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

Abstract

Defective viral genomes of the copy-back type (cbDVGs) are the primary initiators of the antiviral immune response during infection with respiratory syncytial virus (RSV) both in vitro and in vivo. However, the mechanism governing cbDVG generation remains unknown, thereby limiting our ability to manipulate cbDVG content in order to modulate the host response to infection. Here we report a specific genomic signal that mediates the generation of RSV cbDVGs. Using a customized bioinformatics tool, we identified regions in the RSV genome frequently used to generate cbDVGs during infection. We then created a minigenome system to validate the function of one of these sequences and to determine if specific nucleotides were essential for cbDVG generation at that position. Further, we created a recombinant virus that selectively produced a specific cbDVG based on variations introduced in this sequence. The identified sequence was also found as a common site for cbDVG generation during natural RSV infections, and common cbDVGs generated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome are critical determinants of the location of cbDVG generation and, therefore, this is not a stochastic process. Most importantly, these findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome. Author summaryCopy-back defective viral genomes (cbDVGs) regulate infection and pathogenesis of Mononegavirales. cbDVG are believed to arise from random errors that occur during virus replication and the predominant hypothesis is that the viral polymerase is the main driver of cbDVG generation. Here we describe a specific genomic sequence in the RSV genome that is necessary for the generation of a large proportion of the cbDVG population present during infection. We identified specific nucleotides that when modified altered cbDVG generation at this position, and we created a recombinant virus that selectively produced cbDVGs based on mutations in this sequence. These data demonstrate that the generation of RSV cbDVGs is regulated by specific viral sequences and that these sequences can be manipulated to alter the content and quality of cbDVG generated during infection.