The Tiny Epigenetic Addition Plays Big Roles: The RNA Methylation in Both Human and Animal Herpesvirus Infection

Abstract

During human herpesvirus infection, dynamic alterations of N6-methyladenosine (m⁶A) modification have been extensively observed in viral and cellular transcriptomes. This modification plays a crucial role in RNA metabolism, serving as a novel regulator of gene expression alongside DNA and protein modifications. Notably, reversible changes in a single m⁶A modification site can impact viral replication and pathogenicity. Recent studies have reported changes in m⁶A modification-associated epitranscriptomes and their functional analysis during animal herpesvirus infections. This review focuses on the research progress of m⁶A modification on the transcriptome in both human and animal herpesvirus infections within the same family. Specifically, it examines the dynamic alterations of m⁶A modification-associated epitranscriptomes, the expression of m⁶A-machinery proteins, regulatory molecular mechanisms associated with herpesvirus infection, and potential clinical applications. By addressing the gaps in research on m⁶A modification in animal viruses, new insights into the regulatory molecular mechanisms of viral diseases may be uncovered. Furthermore, natural hosts infected with animal herpesvirus serve as valuable biomedical models for studying the regulation of m⁶A modification on viral replication and pathogenesis, thereby supporting the development of novel vaccine and drug targets.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

1. Introduction

The Herpesviridae family encompasses a diverse group of enveloped viruses characterized by large double-stranded DNA genomes, which are further categorized into alpha, beta, and gamma subfamilies [1]. Among the human prototypical herpesvirus are Herpes simplex virus type I (HSV-1), a member of the alpha-herpesvirus subgroup, which commonly causes skin and mucous membrane infections, as well as severe conditions like herpes simplex encephalitis [2]. Human cytomegalovirus (HCMV) belongs to the beta-herpesvirus category and is a prevalent virus known to cause birth defects and neurodevelopmental abnormalities in fetuses, with recurrent infections often proving fatal in transplant recipients [3]. Kaposi’s sarcoma-associated herpesvirus (KSHV), also referred to as human herpesvirus type 8 (HHV-8), is a gamma-herpesvirus associated with diseases such as Kaposi’s sarcoma (KS), primary exudative lymphoma (PEL), and multicentric Castleman’s disease (MCD) [4]. Last, Epstein–Barr virus (EBV), designated as human herpesvirus type 4 (HHV-4), exhibits a unique capability to influence the differentiation of B lymphocytes into a distinct plasma cell-like state [5]. Current research has highlighted the involvement of N6-methyladenosine (m⁶A) modification in the infection processes of these herpesvirus [6, 7]. Nevertheless, conducting experiments outside the natural host environment, namely the human body, presents several limitations, such as variations in extracellular conditions, immune responses, tissue-specific effects, long-term consequences of infection, and responses to therapeutic interventions and drugs.

Pseudorabies virus (PRV) and Marek’s disease virus (MDV) are animal alpha-herpesvirus that can serve as valuable models for understanding alphaherpesvirus-associated pathogenesis and gammaherpesvirus-associated oncogenesis [8, 9]. PRV infection leads to pseudorabies (PR), a highly pathogenic and infectious disease characterized by symptoms like high fever, itchiness, and encephalomyelitis, often resulting in mortality rates reaching up to 100%. PRV is known to infect not only its natural host, pigs, but also other mammals and rodents, including dogs, cats, and mice. Recent findings have shown PRV’s ability to infect human beings [10]. On the other hand, Marek’s disease (MD) caused by MDV is an immunosuppressive disease that triggers rapid tumor development in infected chickens [8]. The MDV life cycle in chickens encompasses four distinct infectious phases: the early cytolytic infection phase (which affects chickens’ B-lymphocytes 3–6 days after inhalation of virus particles), the latency phase (emerging in activated CD4+ T-lymphocytes 7–10 days postinfection), the reactivation phase (stimulating late cytolytic infection), and the transformation phase (resulting in tumors across various organs in latently infected CD4+ T-lymphocytes) [11]. Despite the availability of vaccinations for MD, MDV continues to evolve to enhance its virulence, with the underlying molecular mechanisms not yet fully elucidated. Prior investigations have illustrated how various herpesvirus employ epigenetic modifications, such as DNA methylation and histone modification, to influence gene expression and replication. Recently, the emerging concept of epigenetic modifications on RNA has introduced a new dimension to the regulation of gene expression and viral replication [12].

RNA modifications are a widespread posttranscriptional regulatory mechanism, with more than 170 unique types identified across various RNA species, such as messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), circular RNA (circRNA), and long noncoding RNA (lncRNA) [13]. These modifications play crucial roles in regulating RNA processing and translation by enhancing interactions with reader proteins and translation factors. In tRNA, modifications in the T and D loops influence tRNA folding and stability, while those in the anticodon loop impact codon recognition, decoding efficiency, and accuracy [14]. Conversely, rRNA modifications predominantly occur at functional ribosomal sites, such as the peptidyl transferase center and decoding site, affecting ribosomal biogenesis, structure, and function, consequently, regulating mRNA translation [15]. Methylation modifications like N6-methyladenosine (m⁶A) [16], N1-methyladenosine (m¹A) [17], and 5-methylcytosine (m⁵C) [18] are extensively studied, each exerting specific functions in diverse biological contexts. The most prevalent mRNA modification in eukaryotes is m⁶A modification, with key regulatory proteins, including writers, readers, erasers, and suppressors [19]. The m⁶A modifications are highly conserved and are prevalent on various RNAs, predominantly near mRNA termination codons [20]. Upon surpassing a specific threshold, m⁶A-modified mRNA transcripts undergo proper splicing, translocation, translation, or degradation by reader proteins, participating in diverse physiological processes, including cap-dependent translation, RNA splicing, mRNA stabilization, and microRNA (miRNA) biogenesis. Disruption of these modifications can lead to imbalances in RNA metabolism, impacting various physiological and pathological processes, such as meiosis, biological clock regulation, DNA damage repair, stress response, circadian rhythms, cancer, and viral infections [12, 21–26].

High-throughput sequencing technologies have advanced considerably in the study of RNA modifications, leading to the gradual revelation of the widespread presence of m⁶A methylation marks in viral transcriptomes. This discovery suggests that m⁶A modifications play a significant role in viral replication and pathogenesis by influencing gene expression [27]. Notably, it has been confirmed that m⁶A modification contributes to the epitranscriptome alterations induced by various members of the Herpesviridae family [6, 12]. While several excellent reviews have discussed the regulatory role and molecular mechanisms of m⁶A modification in viral infections, this review focuses primarily on the latest research developments regarding m⁶A modification in human herpesvirus replication and pathogenicity. Additionally, we provide a summary of the most recent research progress on m⁶A modification in animal herpesvirus replication and pathogenicity, a topic explored for the first time. Furthermore, we discuss potential research directions that include investigating the role and molecular mechanisms of m⁶A modification in both human and animal herpesvirus replication and pathogenesis. These insights may illuminate new pathways for vaccine development and the design of antiviral drugs.

2. m⁶A-Machinery Proteins

The regulatory mechanisms of m⁶A modifications can be classified into four main groups as follows: writers, erasers, readers, and suppressors. These groups collectively demonstrate a dynamic and reversible biological process in regulating RNA metabolism as described in Figure 1.

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2.1. m⁶A Modification Writers

The m⁶A modification process is catalyzed by the m⁶A methyltransferase complex (MTC) (m⁶A modification writers), composed of core components, such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), and various regulators, including Wilms’ tumor 1-associating protein (WTAP), vir like m⁶A methyltransferase associated protein (VIRMA/KIAA1429), RNA binding motif protein 15/15B (RBM15/15B), Cbl proto-oncogene like 1 (HAKAI), zinc finger CCCH-type containing 13 (ZC3H13), and methyltransferase like 16 (METTL16) [28, 29]. METTL3, which binds to S-adenosylmethionine (SAM), serves as the central component of the MTC and is responsible for catalyzing the formation of RNA m⁶A modifications. Working in tandem, METTL14 forms a stable heterodimer with METTL3, aiding in the deposition of m⁶A on nuclear RNA in mammalian cells, and thereby heightening the catalytic capacity of METTL3. The combined action of METTL3 and METTL14 results in a significant enhancement of methyltransferase activity compared to their individual roles [28]. WTAP plays a crucial role by interacting with the METTL3-METTL14 complex to localize it to nuclear speckles. In the absence of WTAP, the RNA binding capability of METTL3 diminishes significantly [30]. Additionally, KIAA1429 (VIRMA) proteins interact with WTAP to facilitate the occurrence of m⁶A modifications near the 3′ untranslated region (UTR) and stop codons on mRNA [31]. The RBM15/15B complex recruits the WTAP-METTL3 complex to XIST, leading to the methylation of lncRNA-XIST [32]. ZC3H13 collaborates with the WTAP-VIRILIZER-HAKAI complex to regulate the m⁶A methylation modification levels of embryonic stem cells (ECs) [33]. Lastly, METTL16 participates in modulating SAM homeostasis formation and is involved in the m⁶A modification of nascent RNA in the nucleus [34].

