Effect of mutations in GDNV motif of viral hemorrhagic septicemia virus (VHSV) L protein on polymerase activity, viral growth, and in vivo virulence.

Most Mononegavirales viruses have a GDNQ motif within the L protein, whereas Novirhabdovirus species feature a GDNV motif. This study examined the function of the GDNV motif within the L protein of viral hemorrhagic septicemia virus (VHSV) by modifying its amino acid composition. Substituting the aspartic acid (D) with valine (V) completely abolished polymerase activity in a minigenome assay.

Functional and regulatory diversification of Period genes responsible for circadian rhythm in vertebrates.

The Period genes (Per) play essential roles in modulating the molecular circadian clock timing in a broad range of species, which regulates the physiological and cellular rhythms through the transcription-translation feedback loop. While the Period gene paralogs are widely observed among vertebrates, the evolutionary history and the functional diversification of Per genes across vertebrates are not well known. In this study, we comprehensively investigated the evolution of Per genes at the copy number and sequence levels, including de novo binding motif discovery by comparative genomics.

E3 SUMO ligase SIZ1 splicing variants localize and function according to external conditions.

SIZ1 (SAP and MIZ1) is a member of the Siz/PIAS-type RING family of E3 SUMO (small ubiquitin-related modifier) ligases that play key roles in growth, development, and stress responses in plant and animal systems. Nevertheless, splicing variants of SIZ1 have not yet been characterized. Here, we identified four splicing variants of Arabidopsis (Arabidopsis thaliana) SIZ1, which encode three different protein isoforms.

Generation of Recombinant Snakehead Rhabdovirus (SHRV) Expressing Artificial MicroRNA Targeting Spring Viremia of Carp Virus (SVCV) P Gene and In Vivo Therapeutic Use Against SVCV Infection.

Spring viremia of carp virus (SVCV) is a highly lethal virus in common carp (Cyprinus carpio) and other cyprinid fish species. The aim of the present study was to develop an in vivo therapeutic measure against SVCV using artificial microRNA (AmiRNA) targeting the SVCV P gene transcript. Three candidates of AmiRNAs (AmiR-P1, -P2, and -P3) were selected, and their ability to downregulate SVCV P gene transcript was analyzed by both synthesized AmiRNA mimics and AmiRNA-expressing vector system, in which AmiR-P3 showed the strongest inhibitory activity among the three candidates.

Compensatory mutations in the matrix protein of viral hemorrhagic septicemia virus (VHSV) genotype IVa in response to artificial mutation of two amino acids (D62A E181A).

The matrix (M) protein of rhabdoviruses locates between the inner line of the viral envelope and the nucleocapsids core and plays an important role in viral replication. In the present study, we aimed to rescue a mutant of VHSV genotype IVa that has artificial mutations in the M protein (M-D62A E181A). However, most rescued recombinant viruses unexpectedly showed non-targeted secondary mutations in the M protein.

Effect of CRISPR/Cas9-mediated knockout of either IRF-3 or IRF-5 gene in Epithelioma papulosum cyprini cells on type I interferon response and NF-κB activity.

Transcription factors related to the activation of type I interferons (IFNs) and nuclear factor-kappa B (NF-κB) are known to be critical in innate immune responses. Interferon regulatory factors (IRFs) are a family of transcription factors. IRF-3 is known to act as the primary regulator in type I IFN signaling in response to viral infections, and the upregulation of IRF5 by virus infection has been reported in various fish species.

Intronic long noncoding RNA, RICE FLOWERING ASSOCIATED (RIFLA), regulates OsMADS56-mediated flowering in rice.

Long noncoding RNAs (lncRNAs) are known to play important roles in several plant processes such as flowering, organ development and stress response. However, studies exploring the diversity and complexity of lncRNAs and their mechanism of action in plants are far fewer that those in animals. Here, we show that an intronic lncRNA in rice (Oryza sativa L.

CRISPR/Cas9-mediated generation of auxotrophic Edwardsiella piscicida mutants and immunization in olive flounder (Paralichthys olivaceus).

Edwardsiella piscicida has been a cause of mass mortality in cultured fish. In this study, to produce auxotrophic E. piscicida mutants, a CRISPR/Cas9 system was used instead of the traditional sacB-based allelic exchange method.

