miR-homoHSV of Singapore Grouper Iridovirus (SGIV) Inhibits Expression of the SGIV Pro-apoptotic Factor LITAF and Attenuates Cell Death

<div><p>Growing evidence demonstrates that various large DNA viruses could encode microRNAs (miRNAs) that regulate host and viral genes to achieve immune evasion. In this study, we report that miR-homoHSV, an miRNA encoded by Singapore grouper iridovirus (SGIV), can attenuate SGIV-induced cell death. Mechanistically, SGIV miR-homoHSV targets SGIV ORF136R, a viral gene that encodes the pro-apoptotic lipopolysaccharide-induced TNF-α (LITAF)-like factor. miR-homoHSV suppressed exogenous and endogenous SGIV LITAF expression, and thus inhibited SGIV LITAF-induced apoptosis. Meanwhile, miR-homoHSV expression was able to attenuate cell death induced by viral infection, presumably facilitating viral replication through the down-regulation of the pro-apoptotic gene SGIV LITAF. Together, our data suggest miR-homoHSV may serve as a feedback regulator of cell death during viral infection. The findings of this study provide a better understanding of SGIV replication and pathogenesis.</p> </div

miR-homoHSV inhibits SGIV LITAF-induced apoptosis.

<p>(A) Morphology of FHM cells 48 h after transfection with the empty vector pLL3.7, pLL-homoHSV, pLL3.7 and pEGFP-LITAF, and pLL-homoHSV and pEGFP-LITAF, from left to right. (B) Nuclear morphology of FHM cells 48 h after transfection with the same vectors above. Arrows indicate apoptotic bodies. Many apoptotic bodies were observed in cells co-transfected with pLL3.7 and pEGFP-LITAF. (C) Flow cytometric analysis of dead cells in FHM cells transfected with the vectors above 48 h after transfection. (D) Statistical results for the proportion of PI-stained positive cells 24 h and 48 h after transfection with empty vector pLL3.7, pLL-homoHSV (left panel), pLL3.7 and pEGFP-LITAF, and pLL-homoHSV and pEGFP-LITAF (right panel). Data are means from at least three independent experiments. Error bars indicate SEM. *<i>P</i><0.05.</p

si-LITAF specifically blocks SGIV-induced apoptosis.

<p>(A) si-LITAF efficiently suppresses endogenous SGIV LITAF mRNA (left) and protein (right) expression in FHM cells. (B and C) Morphology (B) and nuclear morphology (C) of FHM cells 48 h after infection with SGIV. FHM cells were transfected with si-con (left) or si-LITAF (right), and infected with SGIV 18 h later. Arrows (bottom panels) indicate apoptotic bodies. Many apoptotic bodies were observed in si-con transfected FHM cells. (D) si-LITAF blocks SGIV-induced apoptosis. PI staining and fluorescence-activated cell sorter analysis of FHM cells 48 h after infection (up). Statistical results of the proportion of PI-stained positive cells 48 h after infection with SGIV (bottom). Data are means of at least three independent experiments; error bars indicate SEM. *<i>P</i><0.05. Flow cytometric analysis of apoptotic cells in FHM cells treated as described for panel B. </p

Expression profiles of endogenous miR-homoHSV and SGIV LITAF.

<p>The relative expression level of miR-homoHSV rose rapidly reaching its peak 6 h p.i. and then decreased (blue). Relative expression level of miR-homoHSV 6 h p.i. was set to 1 for comparison. The U6 gene was used as an internal control. The amount of SGIV LITAF mRNA accumulated stably from 6 to 48 h p.i. (red). Relative expression level of SGIV LITAF 6 h p.i. was set to 1 for comparison. β-Actin was used as an internal control. Data are means from rapidly at least three independent experiments done in duplicate; error bars indicate SEM.</p

miR-homoHSV inhibits exogenous and endogenous SGIV LITAF expression.

