Copy-back defective viral genomes (cbDVGs) are key inducers of antiviral responses during negative-sense RNA virus infection. Once considered byproducts of in vitro viral replication, cbDVGs have since been detected in clinical specimens and implicated in affecting infection outcomes. The molecular mechanism of cbDVG generation remains unclear, thereby hindering our ability to manipulate cbDVG production during infection for therapeutic gain. Previous work showed that respiratory syncytial virus (RSV) cbDVG re-initiation sites cluster in trailer-end hotspots R1, R2, and R3, and that a poly-U mutation in R1 selectively reduced cbDVG formation at the mutated region. Here, we report that a 10U mutation in R2 drastically reduced cbDVGs in this region in both minigenome and recombinant virus systems. Furthermore, during high-multiplicity of infection passaging of the R2-10U virus, we observed delayed detection of cbDVGs with re-initiation sites in R1-R3 (trailer cbDVGs) compared to WT, while no differences in virus titers were observed. Interestingly, we observed the rapid emergence and accumulation of a viral variant bearing a 2-ribonucleotide deletion (R2-8U) within the R2-10U mutation sequence as early as P0. Compared to R2-10U, the R2-8U virus was stable, displayed faster generation and accumulation of trailer cbDVGs, restored cbDVGs with R2 re-initiation sites, and exhibited enhanced genomic replication. Overall, our data identify a sequence in the RSV trailer whose mutation critically modulates both viral replication and the generation/propagation of trailer cbDVGs. Our data also suggest that cbDVG generation, particularly near the trailer, may be an evolutionary tradeoff for more rapid virus genomic replication.IMPORTANCECopy-back defective viral genomes (cbDVGs) are potent inducers of antiviral responses during negative-sense RNA virus infection. They have also been detected in clinical specimens and implicated in modulating infection outcomes. However, the molecular mechanisms governing cbDVG generation remain poorly understood, limiting efforts to manipulate their production for therapeutic benefit. In this manuscript, we focus on cbDVGs generated near the viral trailer end during respiratory syncytial virus (RSV) infection and identify a sequence within the RSV trailer region that, when mutated, critically alters both viral genomic replication and trailer cbDVG generation and propagation. Our observations support the notion that variables promoting enhanced levels of viral replication promote trailer cbDVG emergence and accumulation. Collectively, this work expands the repertoire of RSV genetic tools to manipulate cbDVG composition and kinetics, providing a unique platform to investigate RSV genomic replication, cbDVG-mediated pathogenesis, and the evolutionary significance of cbDVGs.
Brennan et al. (Tue,) studied this question.