2.2. m⁶A Modification Erasers

Two m⁶A demethylases, fat mass and obesity-associated protein (FTO) and ALK B homologue 5 (ALKBH5), have been identified as m⁶A modification erasers. These enzymes rely on divalent iron ions and 2-oxoglutarate for their enzymatic activity, enabling them to catalyze the demethylation of m⁶A-containing mRNA molecules. Generally, ALKBH5 and FTO are predominantly localized within the nucleus [35, 36]. FTO exhibits homology with the DNA repair protein AlkB and is implicated in the oxidative demethylation of 3-methylthymine within single-stranded DNA, as well as the demethylation of 3-methyluracil within single-stranded RNA [37]. Numerous investigations have highlighted the role of FTO as a proficient modulator of nuclear mRNA processing contributing to alternative splicing and the processing of the 3′ end of mRNA [38]. Furthermore, FTO is responsible for modulating the levels of N6, 2′-O-dimethyladenosine (m6Am), leading to selective regulation of m6Am-containing mRNA abundance in cells. Additionally, FTO plays a role in reducing the stability of m6Am mRNA, demonstrating a preference for demethylating m6Am over m⁶A [39]. ALKBH5 is involved in the removal of m⁶A modifications from nuclear RNA, particularly mRNA, both in experimental settings and in living organisms [36]. Moreover, ALKBH5 plays a significant role in the regulation of nuclear RNA export, metabolism, and gene expression, suggesting that reversible m⁶A modifications on RNA have extensive impacts on biological processes [40].

2.3. m⁶A Modification Readers

m⁶A-associated binding proteins, also known as m⁶A “readers,” play a key role in regulating gene expression by interacting with m⁶A sites on RNAs. This family of reader proteins (m⁶A modification readers) includes YTHDF1, YTHDF2, YTHDF3, YTHDC1, and YTHDC2. YTHDF1 functions by directly enhancing translation through binding to the m⁶A modification in the 3′UTR region [22], while YTHDF2 promotes mRNA decay by recruiting the CCR4-NOT deadenylation complex [41]. YTHDF3, acting as a cofactor for both YTHDF1 and YTHDF2, contributes to their regulatory functions [42]. YTHDC1, predominantly found in the nucleus, controls mRNA export from the nucleus to the cytoplasm, and supports the formation of exonic inclusion bodies [43, 44]. On the other hand, YTHDC2 modulates RNA translation by interacting with the 40–80S subunit in the cytoplasm [45]. Recently, Huang et al. [46] identified a new group of insulin-like growth factor two mRNA-binding proteins that exhibit a strong affinity for m⁶A-modified mRNAs via the canonical m⁶A motif GG (A)C, leading to increased mRNA stability. In addition to the YTH protein family, other proteins like eIF3 and HNRNP2AB1 have been recognized for their ability to recognize m⁶A modifications [47, 48]. Specifically, eIF3, a component of the 43S preinitiation complex, aids in protein translation by binding to the m⁶A site in the 5′UTR region of mRNA [47]. HnRNPA2/B1 directly binds to and regulates the processing of m⁶A-modified transcripts, including subset of primary miRNA transcripts by interacting with the miRNA microprocessor complex protein DGCR831 [48].

2.4. m⁶A Modification Suppressors

Exon junction complexes (EJCs) interact with mRNA sequences located upstream of exon junctions and are a prevalent constituent of messenger ribonucleoprotein (mRNP) complexes (m⁶A modification suppressors [49]. The core components of EJCs include three essential proteins: Eukaryotic Translation Initiation Factor 4A3 (EIF4A3), RNA Binding Motif Protein 8A (RBM8A), and Protein Mago Nashi Homolog (MAGOH) [50]. Initial studies suggested that EJCs might influence the nonuniform distribution of m⁶A modifications across the transcriptome by physically hindering them [51]. EJCs package mRNPs, making regions near exon junctions resistant to m⁶A modification and influencing the spatiotemporal distribution of these modifications along nascent RNA transcripts [52]. Through deep learning modeling, researchers found that the absence of pre-mRNA splicing in the host gene increases m⁶A modifications [53]. EJCs inhibit METTL3-mediated m⁶A modifications near splice sites within the coding sequence (CDS), leading to enhanced m⁶A enrichment in the 3′UTR and shaping the landscape of m⁶A modifications [54]. By acting as suppressors of m⁶A methylation, EJC proteins safeguard regions of transcripts lacking m⁶A modifications, guarding RNA proximal to CDS near exons and ultimately affecting mRNA stability through m⁶A modification regulation [49]. However, the role of EJCs in suppressing m⁶A at exon-intron boundaries is partial and limited to specific short internal exons [49].

3. Dynamic Regulation of the m⁶A Modification-Associated Epitranscriptome During Human Herpesvirus Infection

3.1. HSV-1

HSV-1 is a double-stranded DNA virus characterized by its genome structure, which comprises unique long (UL) and unique short (US) regions, flanked by terminal repeat (TR) and internal repeat (IRL) sequences [55]. Research conducted in 1977 first identified m⁶A modifications on HSV-1 mRNAs [56]. Recent advancements in m⁶A modification sequencing methods have led to the identification of at least 12 m⁶A modification peaks within HSV-1 transcripts, encompassing genes such as UL1, UL2, UL12, UL28, UL29, UL38, UL39, UL42, UL46, UL49, US10, US11, and US12. Following HSV-1 infection, an increase in the expression levels of the RNA methyltransferases METTL3 and METTL14, along with the YTHDF family of m⁶A readers (YTHDF1, YTHDF2, and YTHDF3), has been observed at the early stages of infection. Silencing of METTL3 has been shown to inhibit viral replication by downregulating the expression of both early genes (such as ICP0, ICP8, and UL23) and late genes (including VP16, UL44, UL49, and ICP47) [57]. Notably, during HSV-1 infection, METTL3 and METTL14 translocate from the nucleus to the cytoplasm, a process that is facilitated by the viral ICP27 protein [58]. Furthermore, after HSV-1 infection of human oral epithelial cells, a downregulation of the m⁶A demethylases ALKBH5 and FTO occurs, resulting in an increase in the overall m⁶A modification levels within these cells. Silencing of ALKBH5 and FTO has been implicated in the promotion of type I interferon (IFN-I) and interferon-stimulated genes (ISGs) expression, thereby enhancing the antiviral immune response and inhibiting viral replication [59]. Conversely, HSV-1 infection leads to the degradation of m⁶A-containing transcripts by YTHDF proteins, contributing to the downregulation of host m⁶A modifications. In particular, the knockdown of YTHDF proteins has been shown to diminish the expression of viral proteins while upregulating ISGs expression [60]. Notably, in the late stages of HSV-1 infection, the downregulation of YTHDF2 due to protein synthesis shutoff has been linked to an enhancement of the host’s antiviral response. Additionally, METTL3 has been implicated in corneal neovascularization (CNV) during HSV-1 infection, regulated through canonical Wnt and VEGF signaling pathways in both in vitro and in vivo models. Specifically, METTL3 modulates the m⁶A levels of LRP6 mRNA, thereby increasing its stability and protein expression, which fosters angiogenesis in HSV-1-infected human umbilical vein endothelial cells (HUVECs) [61]. Importantly, the m⁶A inhibitor 3-deazaadenosine (3-DAA) has been demonstrated to inhibit HSV-1 replication, suggesting a promising avenue for novel antiviral drug development [57].

m⁶A modification plays a multifaceted role in HSV-1 infection: it serves as both a tool for the virus to hijack host resources and a critical node in host defense. However, the aforementioned studies still leave many questions unresolved. For example, the nuclear-cytoplasmic transport of m⁶A modification enzymes (e.g., METTL3/METTL14) during early infection is mediated by ICP27, but the specific molecular mechanisms (such as whether phosphorylation or acetylation modifications are involved) remain unclear [62]; Addtionally, while HSV-1 infection causes YTHDF proteins to degrade host m⁶A-modified transcripts, it remains unknown whether the virus’s own mRNA evade recognition through a similar mechanism. Futhermore, HSV-1 can establish latent infection in the trigeminal ganglion; does m⁶A modification participate in regulating latent-associated transcripts (such as LAT)? Do dynamic changes in m⁶A during viral reactivation influence the reactivation process? These questions remain to be explored in depth. Future research should focus on elucidating the spatiotemporal dynamics of regulatory mechanisms and developing precision treatment strategies targeting m⁶A to address the challenges posed by HSV-1 infection and its associated pathologies.

3.2. HCMV

HCMV, the prototypical beta-herpesvirus subfamily, has a genome of approximately 250 kb that encodes more than 200 proteins [63]. Within HCMV-encoded lncRNAs, m⁶A modifications have been identified, contributing to their stability and function through interactions with reader proteins [64]. In the context of HCMV infection, there is an upregulation of METTL3 and METTL14, along with increased expression of YTHDF1-3 and YTHDC1. Research has demonstrated that HCMV infection alters the m⁶A modifications of IFNB1 mRNA. Specifically, depletion of METTL14 enhances the accumulation of IFNB1 mRNA, leading to reduced HCMV replication, whereas depletion of ALKBH5 has the opposite effect [65]. Furthermore, HCMV infection has been shown to elevate the total m⁶A modification level in vascular endothelial cells. During HCMV infection, m⁶A modification has been linked to reduced stability and transcriptional termination of ubiquitin carboxy-terminal hydrolases L1 (UCHL1) mRNA, contributing to inflammatory damage in the vascular endothelium [66]. METTL3-mediated m⁶A modification boosts the binding of mitochondrial calcium uniporter (MCU) mRNA to YTHDF3, leading to increased expression and facilitating HCMV-induced apoptosis in vascular endothelial cells. Notably, vitamin D3 downregulates the expression of METTL3 by inhibiting the activation of AMPK, thereby rescuing HCMV-induced apoptosis in vascular endothelial cells [67]. In summary, the alterations in m⁶A modifications induced by HCMV infection impact both viral and host cells, influencing viral infection strategies and virus–host interactions in a significant manner.

