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Withdrawn: Association of N6-methyladenosine with viruses and virally induced diseases
Like histone and DNA, RNA is subject to a number of covalent modifications that can impact its function (1). Post-transcriptional modification of RNA is very common in Eukaryotes, and more than one hundred species are known at present (2). Most of these modifications are found on ribosomal RNA (rRNA) and transfer RNA (tRNA), which modulate RNA structures, functions, translation, as these RNAs are accessory molecules in eukaryotic processes (2, 3). Messenger RNA (mRNA), which is primarily an information-bearing molecule, is also post-transcriptionally modified (2). As early in 1970s, researchers discovered that the presence of N6-methyladenosine (m6A) on cellular mRNA (4). And m6A modification on mRNA was the most common, with 25% of all cellular mRNAs containing generally multiple m6A residues (5, 6). Furthermore, m6A modification mostly occurred at RRACH motif (R=A or G, H=A, C, or U) and m6A sites significantly clustered around the transcription start sites, exonic regions flanking splicing sites, stop codons, the 5’untranslated region (5’UTR) and the 3’untranslated region (3’UTR) (7-9).
m6A modification is dynamically and reversibly regulated by methyltransferases or writers (10, 11), and removed by demethylases or erasers (12); in addition, it exerted its function either directly being recognized by m6A binding proteins or readers, or indirectly by tuning the structure of the modified RNA to regulate RNA reader–protein interactions (6). Emerging evidence has suggested that m6A modification and its related enzyme played an important role in the different steps of the mRNA life, including RNA folding and structure, maturation, stability, splicing, exportation, translation and decay (13). Moreover, the accumulated data has identified that deregulation of m6A modification had recently been correlated with diseases caused by pathogenic virus.
The current review focused on the roles of m6A methylation, and their interactions in cells and organisms. Particularly, we would highlight m6A modification on viral RNA for us to better understand the prognosis of viral diseases, potential therapeutic targets and the development of anti-viral drugs.
Writers could install m6A modification, which was consisted of methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms’ tumor 1-associating protein (WTAP), methyltransferase-like 16 (METTL16), RNA binding motif protein 15 (RBM15), and KIAA1429 (10, 14-16). Bokar et al firstly found a ~200 kDa methyltransferase (MT) complex isolated from HeLa cell nuclear extract that exhibited methyltransferase activity, in which only a 70 kDa protein was identified and named MT-A70 or METTL3 (17). Recently, a study has indicated that METTL14 as another RNA writer formed a stable heterodimer core complex with METTL3 (10). This complex component mediated m6A deposition on nuclear RNA inside mammalian cells. Furthermore, the combination of METTL3/METTL14 dramatically enhanced methyltransferase activity compared to individual protein each. This report also found that WTAP did not possess methylation activity, but it interacted with the METTL3/14 complex to significantly affect cellular m6A deposition, whose functions as a regulatory subunit in the m6A methyltransferase complex. In addition, a study showed that WTAP may function as a regulatory subunit in the m6A methyltransferase complex and might play a critical role in epitranscriptomic regulation of RNA metabolism (11). Besides WTAP, RBM15 and KIAA1429, as a component of another complex, also could regulate the activity of METTL3/14 (18). Depletion of KIAA1429 resulted in a decreasing of m6A level, illustrated that an essential role of KIAA1429 existed during the methylation process (19). Moreover, knockdown of RBM15 caused a significant reduction of m6A level in mRNAs (15). Emerging research reported a new component of writers named METTL16, formed m6A in U6 snRNA and U6-like hairpins of MAT2A mRNA in a C-m6A-G context (14).