Effect of NV gene deletion in the genome of hirame rhabdovirus (HIRRV) on viral replication and the type I interferon response of the host cell.

Hirame rhabdovirus (HIRRV), a member of the genus Novirhabdovirus, causes morbidity and mortality in farmed olive flounder (Paralichthys olivaceus). As no information is available on the role of the NV gene of HIRRV, we produced a recombinant HIRRV with the NV gene deleted (rHIRRV-ΔNV) using reverse genetic technology and investigated whether the NV gene knockout affected HIRRV replication and the type I interferon response of the host cell. The rescue of rHIRRV-ΔNV was successful only when IRF9-gene-knockout Epithelioma papulosum cyprini (ΔIRF9-EPC) cells were used, suggesting that the NV protein of HIRRV might be involved in inhibition of the type I interferon response of the host cell.

A mutation in the pPLA-IIα gene encoding PATATIN-RELATED PHOSPHOLIPASE a causes late flowering in Arabidopsis.

Arabidopsis PATATIN-RELATED PHOSPHOLIPASE 2A (pPLA-IIα) participates in the responses to various growth conditions. The factors affecting pPLA-IIα gene expression and pPLA-IIα protein activity for gycerolipids have been studied thoroughly, but the role of pPLA-IIα during the reproductive phase remains unclear. The effect of pPLA-IIα on flowering time was therefore investigated.

Harnessing snakehead rhabdovirus (SHRV) for gene editing by installment of CRISPR/Cas9 in viral genome.

As there is no risk of viral genome integration into host chromosome, cytoplasmic RNA viruses can be a safer vehicle to deliver CRISPR/Cas system. Snakehead rhabdovirus (SHRV) is a piscine RNA virus belonging to the family Rhabdoviridae, and, in the present study, we evaluated the availability of SHRV as a tool for CRISPR/Cas9 delivery in mammalian cells. SHRV was grown well in baby hamster kidney (BHK-21) cells at 28 °C, and the replication ability was greatly reduced by temperature up-shift to 37 °C.

Generation of Self-Inhibitory Recombinant Viral Hemorrhagic Septicemia Virus (VHSV) by Insertion of Viral P Gene-Targeting Artificial MicroRNA into Viral Genome and Effect of Dicer Gene Knockout on the Recombinant VHSV Replication.

To produce artificial microRNA (amiR)-mediated self-inhibitory viral hemorrhagic septicemia virus (VHSV), we inserted VHSV P gene-targeting amiR sequence (amiR-P) or control amiR sequence (amiR-C) between N and P genes of VHSV genome, and rescued recombinant VHSVs (rVHSV-A-amiR-P and rVHSV-A-amiR-C) using reverse genetic technology. The growth of rVHSV-A-amiR-P was significantly retarded compared to the control virus, rVHSV-A-amiR-C, due to the production of self P gene transcript-attacking microRNAs in infected cells. To enhance the replication of rVHSV-A-amiR-P, we generated the Dicer gene-knockout epithelioma papulosum cyprini (EPC-ΔDicer) cells using a CRISPR/Cas9 system, and evaluated the effect of Dicer knockout on the titer of rVHSV-A-amiR-P.

Mutation of the gene affects heading date in rice.

Aminoacyl-tRNA synthetases play a critical role in protein synthesis by catalyzing the covalent attachment of amino acids to their cognate tRNAs. However, the role of aminoacyl-tRNA synthetases in the transition from vegetative to reproductive growth in plants remains poorly understood. In this study, a rice () glycyl-tRNA synthetase 3, OsGlyRS3, was found to impact heading date in rice.

Effect of miR-155 on type I interferon response in Epithelioma papulosum cyprini cells.

MicroRNA-155 (miRNA-155) is known to play an important role in the regulation of innate and adaptive immune responses in mammals. However, no information is available on the role of miRNA-155 in relation to type I interferon (IFN) responses in fish cells. In the present study, we found that the protein inhibitor of activated STAT 4a (PIAS4a) gene of fathead minnow (Pimephales promelas) was a target of miR-155, which was verified by the inhibitory activity of miR-155 in the expression of reporter gene harboring 3'UTR of PIAS4a of EPC cells.

Enhancing immunogenicity of a reporter protein by fusion to glycoprotein and nucleoprotein of viral hemorrhagic septicemia virus (VHSV) particles.

