Identification of putative packaging signals in the RNA of foot-and-mouth disease virus (FMDV)

Viruses in the picornavirus family comprise a single molecule of positive sense RNA contained within a simple non-enveloped capsid. The mechanism for RNA packaging is not well understood. We have developed a novel and simple approach to identify predicted RNA secondary structures involved in genome packaging in the picornavirus foot-and-mouth disease virus (FMDV). By interrogating deep sequencing data generated from both packaged and unpackaged populations of RNA, we have determined multiple regions of the genome with constrained variation in the packaged population. Predicted secondary structures of these regions revealed stem-loops with conservation of structure and a common motif at the loop. Disruption of these features resulted in attenuation of virus growth in cell culture due to a reduction in assembly of mature virions. To further test the function of these putative packaging signals (PPS), we have developed a trans-encapsidation assay using subgenomic replicons expressing GFP, helper virus and flow cytometry. The results of these studies provide evidence for the involvement of predicted RNA structures in picornavirus packaging and offer readily transferable methodologies for identifying packaging requirements in many other viruses.