Molecular characterization of arenavirus defective viral genomes reveals sequence features associated with their formation.

UNLABELLED: Defective viral genomes (DVGs) are byproducts of replication that arise during infection with diverse RNA viruses and can impact virus infection and disease outcome. To gain insight into DVG generation during arenavirus infection, we serially passaged Tacaribe virus at a high multiplicity of infection, which led to the generation of both deletion DVGs (del-DVGs) and copyback DVGs (cb-DVGs). Interestingly, specific combinations of start/stop breakpoints were highly overrepresented, resulting in certain DVGs being highly enriched within the population. Functional characterization of the most prevalent del-DVGs suggests that they are able to compete for interaction with the viral RNA synthesis machinery and that this ability is length-dependent. A closer analysis of the DVG breakpoints used to generate highly abundant DVGs revealed a role for local sequence identity in the formation of cb-DVGs, while del-DVG formation was associated with the presence of specific nucleotide triplets (i.e., TAG, AGA, and GAA). Taken together with similar findings from other virus families, this then supports the idea that DVG formation is not a random process, but rather that specific mechanisms promote their formation at certain positions. The characterization of these individual arenavirus DVG sequences, and also the identification of sequence elements associated with their production, will facilitate future work examining their impact on arenavirus biology, and also opens up the possibility of using such sequences as a part of antiviral approaches and/or of modulating their production as a part of virus attenuation strategies.

IMPORTANCE: Infection with diverse RNA viruses can generate defective viral genomes (DVGs) that, while unable to support productive virus infection on their own, appear to play a crucial role in determining infection outcome. In light of this apparent biological importance, there is an urgent need to better understand the sequence characteristics of individual DVGs and the molecular mechanisms that regulate their formation to study their biological functions. We have now characterized several DVGs that are highly enriched during infection with the arenavirus Tacaribe virus. Functional analysis of a subset of these DVGs showed length-dependent competition for the viral RNA synthesis machinery, while detailed sequence analysis revealed that DVG formation involves either regions of sequence identity within the genome or the presence of specific nucleotide sequences. Understanding these mechanisms opens up the possibility to leverage DVG generation in support of antiviral and/or vaccine attenuation approaches.