Infection and intracellular transport of white spot syndrome virus require the ESCRT machinery in shrimp.

The cellular endosomal sorting complex required for transport (ESCRT) system comprises five distinct components and is involved in many different physiological processes. Recent studies have shown that different viruses rely upon the host ESCRT system for viral infection. However, whether this system is involved in white spot syndrome virus (WSSV) infection remains unclear. Here, we identified 24 homologs of ESCRT subunits in kuruma shrimp, Marsupenaeus japonicus, and found that some key components were strongly upregulated in shrimp after WSSV infection. Knockdown of key components of the ESCRT system using RNA interference inhibited virus replication, suggesting that the ESCRT system is beneficial for WSSV infection. We further focused on TSG101, a crucial member of the ESCRT-I family that plays a central role in recognizing cargo and activating the ESCRT-II and ESCRT-III complexes. TSG101 colocalized with WSSV in hemocytes. The addition of N16 (a TSG101 inhibitor) markedly decreased WSSV replication. TSG101 and ALIX of the ESCRT system interact with WSSV envelope proteins. The host proteins TSG101, RAB5, and RAB7, the viral protein VP28, and DNA were detected in endosomes isolated from hemocytes of WSSV-infected shrimp. Knockdown of Rab5 and Rab7 expression reduced viral replication. Taken together, these results suggest that the ESCRT system is hijacked by WSSV for transport through the early to late endosome pathway. Our work identified a novel requirement for the intracellular trafficking and infection of WSSV, and provided novel therapeutic targets for the prevention and control of WSSV in shrimp aquaculture. IMPORTANCE: Viruses utilize the ESCRT machinery in a variety of strategies for their replication and infection. This study revealed that the interaction of ESCRT complexes with WSSV envelope proteins plays a crucial role in WSSV infection in shrimp. The ESCRT system is conserved in the shrimp Marsupenaeus japonicus, and 24 homologs of the ESCRT system were identified in the shrimp. WSSV exploits the ESCRT system for transport and propagation via the interaction of envelope proteins with host TSG101 and ALIX in an endosome pathway-dependent manner. Understanding the underlying mechanisms of WSSV infection is important for disease control and breeding in shrimp aquaculture.

Identification of COP9 signalosome (CSN) subunits and antiviral function analysis of CSN5 in shrimp.

The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) typically composing of eight subunits (CSN1-8) mediates the process of deneddylation and deubiquitination. The fifth subunit of COP9 signalosome, CSN5, has special characteristics compared with the other seven subunits, and plays vital roles in the deneddylation activity and diverse cellular processes. However, the role of CSN5 in antiviral immunity is not clear. In this study, we identified 8 subunits (CSN1-8) of COP9 signalosome in shrimp Marsupenaeus japonicus. CSN1-6 were existed in all tested tissues, but CSN7-CSN8 were not detected in hepatopancreas. After WSSV challenged, the expression level of Csn1 to Csn4, and Csn6 to Csn8 were highly decreased, but the expression level of Csn5 was conspicuously increased in shrimp challenged by white spot syndrome virus (WSSV). The CSN5 was recombinantly expressed in Escherichia coli and its polyclonal antibody was prepared. The expression level of CSN5 was conspicuously increased at RNA and protein levels in the shrimp challenged by WSSV. After knockdown of Csn5 by RNA interference, the WSSV replication was obviously increased in shrimp. When injected the recombinant protein of CSN5 with the membrane penetrating peptide into shrimp, WSSV replication was inhibited and the survival rate of shrimp was significantly improved compared with control. We further analyzed the expression of antimicrobial peptides (AMPs) in Csn5-RNAi shrimp, and the results showed that the expression of several AMPs was declined significantly. These results indicate that CSN5 inhibits replication of WSSV via regulating expression of AMPs in shrimp, and the recombinant CSN5 might be used in shrimp aquaculture for the white spot syndrome disease control.

WSSV exploits AMPK to activate mTORC2 signaling for proliferation by enhancing aerobic glycolysis.

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.

Anti-lipopolysaccharide factor D from kuruma shrimp exhibits antiviral activity.

Anti-lipopolysaccharide factors (ALFs) exhibit a potent antimicrobial activity against a broad range of bacteria, filamentous fungi, and viruses. In previous reports, seven groups of ALFs (groups A-G) were identified in penaeid shrimp. Among them, group D showed negative net charges and weak antimicrobial activity. Whether this group has antiviral function is not clear. In this study, the ALF sequences of penaeid shrimp were analyzed, and eight groups of ALF family (groups A-H) were identified. The four ALFs including MjALF-C2, MjALF-D1, MjALF-D2, and MjALF-E2 from kuruma shrimp Marsupenaeus japonicus were expressed recombinantly in Escherichia coli, and the antiviral activity was screened via injection of purified recombinant ALFs into shrimp following white spot syndrome virus (WSSV) infection. Results showed that the expression of Vp28 (WSSV envelope protein) decreased significantly in the MjALF-D2-injected shrimp only. Therefore, MjALF-D2 was chosen for further study. Expression pattern analysis showed that MjAlf-D2 was upregulated in shrimp challenged by WSSV. The WSSV replication was detected in RNA, genomic DNA, and protein levels using VP28 and Ie1 (immediate-early gene of WSSV) as indicators in MjALF-D2-injected shrimp following WSSV infection. Results showed that WSSV replication was significantly inhibited compared with that in the rTRX- or PBS-injected control groups. After knockdown of MjAlf-D2 in shrimp by RNA interference, the WSSV replication increased significantly in the shrimp. All these results suggested that MjALF-D2 has an antiviral function in shrimp immunity, and the recombinant ALF-D2 has a potential application for viral disease control in shrimp aquaculture.

