FIG 4.

Newly identified viral genes targeted by KSHV miRNAs. (A) Repression of 3′UTRs by KSHV miRNAs detected by reporter assays. 3′UTR reporters from KSHV genes with bicistronic and polycistronic transcripts are labeled with an asterisk (*). The miR-K10/ORF-K2 pair, which was independently identified in a HITS-CLIP screening (40), is labeled in red type. The miR-K7/ORF50 and miR-K9/ORF50 pairs identified in previous studies are shown in “red” and used as positive controls (19, 34). (B) Distribution of 3′UTRs from KSHV genes with monocistronic transcripts, and bicistronic and polycistronic transcripts repressed by KSHV miRNAs. (C to F) Confirmation of repression of 3′UTRs by KSHV miRNAs. 3′UTRs were targeted by miR-K3 (C) miR-K8 (D), miR-K10 (E), and miR-K11 (F). 293T cells were cotransfected with 3′UTR reporter plasmids or a reporter vector control and the respective miRNA expression plasmids or a miRNA expression vector control, together with a pSV-β-galactosidase plasmid for 48 h, and the relative luciferase activities were measured after normalization. In the initial screening experiments (A), three rounds of independent screening were performed, each with three repeats. The results of the three rounds of screenings were combined and analyzed. All other experiments (C to F) were repeated at least three times. The results presented as averages and STD by setting the values of miRNA expression vector control as “1”.