These findings suggest that HCMV exploits m⁶A modification as a double-edged sword that promotes viral replication and host damage. However, the aforementioned studies still leave several unresolved questions, including: (1) whether there are significant differences in the m⁶A modification profiles between the lytic and latent phases of HCMV; (2) whether the upregulation of METTL3/METTL14 and the YTHDF family by HCMV depends on virus-encoded proteins directly regulating the host epigenome; (3) whether m⁶A modification of HCMV’s own RNA affects its stability, translation efficiency, or immunogenicity; (4) whether the virus utilizes the host m⁶A modification system to selectively regulate gene expression during distinct infection phases (lytic vs. latent); (5) whether HCMV’s own RNA (e.g., the major immediate-early genes IE1/IE2 that initiate viral replication) carries conserved m⁶A sites; (6) whether these modifications evade innate immunity by inhibiting RIG-I recognition (e.g., through masking the 5′ppp-m⁶A structure) to evade innate immunity. In-depth exploration of these questions is crucial for understanding the interaction between HCMV and its host.

3.3. KSHV

KSHV, a member of the gamma-herpesvirus subfamily, has a complex architectural structure in its mature virion. The virion comprises an envelope, a capsid protein layer, and a nucleocapsid enclosing almost 170 kb of double-stranded DNA. The viral genome exhibits distinct UL and US regions, each bordered by IR regions at the termini. These IR regions contain essential regulatory sequences and a subset of genes crucial for viral function. Within its genome, KSHV encodes around 80 to 90 open reading frames (ORFs) that are involved in various aspects of viral replication, immune evasion, and tumorigenesis. Despite this, the specific roles of the majority of ORFs in viral pathogenesis remain poorly understood, necessitating further investigation in the future to elucidate their functions in the overall course of the infection [68].

In the replication lifecycle of KSHV, abundant m⁶A modifications occur on its transcripts, particularly during the transition from latency to lytic phases [69]. These modifications are notably concentrated within the ORF regions, including ORF46, ORF48, ORF49, and ORF50. They play a crucial role in regulating viral gene expression, with the m⁶A site on ORF50 being especially important [70]. This site enhances the stability of the ORF50 transcript by recruiting Staphylococcal nuclease domain-containing protein 1 (SND1), which binds preferentially to m⁶A-tagged transcripts, particularly in their unspliced form. This interaction promotes early viral gene expression, influencing the viral replication cycle. Depletion of SND1 suppresses early KSHV gene expression, highlighting the significance of m⁶A modification in this context [71]. Further, investigations have shown that methylation induces a conformational shift in the RNA stem-loop structure within ORF50, transitioning it from a closed to a more open state at its apex. This methylation-induced structural change disrupts the energetically favored closed conformation, leading to altered stem-loop openness, which in turn affects transcript accessibility and stability [70]. Additionally, the m⁶A site on ORF50 exerts regulatory control at the posttranscriptional level by recruiting YTHDC1, which facilitates splicing of the precursor mRNA, thereby regulating the expression of KSHV lethal genes [72]. Notably, the reader protein YTHDF2 can also bind to m⁶A-modified viral transcripts, modulating their stability [73]. These findings underscore the intricate regulatory mechanisms mediated by m⁶A modifications in KSHV replication and gene expression.

The latent phase of KSHV infection is characterized by dynamic reprograming of the host cellular epitranscriptome, resulting in a reduction of m⁶A modifications in the 5′UTR and an enhancement in the 3′UTR. These differential m⁶A modifications play a crucial role in cellular transformation processes, epithelial–mesenchymal transitions (EMTs), and various oncogenic pathways. These pathways include signaling cascades triggered by ephrin receptors, integrin-linked kinase (ILK), hypoxic conditions, bone morphogenetic proteins (BMPs), hepatic fibrosis, mammalian target of rapamycin (mTOR) activation, and adherens junction remodeling, which are essential for cancer-related EMT [73]. The transition of KSHV from latency to lytic replication leads to a reshaping of the m⁶A landscape on both viral and host mRNAs. Notably, the expression of G protein-coupled receptor class C group five member A (GPRC5A) is critical for KSHV’s lytic replication, and m⁶A modifications are key in maintaining the stability of GPRC5A mRNA. This expression is induced by replication and transcription activator (RTA), a central regulator that coordinates the transition from latency to lytic replication in KSHV [74].

During KSHV infection, the ORF37-encoded SOX protein primarily degrades the majority of transcripts, although the interleukin-6 (IL-6) transcript has the capability to evade immune-mediated degradation. This evasion is facilitated by the m⁶A modification on the IL-6 transcript, preventing SOX-induced degradation by recruiting YTHDC2. Consequently, the protection and sustained expression of IL-6 are ensured [75]. METTL16 also plays a key role in KSHV lytic replication regulation, as demonstrated by the acceleration of KSHV lytic replication upon METTL16 knockdown, and its restraint upon overexpression. It has been observed that METTL16 influences the production of SAM by modulating the m⁶A modification of the transcript of a crucial enzyme in the SAM cycle, MAT2A. This modulation of the SAM cycle impacts KSHV lytic replication by regulating the balance of intracellular redox and levels of reactive oxygen species [76]. The m⁶A modifications yield diverse regulatory outcomes in specific cellular contexts, potentially enhancing or inhibiting KSHV gene expression. This variability stems from the specificity of m⁶A regulatory mechanisms across various cell types. The life cycle of KSHV is significantly influenced by the m⁶A pathway, with the functional consequences being dependent on the host cell type, highlighting the intricate interplay between the virus and host [77]. Furthermore, changes in m⁶A modification could potentially serve as biomarkers for diagnosing and monitoring KSHV infection. Further research is imperative to elucidate the precise mechanisms underlying m⁶A modification in KSHV infection, aiming to develop more effective prevention and control strategies.

However, the aforementioned studies still have many unresolved issues. the precise regulatory mechanism by which KSHV controls the activity and localization of the METTL3/METTL14 complex and demethylases (e.g., ALKBH5 and FTO) the reason why m⁶A modification of ORF50 promotes gene expression during early lytic stages but is actively cleared by viral nucleases (ORF37/SOX) in late stages—including whether this relates to the requirement for low-methylated RNA during viral packaging; the efficacy of existing m⁶A pathway inhibitors (e.g., 3-DAA) against KSHV; the potential of combining m⁶A regulators with immune checkpoint inhibitors (e.g., anti-PD-1) or oncolytic viruses; whether activating latent KSHV followed by m⁶A targeting could enhance the “activate and kill” strategy. These questions remain to be explored through further research.

3.4. EBV

EBV is a prototypical and common human tumor virus that can lead to the development of various lymphoid and epithelial cell carcinomas. The EBV genome is a linear double-stranded DNA genome approximately 170 to 180 kilobase pairs in length. It is structurally divided into UL and US regions, where most of the coding capacity for viral proteins is located. Flanking these regions are the IR domains situated at both ends of the genome, crucial for viral replication, and gene expression regulation. The EBV genome encodes around 80 to 90 ORFs [78]. However, the roles of the most EBV-encoded proteins remain under investigation, emphasizing the necessity for further research to comprehend their significance in viral biology and disease development.

During the lytic phase of EBV infection, the EBV protein BZLF1 interacts with the promoter of METTL3, leading to suppression of METTL3 expression, resulting in decreased m⁶A modification on KLF4 mRNA. Consequently, the reduced m⁶A modification on KLF4 transcripts evades degradation by YTHDF2, leading to upregulation of KLF4 protein expression, thus, promoting EBV infection in nasopharyngeal cells [79]. YTHDF1 contributes to inhibiting EBV replication through destabilizing the transcripts of BZLF1 and BRLF1 by recruiting RNA decay complexes ZAP, DDX17, and DCP2 [80]. Upon EBV infection, m⁶A modifications of cellular transcripts DTX4 and TYK2 are upregulated due to the downregulation of eraser protein ALKBH5 expression, resulting in diminished IFN production and enhanced viral replication [81]. YTHDFs proteins also suppress EBV replication via PIAS1-mediated SUMOylation. The cleavage of m⁶A pathway molecules by caspase leads to the stabilization of BZLF1 transcripts, therefore, facilitating EBV replication [82]. These findings underscore the pivotal role of m⁶A modification in regulating EBV gene expression.

In the latent phase of EBV infection, the m⁶A modifications that emerged as a consequence of EBV infection were found to be distributed preferentially in the 3′UTR of cellular transcripts. Conversely, the modifications that were lost as a result of EBV infection were distributed preferentially in the CDS region of mRNAs. Notably, viral genes EBNA2 and BHRF1 undergo m⁶A modification. Knockdown of METTL3 results in the reduction of EBNA2 expression [83]. Additionally, EBV infection significantly upregulates METTL14, which subsequently, mediates m⁶A modification and upregulates the expression of the viral latent antigen EBNA3C [84]. It has been demonstrated that EBNA1 degrades METTL3 via the K48-linked ubiquitin–proteasome pathway, resulting in the downregulation of m⁶A modifications on TRL9 mRNA. This downregulation, in turn, reduces TRL9 protein expression and facilitates immune evasion by the EBV [85].

