, an unsaturated fatty acid, increased significantly after WSSV infection, indicating that linoleic acid might be involved in antiviral immunity in shrimp. Further study showed that, after oral administration of linoleic acid, WSSV proliferation decreased evidently in the shrimp, and survival rate of the shrimp increased significantly. Mechanical analysis showed that linoleic acid directly bound to WSSV virions and inhibited the viral replication. Linoleic acid also promoted the expression of antimicrobial peptides and IFN-like gene Vago5 by activating the ERK-NF-kB signaling pathway. Our results indicated that WSSV infection caused metabolomic transformation of intestinal microbiota and that the metabolite linoleic acid participated in the immune response against WSSV in shrimp. [ABSTRACT FROM AUTHOR]

Viruses have developed superior strategies to escape host defenses or exploit host components and enable their infection. The forkhead box transcription factor O family proteins (FOXOs) are reportedly utilized by human cytomegalovirus during their reactivation in mammals, but if FOXOs are exploited by viruses during their infection remains unclear. In the present study, we found that the FOXO of kuruma shrimp (Marsupenaeus japonicus) was hijacked by white spot syndrome virus (WSSV) during infection. Mechanistically, the expression of leucine carboxyl methyl transferase 1 (LCMT1) was up-regulated during the early stages of WSSV infection, which activated the protein phosphatase 2A (PP2A) by methylation, leading to dephosphorylation of FOXO and translocation into the nucleus. The FOXO directly promoted transcription of the immediate early gene, wsv079 of WSSV, which functioned as a transcriptional activator to initiate the expression of viral early and late genes. Thus, WSSV utilized the host LCMT1-PP2A-FOXO axis to promote its replication during the early infection stage. We also found that, during the late stages of WSSV infection, the envelope protein of WSSV (VP26) promoted PP2A activity by directly binding to FOXO and the regulatory subunit of PP2A (B55), which further facilitated FOXO dephosphorylation and WSSV replication via the VP26-PP2A-FOXO axis in shrimp. Overall, this study reveals novel viral strategies by which WSSV hijacks host LCMT1-PP2A-FOXO or VP26-PP2A-FOXO axes to promote its propagation, and provides clinical targets for WSSV control in shrimp aquaculture. • WSSV upregulates expression of LCMT1 which is responsible for PP2A methylation. • WSSV exploits LCMT1-PP2A-FOXO axis to promote its replication during early infection. • WSSV utilizes VP26-PP2A-FOXO axis to promote viral replication during late infection. • FOXO targets Wsv079 to increase transcription of viral genes in WSSV infected shrimp. [ABSTRACT FROM AUTHOR]

C-type lectins (CTLs) are key recognition proteins in shrimp immunity. A few years ago we reviewed sequence information, ligand specificity, expression profiles and specific functions of the shrimp CTLs. Since then, multiple integrated studies that implemented biochemical approaches using both the native and recombinant proteins, functional genetic approaches using RNA interference, and mechanistic studies by analyzing protein-protein interactions were carried out. Results from these rigorous studies revealed the functions and mechanisms of action of selected members of the shrimp CTL family. This review focuses on this new knowledge, that includes unique structural aspects, functions, and mechanisms in host-pathogen interactions, the functional relevance of regions other than the C-type lectin domain, and the regulation of transcription of shrimp CTLs. Thus, this review aims to provide a detailed update of recent studies that have contributed to our better understanding of the shrimp immune events that involve CTL functions. • C-type lectins play important roles in shrimp-pathogen interactions. • Additional regions are essential for the function of shrimp C-type lectins. • This review highlights the in vivo immune-related events of C-type lectins. [ABSTRACT FROM AUTHOR]

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, ΔMjAK, 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. [ABSTRACT FROM AUTHOR]

Lysin motif (LysM) is a peptidoglycan and chitin-binding motif with multiple functions in bacteria, plants, and animals. In this study, a novel LysM and putative peptidoglycan-binding domain-containing protein was cloned from kuruma shrimp ( Marsupenaeus japonicus ) and named as Mj LPBP. The cDNA of Mj LPBP contained 1010 nucleotides with an open reading frame of 834 nucleotides encoding a protein of 277 amino acid residues. The deduced protein contained a Lysin motif and a transmembrane region, with a calculated molecular mass of 31.54 kDa and isoelectric point of 8.61. MjLPBP was ubiquitously distributed in different tissues of shrimp at the mRNA level. Time course expression assay showed that MjLPBP was upregulated in hemocytes of shrimp challenged with Vibrio anguillarum or Staphylococcus aureus . MjLPBP was also upregulated in hepatopancreas after white spot syndrome virus and bacteria challenge. The recombinant protein of Mj LPBP could bind to some Gram-positive and Gram-negative bacteria and yeast. Further study found that r Mj LPBP bound to bacterial cell wall components, including peptidoglycans, lipoteichoic acid, lipopolysaccharide, and chitin. The induction of several antimicrobial peptide genes and phagocytosis-related gene, such as anti-lipopolysaccharide factors and myosin, was depressed after knockdown of MjLPBP . Mj LPBP could facilitate V. anguillarum clearance in vivo. All the results indicated that Mj LPBP might play an important role in the innate immunity of shrimp. [ABSTRACT FROM AUTHOR]

