The broken genome: Merging cutting edge technologies for a molecular understanding of the Flock House virus defective interfering particle.


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Defective RNAs are natural versions of a viral genome that have been truncated or rearranged by non-homologous recombination. While not encoding for functional viruses, they can be amplified and co-passaged with the wild-type virus, effectively parasitizing the normal viral machinery. Some defective RNAs can replicate so successfully so as to subdue the replication of the wild-type virus, forming ‘Defective-Interfering RNAs’ (DI-RNAs). As a result, DI-RNAs may promote the establishment of chronic infections, may prolong the host’s infectious period, and may even be exploited as antiviral therapies or vaccines. Therefore, understanding the mechanisms of how DI-RNAs are formed and what roles they play in infections is important. I have characterized the process of defective-RNA emergence and evolution of Flock House virus in cell culture. Using a combination of short and long read sequencing technologies, ‘ClickSeq’ (to resolve recombination events with nucleotide resolution) and Oxford Nanopore Technologies’ MinION (to characterize full-length and defective genomes) I have characterized the step wise progression of DI-RNA formation and the species distribution of these genomes. I observed a rapid accumulation of mature DI-RNAs suggesting that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ DI-RNAs. These sequencing approaches have allowed me to characterize in detail the genetic makeup of a viral population, identifying samples that are predominantly defective or predominantly wild type. Therefore, I sought to understand how defective genomes affect virus particle structure, whether defective particles display any morphological defects, and if structure can impose selective pressures to the accumulation of defective genomes. Applying cryo-electron microscopy paired with native ultra-high mass spectrometry has shown that there are no structural differences of defective virus particles compared to wild-type particles suggesting that packaging mechanisms play an important role in the selection of defective genomes. Overall, these insights have important consequences for our understanding of viral RNA packaging and assembly, and of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs in RNA viruses.



Defective interfering RNAs, Flock House virus, virus evolution, Next Generation Sequencing, ClickSeq, Nanopore Sequencing