The N79 Rbz is not restricted in its ability to cleave the [−] RNA of positive strand RNA virus constructs when this RNA is in a single-stranded state. (A) Single-stranded [−] RNAs from replicons containing a reverse complemented N79 sequence were generated by in vitro transcription and their cleavage was assessed by gel electrophoresis and image capture. Significant differences between experimental groups are highlighted ([*] P < 0.05; one-way ANOVA; n = 3). (B) RNAs from cells transfected 24 h earlier with YFV or HRV replicons carrying a reverse complemented N79 sequence were collected. They were subsequently subject to treatment designed to release the [−] RNA from its double-stranded state and enable it to fold before Mg2+ was added to activate Rbz activity. Experimental groups lacking Mg2+ addition were included as controls. Cleavage was assessed by northern blot analysis . The arrows represent the position of full length transcripts and arrowheads the position of N79-cleaved product.
