Dynamic molecular architecture and substrate recruitment of cullin3-RING E3 ligase CRL3KBTBD2.

Phosphatidylinositol 3-kinase α, a heterodimer of catalytic p110α and one of five regulatory subunits, mediates insulin- and insulin like growth factor-signaling and, frequently, oncogenesis. Cellular levels of the regulatory p85α subunit are tightly controlled by regulated proteasomal degradation. In adipose tissue and growth plates, failure of K48-linked p85α ubiquitination causes diabetes, lipodystrophy and dwarfism in mice, as in humans with SHORT syndrome. Here we elucidated the structures of the key ubiquitin ligase complexes regulating p85α availability. Specificity is provided by the substrate receptor KBTBD2, which recruits p85α to the cullin3-RING E3 ubiquitin ligase (CRL3). CRL3KBTBD2 forms multimers, which disassemble into dimers upon substrate binding (CRL3KBTBD2-p85α) and/or neddylation by the activator NEDD8 (CRL3KBTBD2~N8), leading to p85α ubiquitination and degradation. Deactivation involves dissociation of NEDD8 mediated by the COP9 signalosome and displacement of KBTBD2 by the inhibitor CAND1. The hereby identified structural basis of p85α regulation opens the way to better understanding disturbances of glucose regulation, growth and cancer.

Identification of fish CMPK2 as an interferon stimulated gene against SVCV infection.

Cytidine/uridine monophosphate kinase 2 (CMPK2) is known as a nucleoside monophosphate kinase in mitochondria to maintains intracellular UTP/CTP, and could be induced by immunostimulants LPS and Poly (I:C) in mammals, suggesting its potential antiviral and antibacterial role. In this study, CMPK2 was cloned and characterized in Fathead minnow (FHM) cells. In vivo analysis of tissue distribution revealed that CMPK2 transcript was detected in all the tissues of zebrafish (Danio rerio) examined in this study, particularly abundant in liver, spleen and kidney. In addition, indirect immunofluorescence showed that CMPK2 was localized in the cytoplasm of FHM cells. Expression of CMPK2 mRNA was significantly up-regulated following challenge with Spring viraemia of carp virus (SVCV), poly(I:C), or zebrafish IFN1 and IFN3 both in vitro and in vivo. Furthermore, overexpression and RNA interference of CMPK2 in SVCV-infected FHM cells showed significantly antiviral effect. In summary, this study for the first time shows the presence and distribution of CMPK2 in different tissues of zebrafish, but also demonstrates its antiviral potential against SVCV infection in vivo. These new findings could contribute to explain the molecular mechanism of the CMPK2 mediated antiviral function.

The antiviral mechanism of viperin and its splice variant in spring viremia of carp virus infected fathead minnow cells.

Viperin is known to play an important role in innate immune and its antiviral mechanisms are well demonstrated in mammals. Fish Viperin mediates antiviral activity against several viruses. However, little has been done to the underlying mechanism. Here, we discovered a novel Viperin splice variant named Viperin_sv1 from viral-infected FHM cells. Spring varimia of carp virus (SVCV) was able to increase the mRNA levels of both Viperin and Viperin_sv1, while poly(I:C) only has effect on Viperin. Viperin functions as an antiviral protein at 24 h post-SVCV infection, but the antiviral activity dramatically declined at late infection stages. However, Viperin_sv1 inhibited SVCV replication significantly at all the tested time. Viperin_sv1, but not Viperin can facilitate the production of type I IFN and IFN stimulate genes (ISGs) through activation of RIG-1, IRF3 and IRF7 signaling cascades. On the other hand, SVCV down-regulated Viperin_sv1 at the protein level through the proteasome pathway to keep itself away from the immune system monitoring. Taken together, these findings provide new insights into the regulation of Viperin from the posttranscriptional modification perspective and the role of splicing variant Viperin_sv1 in virus-host interaction.

14-3-3ß/α-A interacts with glycoprotein of spring viremia of carp virus and positively affects viral entry.

