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Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek’s disease virus in vitro



The newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek’s disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells.


Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection.


In this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.

Peer Review reports


Marek’s disease (MD) induced by Marek’s disease virus (MDV) is a lethal lymphotropic disease of chickens that is characterized by severe immunosuppression, neuronal symptoms and the rapid onset of T-cell lymphoma [1]. Based on its genome structure, MDV belongs to the alphaherpesvirus family but nevertheless, the tumorigenic phenotype induced by MDV is more characteristic of gammaherpesviruses [2]. Genome-wide sequencing has revealed that MDV attenuation is related to viral gene mutations [3] and this has been confirmed in vivo through viral gene deletion mutations [4, 5]. Recently however, epigenetic regulatory factors such as DNA methylation and histone modifications have been shown to play important roles in MD [6].

Non-coding RNAs (ncRNAs) constitute a varied group of RNA molecules that do not encode functional proteins. Amongst these are the long non-coding RNAs (lncRNAs), being defined as ncRNAs more than 200 bp long which function as another layer of epigenetic regulation. Moreover, post-transcriptional RNA modifications of lncRNAs may change the expression and activity of mRNAs, ncRNAs and proteins, resulting in epigenetic changes in infected cells. LncRNAs characteristically fulfil regulatory or structural roles in different biological and pathological activities, which are distinct from protein coding genes [7]. For example, the MDV encoded Latency Associated Transcripts (LAT) lncRNA alters the splicing of the viral microRNA (miRNA) cluster to produce indirect effects on host gene expression [8]. Furthermore, the ERL (edited repeat-long) lncRNA edited by Adenosine Deaminase Acting on RNA 1 (ADAR1) is involved in the innate immunity response during virus infection [9]. Expression profiling of long intergenic non-coding RNA (lincRNAs) has also been previously reported in the chicken bursa following MDV infection. Acting through regulation of the SATB1 gene, the lincRNA linc-satb1 derived from SATB1 was shown to be crucial in the MDV-induced immune response [10]. Other comprehensive work reporting lncRNA expression profiling indicated that five lncRNAs were strongly related to the expression of MDV and host protein coding genes, and these lncRNAs may play significant roles during MDV-induced tumorigenesis [10]. Among them, linc-GALMD1 inhibited tumor formation through regulating both the expression of MDV and host tumor-related genes [11]. However, whether and how lncRNA expression is regulated during MDV replication is unclear.

Extensive RNA modifications were recently discovered to participate in viral infection through post-transcriptional regulation, decorating both host and viral RNA species. To date, more than 100 distinctive chemical RNA modifications have been identified, including pseudouridine, m6A, N1-methyladenosine (m1A), and 5-methylcytosine (m5C) [12,13,14]. All of the RNA modifications are mediated by methyltransferase “writer” complex, which is an enzyme complex containing methyltransferase-like 3 (METTL3), METTL4, Wilms’ tumor 1-associating protein (WTPA) and other uncharacterized proteins. Conversely, demethylase complexes include AlkB Homolog 5 (ALKBH5) and FTO which can reverse RNA modifications, acting as an “eraser”. In addition, m6A-modified RNAs can be recognized and modulated by the m6A-binding protein complex, including YTH N6-Methyladenosine RNA Binding Protein (YTHDF)1, YTHDF2, YTHDF3 and other proteins acting as “readers” [15].

As one of the most abundant and conserved RNA modifications, m6A is known to be involved in various viral infections, suggesting an important regulatory role in viral replication and pathogenesis [16]. Here, we performed transcriptome-wide m6A modification profiling analyses of lncRNAs, comparing MDV-infected with uninfected chicken embryo fibroblast (CEF) cells. Alterations in the m6A signature of lncRNAs suggests that m6A modifications may play important regulatory roles during MDV replication.


Transcriptome-wide m6A modifications in lncRNAs after Md5 (a very virulent MDV strain) infection

RNA-sequencing and transcriptome analyses were performed on mock control and Md5-infected CEF cells following successful construction of cDNA libraries (Fig. 1). To gain further information of transcriptome-wide m6A modifications in the lncRNAs, we then performed Methylated RNA immunoprecipitation sequencing (MeRIP-seq). Altering the m6A sites with fold changes (FCs) > 2 was considered to be unique to specific sites. Using this approach, we identified 363 and 331 m6A peaks in the Md5 and control groups, respectively (Fig. 2a). Furthermore, a total of 294 and 275 annotated genes were mapped to the Md5-infected and control groups, respectively (Fig. 2b). Among them, 277 m6A peaks and 228 m6A modified genes were detected in both the Md5-infected and control groups. Overall, these results indicated that the incidence of the m6A modification in lncRNAs was higher in the Md5 infected group compared to the control group.

