FIG. 9.

SR1f does not decrease the accumulation of negative-strand RNA2 in BYL when the replication phase is separated from the translation phase. (A) First, an RNA1 mutant (R1/m1/d3′SLF) that lacks RNA elements required for negative-strand RNA synthesis and SR1f generation was incubated in BYL at 17°C for 120 min for the production of replicase proteins (thin horizontal arrow). Then, cycloheximide (CHX) was added to shut off the translation of R1/m1/d3′SLF, and RNA2 was incubated with or without a fivefold molar excess of SR1f in BYL at 17°C for an additional 120 min (bold horizontal arrow). (B and C) Total RNA was extracted at the indicated times and used for Northern blotting to detect positive- and negative-strand RNA2. The time when CHX was added was defined as 0 min. The Image Gauge program (Fuji Photo Film, Tokyo, Japan) was used for the quantification of negative-strand RNA2. The error bars indicate standard deviations.