Pseudorabies virus exploits N6-methyladenosine modification to promote viral replication

Abstract

Introduction: Pseudorabies virus (PRV) is the pathogenic virus of porcine pseudorabies (PR), belonging to the Herpesviridae family. PRV has a wide range of hosts and in recent years has also been reported to infect humans. N⁶-methyladenosine (m⁶A) modification is the major pathway of RNA post-transcriptional modification. Whether m⁶A modification participates in the regulation of PRV replication is unknown.

Methods: Here, we investigated that the m⁶A modification was abundant in the PRV transcripts and PRV infection affected the epitranscriptome of host cells. Knockdown of cellular m⁶A methyltransferases METTL3 and METTL14 and the specific binding proteins YTHDF2 and YTHDF3 inhibited PRV replication, while silencing of demethylase ALKBH5 promoted PRV output. The overexpression of METTL14 induced more efficient virus proliferation in PRV-infected PK15 cells. Inhibition of m⁶A modification by 3-deazaadenosine (3-DAA), a m⁶A modification inhibitor, could significantly reduce viral replication.

Results and discussion: Taken together, m⁶A modification played a positive role in the regulation of PRV replication and gene expression. Our research revealed m⁶A modification sites in PRV transcripts and determined that m⁶A modification dynamically mediated the interaction between PRV and host.

Keywords: N6-methyladenosine; m6A regulators; pseudorabies virus; regulation; replication.

Figure 1

The status of m⁶A modification on PRV transcripts. For PRV (MOI = 1) infection, total RNA of PK15 cells was harvested at 24 hpi. (A) Distribution pattern of m⁶A peaks on PRV transcripts was analyzed based on the MeRIP-seq data (NCBI #GSE209949). (B) Density of m⁶A peaks on PRV transcripts. (C) Transcriptome-wide mapping to PRV m⁶A IP reads, input reads and m⁶A peaks based on MeRIP-seq. The m⁶A peaks of PRV transcripts were indicated as blue blocks. The input and PRV IP coverage were indicated with green and red bars, respectively. All genes were shown and overlaid as black arrows in the bottom track. (D) Motif analysis to identify consensus sequences for PRV transcripts. The most prominent motif was shown.

Figure 2

PRV infection affected m⁶A level and expression of m⁶A regulators in PK15 cells. (A) Total RNA was extracted from PRV-infected and uninfected PK15 cells at different time periods, and the m⁶A level of RNA was quantified by ELISA. (B) PK15 cells were infected with PRV for 12 and 24 h. m⁶A regulators were assessed by immunoblotting analysis. β-actin was used as a loading control. (C) RT-qPCR analysis was used to evaluate the mRNA levels of m⁶A regulators at different times of PRV infection. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

PRV infection influenced m⁶A methylome of PK15 cell transcripts. (A) MeRIP-seq of PK15 cells which were infected by PRV (or uninfected as a negative control, i.e., “Mock”) for 24 h. Density of m⁶A peaks on PRV-infected and uninfected cellular transcripts. The m⁶A peaks information was included in our MeRIP-seq data (NCBI #GSE209949). (B) Distribution pattern of m⁶A peaks on PRV-infected (right) and uninfected (left) cellular transcripts. (C) Volcanic map of m⁶A peaks (left was downregulated, right was upregulated by PRV infection). There were 1,286 significantly down-regulated m⁶A peaks, and 260 significantly up-regulated m⁶A peaks induced by PRV infection. (D) GO enrichment analysis of pathways enriched in the hypomethylated (left) and hypermethylated (right) genes (The top 30 enriched pathways are shown.). (E) KEGG analysis of pathways enriched in the hypomethylated genes (left, the top 20 enriched pathways are shown.) and the hypermethylated genes (right, the top 10 enriched pathways are shown.). (F) Motif analysis to identify consensus sequences for PRV-infected (right) and uninfected (left) PK15 cells transcripts. The most prominent motif for each was shown.