<p>(A) miR-homoHSV located in the antisense strand of ORF136R. (B and C) miR-homoHSV inhibits exogenous (B) and endogenous (C) SGIV LITAF protein expression. (D and E) miR-homoHSV does not affect exogenous SGIV MCP expression at the mRNA (D) or protein (E) level. </p

miR-homoHSV attenuates SGIV-induced apoptosis and promotes SGIV replication.

<p>(A) Morphology of FHM cells 48 h after infection with SGIV. FHM cells was transfected with pLL3.7 (left) or pLL-homoHSV (right), and then infected with SGIV. (B) Nuclear morphology of FHM cells 48 h after infection with SGIV. Cells were treated as described for panel A. Arrows indicate apoptotic bodies. Many apoptotic bodies were observed in pLL3.7 transfected FHM cells (left). (C) Flow cytometric analysis of dead cells in FHM cells treated as described for panel A. Top panels are PI staining and fluorescence-activated cell sorter analysis of FHM cells 48 h after infection. Bottom panel shows the statistical results of the proportion of PI-stained positive cells 24 h and 48 h after SGIV infection. Data are means of at least three independent experiments; error bars indicate SEM. *<i>P</i><0.05. (D and E) miR-homoHSV promotes endogenous SGIV ORF016L (D) and SGIV MCP (E) mRNAs (top) and SGIV MCP protein (bottom) expression in FHM cells. (F) miR-homoHSV promotes SGIV replication. FHM cells overexpressing miR-homoHSV/miR-con were infected with SGIV, and virus was collected 48 h p.i. The viral titer was measured using the TCID50 method. Error bars indicate SEM.</p

Comparative Transcriptomic Study of Muscle Provides New Insights into the Growth Superiority of a Novel Grouper Hybrid

<div><p>Grouper (<i>Epinephelus spp</i>.) is a group of fish species with great economic importance in Asian countries. A novel hybrid grouper, generated by us and called the Hulong grouper (Hyb), has better growth performance than its parents, <i>E</i>. <i>fuscoguttatus</i> (Efu, ♀) and <i>E</i>. <i>lanceolatus</i> (Ela, ♂). We previously reported that the GH/IGF (growth hormone/insulin-like growth factor) system in the brain and liver contributed to the superior growth of the Hyb. In this study, using transcriptome sequencing (RNA-seq) and quantitative real-time PCR (qRT-PCR), we analyzed RNA expression levels of comprehensive genes in the muscle of the hybrid and its parents. Our data showed that genes involved in glycolysis and calcium signaling in addition to troponins are up-regulated in the Hyb. The results suggested that the activity of the upstream GH/IGF system in the brain and liver, along with the up-regulated glycolytic genes as well as ryanodine receptors (RyRs) and troponins related to the calcium signaling pathway in muscle, led to enhanced growth in the hybrid grouper. Muscle contraction inducing growth could be the major contributor to the growth superiority in our novel hybrid grouper, which may be a common mechanism for hybrid superiority in fishes.</p></div

qRT-PCR validation of candidate genes.

<p>Nine glycolytic genes (A), RyR and Tn (B) were picked out as representatives of differences between the Hyb and its parents. β-actin was used as the internal control, and each value represents an average of three separate biological replicates. The error bars represent the standard deviation. Asterisk (*) was marked if there was significant difference between the two samples, as assessed by a t-test (<i>P</i> < 0.05).</p

Ca²⁺ signal was activated by RyRs, with downstream troponin proteins up-regulated in the Hyb.

<p>(A) Diagram representing the related Ca²⁺ signaling pathway and the interaction between calcium and troponins. Blue arrows denote the expression differences between the Hyb and paternal Ela. Red arrows denote the expression differences between the Hyb and maternal Efu. Up arrows stand for up-regulation of transcription in the Hyb. The green up arrow denotes the increasing Ca²⁺ concentration in the cytoplasm. (B) Hierarchical cluster analysis of Ca²⁺ regulating genes and troponins. The color key represents RPKM values.</p

GO enrichment in the muscle transcriptome of the Hyb.

<p>The left y-axis indicates the identified GO terms. The right y-axis indicates the three groups of GO enrichment. The down x-axis represents the number of genes in certain category.</p