Mechanism Of M6A Modification Promoting PRV Replication In PK15 Cells

Pseudorabies virus（PRV）is the pathogenic virus of porcine pseudorabies（PR）,belonging to the Herpesviridae family.PRV has a wide range of hosts,which can infect not only pigs but also cattle,sheep,foxes and other animals.In recent years,the virus has also been reported to infect humans.Therefore,the prevention and treatment of PRV is becoming increasingly important.N6-methyladenosine（m6A）modification is the main way of RNA post transcriptional modification.Research has shown that m6A modification is involved in regulating the replication of many viruses and has different regulatory effects on different viruses.However,it is unknown whether m6A modification is involved in regulating PRV replication.This experiment will explore the regulatory effect of m6A modification on PRV replication,laying the foundation for revealing new mechanisms of PRV and epigenetic regulation.Firstly,it was determined through Me RIP-seq that the m6A peaks were mostly located in the CDS region of the viral transcript.PRV infection led to an increase in the m6A peaks content in the CDS region of PK15 cells RNA and a decrease in the UTR region.This change might represent the response of cells to viral infection stress.The significantly up-regulated m6A peaks induced by PRV infection were far lower than the significantly down-regulated m6A peaks.The functional analysis of these m RNAs modified by m6A with differences was performed using Gene Ontology（GO）enrichment analysis and Kyoto Encyclopedia of genes and genomes（KEGG）enrichment analysis.GO enrichment analysis showed that hypomethylated genes were mainly involved in the metabolic process,and hypermethylated genes were mainly involved in protein localization.KEGG pathway analysis showed that the water reabsorption pathway was the first pathway in which hypomethylated genes participated,and the hypermethylated genes mainly participated in the spliceosome pathway.PRV relieved or strengthened these related pathways by regulating m6A modification to facilitate viral replication.After virus infection,the m6A modification level of PK15 cells total RNA was detected,and it was found that the m6A modification level first increased and then decreased.Subsequently,q PCR and Western blot were used to detect the transcription and expression levels of methyltransferase METTL3,METTL14,demethylase FTO,ALKBH5,and specific recognition proteins YTHDF1,YTHDF2,and YTHDF3 in PK15 cells.The results showed that the transcription levels of these m6A regulators significantly decreased24 hpi.Except for YTHDF3,the protein expression levels of all m6A regulators decreased.PRV infection affected the expression of m6A regulators.Using IFA analysis to detect the cell localization of m6A regulators,it was found that the localization of METTL3,FTO,ALKBH5,YTHDF1 and YTHDF2 had changed,which indicated that m6A regulators could shuttle between the nucleus and cytoplasm to respond to the external pressure of PRV infection in a dynamic manner.The PK15 cell model was constructed using the m6A modification inhibitor3-deazaadenosine（3-DAA）for m6A modification level inhibition.After confirming successful inhibition and the use of 3-DAA concentration without significant impact on cell activity,the PRV copies,TCID₅₀,and PRV g E protein expression changes were detected,all of which decreased in a dose-dependent manner with increasing 3-DAA concentration.The m6A modification level of the host positively regulated PRV replication.PRV genome itself does not encode any m6A regulators,so it is speculated that PRV replication may be regulated by host m6A regulators.Knocking down ALKBH5significantly promoted PRV replication,improved viral titer and PRV g E protein expression,while knocking down METTL3 and METTL14 inhibited PRV infection.YTHDF2 and YTHDF3 significantly inhibited PRV replication,while YTHDF1 did not.Overexpression of METTL14 increased the copies and viral titer of PRV,and promoted the expression of PRV g E protein.Therefore,METTL3,METTL14,YTHDF2 and YTHDF3positively regulated PRV replication,while ALKBH5 exhibited a negative regulatory effect.This study revealed that the host m6A mechanism was involved in the regulation of PRV replication process,and demonstrated the dependent coordination between m6A modification and PRV virulence in PK15 cells.