m6A modification on RNA could be removed by at least two erasers, which consisted of the alkB homologue 5 (ALKBH5) and fat mass and obesity-associated (FTO) proteins. It is reported that ALKBH5 and FTO mainly localized to the nuclear compartment (12, 16). FTO, which was homologous to the DNA repair AlkB protein, played an oxidative demethylation of 3-methylthymine role in single-stranded DNA and of 3-methyluracil role in single-stranded RNA (20). An increasing study reported that FTO was a potent regulator of nuclear mRNA processing events such as alternative splicing and 3′ end mRNA processing (21). Moreover, FTO has an additional role as a demethylase to manipulate N6,2′-O-dimethyladenosine (m6Am) level; FTO depletion or FTO overexpression resulted in selective regulation of the abundance of m6Am containing mRNAs in cells, and FTO preferentially demethylated m6Am rather than m6A and reduced the stability of m6Am mRNAs (22). Later, Zheng et al announced the second eraser-ALKBH5 that contributed to the removal of the m6A modification on nuclear RNA (mostly mRNA) both in vitro and in vivo (16). In addition, the demethylation activity of ALKBH5 significantly affected nuclear RNA export and metabolism and gene expression, displaying that there were broad biological roles of the reversible m6A modification on RNA.
Proteins that selectively bind m6A sites could be defined as m6A “readers”, which exert regulatory functions by selecting recognition of methylated RNA. Among all “readers”, the YTH N6-methyladenosine RNA-binding protein family which included YTHDF1, YTHDF2, YTHDF3, and YTHDC1, YTHDC2 was a major group of m6A readers and has been extensively studied. YTHDC1 promoted exon inclusion (23), mRNA m6A in the cytoplasm was recognized by YTHDF1-3. In general, YTHDF1 directly enhanced translation through binding m6A modification in the 3'UTR region (24); YTHDF2 recruited CCR4-NOT de-adenosine complexes to promote mRNA decay (25); YTHDF3 acted as a helper of YTHDF1 and YTHDF2 (26, 27). YTHDF1, 2, and 3 dominantly located in cytoplasm as cytoplasmic proteins, while YTHDC1 mainly localized in the nucleus to regulate mRNA export from nucleus to cytoplasm (28). Besides YTH-containing proteins, a study reported a new family- the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) (29). In contrast to YTH domain family, IGF2BPs were bind to m6A-modified mRNAs through GG(m6A)C, a typical m6A motif, to promote the stability of mRNA. Moreover, other proteins have also been indicated to have a potential function to recognize m6A, including eIF3 and HNRNP2AB1; eIF3 , as part of the 43S pre-translational initiation complex, could be directly linked to the m6A site of mRNA 5'UTR region to facilitate protein translation (22, 30, 31). Therefore, m6A was dynamically regulated by writers, erasers, and readers and other potential proteins that could influence these regulators.
Increasing evidence has shown that m6A modification not only exists in eukaryotic cells but also in the virus. m6A has long been known to be present in the RNA transcripts of viruses to impact on nuclear replication, such as influenza A virus, simian virus 40, Rous sarcoma virus, avian sarcoma virus, and adenovirus (32-35). In recent years, numerous discoveries previously made have uncovered that m6A modification regulates viral life cycles and m6A modification in pathogenic viruses is increasingly favored by researchers. Shreds of observations have revealed that m6A modification is a regulator of key gene expression in viral life. As different type of pathogenic virus, m6A modification plays a role of inhibition or promotion (Table 1). Therefore, exploring the biological functions of m6A methylation in different virus is of great significance to understand the pathogenesis of virus and innovative prevention; relations between m6A and virus and related cancer progression in line with different types of virus were reviewed as follows.
