METTL14 and FTO mediated m⁶A modification regulate PCV2 replication by affecting miR-30a-5p maturity

ABSTRACT

The epigenetic modification of the N6-methyladenosine (m⁶A) methylation plays an important role in virus infection and replication. However, its role in Porcine circovirus type 2 (PCV2) replication has not been well studied. Here, we demonstrated that m⁶A modifications are increased in PK-15 cells after PCV2 infection. In particular, PCV2 infection could increase the expression of methyltransferase METTL14 and demethylase FTO. Moreover, interfering with METTL14 accumulation reduced the m⁶A methylation level and virus reproduction, whereas depleting the FTO demethylase enhanced the m⁶A methylation level and stimulated virus reproduction. Besides, we showed that METTL14 and FTO regulate PCV2 replication by affecting the process of miRNA maturity, especially the miRNA-30a-5p. Taken together, our results demonstrated that the m⁶A modification positively affects PCV2 replication and the role of m⁶A modification in the replication mechanism of the PCV2 virus provides a new idea for the prevention and control of the PCV2.

KEYWORDS:

• Porcine circovirus type 2
• m⁶A modification
• miRNA maturity
• replication

Introduction

The Porcine circovirus type 2 (PCV2) is an emerging swine pathogen causing large economic losses for the global swine industry annually, which was initially identified in 1998 from piglets with the wasting disease and belongs to the genus circovirus, family Circoviridae. The disease syndrome associated with PCV2 was first described as a postweaning multisystemic wasting syndrome (PMWS) and other syndrome diseases collectively known as a porcine circovirus-associated disease (PCVAD) [1–4]. PCV2 DNA is ubiquitous in the environment and can also be found in a variety of non-porcine species including rats, calves, minks, and foxes. However, the pathogenesis of PCV2 is not fully understood.

m⁶A methylation regulation is the post-transcriptional regulation of RNA in higher mammals, plants, yeasts, fruit flies, and other eukaryotes [5]. It was first discovered in 1974 and has aroused great interest in recent years [6]. The internal m6A modification is mainly concentrated in the start sites, stop codon region, and consensus RRACH motif of mRNA [7,8]. It is well known that the m⁶A gene has a variety of biological functions in mammals. m⁶A modification can affect the metabolic process of mRNA, including mRNA processing [9], nuclear transport [10], translation [11], and stability [12], as well as regulation of gene expression at the post-transcriptional level of RNA, which in turn affects a variety of physiological processes [13,14]. Besides, the methylation modification of m⁶A has been proved to be dynamic and reversible [15]. The methylation process is catalysed by a heterodimer formed by the ratio of Methyltransferase-like 3 (METTL3) to methyltransferase-like 14 (METTL14) 1:1 and regulated by the subunit protein Wilms tumour 1–associated protein (WTAP) [16–18]. m⁶A methylation can be removed by m⁶A demethylase composed of alkylated repair homologous protein 5 (ALKBH5) and Fat mass and obesity-associated protein (FTO) [19–21].

The life cycle of a virus depends on the relevant mechanisms and pathways of host cells. Therefore, exploring the process of virus activity from the perspective of epigenetics promotes the study of antiviral mechanisms is extremely important. m⁶A was first detected on the mRNA of adenovirus (Ad) and influenza A virus (IAV) [22,23]. Subsequently, m⁶A was also detected in RNA of herpes simplex virus type 1 (HSV-1), Rolls’ sarcoma virus (RSV), monkey vacuole virus 40 (SV40), B77 sarcoma virus, avian influenza virus, and feline leukaemia virus [22]. These findings prove that m⁶A methylation epigenetic modification plays an important role in virus infection and replication. However, the understanding of m⁶A in PCV2 virus replication is very limited.

In this study, we detected the overall level of m⁶A methylation after PCV2 infection and the results show that the expression of the m⁶A methylation-related genes METTL14 and FTO were significantly up-regulated. Further, we found that METTL14 and FTO regulate the level of m6A methylation in PK-15 cells and the replication of PCV2. Besides, the high level of m⁶A methylation promotes virus replication and vice versa. Finally, we demonstrated that both METTL14 and FTO participate in and mediate the process of pri-miRNA processing into pre-miRNA and miRNA mature bodies, especially the miRNA-30a-5p. Our results discussed the role of m⁶A modification on PCV2 replication and explored the biological mechanism of m⁶A involved in virus replication which further revealed the mechanism of PCV2 infection.