Fig. 1

Flowchart illustrating the construction of cDNA libraries used for RNA sequencing

Fig. 2

Transcriptome-wide m6A modifications in lncRNAs following Md5 infection. a Venn diagram of m6A modification sites identified in lncRNAs from mock control and Md5-infected groups; b Venn diagram of m6A modified lncRNAs from mock control and Md5-infected groups

m6A modification clustering analysis

Results from the methylation heat map and cluster analysis showed that the different clustering could clearly distinguish the m6A modification at the transcriptome level in the Md5-infected group from the control group (Fig. 3a). These findings indicate that the degree of methylation in the Md5-infected group was significantly higher than for the control group (Fig. 3b). In total, 70 m6A modification peaks were identified as being up-regulated (Table 1) with 53 methylation peaks being down-regulated amongst lncRNA genes (Table 2).

Fig. 3

m6A modification clustering analysis. Cluster analysis of the transcriptome (a) and m6A modified lncRNA genes (b) in mock control and Md5-infected groups. The color intensity represents the size of the log-fold enrichment (FE) value; the closer the color is to red, the larger the logFE value

Table 1 Ten top up-methylated m6A peaks
Table 2 Ten top down-methylated m6A peaks

Chromosome visualization of m6A modified lncRNAs

Studying the genomic distribution of m6A methylation sites revealed that lncRNA genes undergoing the m6A modification were scattered on all chromosomes. However, the methylation levels and distribution of m6A of lncRNA genes on each chromosome were different between infected and control groups, a finding which may functionally associate m6A with MDV infection (Fig. 4a and b).

Fig. 4

Differentially methylated N6-methyladenosine peaks in lncRNAs. Both a and b showed that representative upmethylated genes in Md5-infected group relative to mock control group. Highlighted columns show the general locations of differentially methylated peaks

Abundance of m6A peaks and conserved m6A modified motifs in lncRNAs

Regarding the abundance of the m6A peaks in lncRNAs, we found that 77.13% of the lncRNAs in the Md5-infected group contained m6A peaks, which appeared marginally more than the unimodal value calculated at 75.86% in the control group. The respective percentages comparing different numbers of peaks were also determined with two peaks, three peaks, and more than three peaks being 15.81 vs 16.66, 3.92% vs 5.10 and 3.14% vs 2.38%, respectively, for the Md5 infected versus control group (Fig. 5a).

Fig. 5

Abundance of m6A peaks and the conserved m6A modified motif in lncRNAs. a Number of lncRNA harboring different numbers of m6A peaks in the two groups, with the majority harboring only one m6A peak; b The sequence motif of m6A sites in Md5-infected and mock control groups; MeRIP-qPCR analysis of two candidate lncRNAs c ENSGALG00000031400 and d ENSGALG00000030195. * and ** respectively represent the significant difference in gene expression between two groups (* for P-value < 0.05 and ** for P-value < 0.01)

To analyze the conserved motif of m6A modified lncRNAs, we selected the sequences of the first 1000 peaks with the highest enrichment factor in each group (50 bp on both sides of the peak), and scanned the sequences of these peaks using DREME software [17] to determine whether the identified m6A peak contained the RRACH conservative motif sequence (where R represents purine, A represents m6A and H represents non-guanine bases). The sequence of the top ten peaks with the highest enrichment ratio of lncRNA (50 bp on each side of the vertex) was compared with the motif sequence found, and it was found that GGACU sequence was one of the conserved motif sequences of lncRNA (Fig. 5b). GGACU is one of the motif obtained based on E-value. For the peak with GGACU sequence in control group is 202/1000 (202 peaks out of 1000 peaks used for analysis contain this sequence). In Md5-infected group it was 165/1000.

To further confirm the existence and distinctive expression of m6A modified lncRNAs. The relative expression of two lncRNAs were confirmed by m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) (Fig. 5c and d). The results indicated that the results of MeRIP-qPCR are consistent with RNA-Seq.

