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

ABSTRACT: 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.

ORGANISM(S): Porcine epidemic diarrhea virus

PROVIDER: GSE151020 | GEO | 2020/05/22

REPOSITORIES: GEO

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

Project description: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.

2022-12-01 | GSE141291 | GEO

Identification of cell types and transcriptome landscapes of PEDV infected porcine small intestine using single-cell RNA-sequencing

Project description:Swine coronavirus-porcine epidemic diarrhea virus (PEDV) with specific susceptibility to pigs has existed for decades, and recurrent epidemics caused by mutant strains have swept the world again since 2010. Here, single-cell RNA-sequencing was used to perform a systematic analysis of pig small intestines infected with PEDV for the first time. Multiple cell types were identified by representative markers, including the unique marker DNAH11 of tuft cells. Meanwhile, the goblet and tuft cells were also susceptible to PEDV except enterocytes. PEDV infection obviously upregulated REG3G, which significantly inhibited virus replication. Notably, IFN-DELTAs in goblet and enterocyte progenitor cells were increased in virus infected piglet, and IFN-DELTA5 could induce GBP1, ISG15, OAS2 and IFITM1 dramatically raised in IPEC-J2 cells and restricted PEDV replication. Complement molecules were mainly expressed in intestinal cells excepting tuft cells, but PEDV decreased C3, C4A, and C5 in enterocytes, thus escaping the antiviral effect of C3. Finally, enterocytes expressed almost all coronavirus entry factors, and PEDV infection caused significant upregulation of the coronavirus receptor ACE2 in porcine enterocyte cells. In summary, this study systematically studied the response of different cell types in small intestine of pigs after PEDV infection, which deepened the understanding of viral pathogenesis.

2022-04-27 | GSE175411 | GEO

A recombination-resistant genome for live attenuated and stable PEDV vaccines by engineering the transcriptional regulatory sequences

Project description:Porcine epidemic diarrhea virus (PEDV) is a deadly coronavirus for neonatal piglets and no effective vaccines are available. Transcriptional regulatory sequences (TRSs) are critical in regulating coronavirus discontinuous transcription. Also, TRSs contribute to a high recombination rate of coronaviruses, leading to difficulty in developing safe live vaccines. We hypothesize that recoding the TRS core sequences (TRS-CS) of PEDV can make the recombination impossible between the engineered vaccine virus and field strains or wildtype viruses. We used an infectious clone-derived reporter PEDV, dORF3-EGFP, as the backbone to generate a remodeled TRS (RMT) mutant that carries the recoded leader and body TRS-CSs. The RMT and dORF3-EGFP showed comparable replication efficiency in Vero cells. However, the incompatibility between the rewired and wildtype TRS-CSs led to few EGFP in RMT-infected cells. Furthermore, RMT and dORF3-EGFP had a similar attenuated phenotype, replication efficiency, and protective immunogenicity in neonatal pigs. RNA sequencing analysis indicated that EGFP transcription directed by the heterogenous TRS-CSs was significantly reduced to an extremely low level. Meanwhile, recombinant viruses were not detected in Vero cells and in pigs that were co-infected with RMT and a PEDV S-INDEL strain, Iowa106. In vitro and in vivo passaging of the RMT did not result in reversion mutations in the rewired TRS-CSs, introduced gaps, and disrupted wildtype TRSs. In summary, the RMT mutant was resistant to recombination and genetically stable and can be further optimized (e.g., deletion of the EGFP) to serve as a platform to develop safe PEDV live attenuated vaccines.

2023-11-04 | GSE246991 | GEO

Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus

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2019-09-10 | GSE125803 | GEO

Dynamic O-GlcNAcylation of YTHDF proteins controls translation of m6A-modified mRNA

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The cytoplasmic LSm1-7 and nuclear LSm2-8 complexes exert opposite effects on Hepatitis B virus biosynthesis and interferon response

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