Coronavirus replication is regulated by both host and viral RNA N6–methyladenosine modifications

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 depending on the virus. However, whether m6A methylation can modulate replication of coronaviruses, which cause some severe human and animal diseases such as Coronavirus Disease 2019 (COVID-19), remains unknown. Here, we determined that m6A modifications of porcine epidemic diarrhea coronavirus (PEDV) are exclusively located in the N gene at the 3'-end of the genome, and that PEDV infection alters the expression pattern of some 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 the 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 regulate coronavirus replication. Overall design: HMPV virions were purified from supernatants of hMPV-infected Hela and A549 cell lines. Virus derived mRNA as well as host mRNA were purified from total RNA, which were extracted from hMPV infected cells.