METTL3 exerts a positive regulatory effect on pri-miR-BART3-3 p through its interaction with the microprocessor protein DGCR8 in an m⁶A-dependent manner, leading to a reduction in PLCG2 that promotes the proliferative capacity and tumor growth of NK/T cell lymphoma (NKTCL) cells in vitro and in vivo [86]. Furthermore, EBV-circRPMS1 has been shown to promote the progression of EBV-associated gastric cancer (EBVaGC) by recruiting Sam68 to the METTL3 promoter, inducing METTL3 expression [87]. Knockdown of METTL3 or inhibition of methylation using 3-DAA and UZH1a resulted in decreased viability of EBV-positive tumor cells [88]. Additionally, EBV-circRPMS1 can regulate WTAP by affecting the NF-κB signaling pathway, which in turn impacts the proliferation and migration of gastric cancer cells [89]. YTHDF3 recruits DDX5 to inhibit IFITM1 expression, enhancing EphA2-mediated EBV entry into embryonic stem cells (ECs) [90]. FTO plays a critical inhibitory role in EBVaGC metastasis and invasiveness through an m⁶A-FOS-IGF2BP1/2-dependent mechanism [91]. METTL3′s high expression in NKTCL indicates poor prognosis [86]. M⁶A levels are crucial for diagnosing gastric cancer, demonstrating greater sensitivity and specificity than traditional tumor markers carcinoembryonic antigen (CEA) and carbohydrate antigen199 (CA199). Combining m⁶A with these markers can enhance diagnostic accuracy, making m⁶A an effective biomarker for diagnosing and monitoring gastric cancer [92]. These findings underscore the significance of m⁶A modification in the pathogenesis of EBV-associated malignancies. The potential of targeting m⁶A-associated modifying enzymes to treat EBV-associated cancers warrants further investigation.

During the lytic phase and latent phase of EBV infection, m⁶A modification exhibits distinct regulatory mechanisms. Lytic phase: the virus hijacks the m⁶A system to promote replication and immune evasion; latent phase: m⁶A remodels the host epigenetic landscape to sustain oncogenesis.

However, the above studies still leave several unresolved issues. the precise mechanism by which EBV regulates host m⁶A modification enzymes (e.g., METTL3 and ALKBH5) to transition between lytic and latent phases; are there virus-encoded “readers” or “writers” directly involved in regulation? whether virus-encoded “readers” or “writers” directly participate in this regulation; the phase-specificity of dynamic m⁶A changes on viral and host mRNAs during the viral life cycle; the potential roles of viral lncRNAs/miRNAs (in addition to circRNAs) in competitively binding or recruiting m⁶A enzymes; whether EBV has evolutionarily hijacked the host m⁶A system to adapt to immune pressures across different host cells. By addressing these questions, we may gain a more comprehensive understanding of the interaction between EBV and the host m⁶A system, providing a theoretical foundation for developing novel antiviral strategies and cancer diagnostic tools.

4. Dynamic Regulation of the m⁶A Modification-Associated Epitranscriptome During Animal Herpesvirus Infection

4.1. PRV

PRV is classified as a member of the Herpesviridae family and the Alphaherpesvirinae subfamily. The mature PRV particle is composed of an envelope, a layer of tegument proteins, a nuclear capsid, and a double-stranded DNA genome approximately 150 kilobases in length. This genome is organized into a UL region and a US region, both of which are flanked by inverted repeat (IR) regions. Within its genome, PRV contains over 70 ORFs and is estimated to encode between 70 and 100 proteins, the functions of many of which remain unknown [9].

PRV-infected swine testicle (ST) cells displayed a time-dependent decrease in total m⁶A levels, accompanied by a significant reduction in the expression of METTL3, METTL14, and WTAP. Further investigations revealed that during PRV infection, the viral US3 serine/threonine protein kinases phosphorylated METTL3, METTL14, and WTAP. Surprisingly, the viral US3 protein was observed to inactivate the m⁶A MTC in a kinase-independent manner, leading to the dissociation of the complex from chromatin [93]. During PRV infection, YTHDF readers were found to localize to P-bodies, where fewer but larger puncta could be observed in PRV-infected cells, leading to an increased degradation rate of mRNA. The number of P-bodies was also shown to increase during infection. Notably, YTHDF knockdown suppressed PRV protein production and led to an upregulation of IFN expression, suggesting that PRV’s alteration of m⁶A modification serves as a viral immune evasion strategy [60]. Additionally, in a separate study, PRV-infected porcine kidney epithelial (PK15) cells exhibited abundant m⁶A modifications in viral transcripts. Knocking down the expression of writer proteins (METTL3 and METTL14) or reader proteins (YTHDF2 and YTHDF3) was found to inhibit PRV replication, whereas silencing the eraser protein ALKBH5 promoted replication [94].

However, the specific role and mechanisms of m⁶A modification during in vivo PRV infection remain unknown. Key unsolved questions include: whether PRV regulates viral gene silencing/reactivation via m⁶A modification when establishing latency in the peripheral nervous system; the functional significance of m⁶A during latent infection (current studies focus primarily on acute infection); the molecular mechanisms underlying US3-mediated inactivation of the METTL3/METTL14 complex, particularly its kinase-independent pathway; the role of m⁶A modification in PRV-infected pigs (in vivo validation is lacking as most studies use in vitro models); the therapeutic potential of targeting m⁶A-related enzymes (e.g., METTL3 inhibitors or ALKBH5 activators) against PRV. Resolving these questions will deepen our understanding of m⁶A modification in PRV infection and provide new targets for antiviral therapy.

4.2. MDV

MDV belongs to the family Herpesviridae and subfamily Alphaherpesvirinae based on the structure of its double-stranded DNA genome, which is about 180 kb in size [8]. The MDV genome exhibits a similar structure to that of PRV and HSV-1, encoding over 100 genes. Among these genes, those highly homologous and collinear with the genes of HSV-1 and Varicella-zoster virus (VZV) are located in the UL and US regions, while MDV-specific genes are found in the TR and IR regions [95]. There are three MDV serotypes: MDV-1 (Galid herpesvirus 2), MDV-2 (Galid herpesvirus 3), and MDV-3 (Meleagrid herpesvirus 1), with MDV-3 also known as the herpesvirus of Turkey (HVT). It is important to note that, while MDV-1 causes tumors in infected chickens, MDV-2 and HVT are nonpathogenic viruses. Currently, attenuated MDV-1, MDV-2, and HVT strains grown in cell culture can serve as vaccines against virulent virus infections; however, these vaccines are still insufficient in interfering with MDV replication. Hence, there is a need for new research to unravel the regulatory mechanisms underlying MDV pathogenesis and tumorigenesis comprehensively [96, 97].

During MDV replication in cell culture, we discovered a reprograming of m⁶A modifications on lncRNAs and circRNAs through the utilization of Methylated RNA immunoprecipitation sequencing (MeRIP-Seq) paired with bioinformatic analysis [98, 99]. Our findings revealed a close association between lncRNAs m⁶A modifications in MDV-infected chicken embryo fibroblasts and various signaling pathways including ErbB, GnRH, Toll-like receptor, Influenza A, and the MAPK pathway, all of which are linked to MDV infection [98]. Notably, during MDV infection, there was a noticeable reduction in the abundance of circRNAs m⁶A modifications. Subsequent analysis indicated a connection between the regulation of m⁶A modified circRNAs and the insulin signaling pathway [99]. In essence, the results imply that m⁶A modified lncRNAs and circRNAs exert significant regulatory roles in facilitating MDV replication in vitro.

In order to comprehensively understand the regulatory role of m⁶A modification during MDV infection [8], we initially leveraged the natural infectious disease model of MDV-infected chickens. Surprisingly, our investigation revealed that m⁶A modifications occurred on viral transcripts throughout the lytic infection, transformation, and reactivation phases. Subsequently, we delved into the alterations in the cellular m⁶A epitranscriptome across different stages of infection. Through MeRIP-Seq analysis, we identified dynamic changes in transcriptome m⁶A modifications over the MDV replication cycle in vivo, suggestive of MDV-driven reprograming of host transcriptional m⁶A modifications in an infection-cycle-dependent manner. Notably, our comparative analysis of cellular m⁶A modifications among the distinct infectious phases highlighted the extensive m⁶A modification of multiple immunity-associated transcripts. Prior research utilizing transcriptomic sequencing demonstrated that infection by highly virulent MDV leads to changes in the expression of immunity genes, potentially contributing to MDV immune evasion [100, 101]. However, further elucidation is required regarding the role and molecular mechanisms of m⁶A modification in regulating the expression of immunity-associated genes throughout the MDV life cycle. Moreover, our investigation into the expression of m⁶A modification-associated enzymes during MDV replication in chickens revealed altered expression profiles. Interestingly, the ectopic expression of the methyltransferase enzymes METTL3 or METTL14 was found to impact MDV replication, underscoring the pivotal regulatory role of m⁶A modifications in MDV replication and pathogenesis [11].

