FIG 1.

SFV RCs are stable and robust in RNA synthesis in vitro. (A) BHK cells were infected with SFV at an MOI of 50, and PNS was prepared at 4 h p.i. In vitro replication was studied by the incorporation of [³²P]CTP into SFV RNA, indicated by 26S, II, and 42S. After the incubation times indicated, RNA was isolated and analyzed in a denaturing agarose gel. Ribosomal 18S RNA (18S rRNA) was detected by in-gel hybridization from the same gel. (B) Kinetics of the incorporation of [³²P]CTP into 26S (blue) and 42S (green) RNA. Percentages indicate the incorporation compared to the last time point (100%). Data are presented as means ± standard deviations from two independent experiments. (C) Replication assay performed with either undiluted PNS or PNS diluted as indicated. 18S rRNA was detected as described for panel A. For the undiluted sample, 1/10 of the amount of RNA used for other samples was used. (D) Stability of replication activity. PNS was preincubated at 4°C for 3, 24, and 48 h, or PNS was diluted in dilution buffer (DB) or iodixanol and preincubated at 4°C for 24 h before a replication assay. The last two lanes show samples containing 1 mg/ml BSA or 100 μM 3′-dCTP. 18S rRNA was detected by in-gel hybridization from the same gel. (E) Stability of endogenous RNA. To detect endogenous SFV minus and plus strands, total RNA was isolated after the same preincubations as those described for panel D and analyzed by in-gel hybridization with specific probes. (F) Detergent sensitivity. PNS samples containing the indicated detergent concentrations were incubated at 4°C for 1 h and used for either a replication assay or total RNA isolation, followed by in-gel hybridization. After the replication assay, the incorporation of [³²P]CTP into 42S RNA was quantified, and percentages indicate the incorporation compared to the untreated PNS. The lower panel shows the presence of endogenous minus-strand RNA in the untreated and 1% detergent-treated samples detected by in-gel hybridization. Numbers below the lanes indicate the percentage of [³²P]signal compared to that of the untreated sample. Octyl, n-octylglucoside.