Determination of <i>cis</i>-acting RNA elements between the nts −4 and −40 required for (−)- and (+)-strand RNA synthesis.

<p>(A) Deletion mutants of BCoV DI RNA. Dashes denote deleted sequences. (B) The relative levels of (−)-strand DI RNA synthesis. The synthesis of (−)-strand DI RNA from the deletion mutant was quantitated by qRT-PCR and was compared with that from wt BM25. Control A: total cellular RNA from mock-infected cells. Control B: total cellular RNA from BCoV-infected cells. Control C: total cellular RNA from DI RNA-transfected mock-infected cells. Control D: a mixture of BCoV-infected cellular RNA extracted at 10 hpi and 200 ng of BM25A transcript. (C) Upper panel: the synthesis of (+)-strand DI RNA as detected by Northern blot assay. Total cellular RNA was extracted at 48 hpi of VP1 and was subjected to Northern blot assay with N sgmRNA and 18S rRNA as internal controls. Middle panel: the relative levels of the (+)-strand DI RNA synthesis. Lower panel: the sequence of the BCoV DI RNA at 48 hpi of VP1 as determined by RT-PCR and sequencing analysis. The values (B) and (C) represent the mean ±SD of three individual experiments. SD: standard deviation, wt: wild type. **p<0.01.