However, the aforementioned studies still have some unresolved issues. Why are METTL3/METTL14 suppressed during the lytic phase but highly expressed during the tumor phase? Is this differential expression mediated by specific viral proteins? How does m⁶A modification coordinate the promoting viral replication, while suppressing host immunity during the same infection stage? Is there selective regulation of virus RNA-specific methylation sites? During reactivation, are the dynamic changes in m⁶A modification associated with METTL3 nuclear translocation induced by host stress signals (such as the inflammatory factor IL-6)? Does MDV influence the activity of m⁶A modification enzymes by interfering with host metabolic pathways (such as itaconic acid or cholesterol synthesis)?

5. Conclusions and Future Perspectives

In this review, we summarize the latest research on m⁶A modifications during herpesvirus infections in humans (HSV-1, HCMV, KSHV, EBV) and animals (PRV, MDV), revealing its core role in virus–host interactions. Systematic analysis demonstrates that m⁶A functions as a “molecular switch” by dynamically reshaping the epitranscriptomic landscape of viral and host transcriptomes, thereby regulating viral replication cycles, immune evasion, and tumourigenesis.

Viruses hijack host m⁶A machinery to optimize gene expression: HSV-1 mediates the nuclear export of METTL3/14 via ICP27 to promote early gene expression [58]; KSHV utilizes m⁶A-SND1 interactions at ORF50 transcripts to enhance RNA stability, driving lytic reactivation [71]. m⁶A promotes immune evasion by: modulating viral gene immunogenicity (e.g., EBV BZLF1 inhibits METTL3 to evade TLR9 [85]); Reprograming immune signaling (e.g., TLR/MAPK remodeling in MDV infection [98]); suppressing antiviral pathways (e.g., HCMV downregulates IFNB1 via ALKBH5 [65]). m⁶A as a hub for carcinogenesis: latent-phase modifications drive oncogenesis by: stabilizing oncogene transcripts (EBV EBNA3C/EBNA2 [83, 84]); KSHV GPRC5A [74]); regulating noncoding RNAs (e.g., EBV-circRPMS1 [87]), forming “epitranscriptomic memory” to maintain malignancy.

It is worth noting that m⁶A regulation exhibits significant viral specificity and spatiotemporal dynamics: during the lytic phase, it promotes infection spread by enhancing the translation efficiency of viral transcripts or inhibiting host antiviral genes (e.g., HCMV IFNB1 [65]); whereas during the latent phase, m⁶A reprograming drives cellular transformation by stabilizing oncogene transcripts (e.g., EBV EBNA2 [84], KSHV GPRC5A [74]). In animal herpesvirus, the US3 protein of PRV inactivates the host MTC through a dual mechanism involving phosphorylation and kinase-independent mechanisms [93], while the dynamic changes in m⁶A modification during the MDV infection cycle are closely associated with host immune signaling pathways, such as TLR and MAPK [98].

However, existing research still has key limitations:

1. Current understanding of the mechanisms underlying m⁶A modification in herpesvirus infection primarily relies on in vitro models, with a lack of in vivo spatiotemporal resolution data on m⁶A dynamics during latent infection (e.g., HSV-1 in the trigeminal ganglion) and reactivation processes;

2. How virus-specific regulatory elements (e.g., EBV-circRPMS1, MDV lncRNA) modulate host modification networks through competitive binding or epigenetic memory remains unclear;

3. The post-translational modification of m⁶A enzyme activity (e.g., US3-mediated METTL3 phosphorylation) and its coupling mechanism with metabolic reprograming (succinic acid/SAM cycle) have not yet been elucidated.

To deepen our understanding of the role of m⁶A in herpesvirus infection, future research should focus on the following directions:

1. Exploration of virus–host interaction mechanisms: Identify how virus-encoded “hijacking factors” (e.g., EBV BZLF1, PRV US3) regulate the subcellular localization and activity of host m⁶A enzymes (METTL3, ALKBH5) through posttranslational modifications (phosphorylation/ubiquitination); elucidate how viral noncoding RNAs (e.g., KSHV miRNA, EBV lncRNA) modulate host gene exression via m⁶A modification/host pathways by competitive binding to m⁶A reading proteins (YTHDF2) or recruitment of modifying enzymes (e.g., METTL3).

2. High-precision modification profiling: Utilizing spatial transcriptomics combined with m⁶A-CLIP technology to elucidate the synergistic changes in three-dimensional genomic structure and modification sites during the viral “lysis-latent” transition; develop single-base resolution technologies (such as nanopore direct RNA sequencing or APOBEC-Cas13b-mediated site editing) to map the dynamic modification profiles of latent viral transcripts (e.g., HSV-1 LAT) and host non-coding RNAs (e.g., MDV circRNA).

3. Therapeutic strategies: Evaluate the synergistic efficacy of m⁶A inhibitors (3-DAA) combined with immune checkpoint inhibitors (anti-PD-1) or oncolytic viruses (e.g., HSV-1-derived T-VEC), and validate this synergy through humanized animal models (e.g., EBV-infected organoids).

4. Establish humanized animal models to simulate natural infection.

Studies on the regulatory mechanisms of m⁶A modification in herpesvirus infection not only reveal the fundamental principles of virus–host interactions but also provide new insights for vaccine and antiviral drug development. m⁶A modification plays a key role in viral replication cycles, immune evasion, and tumorigenesis by dynamically regulating the epigenetic landscape of the viral and host transcriptomes. Regulating m⁶A modification through inhibitors or agonists targeting enzymes involved in this process (such as methyltransferases and demethylases) offers a potential strategy for modulating viral replication. For example, 3-DAA is an inhibitor of SAH hydrolase [12] that blocks m⁶A modification of mRNA and exhibits broad-spectrum inhibitory effects against both DNA and RNA viruses. Its potential as an antiviral agent against animal viruses warrants further investigation. Additionally, the ability of m⁶A modification to influence mRNA expression provides a promising avenue for vaccine design targeting virus-specific mRNA sequences.

Disclosure

All authors contributed to the final article revision and approved the submitted version.

Author Contributions

Aijun Sun and Guoqing Zhuang contributed to conception, project administration, manuscript revision, and funding support. Xiangqi Qiu and Jiajing Tian wrote and revised the original manuscript. Xuyang Zhao prepared the figure. Lucai Wang, Lele Wang, and Yilin Bai contributed to language polishing. Xiangqi Qiu and Jiajing Tian contributed equally to this work.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (U21A20260), the Key Scientific Research Program of 2025 for Colleges and Universities in Henan Province (25A230004), the Major Scientific and Technological Project of Henan Province (221100110600), the Natural Science Foundation of Henan (232300421160), and the Starting Foundation for Outstanding Young Scientists of Henan Agricultural University (30500690).

References

[1] S. A. Connolly, T. S. Jardetzky, R. Longnecker, "The Structural Basis of Herpesvirus Entry," Nature Reviews. Microbiology, vol. 19 no. 2, pp. 110-121, DOI: 10.1038/s41579-020-00448-w, 2021.

[2] M. J. Bradshaw, A. Venkatesan, "Herpes Simplex Virus-1 Encephalitis in Adults: Pathophysiology, Diagnosis, and Management," Neurotherapeutics, vol. 13 no. 3, pp. 493-508, DOI: 10.1007/s13311-016-0433-7, 2016.

[3] M. Zuhair, G. S. A. Smit, G. Wallis, "Estimation of the Worldwide Seroprevalence of Cytomegalovirus: A Systematic Review and Meta-Analysis," Reviews in Medical Virology, vol. 29 no. 3,DOI: 10.1002/rmv.2034, 2019.

[4] B. Herndier, D. Ganem, "The Biology of Kaposi’s Sarcoma," Cancer Treatment and Research, vol. 104, pp. 89-126, DOI: 10.1007/978-1-4615-1601-9_4, 2001.

[5] P. Mrozek-Gorska, A. Buschle, D. Pich, "Epstein-Barr Virus Reprograms Human B Lymphocytes Immediately in the Prelatent Phase of Infection," Proceedings of the National Academy of Sciences, vol. 116 no. 32, pp. 16046-16055, 2019.

[6] R. Guo, B. E. Gewurz, "Epigenetic Control of the Epstein-Barr Lifecycle," Current Opinion in Virology, vol. 52, pp. 78-88, DOI: 10.1016/j.coviro.2021.11.013, 2022.

[7] Y. Chen, W. Wang, W. Zhang, "Emerging Roles of Biological m 6 A Proteins in Regulating Virus Infection: A Review," International Journal of Biological Macromolecules, vol. 253 no. Pt 3,DOI: 10.1016/j.ijbiomac.2023.126934, 2023.

[8] N. Osterrieder, J. P. Kamil, D. Schumacher, B. K. Tischer, S. Trapp, "Marek’s Disease Virus: From Miasma to Model," Nature Reviews. Microbiology, vol. 4 no. 4, pp. 283-294, DOI: 10.1038/nrmicro1382, 2006.

[9] L. E. Pomeranz, A. E. Reynolds, C. J. Hengartner, "Molecular Biology of Pseudorabies Virus: Impact on Neurovirology and Veterinary Medicine," Microbiology and Molecular Biology Reviews: MMBR, vol. 69 no. 3, pp. 462-500, DOI: 10.1128/MMBR.69.3.462-500.2005, 2005.