Materials and methods

Cell line and virus strain

Cell line: PK-15 cells are preserved by the Shandong Key Laboratory of Animal and Poultry Disease Control and breeding. Virus strain: PCV2 2w3 infectious clone strain was preserved by Shandong Key Laboratory of Livestock and Poultry Disease Control and breeding.

m⁶A semi-quantitative kit to detect the overall level of methylation

The total RNA of normal cultured cells was extracted 72 hours after inoculation with the PCV2 virus of MOI = 1, and the whole m⁶A methylation level was detected by the m⁶A semi-quantitative kit of Amyjet Scientific company. According to the operation instructions, PBS was added to each well, negative control (NC), positive control (PC), and RNA were added, then capture antibody (CA), detection antibody (DA), and enhancer solution (ES) were added for m⁶A capture, and developer solution and stop solution (SS) were added for signal detection. The absorbance value in 450 nm is read and the percentage of m⁶A in the total RNA is calculated by formula.

Transcriptome analysis

72 hours after inoculation with the PCV2 virus of MOI = 1, the cells and the blank control cells were sequenced in the transcriptional group of Lianchuan Biological Co., Ltd. The sequencing results were analysed on Lianchuan biological cloud platform, and the methylation-related enzymes that were significantly different between PCV2-challenged PK-15 cells and uninfected PK-15 cells were screened.

Real-time quantitative PCR

The total RNA of the PK-15 cells was extracted at 48 h or 72 h, and then RNA was inversely transcribed into cDNA. The intracellular methylation-related enzymes METTL14 and FTO were quantitatively analysed by Roche 480 quantitative PCR. The reaction procedure was as follows: 95 °C pre-denaturation, followed by 40 cycles of 95 °C for 30s, 60 °C for 30, and 72 °C for 30s. The primers for RT-qPCR were shown in Table 1.

Table 1. RT-Qpcr Primer sequence.

Primer Name	Sequence (5’-3’)
Sus-METTL14-F	AGAAACTTGCAGGGCTTCCT
Sus-METTL14-R	TCTTCTTCAGGCAAATTTTCTT
Sus-METTL3-F	CTGGGGTTACGAACGGGTAG
Sus-METTL3-R	TGACACCAACCAAGCAGTG
Sus-WTAP-F	GGCGAAGTGTCGAATGCT
Sus-WTAP-R	CCAACTGCTGGCGTGTCT
Sus-FTO-F	GACGGGAGCCTTGGATTACA
Sus-FTO-R	AAACTCGACCTCGTTGTGGAT
Sus-ALKBH5-F	CCCGAGGGCTTCGTCAACA
Sus-ALKBH5-R	CGACACCCGAATAGGCTTGA
Sus-YTHDF2-F	GGCAGCACTGAAGTTGGG
Sus-YTHDF2-R	CTATTGGAAGCCACGATGTTA
β-actin-F	TCTGGCACCACACCTTCT
β-actin -R	ATCTGGGTCATCTTCTCAC

Silent expression and overexpression of METTL14 and FTO methylase

The small interference RNA (siRNA) of METTL14 and FTO were designed and synthesized by Jinan Boshang Biotechnology Co., Ltd. Si-METTL14 and si-FTO designed three targets siMETTL14–1, siMETTL14–2, siMETTL14–3, siFTO-1, siFTO-2, and siFTO-3. The small interference RNA sequence is shown in Table 2. The overexpression plasmids pc3.1-METTL14 and pc3.1FTO with His tag at the C-terminus were designed and synthesized by Jima Gene of Jinan Boshang Biotechnology Co., Ltd. The length of the pc3. -METTL1 plasmid is 1407bp, and the sequence length of the pc3.1-FTO plasmid is 1554bp

Table 2. siRNA target sequence.

Primer Name	Sequence (5’-3’)
FTO-sus-143	GGATCCAAGGCAAAGATTTGC
FTO-sus-1260	GCTGTGCTTCGTGAAGTTAGA
FTO-sus-735	GCGTGTATAGGTCCAGATTTC
METTL14-sus-255	GCATTGGTGCTGTGTTAAATA
METTL14-sus-854	GGACTTGGGATGACATCATGA
METTL14-sus-1379	GGGAATCAAAGGAACTGTTAA

According to Lipofectamine RNAi MAX transfection’s instructions, siFTO-1, siFTO-2, siFTO-2, siMETTL14–1, siMETTL14–2, siMETTL14–3, and Negative control siRNA were transfected into the PK-15 cells. According to the instructions of the lipofectamine 3000 transfection reagent, the overexpression plasmids and empty plasmids of pc3.1-METTL14 and pc3.1-FTO were transfected into the cells. METTL14 and FTO methylase expression were detected by fluorescence quantitative PCR and WB at 48 h and 72 h, respectively.