Metabolomic Investigation of Ultraviolet Ray-Inactivated White Spot Syndrome Virus-Induced Trained Immunity in Marsupenaeus japonicus.

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.

Acronyrones A-C, unusual prenylated acetophenones from Acronychia pedunculata.

Two novel prenylated acetophenones with new carbon skeletons, acronyrones A and B (1 and 2), and a new analogue, acronyrone C (3), together with two known compounds (4 and 5) were isolated from the leaves of Acronychia pedunculata. Their structures with absolute configurations were identified by interpretation of spectroscopic data, single crystal X-ray diffraction, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 represent the first example of prenylated acetophenones possessed a C7 (1) and a C6 (2) side chain, forming a 4-isobutylchroman-2-one unit and a 3-(2-methylpropylidene)benzofuran-2(3H)-one moiety with the acetophenone core, respectively. In addition, compound 4 exhibited significant dose-dependent transcriptional activation effect against retinoid X receptor-α (RXRα), and could be regarded as a new type of non-classical RXR ligand.

Ji-Chuan decoction ameliorates slow transit constipation via regulation of intestinal glial cell apoptosis.

BACKGROUND: Slow transit constipation (STC) is a common intestinal disease with increasing incidence. STC results from various factors, such as the enteric nervous system and metabolic changes. As a classical formula of traditional Chinese medicine, Ji-Chuan decoction (JCD) has been extensively and effectively used in STC treatment, yet its pharmacological mechanism remains unclear. AIM: To explore the integrated regulatory pattern of JCD against STC through hyphenated techniques from metabolism, network pharmacology and molecular methods. METHODS: STC model mice were generated by intragastric administration of compound diphenoxylate (10 mg/kg/d) for 14 d. The STC mice in the low dose of JCD (3.04 g/kg), middle dose of JCD (6.08 g/kg) and high dose of JCD (12.16 g/kg) groups were orally administered JCD solution once a day for 2 wk. The acetylcholine (ACH) level was examined by enzyme-linked immunosorbent assay. The pathological features of colon tissue were observed by hematoxylin and eosin staining. The differentially expressed metabolites and metabolic pathways were tested by nontargeted metabolomics. The main targets and core ingredients of JCD were identified by network pharmacology, and the expression of AKT was confirmed by immunohistochemistry. Finally, the pathways involved in JCD treatment were predicted using a combination of differentially expressed metabolites and targets, and intestinal glial cell apoptosis was demonstrated by immunofluorescence. RESULTS: JCD significantly promoted intestinal motility, increased the levels of the excitatory neurotransmitter ACH and reduced intestinal inflammation in STC mice. Untargeted metabolomics results showed that JCD significantly restored metabolic dysfunction and significantly affected taurine and hypotaurine metabolism. Network pharmacology and molecular experiments showed that JCD regulates AKT protein expression, and the core component is quercetin. Combined analysis demonstrated that apoptosis may be an important mechanism by which JCD relieves constipation. Further experiments showed that JCD reduced enteric glial cell (EGC) apoptosis. CONCLUSION: This work demonstrated that reducing EGC apoptosis may be the critical mechanism by which JCD treats STC. These findings call for further molecular research to facilitate the clinical application of JCD.

Alkylglyceronephosphate synthase (AGPS) alters lipid signaling pathways and supports chemotherapy resistance of glioma and hepatic carcinoma cell lines.

Chemotherapy continues to be a mainstay of cancer treatment, although drug resistance is a major obstacle. Lipid metabolism plays a critical role in cancer pathology, with elevated ether lipid levels. Recently, alkylglyceronephosphate synthase (AGPS), an enzyme that catalyzes the critical step in ether lipid synthesis, was shown to be up-regulated in multiple types of cancer cells and primary tumors. Here, we demonstrated that silencing of AGPS in chemotherapy resistance glioma U87MG/DDP and hepatic carcinoma HepG2/ADM cell lines resulted in reduced cell proliferation, increased drug sensitivity, cell cycle arrest and cell apoptosis through reducing the intracellular concentration of lysophosphatidic acid (LPA), lysophosphatidic acid-ether (LPAe) and prostaglandin E2 (PGE2), resulting in reduction of LPA receptor and EP receptors mediated PI3K/AKT signaling pathways and the expression of several multi-drug resistance genes, like MDR1, MRP1 and ABCG2. ß-catenin, caspase-3/8, Bcl-2 and survivin were also found to be involved. In summary, our studies indicate that AGPS plays a role in cancer chemotherapy resistance by mediating signaling lipid metabolism in cancer cells.