Figure 5

The genome of PRRSV was not the target of miR-26a

. Fragments of the PRRSV genome were inserted into the 3`-UTR of the luciferase gene in the luciferase expression vector pRL-TK (Promega) (A). Then, co-expression of 1 μg miR-26a (or miR-Ctr) with the TK-ORFs in MARC-145 cells and luciferase activity assays were performed (B). (C) IFA was performed with an anti-myc monoclonal antibody to determine the expression efficiency in MARC-145 cells transfected with plasmids encoding 1 μg miRNAs (miR-26a or miR-Ctr) and the recombinant plasmids encoding the non-structural and structural proteins of PRRSV (including nsp2, nsp9, Gp2a, E, Gp3, Gp4, Gp5, M and N) with a myc-tag at their N-terminus. (D) MARC-145 cells were first individually transfected with plasmids expressing miR-26a or miR-Ctr or mock transfected followed by PRRSV VR-2332 infection at 10 MOI or mock infection and the mRNA level of each of the ORFs in the PRRSV genome was measured by real-time PCR at 6 hpi.