Western Blotting

At 48- and 72-hours post-transfection, cells were washed with PBS and lysed on ice with cell lysis buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethanesulfonyl fluoride (PMSF) (Beyotime, Shanghai, China). The cell lysates were centrifuged at 12,000×g for 15 min at 4°C. The supernatant was collected and determined with the BCA protein assay reagent (Beyotime, Shanghai, China). Proteins were separated by SDS-PAGE and blot onto polyvinylidene fluoride membranes, and then blocked with EveryBlot Blocking Buffer (Bio-Rad, American) for 5 min at room temperature. The membranes were probed with primary antibodies overnight at 4°C. After washing with TBST three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Immunoreactive bands were visualized using enhanced chemiluminescence (ECL) detection kits.

Estimation of miRNA and pri-miRNA by Qpcr

The purpose of miRNA and pri-miRNAquantitative detection is to detect the expression of miRNA and pri-miRNA based on silencing and overexpression of METTL14 and FTO. The siRNA and overexpression plasmids of METTL14 and FTO were transfected into the cells and inoculated with PCV2 virus solution 6 hours later. After 72 hours, the total RNA of the cells was extracted, and the RNA was detected by reverse transcription and qPCR. For miRNA and pri-miRNA quantification, actin was used as the internal reference, and cDNA was synthesized using downstream primers by reverse transcription. The primer sequence is shown in Tables 3 and 4.

Table 3. miRNA quantitative analysis primer sequence.

Primer Names		Sequence (5’-3’)
miR-328	Sense	GCTGGCCCTCTCTGCCC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-504	Sense	GCGAGACCCTGGTCTGCACT
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-7134-5p	Sense	CGATGTCCGCGGGTTCC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-30b-3p	Sense	GCGCTGGGAGGTGGATGT
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-125a	Sense	GCGTCCCTGAGACCCTTTAA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-92b-3p	Sense	GCGTATTGCACTCGTCCCG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-10b	Sense	CGCGTACCCTGTAGAACCGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-155-5p	Sense	GCGCGTTAATGCTAATTGTGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-125a	Sense	GCGTCCCTGAGACCCTTTAA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-92b-3p	Sense	GCGTATTGCACTCGTCCCG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-10b	Sense	CGCGTACCCTGTAGAACCGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-155-5p	Sense	GCGCGTTAATGCTAATTGTGA
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-21	Sense	CGCGCGTAGCTTATCAGACTG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-29b	Sense	CGCGTAGCACCATTTGAAATC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-361-3p	Sense	GCCCCCAGGTGTGATTCTG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-15a	Sense	GCGCGTAGCAGCACATAATG
	Anti-Sense	AGTGCAGGGTCCGAGGTATT
miR-30a-5p	Sense	CGCGTGTAAACATCCTCGAC
	Anti-Sense	AGTGCAGGGTCCGAGGTATT

Table 4. Pri-miRNA quantitative analysis primer sequence.

