Figure 2

The 5′- and 3′-terminal UTRs of TSWV S cRNA are necessary and sufficient for TSWV RNA synthesis. (a) Deletion analysis of TSWV S RNA. Ten mutant RNAs lacking the indicated regions (d1–d10) were expressed in yeast with L and N proteins. S: unmodified TSWV S RNA. RNA was detected by Northern blot hybridization. Bands observed above the S cRNA and its derivatives likely represent uncleaved transcripts. Lanes represent independent colonies. (b) An RNA that contains 5′- and 3′-terminal UTRs of TSWV S cRNA can be replicated with the aid of both L and N proteins in yeast. In the panels marked YFPrv and YFPfw, expressed and anti-expressed RNA strands were detected, respectively. (c) The 3′ UTR of S cRNA has a stronger signal for complementary strand synthesis than the 3′ UTR of S vRNA. The UTRs were fused to the YFP sequence as shown in the figure and YFP-related RNAs were analyzed. Band intensity was quantified and the ratio of the intensity of the anti-expressed strand (YFPfw) to that of the expressed strand (YFPrv) is shown below the panel. (d) Quantification of YFP fluorescence in yeast cells expressing the YFP replicon (Yrep), N protein, and the L or L(SAA) mutant protein in the indicated combinations. As a positive control, YFP was expressed from the same vector as Yrep. Values are means of cultures from four independent colonies. Error bars represent standard deviation. A.U.: arbitrary unit showing YFP values normalized by optical density at 595 nm.