FIG 2.

ttsgR is unlikely a product of 5′-to-3′ degradation of longer TCV RNAs. (a) Schematic depiction of Δsg1 and Δsg2 mutants of TCV_sg2R. The bottom portion of the stem-loop structure identified previously as the sgRNA1 promoter is shown on the top left. The Δsg1 mutant, with the five single-nucleotide mutations shown in red letters, is represented to the right. Similarly, the partial sgRNA2 promoter stem-loop is at the bottom left, with the corresponding Δsg2 mutant shown to the right, in which a deletion of 12 nt is denoted as a red line. (b) ttsgR accumulation was not abolished in Δsg1, Δsg2, and Δsg1&2 mutants, even though the levels of the corresponding sgRNAs were drastically diminished, as evidenced by Northern blotting results. (c) Schematic representation of three template constructs that transcribe 3′-coterminal TCV (TCV_sg2R)-derived RNAs of various lengths, with none of them capable of translating the replication proteins. (d) The three defective templates templated the production of ttsgR, in addition to RNAs of template lengths, in the presence of trans-supplied p28 and p88, but not p28 and p88mGDD. Note that the [p28stop]_sg2R template accumulated very low levels of full-length RNA but an easily detectable level of sgRNA2 and ttsgR. This outcome was expected, as overexpressed p88 complemented the replication of TCV gRNA poorly. (e) Preliminary mapping of the 5′ terminus of ttsgR by Northern blotting with four different probes.