Primer Names		Sequence (5’-3’)
Sus-pri-miR-328	Sense	ATGGAAGCTCTGGTCTTGGC
	Anti-Sense	GGCACACACTTTCTCCCTGA
Sus-pri-miR-504	Sense	ACAGCCATTTACACGGGGTT
	Anti-Sense	TCAGTCTCCGCAAAAAGGCA
Sus-pri-miR-7134-5p	Sense	AGGTGAGACTTGAGTGAGGAGA
	Anti-Sense	GCCCTTATGCAAGACAGCGG
Sus-pri-miR-30b-3p	Sense	AGTCTCAGGTAGAGGGCCAG
	Anti-Sense	ACGGTTGATATTCCAAGTCAGC
Sus-pri-miR-125a	Sense	TTGTGCCGCAGCCCC
	Anti-Sense	GGTTTTCAGGGGAGGAAGGG
Sus-pri-miR-92b-3p	Sense	CGCGGTGCAGTGTTGTTCTTT
	Anti-Sense	GCGGCGACACTCCAGTCTAT
Sus-pri-miR-10b	Sense	ACAGCCAGAATCGGAAGTGG
	Anti-Sense	TTTTGGCCTTGCATTCCTGC
Sus-pri-miR-128	Sense	ACTGTGTTGTCTGTTGAAGCC
	Anti-Sense	TCACTGCTTATATGGCTCCAGT
Sus-pri-miR-155-5p	Sense	CTCTTGCAGGTGGTGCAAAC
	Anti-Sense	TGTAGGAGTCAGACGGAGGT
Sus-pri-miR-21	Sense	TCCTTTGGGATGTTTCTGGTTGA
	Anti-Sense	TCACGACGGTAGGCAAAACA
Sus-pri-miR-29b	Sense	GCCAGCTTATTGTAGGGAAGCA
	Anti-Sense	CCTTCAAAGTCTTCAGGACCG
Sus-pri-miR-31-3p	Sense	CTGGGATTTGGGAGCTTATCA
	Anti-Sense	GTAGAATGGGTACATGGAGATCAG
Sus-pri-miR-15a	Sense	TATCTTTAGGTGGTAAGGTGTGTT
	Anti-Sense	AGGCACTGCTGACATTGCTA
Sus-pri-miR-30a-5p	Sense	ACCCTTCTCCAGGGTAACCA
	Anti-Sense	AACATCCGACTGAAAGCCCG
Sus-pri-miR-122	Sense	TATGGTGGAATGTGCAGGTGAA
	Anti-Sense	AACCTTCTTGCCCATACCAAAC
Sus-pri-miR-98	Sense	CTGTCGTCAGGTAAAATGGCTC
	Anti-Sense	TGATGGAGGCAGCGGTATCAA

Synthesis and transfection of microRNA

Mimic targeting miR-30a-5p was designed and synthesized by Shanghai Jima Biology Co., Ltd. The mimic sequence of miR-30a-5p is shown in Table 5, and a native control was set up at the same time. The method of mimics’ transfection was the same as that of siRNA.

Table 5. The mimic sequence of miR-30a-5p is shown in the table.

Names		Sequence (5’-3’)
miR-30a-5p mimics	Sense	UGUAAACAUCCUCGACUGGAAG
	Anti-Sense	CUUCCAGUCGAGGAUGUUUACA
NC	Sense	UCACAACCUCCUAGAAAGAGUAGA
	Anti-Sense	UCUACUCUUUCUAGGAGGUUGUGA

Statistical analysis

The quantitative data in the experimental results are expressed by mean ±standard deviation (x ±s), and the data differences are compared by t-test; the results are analysed by GraphPad Prism statistical software and t-test. Data were considered statistically significant as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.

Results

PCV2 infection affects the expression level of m⁶A methylation in PK-15 cells

In the present study, transcriptomes of PK-15 cells inoculated with PCV2 at the early stage of infection were sequenced using the Illumine HiSeq 4000 platform. The RNA-Seq results showed that the expression of the m⁶A methylation-related genes up-regulated, namely, METTL3, METTL14, ALKBH5, FTO, and WTAP in PK-15 cells, except for YTHDC1 (Figure 1a,b). Among them, two genes with the opposite catalytic activity of m⁶A, METTL14, and FTO, were significantly up-regulated, while the other genes were not significantly expressed (Figure 1a,b). Next, the m⁶A RNA methylation was estimated to identify the overall level of m⁶A in the total RNAs of PK-15 cells in infected and uninfected cells. It was found that the level of m⁶A in PK-15 cells was up-regulated after PCV2 infection (Figure 1c). These results demonstrated that the level of m⁶A methylation in PK-15 cells was significantly up-regulated after PCV2 infection. Besides, we used RT-qPCR and WB to detect the expression level of m⁶A methylation-related genes before and after PCV2 infection. The expression level of m⁶A methylation-related genes is consistent with the transcriptome analyses (Figure 1d,e). The methyltransferase METTL14 was the most significantly up-regulated gene, followed by the demethylase FTO. No significant difference was observed for other methylation-related genes (Figure 1). These results showed that PCV2 infection could significantly increase m⁶A levels of cellular RNA.

Figure 1. PCV2 infection affects the expression level of m⁶A methylation in PK-15 cells.

