In vitro RdRp activity assay was performed by using DI-72(2) or (+) RNA template transcribed in vitro by T7 transcription [14] . To measure the effect of host proteins in the replicase assay with DI72(2) RNA containing short double-stranded region at the 39end, a heat denatured RNA transcript (94uC for 2 min) was annealed with a 21-nt oligodeoxynucleotide (#20) (in 1:10 molar ratio) complementary to the 39 end of DI-72(2)RNA in STE buffer (10 mM TRIS, pH 8.0, 1 mM EDTA, and 100 mM NaCl) and then slowly (in 30 min) cooled them down to 25uC . RNase ONE digestion to remove single-stranded 32 P-labeled RNA was performed at 37uC for 30 min in a 16 RNase ONE buffer containing 0.1 ml of RNase ONE (Promega).