Bioinformatics analysis and protein interaction of ISKNV ORF111L.

(A) ISKNV ORF111L domain architecture was predicted by SMART program. (B) Phylogenetic tree of ISKNV ORF111L with other TRAF family proteins. The Bootstrap test of phylogeny was calculated 1000 replicates. The numbers at the nodes indicate bootstrap values. (C) Homology model structures analysis of TRAF domain from ISKNV ORF111L (panel a) and Homo sapiens (panel b). (D) Multiple sequence alignments of TRAF domain from different TRAF proteins. PTH, predicted TRAF homology; TD, TRAF domain. (E) Expression of GST proteins (c, lane 1), GST-111L fusion proteins (c, lane 2) and MYC-TRADD fusion proteins (d, lanes 1 and 2) were effective. After the GST pull down assay, MYC-TRADD fusion proteins were detected in the GST-111L sample (e, lane 2) but not in the GST control sample (e, lane 1), indicating the interaction between ISKNV ORF111L and zebrafish TRADD.

ISKNV ORF111L overexpression resulted in abnormal phenotype in zebrafish embryo.

(A–B) The GFP and 111L-GFP expression in pEGFP-N3- and p111L-GFP-injected embryos were shown. (C–D) Empty vector pEGFP-N3-injected embryo did not show any phenotypic abnormality, while the yolk sac was not completely developed in 111L-GFP overexpressing embryo. (E–F) Hematoxylin-eosin staining assay was performed in GFP- and 111L-GFP-overexpressing embryo.

ISKNV ORF111L overexpression resulted in evident apoptosis in zebrafish embryo.

(A–B) The RFP and RFP-111L expression in pdsRed2-C1- and pRFP-111L-injected embryos were shown. (C–D) No obvious apoptotic signal was found in RFP overexpressing embryo, while increased apoptosis signal were clearly observed in RFP-111L-overexpressing embryo. (E) Statistics analysis of fluorescent, abnormality and apoptosis of embryos. Embryos shown above were at 3 dpf stage and represented the typical phenotype in three individual microinjection experiments. The significance of differences are calculated by the t-test (** indicates p<0.01). (F–G) TUNEL assay was performed in wild type and ISKNV ORF111L mRNA-injected embryos. Apoptotic cells were shown (arrow head). Figure F and G is the enlarged figure from panel f and g, respectively. (H) After the NBT/BCIP staining, the number of apoptotic cells was counted in wild type and ISKNV ORF111L mRNA-injected embryos.

ISKNV ORF111L-induced apoptosis was mediated by caspase 8.

(A) The caspase 8 expression was tested by RT-qPCR assay. The expression level of β-actin was set as 1, and values were normalized to the corresponding β-actin values to determine the relative copy number. All data are presented as means from three individual injection experiments. (B) The caspase 8 activity was tested in wild type, pdsRed2-C1- and pRFP-111L-injected embryos. (C) Knockdown efficiency of caspase 8^Spl-MO in zebrafish embryos. Schematic representation of normal transcription of caspase 8 and morpholino splicing blocking of caspase 8 was shown. The caspase 8^Spl-MO interferes with splicing junction at exon 3/intron 3, resulting in retention of the intron 3. The retention of intron 3 resulted in a frame-shift and the truncation of protein translation. Knockdown effect of caspase 8^Spl-MO was tested in by RT-PCR. Forward primer is located in exon 2, and the reverse primer is located in exon 4. Result showed that caspase 8^Spl-MO strongly depletes the wild type caspase 8 mRNA and had a high gene knockdown effect. (D) Caspase 8 knockdown effectively blocked ISKNV ORF111L-induced apoptosis. The caspase 8^Spl-MO and pRFP-111L were co-injected into 1–2 cell stage embryos. The yolk sac of embryo was healthily developed (panel a). The expression of RFP-111L fusion proteins were clear (panel b), and no obvious apoptosis signal were found in TUNEL assay (panel c), indicating that knockdown the expression of caspase 8 could effectively block ISKNV ORF111L-induced apoptosis. Embryos shown above are the typical phenotype in three independent experiments.

ISKNV ORF111L and zebrafish TRAF2 overexpression resulted in caspase 8 and caspase 3 upregulation.

(A–B) The caspase 8 or caspase 3 expression was shown in wild type embryos by whole mount RNA in situ hybridization assay. (C–D) ISKNV ORF111L overexpression resulted in significant caspase 8 (C, arrow) and caspase 3 (D, arrow head) upregulation. (E–F) Zebrafish TRAF2 overexpression induced modest caspase 8 (E, arrow) and caspase 3 (F, arrow head) upregulation compared with those of ORF111L. All embryos shown represent the typical staining and are lateral views with anterior to the left at 3 dpf.