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. 2019 Apr 17;15(4):e1007707. doi: 10.1371/journal.ppat.1007707

Fig 4. Specific nucleotide composition determines the position for cbDVG rejoin.

Fig 4

(A) AU-content (top, purple) over the entire genome and major rejoin region (inset, purple) of the RSV strain A2 antigenome. AU-content is plotted centered on a 40 base-pair sliding window in both plots. Grey box indicates the position of Rejoin1 and Trailer. (B) cbDVG-like fragments generated in mutant GC>Us and mutant AU>GCs were detected by RT-PCR using the DI-F1 and DI-R primer set (S1 Table). Confirmed cbDVG-like fragments are indicated with asterisks next to the gel. (C) Schematic summary of all identified cbDVG rejoin points within positions 14998–15105 of the genome (RSV A2, reference genome NCBI KT992094.1) generated from WT Pair1 and Pair1 mutant minigenomes. This region includes gene end of L and the beginning of Trailer. Different colored arrows indicate rejoin points from cbDVGs detected by different primer sets as indicated in the figure. Primer sets are composed of the identical reverse primer, DI-R, and one of either three different forward primers, DI-F1, DI-F2, and DI-F3. The numbers near the arrows indicated the number of times (>1) the same cbDVG was observed at this location (out of three independent experimental repeats with 1–3 clones sequenced each time). The thick black line marks the sequence of rejoin region selected for mutation studies. The antigenomic position range of the mutations is indicated at the bottom of the figure and the actual RNA sequences at the mutation sites in WT and five mutants are indicated. The numbers on top of the WT RNA sequence indicate their positions from the 3’ end of the antigenome. Grey shade marks the area where mutants GC>Us, C186U, and AU, did not have the rejoin points. (D) cbDVG fragments generated in mutant GC>Us and mutant AU>GCs were detected by DI-F2/DI-R and DI-F3/DI-R through DVG specific RT-PCR. The DVG-like bands labeled by asterisks were confirmed to contain the same cbDVG-like fragment detected by DI-F1/DI-R in B (only band labeled with an asterisk). (E) cbDVG fragments generated in mutants C188U, C186U, and AU were detected by DVG specific RT-PCR using the indicated primers. Confirmed cbDVG-like fragments are indicated with asterisks. Band labeled with an arrowhead was confirmed not to be a DVG product. Sequences of all the confirmed cbDVG-like fragments are shown in S3B–S3F Fig.