FIG. 5.

p27 recruited RNA2 to the membrane fraction via a p27-YRE interaction in BYL. (A) Fractionation of replicase proteins and positive- and negative-strand RNA2 by centrifugation. RNA2 or RNA2-SL8LoopM was incubated with R1-p27F-R1 and R1-m1/dSLF (which are a source of replicase proteins) in BYL at 17°C for 2 h. RNA2 was also incubated alone in BYL. After incubation, samples (total [T]) were centrifuged at 20,000 × g for 20 min, to obtain supernatant (S) and membrane-containing pellet (P) fractions. Aliquots of these fractions were used in Northern and Western blotting experiments. Western blotting was performed using an anti-p27 antiserum. Northern blotting was performed using digoxigenin-labeled RNA probes that were complementary to the 3′ UTR of positive-strand RNA2 [RNA2(+)] and to full-length negative-strand RNA2 [RNA2(−)]. The asterisks indicate cross-reacting soluble cellular proteins. (B) Quantification of the relative accumulations of positive-strand RNA2 and RNA2-SL8LoopM in the pellet fractions shown in panel A by using the Image Gauge program (Fuji Photo Film, Tokyo, Japan). The accumulation level was calculated as 100 × (RNA2 in pellet)/(RNA2 in supernatant + pellet). The error bars represent the standard deviations from the means of at least three independent experiments. (C) Membrane flotation analysis of replicase proteins and RNA2. RNA2 or RNA2-SL8LoopM was incubated with R1-p27F-R1 in BYL at 17°C for 2 h. RNA2 was also incubated alone in BYL. These samples were fractionated into top (T), middle (M), and bottom (B) fractions by using the membrane flotation analysis described in Materials and Methods. Western blotting was performed using an anti-p27 antiserum. Northern blotting was performed using digoxigenin-labeled RNA probes that were complementary to the 3′ UTR of positive-strand RNA2.