Virus/cancer type | Molecule | Change | Sample source | Target | Biological function | Reference |
---|---|---|---|---|---|---|
HIV | METTL3 |
Down | CD4+T cell | REV | Suppress virus replication | (37) |
ALKBH5 | Down | CD4+T cell | REV | Promote virus replication | ||
YTHDF1-3 | Up | CEM-SS T cell | -- | Promote virus replication | (1, 37) | |
HeLa/CD4 cell | Gag | Suppress virus replication | (38) | |||
EV71 | METTL3 | Down | Vero cells | RdRp 3D | Decrease in virus titer | (41) |
FTO | Down | Vero cells | -- | Promote in virus titer | ||
YTHDF2/3 | Down | Vero cells | -- | Suppress virus replication | ||
IAV | METTL3 | Down | A549 cell | -- | Suppress IAV replication | (44) |
YTHDF2 | Up | A549 cell | -- | Promote IAV replication | ||
KSHV | METTL3 | Down | BCBL1 cell | ORF50 | Suppress viral lytic replication | (50) |
FTO | Down | BCBL1 cell | ORF50 | Promote viral lytic replication | ||
YTHDC1 | Up | BCBL1 cell | ORF50 | Promote ORF50 pre-mRNA splicing | ||
YTHDF2 | Down | iSLK.219/ iSLK.BAC16 cell | ORF50 | Suppress transcription in ORF50 | (52) | |
HBV | METTL3/14 | Down | HepAD38 cell | -- | Promote HBc/s protein expression and the half-life of pgRNA | (55) |
FTO/ALKBH5/YTHDF2-3 | Down | HepAD38 cell | -- | Suppress HBc/s protein expression and the half-life of pgRNA | ||
HCV | METTL3/14 | Down | Huh7 cell | -- | Enhance titer of HCV | (56) |
FTO | Down | Huh7 cell | -- | Decrease viral titer | ||
YTHDF1-3 | Up | Huh7 cell | -- | Suppress HCV replication | ||
HCC | METTL3 | Down | Patient sample/ HepG2, Huh-7 and MHCC97L | SOCS2 | Attenuate SOCS2 mRNA stability | (58) |
METTL14 | Down | Patient sample/ tumor tissues/ SMMC-7721, Hep3B and HepG2 | miR-126 | Regulate processing of miR-126 by DGCR8 | (59) | |
CC | FTO | Up | Patient sample/ SiHa cell | -- | Induce poor prognosis | (62, 63) |
ZIKV | METTL3/14 | Down | Vero cell | -- | Increase viral titer | (68) |
ALKBH5/FTO | Down | Vero cell | -- | Decrease viral titer | ||
YTHDF2 | Up | Vero cell | -- | Suppress ZIKV replication | ||
SV40 | METTL3 | Down | BCS40 cell | -- | Reduce SV40 replication | (32) |
YTHDF2 | Up | BCS40 cell | -- | Enhance replication of SV40 | ||
EBV | METTL14 | Up | LcLs and Akata cells | EBNA3C | Promote growth and proliferation of |
(73) |
Human immunodeficiency virus I (HIV-1) is the prototype member of the retroviral family of lentiviruses and is the etiologic agent of acquired immunodeficiency syndrome (AIDS). People infected with HIV-1 need long-term antiviral treatment, once the treatment interrupted, it would lead to rapid rebound viremia (36). Therapies for viral infections are mainly dependent on vaccine defense and drug treatment. Nevertheless, completely effective cure has not been reported so far. Further exploration is expected for such a disease to seek more effective and safe strategy for treatment.
The study of m6A modification on HIV was lagged behind another virus such as influenza virus, adenovirus, Rous sarcoma virus, and simian virus 40 known for almost 40 years (32-35). Currently, Lichinchi et al first discovered m6A modification on HIV-1 RNA, and deeply explored host RNA and HIV m6A molecular features, topology and function during CD4+ T cell infection (37). They used HIV infected CD4+ T cells as experimental subjects and analyzed their RNA by methylated RNA immunoprecipitation sequening (MeRIP-seq). Data showed that the level of m6A modification was greatly improved after HIV-1 infection. Knockdown of METTL3/14 resulted in reduction of virus replication, while knocking down ALKBH5 contributed to an increasing. Subsequently, they discovered that m6A enrichment region mainly located at the 5’UTR and 3’UTR of HIV genomic RNA, this finding was consistent with Tirumuru et al (38); however, Kennedy et al reported that enrichment of m6A merely located at the 3′UTR of HIV-genomic RNA by photo-crosslinking-assisted m6A sequencing (PA-m6A-seq) technique (1).
