Coronavirus replication is controlled by both host and viral RNA m6A methylations

N6–methyladenosine (m6A) is the most abundant internal mRNA modification in eukaryotes, and it plays an important role in RNA metabolism and function. Recent studies have revealed that viral RNA m6A modification can play an anti-viral or pro-viral role in virus life cycle. However, whether m6A methylation can modulate replication of coronaviruses, which contain the largest known RNA genomes, remains elusive. Here, we determined that m6A modifications of porcine epidemic diarrhea coronavirus (PEDV) are exclusively located in the N gene at the 3’-end of genome, and that PEDV infection alters the expression pattern of host proteins involved in m6A modification. Depletion of m6A demethylases significantly increased PEDV replication and gene expression whereas knockdown of m6A methyltransferases slightly decreased PEDV infection. Interestingly, m6A binding proteins YTHDF 2 and 3 significantly inhibited PEDV replication whereas YTHDF1 has an opposite effect. When the major m6A sites in the N gene were mutated, the resultant recombinant PEDVs and PEDV replicons had a significant increase in replication and gene expression. This study illustrates that (i) addition of m6A to PEDV RNA inhibits viral replication, and (ii) both host and viral m6A machinery regulates coronavirus replication. MeRIP-seq of poly(A)-RNA from Mock or PEDV-infected MARC-145 and S10-3 cells in two replicates.