GO enrichment analysis

To explore the potential function of m6A in CEF cells and infected cells, we carried out GO enrichment analysis of differentially m6A-methylated genes of lncRNAs. The GO Project has developed a structured, controlled vocabulary for annotating genes, gene products and sequences divided into three parts: molecular function (MF), biological process (BP) and cellular component (CC). GO function analysis performed against the differentially methylated lncRNAs showed no significant enrichment but when analysis was performed on the input sequencing data, only the up-regulated methylated sites were found.

The BP data showed enrichment in steroid hormone receptor activity, sequence-specific DNA binding RNA polymerase II transcription factor activity and DNA binding (Fig. 6a). CC data showed mainly enrichment for nucleosome, DNA packaging complex and DNA bending complex (Fig. 6b). The MF outputs showed the genes with increased methylation were notably enriched in the steroid hormone mediated signaling pathway, response to retinoic acid, nucleosome organization, nucleosome assembly, hindbrain development, DNA packaging, chromatin assembly and cellular response to steroid hormone stimulus (Fig. 6c).

Fig. 6

GO analysis of coding genes harboring differentially methylated m6A sites. The top ten GO terms for a biological processes; b molecular functions; and c cellular components significantly enriched for the up-methylated transcriptome in Md5-infected versus mock control groups

KEGG pathway analysis

KEGG analyses map molecular data sets from genomics, transcriptome, proteomics and metabolomics to explore associated biological functions. KEGG pathway analyses indicated significant gene enrichments associated with five up-regulated pathways, including ErbB signaling, GnRH signaling and Toll-like receptor signaling pathways along with Influenza A and MAPK signaling (Fig. 7a). Two significantly down-regulated pathways involved ABC transporters and Notch signaling (Fig. 7b).

Fig. 7

KEGG analysis and gene set enrichment analysis (GSEA) of differentially methylated genes in Md5-infected and control groups; a Pathway analysis of up-methylated; b down-methylated genes


The transcriptome-wide m6A modification is important in virus infection

MD is a highly contagious tumor-causing disease which threatens all poultry-raising countries across the globe [18]. The pathogenesis of MD is complex with apparent genetic changes, heritable gene expression changes and chromatin tissue being shown to promote tumor initiation and progression. Additionally, it is now emerging that epigenetic changes, particularly those associated with reversible chemical modifications of RNA, fulfil important roles in the life cycle of viruses and therefore also in viral pathologies. For example, HIV infection increases the levels of m6A modification in both viral and host transcripts, and moreover, m6A modified-HIV transcripts display enhanced binding ability to viral proteins. Instructively, knockdown of the ALKBH5 demethylase or alternatively the METTL3/14 methylase to alter the level of HIV m6A modifications either promotes or inhibits viral replication, respectively [19]. Furthermore, twelve m6A modified sites have been found in ZIKV genomic RNA but in contrast to HIV, demethylase knockout inhibits ZIKV replication, while methylase knockout increases ZIKV replication rates. However, the impact of the m6A modification in MVD is yet to be determined [20].

MDV infection increased lncRNAs m6A modification

In the present study, we investigated how the m6A modification in lncRNAs was affected by MDV infection. The results obtained in CEF cells showed that the abundance and distribution of m6A in Md5-infected and control groups were different albeit not significantly. Interestingly, we found that some of the lesser expressed genes in the control group were not only highly expressed in the infected group, but also displayed increased levels of m6A modification. Interestingly, there were significantly higher expressions of METTL14 and ALBHK5 in MDV infected CEF cells comparing to mock-infected control (Data not shown). This suggests MDV might control lncRNAs m6A modification through regulating activities of methyltransferase and demethylase, and even reader proteins. It is of great importance to determine the detailed mechanism of how MDV affect and regulate the lncRNAs m6A modification in the future. Alternatively, the role of m6A modified lncRNAs on MDV replication also need to be further investigated.

MDV infection altered lncRNAs m6A modification associated with genes function

GO analysis of the m6A modified genes showed that most are up-regulated methylated sites. For BP, CC and MF, up-regulated methylated genes were notably enriched in steroid hormone mediated signaling pathway, nucleosome organization, nucleosome assembly, DNA packaging, DNA binding complex, chromatin assembly and cellular response to steroid hormone stimulus. Most of these biological activities are related to virus replication, suggesting lncRNA may change structural and regulatory roles after m6A modification.

