Figure 1
From: The 3' sequences required for incorporation of an engineered ssRNA into the Reovirus genome
Survey of the minimum 3' terminal s2 ssRNA nucleotides required to direct a ssRNA into the reovirus genome using 50-nucleotide deletions, single-nucleotide deletions and nucleotide deletions using ssRNAs with extended 5' sequences. At the top, are the sequences of the 5' nucleotides of the ssRNAs produced using the T7 RNA polymerase promoter and cDNA template pS2CAT198 and pS2CAT96. The top two sequences include the first 18 nucleotides of the CAT coding sequence but do not show the 3' end of the ssRNA. The 3' sequence of these ssRNAs is shown in its entirety from the CAT stop codon to the authentic reovirus 3' terminus directly below the 5' ssRNA sequences. A line connects the last retained s2 nucleotide in the displayed sequence to the named cDNA plasmid, below which are displayed autoradiogram panels. Within each panel, the ssRNA, dsRNA and CAT dsRNA panels are Northerns analyzing RNA extracted from cells lipofected 12 hours earlier with 9 wildtype ssRNAs and the ssRNA obtained following transcription of the indicated cDNA template. The fourth and bottom panel of each set is an autoradiogram generated by in vivo labeling with 32P of the dsRNA genome segments of an isolated progeny virus containing the indicated mutated-S2 dsRNA. Progeny virus generated following lipofection was first triple-plaque purified. Deletions were initially made in blocks of 50 nucleotides. Based on the sequences required to incorporate a ssRNA into a reovirus using the ssRNAs generated from these templates, additional cDNA templates were constructed deleting ten, five and individual nucleotides until the minimal 3' sequence had been determined. Left and center panels. To test the possibility that increasing the 5' s2 sequence from 96 to 198 nucleotides might reduce the length of the 3' sequence required, a number of the 3' deleted cDNA templates were altered to include a the 198 5' sequence and retested. The ability to incorporate these ssRNAs into the genomes of reoviruses is summarized in the right panel. As described in the Materials and Methods, all ssRNAs were sequenced/analyzed to confirm the 5' and 3' ends of these RNAs.