FIGURE 7.

The effect of a 3-nt bulge plus the presence (or not) of the 5′ pppN within dsRNA of modified Junin virus RNA on PAMP function. A, the sequence of the 5′ 61 nucleotides of the Junin virus S genome RNA is shown (5′ to 3′, top line), in which capital letters indicate uridines that were changed to the nucleotides shown to generate Jun −1/60^mod RNA. The bottom line shows the sequence of the 3′ terminal 20 nucleotides of the S genome RNA (3′ to 5′). The mismatches in the dsRNA panhandle structure are indicated. B, 5′ ppp Jun −1/60^mod RNA, RNase III treated (light gray) or not (dark gray), was annealed (or not, lane 7) with synthetic oligonucleotides representing positions −1 or +1 to 20 of the S genome panhandle as complementary RNA, maintaining the 3-nt bulge (lines 8 and 9, respectively), or oligonucleotides in which positions 5 and 7 were changed so that a perfect dsRNA panhandle would be formed to resemble to L segment (lines 10 and 11, respectively). The hybridized RNA (see supplemental Fig. S5B) were then transfected into A549 cells, and their ability to activate the IFNβ promoter was determined. 1 μg of 5′ ppp Jun −1/60^mod RNA was transfected in all cases. Lane 1 (Ctl) shows the level of IFNβ activation in untransfected cells. Transfection of tRNA by itself is shown in lane 6. Transfection of the oligonucleotides by themselves is shown in lanes 2–5.