Understanding off-target growth defects introduced to influenza A virus by synonymous recoding.

CpG dinucleotides are under-represented in the genomes of most RNA viruses. Synonymously increasing CpG content of a range of RNA virus genomes reliably causes replication defects due to the recognition of CpG motifs in RNA by cellular Zinc-finger Antiviral Protein (ZAP). Prior to the discovery of ZAP as a CpG sensor, we described an engineered influenza A virus (IAV) enriched for CpGs in segment 5 that displays the expected replication defects. However, we report here that this CpG-high ('CpGH') mutant is not attenuated by ZAP. Instead, a pair of nucleotide changes, introduced as compensatory changes to maintain base frequencies, were found to be responsible. These mutations resulted in the encoding of a stretch of eight consecutive adenosines (8A), a phenomenon not seen in natural IAV isolates. Viral polymerase slippage occurs at this site, resulting in the production of aberrant peptides and type I interferon induction. When the nucleotides in either one of these two positions were restored to wildtype sequence, no viral attenuation was seen, despite the 86 extra CpGs encoded by this virus. Conversely, when these two adenosines were introduced into wildtype virus (thereby introducing the 8A tract), viral attenuation, polymerase slippage, aberrant peptide production and type I interferon induction were all apparent. That a single nucleotide change can offset the growth defects in a virus designed to have a formidable barrier to wild-type reversion highlights the importance of understanding the processes underlying viral attenuation. PolyA tracts are a correlate for the emergence of polybasic cleavage sites in avian IAV haemagglutinins to produce highly pathogenic strains, and we found that slippage occurs preferentially on polyU over polyA tracts, thereby uncovering possible insights into the intermediary events of this important evolutionary process.