MDV infection altered lncRNAs m6A modification associated with signaling pathways

LncRNA expression can be variously regulated by histone modification, DNA methylation or through changes in the expression of the responsible transcription factors. In this study, many differentially expressed m6A modification sites were found, among which the unique m6A modification related genes were only found in Md5-infected group. These results suggest that some of the m6A modification sites are changed by Md5 virus infection. Furthermore, KEGG pathway analyses implicate roles for m6A-modified lncRNAs in biological pathways known to be associated with viral infection, namely ErbB signaling, GnRH signaling, Toll-like receptor signaling, Influenza A and the MAPK signaling pathway. Notably the ErbB gene encoding tyrosine kinases of the epidermal growth factor (EGF) receptor family can promote herpesvirus replication [21] while the Toll-like receptor signaling pathway is also upregulated by MDV infection in vitro [22]. The mitogen-activated protein kinase (MAPK) upstream of intracellular signaling pathways also participates in HSV-1 cell-to-cell spreading. Indeed, MDV infection alters MAPK signaling in vitro and in vivo, suggesting a key role in herpesvirus replication and even pathogenesis [23, 24]. Furthermore, influenza A virus (IAV) infection activates multiple signaling pathways to overcome the innate immunity barrier where IAV is recognized by the pathogen recognition receptor RIG-I to control type I IFN production [25]. Notably, it has been demonstrated that AIV expresses m6A modified transcripts and that inhibition of m6A could decrease gene expression and inhibit AIV replication [26]. Moreover, mutations in AIV transcripts to alleviate m6A modifications reduced viral pathogenicity thereby confirming this important regulatory role. Thus overall, there is evidence that up-regulation of m6A modified transcripts might be a common feature for both DNA and RNA viruses that helps facilitate viral replication through regulating host RNA regulatory pathways [27].


In this study, we employed MeRIP-seq to evaluate differential lncRNA m6A modifications following Md5 infection. Comparing MDV infected and control cells we identified the abundance of m6A modifications and the genome wide utilization of the conserved motif. Tellingly, we observed increased lncRNA m6A modifications following Md5 infection, clearly suggesting a relationship between lncRNA m6A modifications and viral infection. In support, GO and KEGG analyses showed genes with up-regulation of methylation were associated with host cell signaling pathways known to contribute to viral infection. However, further investigations are required to dissect the molecular mechanisms linking m6A-modified lncRNAs with MDV pathogenesis and tumorigenesis.


Cells and virus

CEF cells were isolated and prepared from 9-day-old specific-pathogen-free (SPF) embryonated white leghorn chicken (Boehringer Ingelheim, Beijing, China) as previously described [28]. CEF cells were maintained in Dulbecco’s modified essential medium (DMEM) (Solarbio, Beijing, China) containing 5% fetal bovine serum (FBS) (Gibco, CA, USA).

A very virulent MDV strain, Md5 (Genbank accession no: NC_002229.3) was used in the present study. For virus infection assay, secondary CEF cells were seeded to 80–90% confluence in T75 culture dishes and separated into mock-infected and infected groups with three repeats in each group. The infected group was inoculated with 106 plaque formation units (PFU) of the Md5 strain (passage two) and cells harvested 7 days post-inoculation when the cytopathic effects (CPE) became clearly visible in about 80% of infected cells.

RNA extraction

Total RNA was extracted using Trizol reagent (Invitrogen Corporation, Carlsbad, CA) according to the manufacturer’s instruction, with DNase treatment. RNA concentrations were quantified using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

cDNA library construction

RNA samples were fragmented into 100 bp using fragmentation buffer and then incubated with anti-m6A polyclonal antibody (Synaptic Systems, 202,003, Germany) in immunoprecipitation (IP) buffer for 2 h at 4 °C. The mixture was then immunoprecipitated by incubation with protein-A beads (Thermo Fisher Scientific, Waltham, MA, USA) at 4 °C for an additional 2 h. Then, bound RNA was eluted from the beads with N6-monophosphate (BERRY & ASSOCIATES, PR3732) in IP buffer and then extracted with Trizol reagent. Purified RNA was used for RNA-seq library generation with NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England Biolabs, USA) following the manufacturer’s instructions. Both the input sample without immunoprecipitation and the m6A IP samples were subjected to 150 bp paired-end sequencing on an Illumina HiSeq 4000 sequencer [14].

