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. 2014 Jul 30;6(8):2938–2959. doi: 10.3390/v6082938

Figure 2.

Figure 2

Identification of the cis-acting elements within the 5' and 3' UTRs of sgmRNA that are required for (−)-strand sgmRNA synthesis. (A) Illustration of the constructs with deletions in the 5' UTR (construct sNL) and 3' UTR (constructs sΔB, sΔP, and s3'Δ55). SL: stem-loop; BSL: bulged stem-loop; PK: pseudoknot; HVR: hypervariable region. (B) The relative efficiency of (−)-strand sgmRNA synthesis, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sNL -transfected mock-infected cells. (C) The relative efficiency of (−)-strand sgmRNA synthesis, as measured by RT-qPCR with (left panel) or without (right panel) head-to-tail ligation. Left panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 8 hpt and 200 ng of sBM25A transcript. Right panel: Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from sBM25A-transfected mock-infected cells. Control D: total cellular RNA from sΔB-transfected mock-infected cells. Control E: total cellular RNA from sΔP-transfected mock-infected cells. Control F: total cellular RNA from s3'Δ55-transfected mock-infected cells. (D) Measurements of reporter-containing sgmRNA, 18S rRNA and M sgmRNA (from helper virus) at 8 hpt of VP0 by Northern blot assay. M sgmRNA rather than N sgmRNA was chosen to represent helper virus here and in the following figures because the reporter-containing (+)-strand sgmRNA and BCoV N sgmRNA (sgmRNA 7) migrated to the same position. The values (B) and (C) represent the mean ± SD of three individual experiments. ** p < 0.01, *** p < 0.001.