The introduction of reverse genetic technology to generate recombinant VHSVs (rVHSVs) has contributed to the uncovering of functional roles of viral genes and to the development of attenuated prophylactic vaccines. In this study, to assess the possible use of rVHSVs as a tool of combined vaccines, we newly rescued rVHSVs that harbor viral envelop-studded eGFP (rVHSV-A-SGT) or nucleoprotein-fused eGFP (rVHSV-A-NLG), and the ability of these rVHSVs to induce adaptive humoral immunity in olive flounder (Paralichthys olivaceus) was compared with that of rVHSV-A-eGFP that expresses eGFP as a soluble form in the cytoplasm of infected cells. The results showed that antibodies against eGFP were efficiently induced by the immunization of olive flounder with rVHSV-A-SGT and rVHSV-A-NLG, while rVHSV-A-eGFP was poor in the ability to induce antibody response against eGFP.

E3 SUMO ligase AtSIZ1 regulates the cruciferin content of Arabidopsis seeds.

Arabidopsis thaliana E3 SUMO ligase SIZ1 (AtSIZ1) controls vegetative growth and development, including responses to nutrient deficiency and environmental stresses. Here, we analyzed the effect of AtSIZ1 and its E3 SUMO ligase activity on the amount of seed proteins. Proteomic analysis showed that the level of three major nutrient reservoir proteins, CRUCIFERIN1 (CRU1), CRU2, and CRU3, was reduced in the siz1-2 mutant compared with the wild type.

Generation of microRNA-30e-producing recombinant viral hemorrhagic septicemia virus (VHSV) and its effect on in vitro immune responses.

MicroRNAs (miRNAs) are non-coding small RNAs involved in the regulation of gene expression. In the present study, we firstly reported the use of a fish RNA virus, viral hemorrhagic septicemia virus (VHSV), as a delivery vehicle of a miRNA-30e, and the effect of miR-30e produced by the recombinant VHSV on the immune responses of Epithelioma papulosum cyprini (EPC) cells was investigated. The expression of functional miR-30e using a CMV promoter-driven vector was verified by the significantly lower eGFP expression in cells transfected with a vector containing miR-30e sponge sequence than that in cells transfected with a control vector that had mutated miR-30e sponge sequence.

Generation of a recombinant viral hemorrhagic septicemia virus (VHSV) expressing olive flounder (Paralichthys olivaceus) interferon-γ and its effects on type I interferon response and virulence.

Rhabdoviruses including viral hemorrhagic septicemia virus (VHSV) are highly susceptible to type I interferon (IFN) responses, and IFN-γ that is belonging to the type II IFN has been known to enhance type I IFN responses in mammals. In this study, we generated a recombinant VHSV that can express olive flounder IFN-γ (rVHSV-A-IFNγ) using reverse genetics technology, and analyzed the effect of rVHSV-A-IFNγ infection on type I IFN response in Epithelioma papulosum cyprini (EPC) cells. Furthermore, the virulence of rVHSV-A-IFNγ was evaluated by infection to olive flounder (Paralichthys olivaceus).

Post-translational modifications of FLOWERING LOCUS C modulate its activity.

Flowering Locus C (FLC) is a key floral repressor that precisely controls flowering time. The role of FLC has been extensively studied at the transcriptional level using molecular biological and epigenetic approaches. However, how FLC functions and how its stability is controlled at the post-translational level are only beginning to be understood.

Arabidopsis HIGH PLOIDY2 Sumoylates and Stabilizes Flowering Locus C through Its E3 Ligase Activity.

Flowering Locus C (FLC), a floral repressor, plays an important role in flowering. The mechanisms regulating FLC gene expression and protein function have been studied extensively; however, post-translational regulation of FLC remains unclear. Here, we identified Arabidopsis HIGH PLOIDY2 (HPY2) as an E3 SUMO ligase for FLC.

The E3 SUMO ligase AtSIZ1 functions in seed germination in Arabidopsis.

Seed germination is an important stage in the lifecycle of a plant because it determines subsequent vegetative growth and reproduction. Here, we show that the E3 SUMO ligase AtSIZ1 regulates seed dormancy and germination. The germination rates of the siz1 mutants were less than 50%, even after a short period of ripening.