[10] Q. Liu, Y. Kuang, Y. Li, "The Epidemiology and Variation in Pseudorabies Virus: A Continuing Challenge to Pigs and Humans," Viruses, vol. 14 no. 7,DOI: 10.3390/v14071463, 2022.

[11] G. Zhuang, X. Zhao, J. Jin, "Infection Phase-Dependent Dynamics of the Viral and Host N6-Methyladenosine Epitranscriptome in the Lifecycle of an Oncogenic Virus in Vivo," Journal of Medical Virology, vol. 95 no. 1,DOI: 10.1002/jmv.28324, 2023.

[12] K. Tsai, B. R. Cullen, "Epigenetic and Epitranscriptomic Regulation of Viral Replication," Nature Reviews Microbiology, vol. 18 no. 10, pp. 559-570, DOI: 10.1038/s41579-020-0382-3, 2020.

[13] B. S. Zhao, I. A. Roundtree, C. He, "Post-Transcriptional Gene Regulation by mRNA Modifications," Nature Reviews Molecular Cell Biology, vol. 18 no. 1, pp. 31-42, DOI: 10.1038/nrm.2016.132, 2017.

[14] T. Pan, "Modifications and Functional Genomics of Human Transfer RNA," Cell Research, vol. 28 no. 4, pp. 395-404, DOI: 10.1038/s41422-018-0013-y, 2018.

[15] K. E. Sloan, A. S. Warda, S. Sharma, K.-D. Entian, D. L. J. Lafontaine, M. T. Bohnsack, "Tuning the Ribosome: The Influence of rRNA Modification on Eukaryotic Ribosome Biogenesis and Function," RNA Biology, vol. 14 no. 9, pp. 1138-1152, DOI: 10.1080/15476286.2016.1259781, 2017.

[16] E. Sendinc, Y. Shi, "RNA m6A Methylation Across the Transcriptome," Molecular Cell, vol. 83 no. 3, pp. 428-441, DOI: 10.1016/j.molcel.2023.01.006, 2023.

[17] S. Oerum, C. Dégut, P. Barraud, C. Tisné, "m1A Post-Transcriptional Modification in tRNAs," Biomolecules, vol. 7 no. 1,DOI: 10.3390/biom7010020, 2017.

[18] Y.-S. Chen, W.-L. Yang, Y.-L. Zhao, Y.-G. Yang, "Dynamic Transcriptomic m 5 C and Its Regulatory Role in RNA Processing," WIREs RNA, vol. 12 no. 4,DOI: 10.1002/wrna.1639, 2021.

[19] H. Shi, J. Wei, C. He, "Where, When, and How: Context-Dependent Functions of RNA Methylation Writers, Readers, and Erasers," Molecular Cell, vol. 74 no. 4, pp. 640-650, DOI: 10.1016/j.molcel.2019.04.025, 2019.

[20] K. D. Meyer, Y. Saletore, P. Zumbo, O. Elemento, C. E. Mason, S. R. Jaffrey, "Comprehensive Analysis of mRNA Methylation Reveals Enrichment in 3′ UTRs and Near Stop Codons," Cell, vol. 149 no. 7, pp. 1635-1646, DOI: 10.1016/j.cell.2012.05.003, 2012.

[21] D. Dominissini, S. Moshitch-Moshkovitz, S. Schwartz, "Topology of the Human and Mouse m6A RNA Methylomes Revealed by m6A-Seq," Nature, vol. 485 no. 7397, pp. 201-206, DOI: 10.1038/nature11112, 2012.

[22] X. Wang, B. S. Zhao, I. A. Roundtree, "N6-Methyladenosine Modulates Messenger RNA Translation Efficiency," Cell, vol. 161 no. 6, pp. 1388-1399, DOI: 10.1016/j.cell.2015.05.014, 2015.

[23] J.-M. Fustin, M. Doi, Y. Yamaguchi, "RNA-Methylation-Dependent RNA Processing Controls the Speed of the Circadian Clock," Cell, vol. 155 no. 4, pp. 793-806, DOI: 10.1016/j.cell.2013.10.026, 2013.

[24] Y. Xiang, B. Laurent, C.-H. Hsu, "RNA m6A Methylation Regulates the Ultraviolet-Induced DNA Damage Response," Nature, vol. 543 no. 7646, pp. 573-576, DOI: 10.1038/nature21671, 2017.

[25] T. Sun, R. Wu, L. Ming, "The Role of m6A RNA Methylation in Cancer," Biomedicine & Pharmacotherapy, vol. 112,DOI: 10.1016/j.biopha.2019.108613, 2019.

[26] K. D. Meyer, S. R. Jaffrey, "Rethinking m 6 A Readers, Writers, and Erasers," Annual Review of Cell and Developmental Biology, vol. 33 no. 1, pp. 319-342, DOI: 10.1146/annurev-cellbio-100616-060758, 2017.

[27] B. Tan, S.-J. Gao, "RNA Epitranscriptomics: Regulation of Infection of RNA and DNA Viruses by N 6 -Methyladenosine (m 6 A)," Reviews in Medical Virology, vol. 28 no. 4,DOI: 10.1002/rmv.1983, 2018.

[28] J. Liu, Y. Yue, D. Han, "A METTL3-METTL14 Complex Mediates Mammalian Nuclear RNA N6-Adenosine Methylation," Nature Chemical Biology, vol. 10 no. 2, pp. 93-95, DOI: 10.1038/nchembio.1432, 2014.

[29] A. S. Warda, J. Kretschmer, P. Hackert, "Human METTL16 is a N 6 -Methyladenosine (m 6 A) Methyltransferase That Targets Pre-mRNAs and Various Non-Coding RNAs," EMBO Reports, vol. 18 no. 11, pp. 2004-2014, DOI: 10.15252/embr.201744940, 2017.

[30] X. L. Ping, B. F. Sun, L. Wang, "Mammalian WTAP Is a Regulatory Subunit of the RNA N6-Methyladenosine Methyltransferase," Cell Research, vol. 24 no. 2, pp. 177-189, 2014.

[31] S. Schwartz, M. R. Mumbach, M. Jovanovic, "Perturbation of m6A Writers Reveals Two Distinct Classes of mRNA Methylation at Internal and 5′ Sites," Cell Reports, vol. 8 no. 1, pp. 284-296, DOI: 10.1016/j.celrep.2014.05.048, 2014.

[32] D. P. Patil, C.-K. Chen, B. F. Pickering, "m6A RNA Methylation Promotes XIST-Mediated Transcriptional Repression," Nature, vol. 537 no. 7620, pp. 369-373, DOI: 10.1038/nature19342, 2016.

[33] J. Wen, R. Lv, H. Ma, "Zc3h13 Regulates Nuclear RNA m6A Methylation and Mouse Embryonic Stem Cell Self-Renewal," Molecular Cell, vol. 69 no. 6, pp. 1028-1038.e6, DOI: 10.1016/j.molcel.2018.02.015, 2018.

[34] K. E. Pendleton, B. Chen, K. Liu, "The U6 snRNA m6A Methyltransferase METTL16 Regulates SAM Synthetase Intron Retention," Cell, vol. 169 no. 5, pp. 824-835.e14, DOI: 10.1016/j.cell.2017.05.003, 2017.

[35] G. Jia, Y. Fu, X. Zhao, "N6-Methyladenosine in Nuclear RNA Is a Major Substrate of the Obesity-Associated FTO," Nature Chemical Biology, vol. 7 no. 12, pp. 885-887, DOI: 10.1038/nchembio.687, 2011.

[36] G. Q. Zheng, J. A. Dahl, Y. M. Niu, "ALKBH5 Is a Mammalian RNA Demethylase That Impacts RNA Metabolism and Mouse Fertility," Molecular Cell, vol. 49 no. 1, pp. 18-29, 2013.

[37] G. Jia, C.-G. Yang, S. Yang, "Oxidative Demethylation of 3-Methylthymine and 3-Methyluracil in Single-Stranded DNA and RNA by Mouse and Human FTO," FEBS Letters, vol. 582 no. 23-24, pp. 3313-3319, DOI: 10.1016/j.febslet.2008.08.019, 2008.

[38] M. Bartosovic, H. C. Molares, P. Gregorova, D. Hrossova, G. Kudla, S. Vanacova, "N6-Methyladenosine Demethylase FTO Targets Pre-mRNAs and Regulates Alternative Splicing and 3′-End Processing," Nucleic Acids Research, vol. 45 no. 19, pp. 11356-11370, DOI: 10.1093/nar/gkx778, 2017.

[39] J. Mauer, X. Luo, A. Blanjoie, "Reversible Methylation of m6Am in the 5′ Cap Controls mRNA Stability," Nature, vol. 541 no. 7637, pp. 371-375, DOI: 10.1038/nature21022, 2017.

[40] C. Tang, R. Klukovich, H. Peng, "ALKBH5-Dependent m6A Demethylation Controls Splicing and Stability of Long 3′-UTR mRNAs in Male Germ Cells," Proceedings of the National Academy of Sciences, vol. 115 no. 2, pp. e325-e33, DOI: 10.1073/pnas.1717794115, 2018.

