(A) Target sequences complementary to two distinct miRNAs (let-7a and miR124a) were inserted into two locations within the poliovirus genome. The 5’ site is located in the “variable” segment between the poliovirus internal ribosome entry site (IRES) and the start codon. The 3’ site is located between the structural and nonstructural genes.
(B) Perfect sequence complementarity between the endogenous miRNA and the target sequence inserted into the poliovirus genome is illustrated. Silence mutations were engineered into the target sequences to create virus controls that disrupt base pairing with the endogenous miRNA, while conserving the wild type encoded amino acid sequence.
(C) Northern Blot analysis of let-7a and miR-124a miRNA expression levels observed in cell lines (HeLa and NTERA-2, lanes 1 and 2) and mouse tissues (lanes 3–7).