To determine the contribution of the individual residues of the sh-B971 sequence, we introduced a series of point mutations into the shRNA and tested them for IFN induction. We changed the first nucleotide from A to G, C, or T while maintaining base-pairing between nucleotides +1 and +47. These mutant shRNAs lacked the ability to activate IFN production (Table 1) . Changing the +1 nucleotide to G while leaving the +47 nucleotide intact also abolished IFN activation by the shRNA (A1/G), as did the reciprocal mutation U47/C. The importance of the first nucleotide was further confirmed by the inability of sh-B971+1 to activate IFN. The target of sh-B971+1 was shifted 1 nt downstream on the CyPB mRNA, producing an shRNA starting with a G at the +1 position. The presence of an A at the +1 position was not, however, sufficient to render a shRNA competent for IFN activation, as replacing the first nucleotide of the sh-NTC with an A did not generate an IFN-inducing shRNA (NTC-A and NTC+1). These results indicate that a protruding/unpaired A at the end of the hairpin or the RNA duplex, a potential result of 'breathing' at the end of the dsRNA, is not sufficient to trigger IFN induction as previously suggested (38) .