Figure 4

Depletion of methyltransferases METTL3 and METTL14 suppressed PRV replication. (A) PK15 cells were transfected with the specified siRNAs (60 nM) for 24 h. METTL3 and METTL14 were assessed by immunoblotting analysis. β-actin was used as a loading control. (B) PK15 cells were transfected with the specified siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 12 and 24 h. PRV DNA copies were evaluated by RT-qPCR analysis. (C) PK15 cells were transfected with the specified siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV titers were assessed by TCID₅₀ analysis. (D) PK15 cells were transfected with the indicated siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV gE was assessed by immunoblotting analysis. β-actin was used as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5

Overexpression of METTL14 promoted PRV proliferation. (A) PK15 cells were transfected with pEGFP-C3 and pEGFP-C3-METTL14 (2.5 μg) for 6 h, and then cultured with fresh maintenance medium for 24 h. METTL14 was assessed by immunoblotting analysis. β-actin was used as a loading control. (B) PK15 cells were transfected with pEGFP-C3 and pEGFP-C3-METTL14 (2.5 μg) for 6 h, and then cultured with fresh maintenance medium for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 12 and 24 h. PRV DNA copies were evaluated by RT-qPCR analysis. (C) PK15 cells were transfected with pEGFP-C3 and pEGFP-C3-METTL14 (2.5 μg) for 6 h, and then cultured with fresh maintenance medium for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV titers were assessed by TCID₅₀ analysis. (D) PK15 cells were transfected with pEGFP-C3 and pEGFP-C3-METTL14 (2.5 μg) for 6 h, and then cultured with fresh maintenance medium for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV gE was assessed by immunoblotting analysis. β-Actin was used as a loading control. *p < 0.05, **p < 0.01.

Figure 6

Demethylase FTO and ALKBH5 promoted PRV proliferation. (A) PK15 cells were transfected with the specified siRNAs (60 nM) for 24 h. FTO and ALKBH5 were assessed by immunoblotting analysis. β-actin was used as a loading control. (B) PK15 cells were transfected with the specified siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 12 and 24 h. PRV DNA copies were evaluated by RT-qPCR analysis. (C) PK15 cells were transfected with the indicated siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV titers were assessed by TCID₅₀ analysis. (D) PK15 cells were transfected with the indicated siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV gE was assessed by immunoblotting analysis. β-actin was used as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Specific recognition protein YTHDF2 and YTHDF3 inhibited PRV proliferation. (A) PK15 cells were transfected with the specified siRNAs (60 nM) for 24 h. YTHDF1, YTHDF2 and YTHDF3 were assessed by immunoblotting analysis. β-actin was used as a loading control. (B) PK15 cells were transfected with the specified siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 12 and 24 h. PRV DNA copies were evaluated by RT-qPCR analysis. (C) PK15 cells were transfected with the indicated siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV titers were assessed by TCID₅₀ analysis. (D) PK15 cells were transfected with the specified siRNAs and were mock transfected (MT) with transfection reagent alone for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.1) for 24 h. PRV gE was assessed by immunoblotting analysis. β-actin was used as a loading control. *p < 0.05, **p < 0.01.

Figure 8

Inhibition of PRV infection by methylation inhibitor 3-deazaadenosine (3-DAA). (A) PK15 cells were treated with the specified concentrations of 3-DAA for 24 h. m⁶A level quantification was performed by ELISA assays. (B) PK15 cells were treated with the specified concentrations of 3-DAA for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.01) for 24 h, and images of cytopathic effects were recorded (200×). (C) PK15 cells were treated with the specified concentrations of 3-DAA for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.01) for 24 h. PRV DNA copies were evaluated by RT-qPCR analysis. (D) PK15 cells were treated with the specified concentrations of 3-DAA for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.01) for 24 h. PRV titers were assessed by TCID₅₀ analysis. (E) PK15 cells were treated with the specified concentrations of 3-DAA for 24 h. PK15 cells were infected with PRV-FJ01 (MOI = 0.01) for 24 h. PRV gE was assessed by immunoblotting analysis. β-actin was used as a loading control. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 9

Schematic representation of m⁶A regulation of PRV replication. Upon viral infection, virions first attach to the host cell surface, subsequently enter the cell, and finally the viral genome is released into the host cell nucleus. In the nucleus, the methyltransferases METTL3/14 co-induce the methylation of multiple viral mRNAs, whereas the demethylases FTO and ALKBH5 regulate the demethylation process. The methylation of viral mRNA promotes its own nuclear export. In the cytoplasm, YTHDF1 and YTHDF3 synergistically promote mRNA stability and translation, and YTHDF3 cooperates with YTHDF2 to promote mRNA degradation. Ultimately, the expression of PRV proteins is promoted by the cooperation of YTHDF1/2/3, and these products are transported back into the nucleus, where they complete the viral nucleocapsid assembly and eventually release more viral particles.