β-Thymosins participate in numerous biological activities, including cell proliferation and differentiation, wound healing, and anti-inflammatory and antimicrobial activities. Many studies have investigated vertebrate β-thymosins, whereas few reports have focused on invertebrate β-thymosins. In this study, nine isoforms of β-thymosins ( Pc Thy-1 to Pc Thy-8) were identified from the red swamp crayfish Procambarus clarkii . The isoforms contained different numbers of the thymosin β actin-binding motif. Pc Thy-1 contained one thymosin β actin-binding motif, whereas Pc Thy-8 contained eight motifs. Western blot analysis with anti- Pc Thy-4 antibody showed that three to six isoforms were present in one tissue, and Pc Thy-4, Pc Thy-5, Pc Thy-6, and Pc Thy-7 were the main isoforms in several tissues. Time course expression analysis of Pc Thys at the protein level showed that Pc Thy-4 was upregulated in hemocytes and gills after white spot syndrome virus (WSSV) challenge. Pc Thy-4, which contained four thymosin β actin-binding motifs, was selected for further research. Tissue distribution analysis by quantitative real-time PCR showed that PcThy-4 was present in tissues of the hemocytes, heart, hepatopancreas, gills, stomach, and intestine at the transcriptional level. Transcriptional expression profiles showed that PcThy-4 was upregulated after WSSV challenge. In vivo RNAi and protein injection assay results showed that Pc Thy-4 inhibited the replication of WSSV in crayfish and enhanced the survival rate after WSSV infection. Furthermore, Pc Thy-4 promoted hemocyte phagocytosis of WSSV. Overall, results suggested that Pc Thys protected crayfish from WSSV infection and played an important role in antiviral immune response. [ABSTRACT FROM AUTHOR]

White spot syndrome virus (WSSV) mainly infects crustaceans through the digestive tract. Whether C-type lectins (CLs), which are important receptors for many viruses, participate in WSSV infection in the shrimp stomach remains unknown. In this study, we orally infected kuruma shrimp Marsupenaeus japonicus to model the natural transmission of WSSV and identified a CL (designated as M. japonicus stomach virus-associated CL [MjsvCL]) that was significantly induced by virus infection in the stomach. Knockdown of MjsvCL expression by RNA interference suppressed the virus replication, whereas exogenous MjsvCL enhanced it. Further analysis by GST pull-down and coimmunoprecipitation showed that MjsvCL could bind to viral protein 28, the most abundant and functionally relevant envelope protein of WSSV. Furthermore, cell-surface calreticulin was identified as a receptor of MjsvCL, and the interaction between these proteins was a determinant for the viral infection-promoting activity of MjsvCL. The MjsvCL-calreticulin pathway facilitated virus entry likely in a cholesterol-dependent manner. This study provides insights into a mechanism by which soluble CLs capture and present virions to the cell-surface receptor to facilitate viral infection. [ABSTRACT FROM AUTHOR]

Dyslexia-associated protein KIAA0319, with immunoglobulin-like polycystic kidney disease (PKD) domains, is a dyslexia susceptibility protein. KIAA0319 participates in neuronal migration and is involved in the interaction between neurons and glial fibers. Recent studies found that KIAA0319 acts as a viral receptor via an interaction between its PKD domains with viruses. KIAA0319 mediates viral internalization via a clathrin-dependent pathway involving interaction with adaptor protein 2 (AP2). In the present study, we identified a cDNA encoding a dyslexia-associated KIAA0319-like protein from Marsupenaeus japonicus (designated as Mj KIAA0319L). MjKIAA0319L was upregulated in shrimp challenged by white spot syndrome virus (WSSV). After knockdown of MjKIAA0319L using RNA interference, followed by WSSV infection in shrimp, WSSV replication increased and the shrimp survival rate decreased significantly. Further study found that the extracellular domain of Mj KIAA0319L expressed in Escherichia coli bound WSSV particles by interacting with the WSSV envelope protein VP28. Fluorescent immunocytochemical analysis showed that Mj KIAA0319L was internalized into the cytoplasm of hemocytes during WSSV infection. The intracellular domain of Mj KIAA0319L, comprising a YXXΦ motif, interacted with AP2μ, suggesting that Mj KIAA0319L binds to WSSV and induces endocytosis in a clathrin-dependent manner. Therefore, Mj KIAA0319L recognizes WSSV via its extracellular domain and induces endocytosis of WSSV through its intracellular domain in shrimp. Thus, the Mj KIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV to restrict viral infection in shrimp. • KIAA0319-like mRNA expression was upregulated in kuruma shrimp challenged by WSSV. • KIAA0319L binds to WSSV via an interaction with viral envelope protein VP28. • The intracellular region of KIAA0319L interacts with AP2 and induces endocytosis. • The KIAA0319L-AP2-clathrin pathway is involved in the phagocytosis of WSSV. [ABSTRACT FROM AUTHOR]