Note: (a) Heat map of intracellular m⁶A methylation-related gene analysis after PCV2 infection. (b) The expression changes of each methylase in PK-15 cells after PCV2 infection. (c) The m⁶A content of total RNA in PK-15 cells infected with PCV2 was compared with that in normal PK-15 cells (p < 0.05). (d) RT-qPCR was used to detect the expression of m⁶A regulation-related genes in PK-15 cells infected with the PCV2 virus. β-actin was used as an internal reference. (e) m⁶A regulation-related genes expression of PCV2 virus at 72 h in PCV2 virus-infected and uninfected groups and ACTB as an internal control. The ratios to ACTB from three independent experiments are shown (*, P, 0.05; **, P, 0.01; ***, P, 0.001). ImageJ was used to quantify the level of protein.

METTL14 and FTO regulate the level of m⁶A methylation in PK-15 cells

Given that m⁶A, METTL14, and FTO levels are significantly increased after PCV2 infection in PK-15 cells and that METTL14 and FTO play a pivotal role in mediating m⁶A formation. We assessed whether METTL14 and FTO could lead to the different m⁶A modifications in PK-15 cells. We inhibited METTL14 and FTO expression by transfecting METTL14 and FTO siRNA into PK-15 cells (Figure 2a,b) and then examined m⁶A levels in the cells. The results show that METTL14 and FTO knockdown resulted in the opposite effect on m⁶A levels in PK-15 cell lines (Figure 2c). Moreover, METTL14 overexpression restored the reduction in m⁶A levels induced by METTL14 depletion (Figure 2c) and FTO overexpression decreased the m⁶A levels in PK-15 cells Figure 2c). Collectively, these findings indicated that METTL14 and FTO were the main factors involved in aberrant m⁶A modification in PK-15 cells.

Figure 2. METTL14 and FTO regulate the level of m⁶A methylation in PK-15 cells. (a, b) METTL14 and FTO knockdown efficiency in each of three siRNA-transfected PK-15 cells at 48 h and 72 h post-transfection. β-actin served as a loading control in the western blot. Western blotting of METTL14 and FTO. (c).

Simultaneously, we demonstrated that there was an interaction between METTL14 and FTO. When we knocked down or overexpressed FTO, no significant change was observed in the expression of METTL14 (Figure 2d). In contrast, the expression of FTO was down-regulated in the cells overexpressed by METTL14 and up-regulated in the silent METTL14 (Figure 2d). These results indicate that methyltransferase METTL14 affects the expression of FTO, but FTO does not affect the expression of METTL14 in PK-15 cells.

METTL14 and FTO regulate PCV2 replication in PK-15 cells

To identify the function of METTL14 and FTO in PCV2 replication, we overexpressed and silenced the METTL14 and FTO, respectively. Our quantitative analysis of viral genes in the supernatant after infection showed that the viral load was markedly increased by METTL14 overexpression and decreased by FTO overexpression (Figure 3a). Besides, the viral load was reduced in METTL14-depleted and increased by FTO-depleted in PK-15 cells during PCV2 infection (Figure 3b). Furthermore, viral Cap protein expression was confirmed that the viral gene content was greatly reduced in METTL14-depleted and FTO-overexpressed cells after viral infection (Figure 3c). These results indicate that suppressing m⁶A by depletion of methyltransferases METTL14 or overexpressing FTO reduced the PCV2 viral load in infected cells.

Figure 3. METTL14 and FTO regulate PCV2 replication in PK-15 cells. (a) RT-qPCR detection of PCV2 mRNA expression at 48 h and 72 h in the overexpression group (b) RT-qPCR detection of PCV2 virus in the siRNA group, (c) Cap protein expression of PCV2 virus at 48 h and 72 h in oe-pc3.1-METTLE14, oe-pc3.1-FTO, si-METTLE14, si-FTO and PK-15 cells of the control group, (d) RT-qPCR detection of miRNA expression at 72 h in the PCV2 virus-infected and uninfected groups.

METTL14 and FTO regulate the process of miRNA maturity

Previously studies have shown the important role of m⁶A modification in RNA processing, including miRNAs and pri-miRNAs [24]. Thus, we screened 10 miRNAs that were differentially expressed after PCV2 infection according to previous studies [25–29], which were miR-30b-3p, miR-125a, miR-92b-3p, miR-10b, miR-155p, miR-21, miR-29b, miR-361-3p, miR-15a, miR-30a-5p. To determine the expression level of these miRNAs after PCV2 infection, we performed RT-qPCR assays. The experiment confirmed that the expression of all these ten mature miRNAs was up-regulated 48 h after virus infection (Figure 3d).