[41] H. Du, Y. Zhao, J. He, "YTHDF2 Destabilizes m6A-Containing RNA Through Direct Recruitment of the CCR4-NOT Deadenylase Complex," Nature Communications, vol. 7 no. 1,DOI: 10.1038/ncomms12626, 2016.

[42] H. Shi, X. Wang, Z. Lu, "YTHDF3 Facilitates Translation and Decay of N6-Methyladenosine-Modified RNA," Cell Research, vol. 27 no. 3, pp. 315-328, DOI: 10.1038/cr.2017.15, 2017.

[43] I. A. Roundtree, G. Z. Luo, Z. Zhang, "YTHDC1 Mediates Nuclear Export of N6-Methyladenosine Methylated mRNAs," Elife, vol. 6, 2017.

[44] I. A. Roundtree, C. He, "Nuclear m6A Reader YTHDC1 Regulates mRNA Splicing," Trends in Genetics, vol. 32 no. 6, pp. 320-321, DOI: 10.1016/j.tig.2016.03.006, 2016.

[45] P. J. Hsu, Y. Zhu, H. Ma, "Ythdc2 Is an N6-Methyladenosine Binding Protein That Regulates Mammalian Spermatogenesis," Cell Research, vol. 27 no. 9, pp. 1115-1127, DOI: 10.1038/cr.2017.99, 2017.

[46] H. Huang, H. Weng, W. Sun, "Recognition of RNA N(6)-Methyladenosine by IGF2BP Proteins Enhances mRNA Stability and Translation," Nature Cell Biology, vol. 20 no. 3, pp. 285-295, 2018.

[47] K. D. Meyer, D. P. Patil, J. Zhou, "5′ UTR m6A Promotes Cap-Independent Translation," Cell, vol. 163 no. 4, pp. 999-1010, DOI: 10.1016/j.cell.2015.10.012, 2015.

[48] C. R. Alarcón, H. Goodarzi, H. Lee, X. Liu, S. Tavazoie, S. F. Tavazoie, "HNRNPA2B1 Is a Mediator of m6A-Dependent Nuclear RNA Processing Events," Cell, vol. 162 no. 6, pp. 1299-1308, DOI: 10.1016/j.cell.2015.08.011, 2015.

[49] P. C. He, J. Wei, X. Dou, "Exon Architecture Controls mRNA m 6 A Suppression and Gene Expression," Science, vol. 379 no. 6633, pp. 677-682, DOI: 10.1126/science.abj9090, 2023.

[50] T. Tange, T. Shibuya, M. Jurica, M. Moore, "Biochemical Analysis of the EJC Reveals Two New Factors and a Stable Tetrameric Protein Core," RNA, vol. 11 no. 12, pp. 1869-1883, DOI: 10.1261/rna.2155905, 2005.

[51] A. Uzonyi, D. Dierks, R. Nir, "Exclusion of m6A From Splice-Site Proximal Regions by the Exon Junction Complex Dictates m6A Topologies and mRNA Stability," Molecular Cell, vol. 83 no. 2, pp. 237-251.e7, DOI: 10.1016/j.molcel.2022.12.026, 2023.

[52] P. C. He, C. He, "MRNA Accessibility Within mRNPs as a Determinant of Gene Expression," Trends in Biochemical Sciences, vol. 49 no. 3, pp. 199-207, DOI: 10.1016/j.tibs.2023.11.003, 2024.

[53] Z. Luo, Q. Ma, S. Sun, "Exon-Intron Boundary Inhibits m6A Deposition, Enabling m6A Distribution Hallmark, Longer mRNA Half-Life and Flexible Protein Coding," Nature Communications, vol. 14 no. 1,DOI: 10.1038/s41467-023-39897-1, 2023.

[54] X. Yang, R. Triboulet, Q. Liu, E. Sendinc, R. I. Gregory, "Exon Junction Complex Shapes the m6A Epitranscriptome," Nature Communications, vol. 13 no. 1,DOI: 10.1038/s41467-022-35643-1, 2022.

[55] M. L. Szpara, D. Gatherer, A. Ochoa, "Evolution and Diversity in Human Herpes Simplex Virus Genomes," Journal of Virology, vol. 88 no. 2, pp. 1209-1227, DOI: 10.1128/JVI.01987-13, 2014.

[56] B. Moss, A. Gershowitz, J. R. Stringer, L. E. Holland, E. K. Wagner, "5′-Terminal and Internal Methylated Nucleosides in Herpes Simplex Virus Type 1 mRNA," Journal of Virology, vol. 23 no. 2, pp. 234-239, DOI: 10.1128/jvi.23.2.234-239.1977, 1977.

[57] Z. Feng, F. Zhou, M. Tan, "Targeting m6A Modification Inhibits Herpes Virus 1 Infection," Genes & Diseases, vol. 9 no. 4, pp. 1114-1128, DOI: 10.1016/j.gendis.2021.02.004, 2022.

[58] K. P. Srinivas, D. P. Depledge, J. S. Abebe, S. A. Rice, I. Mohr, A. C. Wilson, "Widespread Remodeling of the m6A RNA-Modification Landscape by a Viral Regulator of RNA Processing and Export," Proceedings of the National Academy of Sciences, vol. 118 no. 30, 2021.

[59] J. Xu, Y. Qi, Q. Ju, "Promotion of the Resistance of Human Oral Epithelial Cells to Herpes Simplex Virus Type I Infection via N6-Methyladenosine Modification," BMC Oral Health, vol. 23 no. 1,DOI: 10.1186/s12903-023-02744-2, 2023.

[60] R. J. J. Jansens, A. Olarerin-George, R. Verhamme, A. Mirza, S. Jaffrey, H. W. Favoreel, "Alphaherpesvirus-Mediated Remodeling of the Cellular Transcriptome Results in Depletion of m6A-Containing Transcripts," iScience, vol. 26 no. 8,DOI: 10.1016/j.isci.2023.107310, 2023.

[61] W. Wang, W. Ye, S. Chen, "METTL3-Mediated m 6 A RNA Modification Promotes Corneal Neovascularization by Upregulating the Canonical Wnt Pathway during HSV-1 Infection," Cellular Signalling, vol. 109,DOI: 10.1016/j.cellsig.2023.110784, 2023.

[62] Y. Li, X. He, X. Lu, "METTL3 Acetylation Impedes Cancer Metastasis via Fine-Tuning Its Nuclear and Cytosolic Functions," Nature Communications, vol. 13 no. 1,DOI: 10.1038/s41467-022-34209-5, 2022.

[63] Y. Cohen, N. Stern-Ginossar, "Manipulation of Host Pathways by Human Cytomegalovirus: Insights From Genome-Wide Studies," Seminars in Immunopathology, vol. 36 no. 6, pp. 651-658, DOI: 10.1007/s00281-014-0443-7, 2014.

[64] S. Lee, H. Kim, A. Hong, "Functional and Molecular Dissection of HCMV Long Non-Coding RNAs," Scientific Reports, vol. 12 no. 1,DOI: 10.1038/s41598-022-23317-3, 2022.

[65] R. M. Rubio, D. P. Depledge, C. Bianco, L. Thompson, I. Mohr, "RNA m 6 A Modification Enzymes Shape Innate Responses to DNA by Regulating Interferon β," Genes & Development, vol. 32 no. 23-24, pp. 1472-1484, DOI: 10.1101/gad.319475.118, 2018.

[66] W. Zhu, W. Zhu, S. Wang, S. Liu, H. Zhang, "UCHL1 Deficiency Upon HCMV Infection Induces Vascular Endothelial Inflammatory Injury Mediated by Mitochondrial Iron Overload," Free Radical Biology and Medicine, vol. 211, pp. 96-113, DOI: 10.1016/j.freeradbiomed.2023.12.002, 2024.

[67] W. Zhu, H. Zhang, S. Wang, "Vitamin D3 Suppresses Human Cytomegalovirus-Induced Vascular Endothelial Apoptosis via Rectification of Paradoxical m6A Modification of Mitochondrial Calcium Uniporter mRNA, Which Is Regulated by METTL3 and YTHDF3," Frontiers in Microbiology, vol. 13,DOI: 10.3389/fmicb.2022.861734, 2022.

[68] M. M. Gaglia, "Kaposi’s Sarcoma-Associated Herpesvirus at 27," Tumour Virus Research, vol. 12,DOI: 10.1016/j.tvr.2021.200223, 2021.

[69] Y. Tang, K. Chen, B. Song, "m6A-Atlas: A Comprehensive Knowledgebase for Unraveling the N6-Methyladenosine (m6A) Epitranscriptome," Nucleic Acids Research, vol. 49 no. D1, pp. D134-D143, DOI: 10.1093/nar/gkaa692, 2021.

[70] K. Röder, A. M. Barker, A. Whitehouse, S. Pasquali, S.-J. Chen, "Investigating the Structural Changes due to Adenosine Methylation of the Kaposi’s Sarcoma-Associated Herpes Virus ORF50 Transcript," PLOS Computational Biology, vol. 18 no. 5,DOI: 10.1371/journal.pcbi.1010150, 2022.

[71] B. Baquero-Perez, A. Antanaviciute, I. D. Yonchev, I. M. Carr, S. A. Wilson, A. Whitehouse, "The Tudor SND1 Protein Is an m6A RNA Reader Essential for Replication of Kaposi’s Sarcoma-Associated Herpesvirus," Elife, vol. 8, 2019.