Prohibitins (PHBs) are ubiquitously expressed conserved proteins in eukaryotes that are associated with apoptosis, cancer formation, aging, stress responses, cell proliferation, and immune regulation. However, the function of PHBs in crustacean immunity remains largely unknown. In the present study, we identified a PHB in Procambarus clarkii red swamp crayfish, which was designated PcPHB1. PcPHB1 was widely distributed in several tissues, and its expression was significantly upregulated by white spot syndrome virus (WSSV) challenge at the mRNA level and the protein level. These observations prompted us to investigate the role of PcPHB1 in the crayfish antiviral response. Recombinant PcPHB1 (rPcPHB1) significantly reduced the amount of WSSV in crayfish and the mortality of WSSV-infected crayfish. The quantity of WSSV in PcPHB1 knockdown crayfish was increased compared with that in the controls. The effects of RNA silencing were rescued by rPcPHB1 reinjection. We further confirmed the interaction of PcPHB1 with the WSSV envelope proteins VP28, VP26, and VP24 using pulldown and far-Western overlay assays. Finally, we observed that the colloidal gold-labeled PcPHB1 was located on the outer surface of the WSSV, which suggests that PcPHB1 specifically binds to the envelope proteins of WSSV. VP28, VP26, and VP24 are structural envelope proteins and are essential for attachment and entry into crayfish cells. Therefore, PcPHB1 exerts its anti-WSSV effect by binding to VP28, VP26, and VP24, preventing viral infection. This study is the first report on the antiviral function of PHB in the innate immune system of crustaceans. [ABSTRACT FROM AUTHOR]

Highlights: [•] Immune deficiency (FcIMD and PcIMD) were identified from F. chinensis and P. clarkii. [•] FcIMD was upregulated by white spot syndrome virus challenge. PcIMD was upregulated by V. anguillarum challenge. [•] IMD pathway might be involved in regulating the expression of five antimicrobial peptides in crayfish challenged by V. anguillarum. [•] IMD pathway may have different functions in shrimp and crayfish immunity. [Copyright &y& Elsevier]

Abstract: BAX inhibitor-1 (BI-1) was originally described as an anti-apoptotic protein in both animal and plant cells. BI-1 overexpression suppresses ER stress–induced apoptosis in animal cells. Inhibition of BI-1 activity could induce the cell death in mammals and plants. However, the function of BI-1 in crustacean immunity was unclear. In this paper, the full-length cDNA of a BI-1 protein in red swamp crayfish, Procambarus clarkii (PcBI-1) was cloned and its expression profiles in normal and infected crayfish were analyzed. The results showed that PcBI-1 was expressed in hemocytes, heart, hepatopancreas, gills, stomach, and intestines of the crayfish and was upregulated after challenged with Vibrio anguillarum and with white spot syndrome virus (WSSV). To determine the function of PcBI-1 in the innate immunity of the crayfish, the RNA interference against PcBI-1 was performed and the results indicated the hemocyte programmed cell death rate was increased significantly and WSSV replication was declined after PcBI-1 knocked down. Altogether, PcBI-1 plays an anti-apoptotic role, wherein high PcBI-1 expression suppresses programmed cell death, which is beneficial for WSSW replication in crayfish. [Copyright &y& Elsevier]

Abstract: White spot syndrome virus (WSSV) infects all shrimp species and is the greatest detriment to shrimp culture. To better understand the mechanism of molecular responses to WSSV infection in Chinese white shrimp Fenneropenaeus chinensis, two dimensional electrophoresis (2-DE) was used. Differentially expressed proteins in the hepatopancreas of control and WSSV-injected Chinese white shrimp between (6, 12 and 24 h post-injection) were screened. Quantitative intensity analysis and mass spectrometry revealed that 54 spots of 47 proteins were significantly up-regulated, including some immune-related proteins, such as Toll receptor precursor, Leu-rich repeat protein, peroxinectin and serine proteinase-like protein. Fourteen spots of 13 proteins, such as heat shock protein, ATP synthase sub-unit beta and thrombospondin, were down-regulated in WSSV-infected shrimps. Protein expression patterns of the infected shrimp were drastically altered by WSSV infection. Some of the altered proteins were further investigated at the mRNA level using semi-quantitative reverse transcript PCR. These data may provide some information about shrimp proteins that participate in the WSSV infection process. [Copyright &y& Elsevier]