Besides writers and erasers, previous studies above also displayed that three YTHDF proteins could interact with HIV methylated RNA, but only two of these studies showed that overexpression of YTHDF promoted HIV replication, while silencing inhibited replication (1, 37). Kennedy et al testified that overexpression of YTHDF1/3 protein in HIV infected HEK 293T cells enhanced the level of HIV-1 mRNA and protein expression, while overexpression or knockdown of YTHDF2 in CEM-SS T cells either increased or decreased HIV-1 replication and protein expression, respectively. In contrast, Tirumuru et al verified that three YTHDFs were of an inhibitory effect on HIV replication, and the detail mechanism why there were so many differences is needed to be further explored. Moreover, Lichinchi et al verified that m6A modification level of 56 host genes from infected T cells using MeRIP-seq. These 56 genes were necessary to viral replication, this illustration unraveled that m6A modification could also enhanced virus replication by regulate the expression of 56 genes.
The Rev response element (RRE) is a structurally and functionally well-characterized RNA element within the HIV-1 env gene. After translation, Rev proteins were imported back into the nucleus where they assembled at the RRE to form active nuclear export complexes that facilitated the transit of viral transcripts into the cytoplasm (39). This is an essential step for viral replication. Lichinchi et al displayed that m6A modification on viral RNA affected the interaction between HIV Rev protein and RRE. Overall, the links between m6A related proteins and HIV-1 may identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system.
Enterovirus 71 (EV71), a single-stranded RNA virus with approximately 7.5 kb of genome in length, belongs to the genus enterovirus within the family Picornaviridae that has three genotypes (A, B and C) and several sub-genotypes, and usually is associated with serious infectious diseases affecting millions of people around the world (40). Since the discovery of the EV71 for the first time in 1969, innumerable outbreaks and epidemics have been caused worldwide, especially in Asia and the Pacific, such as China, Korea, Singapore, Japan and Vietnam. Unfortunately, there are no effective antiviral drugs being currently available for the treatment of this kind of infection so far.
Recently, with the rapid development of sequencing technology, Hao et al. ascertained that EV71 RNA also contained m6A modification which was located in the VP, 3D and 2C coding region, and the expression and localization of m6A writers, erasers, and readers were affected upon virus infection (41). In addition, writers, erasers, and readers in host cells also had impact on viral replication. Similarly, mutation of the m6A modification sites in the infectious clone decreased EV71 progeny virus production and protein expression. Knocking down endogenous METTL3 and FTO in Vero cells using shRNA stated clearly that abundance of m6A in EV71 RNA decreased by silencing METTL3 gene and increased by FTO depletion. Furthermore, silencing METTL3 gene resulted in a significant decrease in virus titer and copy numbers of EV71 RNA, while depletion of FTO got come out a converse phenomenon. All data above suggested that METTL3 and FTO would have impacts on efficiency EV71 replication. Meanwhile, Knockdown of YTHDF2 and YTHDF3 in Vero cells also led to a decrease in viral replication. Then, investigators established an experiment with IP method, and confirmed that METTL3 could interact with viral RNA-dependent RNA polymerase (RdRp) 3D, which was increased as overexpression of METTL3 and regulated its modification to modulate viral replication. METTL3 played a positive regulator in EV71 replication in this research Hao and their groups did. Whether suppression of METTL3 could be used as a new therapeutic target remains to be further elucidated.
Influenza A virus (IAV) is a major cause of upper and lower respiratory tract infection, and poses a continuing threat to global health. Current influenza prevention and treatment strategies to limit the pathogenesis associated with influenza virus infections include annual vaccination and antiviral drugs (42). However, frequent changes in influenza virus surface antigens due to the antigenic shift and drift allow influenza viruses to escape antibody-mediated immunity following vaccination (43). Therefore, there is an urgent need to explore drugs for the influenza virus itself which can replace the traditional method for the prevention and treatment of influenza.
