Figure 5.

The RABV N gene-start sequence serves as internal and terminal initiation signals for RABV RdRp. (A) In vitro oligo-RNA synthesis was performed with RABV L, P, and the indicated oligo-RNA templates as in Figure 4A. Relative RNA synthesis activities of the templates were expressed as percentages of the activity of RABV Le(–)20 (WT) (lane 2). Partial oligo-RNA sequences (mutated bases shown in red) are written in the 3′ to 5′ direction. Two independent transcription initiation sites were identified at positions 1 and 7 in RABV Le(–)20 (bottom). (B) RABV LeRNA and capped N mRNA are synthesized from the 3′-terminal Le promoter and internal gene-start sequence, respectively. The gene-start-like sequence in the RABV Le(–)20 template functions as an internal initiation site (see panel A). To examine whether the N gene-start sequence serves as internal and/or terminal initiation sites, the indicated oligo-RNAs with the N gene-start sequence were designed. The Le(–)6-GSN14 G11A, Le(–)6-GSN14 1UG-GU + G11A, and GSN14 G5A templates are called templates (i), (ii), and (iii), respectively, in Figure 6. (C) The indicated templates were subjected to transcription with ATP and [α-³²P]CTP, and RABV RdRp (L and P) as in Figure 4A. Relative RNA synthesis activities of the templates were expressed as percentages of the activity of RABV Le(–)20 (lane 2).