On the stability of sequences inserted into viral genomes

Willemsen, Anouk; Zwart, Mark P

The ssRNA(þ) viruses range in genome size from 2.3 to 31 kbp. It has been shown that both animal and plant ssRNA(þ) viruses can express inserted foreign genes. However, the nature of the ssRNA(þ) genomes poses several limitations to efficient expression and maintenance of the insert. Most ssRNA(þ) genomes used for the expression of foreign genes code for a polyprotein, a single ORF that is further processed after translation into different mature peptides. The processing occurs through autocatalytic cleaving at specific cleavage sites located between the different proteins to be expressed. Insertions should therefore be carefully engineered, including proper cleavage sites corresponding to the site of insertion. Even when respecting these design rules, inserts may impose restriction on viral replication due to the failure of proper protease cleavage due to conformational constraints. In addition, the genomes of RNA viruses tend to be composed of overlapping genes (Belshaw, Pybus, and Rambaut 2007) , which limits their adaptive capacity (Simon-Loriere, Holmes, and Pagá n 2013). Overlap can form an impediment to engineering, and perhaps to the likelihood an inserted sequence is maintained, as insertions will often affect multiple genes. We focus exclusively on engineered viruses, given that there are many examples for this virus group.