Mutations in the TRS Region Alter Coronavirus (Porcine Epidemic Diarrhea Virus) Replication and Interferon Induction by Modulating Subgenomic RNA Transcription

Coronaviruses frequently cause epidemics of respiratory or enteric disease in humans and livestock. CoVs generate a nested set of subgenomic messenger RNAs (sgmRNAs) through discontinuous transcription mediated by transcription regulatory sequences (TRSs). We previously developed a porcine epidemic diarrhea virus (PEDV) mutant, RMT, by remodeling TRS-core sequences (TRS-CSs). Unexpectedly, plasmid mutagenesis introduced a 189-nucleotide insertion upstream of the E gene TRS-CS and a guanine deletion in the N gene TRS-CS. RMT displayed markedly increased sgmRNA-E tran-scription, which is normally the least abundant in wild-type PEDV. To assess the effects of TRS remodeling and the additional mutations, we generated PEDV mutants with various modification combinations and characterized them in vitro. TRS-CS remodeling alone impaired replication, reduced sgmRNA-N abundance, and increased non-canonical transcription, which correlated with strong interferon responses. The insertion upstream of E gene TRS-CS enhanced sgmRNA-E transcription in a size-dependent manner, with the 189-nt insertion more effective than 94-nt insertion. The 189-nt insertion also boosted other canonical sgmRNA transcription, reduced transcriptional noise and partially re-stored replication, while the 94-nt insertion did not. These results demonstrate that TRS-CSs and their flanking sequences critically regulate CoV transcription, replication, and immune responses, providing insights for designing recombination-resistant live attenuated vaccines through TRS remodeling.