Signal sequences in the genome of Mononegavirales regulate the generation of copy-back defective viral genomes

Copy-back defective viral genomes (cbDVGs) are the primary initiators of antiviral immunity during infection with respiratory syncytial virus (RSV), parainfluenza virus, and other common pathogens. However, the mechanism governing cbDVG generation remains unknown limiting our ability to manipulate their content during infection. Here we developed VODKA (Viral Opensource DVG Key Algorithm) to identify cbDVGs from RNA-Seq data from infected samples and used these DVG sequences to identify signals that regulate cbDVG generation. After validation using data from an in vitro Sendai virus infection and Ebola virus-infected non-human primates, we applied VODKA to datasets from RSV-positive patients. VODKA identified common cbDVGs across multiple samples, and predicted specific genomic regions that mediate RSV cbDVG formation. Using an RSV minigenome system, we demonstrate that these viral sequences drive cbDVG wroduction. Importantly, the region involved in polymerase rejoin and reinitiation was highly conserved and its nucleotide composition determined the quality of cbDVGs generated. Our results indicate that generation of cbDVGs during natural infection is regulated by specific viral sequences and open the possibility to manipulate DVG formation during infection.