A specific sequence in the genome of respiratory syncytial virus regulates the generation of copy-back defective viral genomes
Fig 1
VODKA identified hotspots for polymerase break and rejoin during cbDVG formation.
(A) Schematic representation of cbDVG generation from negative strand viruses and general principle behind VODKA identification of cbDVGs. The red dashed square marks the unique junction region that distinguishes cbDVGs from full-length viral genomes. This junction region was used by VODKA to identify cbDVGs. (B) A549 cells were mock infected or infected with SeV Cantell at a MOI of 1.5 TCID50/cell and harvested at 2, 6, 12, and 24 h post infection followed by detection of cbDVGs by RT-PCR using primers SeV DI1 and gSeV DI1 (S1 Table). (C) Alignment of SeV cbDVG junction reads obtained from VODKA to the last 3kb nucleotides of the SeV antisense genome (top) or the DVG-546 junction sequence (bottom). Blue histogram shows a synopsis of total coverage at any given position of the last 3kb of the SeV reference genome (top) or the DVG-546 junction sequence (bottom). The numbers on the left-side axis of the graphs represent the total number of reads at the position with the highest coverage. Colored nucleotides beneath the blue histogram represent the consensus sequences of cbDVG junction identified by VODKA. The size of the letter represents the degree of conservation at the site and if there were mutations at a certain position the most representative nucleotide was listed on top. Black nucleotides beneath colored nucleotides indicate the sequence of the DVG-546 junction region obtained by Sanger sequencing. The grey boxed areas in the graphs mark the region after the rejoin point and the pink boxed area the DVG region before the break point. (D) A549 cells were infected with RSV stocks1-7. For stocks1-6, RNA was extracted from the supernatants of the infected cells at 48 h post infection, followed by RNA-seq and VODKA screening. DVG junction reads were then mapped to the antigenome of the RSV A2 reference strain. Blue histogram shows synopsis of total coverage at any given position of the last 3kb of the RSV reference genome. The number on the right side of the graph represents the total reads at the position with highest coverage. Based on the VODKA output, individual peaks were identified as break or rejoin regions indicated by red or black facing-down arrows, respectively. For stock7, RNA was extracted from infected cells at 24 h post infections, followed by DVG specific RT-PCR using primer DI1 and DI-R (see S1 Table) that captures cbDVGs larger than 453 bp and with the break after DI1 and the rejoin after DI-R. Based on conventional cloning and Sanger sequencing of PCR products, the break and rejoin points of cbDVGs detected in this stock were marked by red and black facing upwards arrows, respectively. Dashed lines indicate join sequences from individual cbDVGs in stock7. Light and dark grey shades represented the rejoin and break regions that were cloned into RSV minigenome backbone for further analysis in Fig 2A. Reference genome is shown at the bottom of the figure and the corresponding positions of DVG break/rejoin regions from all stocks, Break1, Rejoin1, and Trailer are also indicated.