Figure 3. Mutation of the 3A box promoter elements inhibits the production of mature miRNAs.

(A) RLM-RT-PCR of the viral miRNAs of TMER-1 and TMER-5. 293 cells were either infected with γHV68 or γHV68Δ9473 or transfected with the various Left End plasmid constructs followed by total RNA isolation and RLM-RT-PCR analysis. The human miRNA hsa-miR-15a is an endogenous cellular control for the RLM-RT-PCR assay. NTC – non-template control. (B) Schematics of the pGL3 firefly luciferase reporter system. A single copy of the miR-M1-1, miR-M1-10, miR-M1-7-3p, and miR-M1-12 were each individually cloned into a XbaI cut site located between the firefly luciferase coding region and the polyA signal in the pGL3-Control plasmid. (C) Dual luciferase reporter analysis with the A box mutant plasmids in 3T3 and 293 cells. The pGL3 luciferase reporter target is labeled below each graph. In infected samples, results are shown as a ratio of firefly luciferase expression in γHV68 infected cells divided by firefly luciferase expression in γHV68Δ9473 infected cells. In transfected samples, results are shown as a ratio of firefly luciferase expression in pLE-WT or A box mutant plasmids divided by the firefly luciferase expression in the pLE-KO transfected cells. All firefly luciferase values were normalized to the renilla luciferase transfection control readings prior to the comparative analysis shown on the graphs. Readings below the dashed line represent translational repression by the corresponding viral miRNA. Data represents the average of two experiments with each experiment conducted in triplicate. Error bars represent one standard of deviation.