Date Published: April 17, 2019
Publisher: Public Library of Science
Author(s): Yan Sun, Eun Ji Kim, Sébastien A. Felt, Louis J. Taylor, Divyansh Agarwal, Gregory R. Grant, Carolina B. López, Marco Vignuzzi.
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 a subset of RSV cbDVG species. 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 unable to produce a subset of cbDVGs due to mutations introduced in this sequence. The identified sequence was also found as a site for cbDVG generation during natural RSV infections, and common cbDVGs originated at this sequence were found among samples from various infected patients. These data demonstrate that sequences encoded in the viral genome determine the location of cbDVG formation and, therefore, the generation of cbDVGs is not a stochastic process. These findings open the possibility of genetically manipulating cbDVG formation to modulate infection outcome.
Defective viral genomes (DVGs), which are generated during the replication of most RNA viruses, potentiate the host innate immune response [1–5] and attenuate the infection in vitro and in vivo [4, 6–9]. Importantly, in naturally infected humans, the presence of DVGs correlates with enhanced antiviral immune responses during RSV infection  and reduced disease severity in influenza virus infection . Significant effort is currently invested in harnessing DVGs as antivirals due to their strong immunostimulatory activity and ability to interfere with the replication of the standard virus. However, despite over 50 years of appreciating their critical functions in multiple aspects of viral infections, the molecular mechanisms that drive DVG generation remain largely unknown. This lack of understanding hampers our ability to effectively harness DVGs for therapeutic purposes and limits our capacity to generate tools to elucidate their mechanism of action and impact during specific viral infections.
DVGs are critical regulators of viral replication and pathogenesis in multiple RNA virus infections, but the mechanisms modulating their generation are unknown. Historically, DVGs were thought to result from random errors introduced by the viral polymerase during replication. However, mounting evidence indicates that the generation of cbDVGs is not totally stochastic. Specifically, we show that during RSV infection discrete hotspots in the viral genome mark sites for the viral polymerase to release and rejoin during cbDVG formation, both in vitro and during natural RSV infections in humans. Moreover, we show that the content of C nucleotides, and possibly G nucleotides, within the major rejoin hotspot critically impacts the generation of cbDVGs at that position. We also identified specific nucleotides that, when mutated, altered the ability of recombinant viruses to generate diverse species of DVGs. The identification of a specific sequence involved in cbDVG formation opens the unprecedented possibility of genetically manipulating the content of cbDVGs during infection. This possibility may significantly impact our ability to generate tools to further understand the role of these viral products in virus pathogenesis, as well as potentially manipulate the cbDVG content with antiviral and/or therapeutic purposes.