Spring viremia of carp virus (SVCV) is a fierce pathogen causing high mortality in the common carp. The glycoprotein (G protein) of SVCV is a pivotal component of the viral structure, located in the surface of the virion, and plays a key role in viral endocytosis. In this study, tandem affinity purification (TAP) followed by mass spectrometry analysis (LC-MS/MS) was carried out to search for novel host molecules that interact with SVCV G protein and a 14-3-3ß/α-A protein was identified. The level of 14-3-3ß/α-A mRNA expression was dramatically down regulated by SVCV infection. Furthermore, over expression of 14-3-3ß/α-A results in a significantly increased SVCV attachment and entry in FHM cells. This study reveals an important role of 14-3-3 protein in regulating the early stage of SVCV infection, which offers a potential target for development of anti-SVCV therapies.

Comparative transcriptome analysis of zebrafish (Danio rerio) brain and spleen infected with spring viremia of carp virus (SVCV).

Spring viremia of carp virus (SVCV) is the pathogen of spring viremia of carp (SVC) and often causes acute hemorrhagic symptoms in various kinds of cyprinids and induces serious environmental and economic losses. However, the molecular mechanisms of infection remain poorly understood, especially at the individual level. In this study, zebrafish was employed as the infection model to explore the pathogenesis of SVCV. 4 groups of zebrafish tissues were set and RNA sequencing (RNA-Seq) technology was employed to analyze the differentially expressed genes (DEGs) after SVCV-infection. A total of 360,971,498 clean reads were obtained from 12 samples, 382 DEGs in the brain and 926 DEGs in the spleen were identified. These DEGs were annotated into three ontologies after gene ontology (GO) enrichment analysis. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that these DEGs were primarily related to Influenza A pathway and Herpes simplex infection pathway in brain and Tuberculosis and Toxoplasmosis pathways in spleen, and all of these pathways may be involved in response to pathogen invasion. At the same time, 3' and 5' alternative splicing (AS) events were significantly up-regulated in the spleen. The transcriptome analysis results demonstrated changes and tissue-specific influences caused by SVCV in vivo, which provided us with more information to understand the complex relationships between SVCV and its host.

Characterization and biological function analysis of the TRIM47 gene from common carp (Cyprinus carpio).

The TRIM family protein was known to play an important role in many cellular processes, including potential antiviral activity, which has attracted lots of attention. In this study, a TRIM47 homolog from common carp (Cyprinus carpio) was cloned and the full length coding DNA sequence (CDS) of this gene was analyzed, results showed that there was a 97% similarity between common carp and zebrafish (Danio rerio), but only 18% similarity with that of human (Homo sapiens) and mouse (Mus musculus). The tissue distribution analysis showed TRIM47 had the highest mRNA level in the brain, a few immune related organs such as liver and kidney also had a relatively high level of TRIM47 expression. SVCV infection decreased TRIM47 mRNA level significantly both in vitro and in vivo, but its expression was not affected by the virus at the protein level. The recombinant plasmid pcDNA4-TRIM47-His was constructed, the subcellular localization in FHM cells showed that TRIM47 uniformly distributed in the cytoplasm at the form of tiny spots, and partially localized in the mitochondria. Overexpression TRIM47 in FHM cells significantly decreased the mRNA level of SVCV-G gene, and it was accompanied with the increasing of IFN1, a member of type I IFN, at the case of SVCV stimulation. In summary, our results had first demonstrated that TRIM47 of the common carp played an important role in viral resistance processes as well as the regulation of IFN signaling pathway.

Molecular Characterization, Tissue Distribution and Expression, and Potential Antiviral Effects of TRIM32 in the Common Carp (Cyprinus carpio).

Tripartite motif-containing protein 32 (TRIM32) belongs to the tripartite motif (TRIM) family, which consists of a large number of proteins containing a RING (Really Interesting New Gene) domain, one or two B-box domains, and coiled coil motif followed by different C-terminal domains. The TRIM family is known to be implicated in multiple cellular functions, including antiviral activity. However, it is presently unknown whether TRIM32 of common carp (Cyprinus carpio) has the antiviral effect. In this study, the sequence, expression, and antiviral function of TRIM32 homolog from common carp were analyzed. The full-length coding sequence region of trim32 was cloned from common carp. The results showed that the expression of TRIM32 (mRNA) was highest in the brain, remained stably expressed during embryonic development, and significantly increased following spring viraemia of carp virus (SVCV) infection. Transient overexpression of TRIM32 in affected Epithelioma papulosum cyprinid cells led to significant decrease of SVCV production as compared to the control group. These results suggested a potentially important role of common carp TRIM32 in enhancing host immune response during SVCV infection both in vivo and in vitro.