[72] F. Ye, E. R. Chen, T. W. Nilsen, "Kaposi’s Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N 6 -Adenosine Methylation To Promote Lytic Replication," Journal of Virology, vol. 91 no. 16,DOI: 10.1128/JVI.00466-17, 2017.

[73] B. Tan, H. Liu, S. Zhang, "Viral and Cellular N6-Methyladenosine and N6,2′-O-Dimethyladenosine Epitranscriptomes in the KSHV Life Cycle," Nature Microbiology, vol. 3 no. 1, pp. 108-120, 2018.

[74] O. Manners, B. Baquero-Perez, T. J. Mottram, "m 6 A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication," Viruses, vol. 15 no. 6,DOI: 10.3390/v15061381, 2023.

[75] D. Macveigh-Fierro, A. Cicerchia, A. Cadorette, V. Sharma, M. Muller, "The m6A Reader YTHDC2 Is Essential for Escape From KSHV SOX-Induced RNA Decay," Proceedings of the National Academy of Sciences, vol. 119 no. 8, 2022.

[76] X. Zhang, W. Meng, J. Feng, "METTL16 Controls Kaposi’s Sarcoma-Associated Herpesvirus Replication by Regulating S-Adenosylmethionine Cycle," Cell Death & Disease, vol. 14 no. 9,DOI: 10.1038/s41419-023-06121-3, 2023.

[77] C. R. Hesser, J. Karijolich, D. Dominissini, C. He, B. A. Glaunsinger, D. P. Dittmer, "N6-Methyladenosine Modification and the YTHDF2 Reader Protein Play Cell Type Specific Roles in Lytic Viral Gene Expression During Kaposi’s Sarcoma-Associated Herpesvirus Infection," PLOS Pathogens, vol. 14 no. 4,DOI: 10.1371/journal.ppat.1006995, 2018.

[78] L. S. Young, A. B. Rickinson, "Epstein-Barr Virus: 40 Years on," Nature Reviews Cancer, vol. 4 no. 10, pp. 757-768, DOI: 10.1038/nrc1452, 2004.

[79] D.-L. Dai, X. Li, L. Wang, "Identification of an N6-Methyladenosine-Mediated Positive Feedback Loop that Promotes Epstein-Barr Virus Infection," Journal of Biological Chemistry, vol. 296,DOI: 10.1016/j.jbc.2021.100547, 2021.

[80] T. L. Xia, X. Li, X. Wang, "N6-Methyladenosine-Binding Protein YTHDF1 Suppresses EBV Replication and Promotes EBV RNA Decay," EMBO Reports, vol. 22 no. 4, 2021.

[81] D. Bose, X. Lin, L. Gao, Z. Wei, Y. Pei, E. S. Robertson, "Attenuation of IFN Signaling due to m6A Modification of the Host Epitranscriptome Promotes EBV Lytic Reactivation," Journal of Biomedical Science, vol. 30 no. 1,DOI: 10.1186/s12929-023-00911-9, 2023.

[82] K. Zhang, Y. Zhang, Y. Maharjan, "Caspases Switch off the m 6 A RNA Modification Pathway to Foster the Replication of a Ubiquitous Human Tumor Virus," mBio, vol. 12 no. 4,DOI: 10.1128/mBio.01706-21, 2021.

[83] X. Zheng, J. Wang, X. Zhang, "RNA m 6 A Methylation Regulates Virus-Host Interaction and EBNA2 Expression during Epstein-Barr Virus Infection," Immunity, Inflammation and Disease, vol. 9 no. 2, pp. 351-362, DOI: 10.1002/iid3.396, 2021.

[84] F. Lang, R. K. Singh, Y. Pei, S. Zhang, K. Sun, E. S. Robertson, "EBV Epitranscriptome Reprogramming by METTL14 Is Critical for Viral-Associated Tumorigenesis," PLoS Pathogens, vol. 15 no. 6,DOI: 10.1371/journal.ppat.1007796, 2019.

[85] X. Zhang, Z. Li, Q. Peng, "Epstein-Barr Virus Suppresses N6-Methyladenosine Modification of TLR9 to Promote Immune Evasion," Journal of Biological Chemistry, vol. 300 no. 5,DOI: 10.1016/j.jbc.2024.107226, 2024.

[86] S. Wu, H. Wang, Q. Yang, "METTL3 Regulates M6A Methylation-Modified EBV-Pri-MiR-BART3-3p to Promote NK/T Cell Lymphoma Growth," Cancer Letters, vol. 597,DOI: 10.1016/j.canlet.2024.217058, 2024.

[87] J.-Y. Zhang, Y. Du, L.-P. Gong, "Ebv-circRPMS1 Promotes the Progression of EBV-Associated Gastric Carcinoma via Sam68-Dependent Activation of METTL3," Cancer Letters, vol. 535,DOI: 10.1016/j.canlet.2022.215646, 2022.

[88] Y. Yanagi, T. Watanabe, Y. Hara, Y. Sato, H. Kimura, T. Murata, "EBV Exploits RNA m6A Modification to Promote Cell Survival and Progeny Virus Production During Lytic Cycle," Frontiers in Microbiology, vol. 13,DOI: 10.3389/fmicb.2022.870816, 2022.

[89] H. Xiao, Y. Zhang, L. Sun, Z. Zhao, W. Liu, B. Luo, "EBV Downregulates the m6A “Writer” WTAP in EBV-Associated Gastric Carcinoma," Virus Research, vol. 304,DOI: 10.1016/j.virusres.2021.198510, 2021.

[90] Y. Yang, T. Ding, Y. Cong, "Interferon-Induced Transmembrane Protein-1 Competitively Blocks Ephrin Receptor A2-Mediated Epstein-Barr Virus Entry Into Epithelial Cells," Nature Microbiology, vol. 9 no. 5, pp. 1256-1270, DOI: 10.1038/s41564-024-01659-0, 2024.

[91] Y.-Y. Xu, T. Li, A. Shen, "FTO up-Regulation Induced by MYC Suppresses Tumour Progression in Epstein-Barr Virus-Associated Gastric Cancer," Clinical and Translational Medicine, vol. 13 no. 12,DOI: 10.1002/ctm2.1505, 2023.

[92] L. Ge, N. Zhang, Z. Chen, "Level of N6-Methyladenosine in Peripheral Blood RNA: A Novel Predictive Biomarker for Gastric Cancer," Clinical Chemistry, vol. 66 no. 2, pp. 342-351, DOI: 10.1093/clinchem/hvz004, 2020.

[93] R. J. J. Jansens, R. Verhamme, A. H. Mirza, "Alphaherpesvirus US3 Protein-Mediated Inhibition of the m6A mRNA Methyltransferase Complex," Cell Reports, vol. 40 no. 3,DOI: 10.1016/j.celrep.2022.111107, 2022.

[94] P. L. Yu, R. Wu, S. J. Cao, "Pseudorabies Virus Exploits N6-Methyladenosine Modification to Promote Viral Replication," Frontiers in Microbiology, vol. 14, 2023.

[95] E. R. Tulman, C. L. Afonso, Z. Lu, L. Zsak, D. L. Rock, G. F. Kutish, "The Genome of a Very Virulent Marek’s Disease Virus," Journal of Virology, vol. 74 no. 17, pp. 7980-7988, DOI: 10.1128/JVI.74.17.7980-7988.2000, 2000.

[96] S. M. Reddy, Y. Izumiya, B. Lupiani, "Marek’s Disease Vaccines: Current Status, and Strategies for Improvement and Development of Vector Vaccines," Veterinary Microbiology, vol. 206, pp. 113-120, DOI: 10.1016/j.vetmic.2016.11.024, 2017.

[97] L. D. Bertzbach, A. M. Conradie, Y. You, B. B. Kaufer, "Latest Insights Into Marek’s Disease Virus Pathogenesis and Tumorigenesis," Cancers, vol. 12 no. 3,DOI: 10.3390/cancers12030647, 2020.

[98] A. Sun, X. Zhu, Y. Liu, "Transcriptome-Wide N6-Methyladenosine Modification Profiling of Long Non-Coding RNAs During Replication of Marek’s Disease Virus in Vitro," BMC Genomics, vol. 22 no. 1,DOI: 10.1186/s12864-021-07619-w, 2021.

[99] A. Sun, R. Wang, S. Yang, "Comprehensive Profiling Analysis of the N6-Methyladenosine-Modified Circular RNA Transcriptome in Cultured Cells Infected With Marek’s Disease Virus," Scientific Reports, vol. 11 no. 1,DOI: 10.1038/s41598-021-90548-1, 2021.

[100] A. J. Sarson, M. F. Abdul-Careem, H. Zhou, S. Sharif, "Transcriptional Analysis of Host Responses to Marek’s Disease Viral Infection," Viral Immunology, vol. 19 no. 4, pp. 747-758, DOI: 10.1089/vim.2006.19.747, 2006.

[101] Z.-J. Zhu, M. Teng, Y. Liu, "Immune Escape of Avian Oncogenic Marek’s Disease Herpesvirus and Antagonistic Host Immune Responses," NPJ Vaccines, vol. 9 no. 1,DOI: 10.1038/s41541-024-00905-0, 2024.