To further explore the role of METTL14 and FTO in miRNA processing during PCV2 infection, we next overexpressed and silence the METTL14 and FTO, respectively. Notably, METTL14 interference and FTO overexpression significantly suppressed the miRNA and pri-miRNA expression after PCV2 infection in PK-15 cells, especially the miR-30a-5p (Figure 4 and Figure 5). Consistently, the overexpression of METT14 and silenced FTO indicate that METTL14 and FTO influence PCV2 replication by targeting metastasis-related miRNAs in an m6A-dependent manner (Figure 4 and Figure 5). These results showed that methylation-related enzymes METTL14 and FTO are needed to mediate the processing of pri-miRNA into pre-miRNA and miRNA maturity.

Figure 4. miRnas were quantified by RT-qPCR upon METTL14 and FTO depletion or overexpression in PK-15 cells after PCV2 infection.

Figure 5. Pri-miRnas were quantified by RT-qPCR upon METTL14 and FTO depletion or overexpression in PK-15 cells after PCV2 infection.

MiR-30a-5p promotes PCV2 replication

To further explore the role of miR-30a-5p as a downstream target of METTL14 and FTO in PCV2 replication. We first assessed whether miR-30a-5p was required for PCV2 replication. The results showed that the expression of PCV2 was up-regulated after transfecting miR-30a-5p mimics, indicating that miR-30a-5p can promote the replication of PCV2 (Figure 6a). Then we treated METTL14-silenced cells and FTP-overexpressing cells with miR-30a-5p mimic. We found that miR-30a-5p mimics could remedy the down-regulation of METTL14 and up-regulation of FTO on the inhibition of PCV2 virus replication (Figure 6b). Taken together, the results show that miR-30a-5p was regulated by these two methylation-related enzymes and participate in the replication process of the PCV2 virus

Figure 6. MiR-30a-5p promotes PCV2 replication. (a) western blot detection of cap protein expression in PK-15 cells infected with PCV2 under the action of miR-30a-5p mimics. (b) Western blot detection of cap protein expression in PK-15 cells infected with PCV2 under the action of miR-30a-5p mimics, oe-FTO, and si-METTL14.

Discussion

RNA m⁶A modification is considered to be another layer of epigenetic regulation similar to DNA histone modification and has emerged as a widespread regulatory mechanism that controls a variety of cellular processes, such as RNA splicing, protein translation, and stem cell renewal. The abnormal occurrence of m⁶A modification can lead to tumorigenesis and affect the replication of the pathogenic virus and the expression of related important genes. Recent studies have shown that virus replication and host immune response to the virus infection was affected by m⁶A methylation. However, few studies have directly focused on the role of m⁶A modification in animal diseases.

In the previous work, we confirmed that there were differences in m⁶A modification in PK-15 cells infected with the PCV2 virus. We found that the content of m6A in the exposed cells was higher than that in the unexposed cells (Figure 1c). This shows that infection with the virus increases the body’s methylation level. We then performed transcriptome sequencing, and analysis of the results revealed significant upregulation of the methyltransferase METTL14 and demethyltransferase FTO (Figure 1). Therefore, this part of the work is mainly focused on the function and mechanism of METTL14 and FTO regulating the whole m6A pathway in PK-15 cells inoculated with PCV2.

m⁶A modification, as an epigenetic modification, has an opposite regulatory effect on the replication of different viruses. Lichinchi [30] found that during HIV-1 infection, the silencing of METTL3 and METTL14 decreased the expression of GP120 and p24. In contrast, ALKBH5 silencing significantly increased the expression of GP120 and p24, indicating that the abundance of m⁶A modification was positively correlated with the expression of GP120 and p24. And our results demonstrated that a high methylation level promotes virus replication, while a low methylation level inhibits PCV2 replication. (Figure 3). Similarly, it has been confirmed that there is a positive regulatory relationship between m⁶A and virus in the study of IVA [31], SV40 [32], and KSHV [33]. But for ZIKV [34], when the expression of METTL3 and METTL14 decreases, virus replication increases, while silencing ALKBH5 and FTO reduces ZIKV replication. In addition, some studies have shown that there is a negative regulatory relationship between m⁶A and HCV [35] virus and AMV [36] virus. In summary, studies have shown that m⁶A modification plays an important role in the replication and pathogenesis of pathogenic viruses, whether positive or negative regulation.