Sequencing and data analysis

Paired-end reads were harvested for image and base recognition with Q30 used as the quality control standard, with the sequencing quality of Q30 being usually over 80%. After 3′ adaptor-trimming and low-quality reads removing by cutadapt software (v1.9.3), the reads were aligned to the chicken reference genome (Gal5; GCA_000002315.3) with Hisat2 software (v2.0.4). The expressed lncRNAs were identified using Input reads and the methylated sites on lncRNAs identified using the MeTPeak package in R software. Differentially methylated sites were identified by MeTDiff package in R. The Gene Ontology (GO) ( and pathway enrichment analysis were performed for the differentially methylated genes. The read alignments on genome were visualized using the interactive analysis tool Integrative Genomics Viewer (IGV).

To define the possible roles of the differentially methylated genes, the GO functions were analyzed using the corresponding lncRNA genes as inputs. GO terms providing P-values ≤0.05 were considered to be statistically significant. In concert, Kyoto Encyclopedia of Genes and Genomes (KEGG) [29] analyses of the genes associated with differentially methylated lncRNAs were used as inputs to derive significantly altered pathways. P-values < 0.05 were taken as the threshold for significant enrichment.

m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR)

We selected two differentially methylated RNA sites (ENSGALG00000031400 and ENSGALG00000030195) to design specific primers for MeRIP-qPCR using NCBI Primer-Blast [30]. The forward primer (5′-TCATGGCCTGATTCTTTGAGC-3′) and reverse primer (5′-TGCTGTGGATTGGCTTGGAA-3′) designed to amplify 100 bp of ENSGALG00000031400, and the forward primer (5′-CAGCTGCCTGAACAAGGAGA-3′) and reverse primer (5′-ACATACTGCTAAAGCTCAGGAA-3′) designed to amplify 101 bp of ENSGALG00000030195 were synthesized by Sangon Biotech Co. (Shanghai, China). Then reverse transcribed IP RNA and input RNA by PrimeScriptTM RT Reagent Kit and gDNA Eraser Kit (TAKARA, Shiga, Japan) to get cDNA, and qPCR was performed on QuantStudio™ 5 System.

Availability of data and materials

All data generated or analyzed during this study are included in this submitted manuscript. The datasets generated and/or analyzed during the current study are available in the NCBI repository ( The data is accessible via NCBI GEO submission ID: GSE166240. To review GEO accession GSE166240: Go to Enter token klufyeaednulxgb into the box.



Marek’s disease virus


Marek’s disease




Chicken embryo fibroblast


Methylated RNA immunoprecipitation sequencing


Gene ontology


Kyoto encyclopedia of genes and genomes


Adenosine Deaminase Acting on RNA 1


Latency Associated Transcripts


long intergenic non-coding RNA


Unique long region


Unique short region


MDV serotype 1


MDV serotype 2


MDV serotype 3


non-coding RNAs




long non-coding RNAs


Specific pathogen-free


Methyltransferase like protein 3


Methyltransferase like protein 14


Wilms’ tumor 1-associating protein


Fat mass and obesity-associated protein


AlkB homolog 5 RNA demethylase


YT521-B homology


messenger RNA


N1-adenylate methylation


Cytosine hydroxylation


Specific pathogen free


Epidermal growth factor


Mitogen-activated protein kinase


influenza A virus


Dulbecco’s modified essential medium


Fetal bovine serum


Plaque formation units


Cytopathic effects




Integrative Genomics Viewer


Biological processes


Molecular functions


Fold change


Cellular components


Fold enrichment


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This work is supported by Grants of the Starting Foundation for Outstanding Young Scientists of Henan Agricultural University (No 30500690); The Henan province advanced program of 2020 for returned overseas scholar (No 30602136); The grants of National Natural Science Foundation of China (No 31802160 and U1604232); The Henan Thousand Talents Program-Leading Talents in Basic Research (2019–2020); The Natural Science Foundation of Henan Province (2021); and The Key R&D and Promotion Project of Henan Province (2021). The grants above were used in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

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AJS and GQZ designed the experiments. XJZ, YL, RW, SKY, and LUZ performed the experiments. AJS, MT, and JL analyzed the data. AJS, XJZ and GQZ drafted the manuscript. GQZ and GPZ revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Guoqing Zhuang.

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Sun, A., Zhu, X., Liu, Y. et al. Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek’s disease virus in vitro. BMC Genomics 22, 296 (2021).

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  • Marek’s disease virus
  • Long non-coding RNA
  • m6A
  • MeRIP-Seq
  • KEGG