ISKNV-miR-1 locates at Kaempferol-3-rutinoside ISKNV genomic coordinate 7146–7165, just on the complementary strand of the VP08R-coding sequence. To investigate the modulatory effect of ISKNV-miR-1 on VP08R expression, dual-luciferase reporter assays were performed on HEK293T cells. Compared with the control, the ISKNV-miR-1 mimics could down-regulate 29.8% of the activity of VP08R-fused luciferase but showed no effect on the ISKNV-miR-1 seed-complementary site mutated VP08R (mVP08R)-fused luciferase (Fig. 2A and B), suggesting ISKNV-miR-1 could modulate VP08R expression. The effect of ISKNV-miR-1 on VP08R expression was also analyzed at protein level. Western-blot analyses exhibited that ISKNV-miR-1 could reduce the protein level of ectopic-expressed VP08R but not mVP08R in HEK293T cells (Fig. 2C). The modulatory effect of ISKNV-miR-1 on virus-expressed VP08R was further examined. Western-blot and the subsequent gray value analysis showed that compared with those in the control mimics or inhibitors treated cells, the VP08R expression was significantly decreased in ISKNV-miR-1 mimics-transfected cells but increased in the inhibitors-transfected cells (Fig. 2D), confirming that ISKNV-miR-1 could target VP08R.
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