We found that METTL14 could not affect the expression of FTO in cell lines, but FTO could affect the expression of METTL14 in cell lines. This indicates that the upregulation of METTL14 in PK-15 cells infected with PCV2 leads to the upregulation of m⁶A modification of total RNAs, while the upregulation of FTO is a negative feedback regulation response to the upregulation of m⁶A. Ma [37] also concluded that METTL14 could not affect the expression of FTO in HCC cells. In addition, it has been reported that the overexpression of the METTL13 gene significantly inhibits the expression of the FTO gene in porcine subcutaneous adipocytes. A reasonable explanation for this phenomenon may be that methylases have complex regulatory networks, and their expression is affected by more factors.

m⁶A labelling is the key post-transcriptional modification to promote the biogenesis of miRNA. Professor Dr.Tavazoie of Rockefeller University has found that in mammalian cells, the absence of METTL3 reduces the combination of DGCR8 and pri-miRNA, decreasing mature miRNA and the accumulation of unprocessed pri-miRNA [38]. Besides, Ma et al confirmed that METTL14 interacts with microprocessor protein DGCR8 and positively regulates the process of primary miR-126 in an m⁶A-dependent manner. Further experiments showed that miR-126 inhibited the inhibitory effect of METTL14 on tumour metastasis [37]. Moreover, miRNAs can directly exert their antiviral effect through viral RNA binding, regulate host factors, affects the function of virus RNA, restricts virus replication, and even inactivates the viruses. Previous studies have shown that miR-15a can prolong the G0/G1 cell cycle phase in PCV2, thus hindering the progress of the cell cycle [28,39,40]. Our studies found that during PCV2 infection overexpression of METTL14 can promote the expression of miRNA and promote the process of pri-miRNA forming miRNA. On the contrary, FTO plays an inhibitory role in the formation of miRNA maturity (Figure 4 and 5). This study provides evidence that PCV2 replication is significantly promoted by the miRNA maturity.

It has been reported that miR-30a-5p overexpression significantly enhanced PCV2 infection and 3D4/21 cell autophagy, and blocking miR-30a-5p could significantly reduce PCV2 replication. miR-30a-5p directly interacts with 14-3-3 to promote cell cycle arrest in the G2 phase, thus regulating PCV2 replication and autophagy [28]. Our studies also verified that m⁶A modification is regulated by METTL14 and FTO-mediated m⁶A modification in miR-30a-5p, and m⁶A modification can promote the production of mature miR-30a-5p. After transfecting miR-30a-5p mimics into PK-15 cells and inoculating the PCV2 virus solution, we found that miR-30a-5p could promote the replication of the PCV2 virus as in previous studies, and found that mimics could remedy PCV2 replication in cells with low m⁶A level formed by METTL14 deletion and FTO overexpression (Figure 6). This shows that the low expression of miRNA caused by low m⁶A levels can be remedied.

To sum up, this study is the first to investigate the effect and mechanism of m⁶A methylation modification on PCV2 replication. m⁶A methylation modification showed an upward trend in PK-15 cells infected with PCV2, and METTL14 and FTO were the key genes leading to m⁶A disorder.;Depletion of METTL14 or overexpressing FTO inhibit the replication of PCV2, and high level of m⁶A methylation promotes the replication of PCV2 in PK-15 cell; METTL14 and FTO affect the process of miRNA maturity, especially the miR-30a-5p. This study provides an important theoretical basis for further elucidating the mechanism of PCV2 replication and pathogenesis, and for using host protein to improve self-immunity to resist virus infection and to find a general therapeutic target of the virus.

Disclosure statement

No potential conflict of interest was reported by the authors.

Data Availability statement

The data that support the findings of this study are available from the corresponding author, [[email protected]], upon reasonable request.

Additional information

Funding

This work was supported by grants from the Major Scientific and Technological Innovation Project (MSTIP) (grant no. 2019JZZY010720), the Shandong Province Pig Industry Technology System (grant no. SDAIT-08-06), the Shandong Province agricultural applications of major innovation projects (grant no. SD2019XM003, SD2019XM006), the Agricultural Science and Technology Innovation Project of Shandong Academy of Agricultural Sciences (grant no. CXGC2018E10, CXGC2023G03, CXGC2023E02 and CXGC2023A21), and The Natural Fund of Shandong Province (grant no. ZR2022MC011).