It has previously been stated that influenza virus mRNAs contained internal m6A residues. Influenza viral mRNA was tested for the presence of internal m6A since the cell nucleus was required for viral replication and a nuclear, as well as a cytoplasmic phase, occurs during virus replication (35). Recently, Courtney and their group reported that m6A affected IAV gene expression and replication (44). In this study, they used A549 lung cancer cells as the main research object. After depletion of METTL3 using 3DAA, which has been reported to inhibit the presence of m6A residues in mRNA transcripts by inducing the depletion of SAM, or CRISPR/Cas technology, the viral mRNA level was reduced. Additionally, IAV replication ability was also reduced. Conversely, overexpression of YTHDF2 in A549 cells brought about the promotion of IAV replication and infectious particles, while overexpression of YTHDF1 and YTHDF3 had little effect on virus replication and virus production. Subsequently, they found that m6A modification sites not only existed on the positive strands of IAV mRNA/cRNA and negative strands of vRNA, but high level of m6A modification on the mRNAs of HA, NA, M1/M2 and NP encoded by the virus, using PAR-CLIP and PA-m6A-seq sequencing technology. Finally, they performed silencing mutations at the m6A site in the sense and antisense RNA strands of the hemagglutinin segment. The hemagglutinin protein levels and viral replication were reduced after mutation, and the IAV pathogenicity of the virus-infected mice was also declined. All these findings implied that m6A modification might be critical for the expression of important viral genes, viral replication and production and also expand our understanding of m6A role in IAV development.
The herpesviridae family is made up of the most prevalent human pathogens, and 90% of adults were infected at least one of the eight herpesvirus subtypes (45). In healthy seropositive individuals, Kaposi’s sarcoma-associated herpesvirus (KSHV) causes persistent infection by establishing latency in CD19+ peripheral B-lymphocytes (46). KSHV is an oncogenic virus linked to multiple malignancies including Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD) (47-49). Latency is a hallmark of all herpes viruses. A recent study discovered that KSHV reactivation stalled if the newly transcribed viral RNAs fail to undergo post-transcriptional m6A (50).
When the latent virus reactivates, it causes epigenetic changes that lead to transactivation of the viral genome (51). Therefore, understanding of the interaction between KSHV and m6A can develop new therapeutic approaches for KSHV-induced diseases.
A study by Fengchun Ye et al reported that the level of m6A-modified mRNA (m6A-mRNA) for a given viral transcript increased substantially when infected cells were stimulated to lytic replication (50), Charles R et al also authenticated this in cells infected with KSHV (52). KSHV replication transcription activator (RTA), which is an immediate early protein encoded by Open reading frame 50 (ORF50), is an essential mediator of KSHV lytic replication and halts virion production. Fengchun Ye et al stimulated KSHV-infected cells to lytic replication in the presence of 3-deazaadenosine (DAA), and then they affirmed that DAA strongly inhibited splicing of the pre-mRNA. Later studies announced that knockdown of METTL3 or inhibition of methyltransferase complexes with 3DAA in BCBL1 cells reduced viral lytic replication, while knockdown of FTO moderately increased viral lytic replication. These observations indicated that KSHV not only utilized but also manipulated the host m6A machinery to promote lytic gene expression and replication. Then they concluded that YTHDC1 was also critical for splicing of ORF50 pre-mRNA, which bound to ORF50 pre-mRNA and promoted ORF50 pre-mRNA splicing by recruiting the splicing factors SRSF3 and SRSF10. At the same time, the expression of RTA could promote the increasing of m6A level, which in turn could mediate the splicing of pre-mRNA.
Charles R et al promulgated that YTHDF2 played an important role in KSHV replication based on KSHV iSLK.219 and iSLK.BAC16 reactivation models. They used si-RNA to knock down METTL3 and YTHDF2, results exhibited that a small number of virions were produced by knocking down METTL3, while deletion of YTHDF2 displayed almost no virions. Subsequent experiments showed that the deletion of YTHDF2 also affected the expression of immediate early lytic gene and viral activation. ORF50 acted as a viral transcriptional transactivator whose expression was critical for driving the KSHV lytic gene expression cascade (53). Then, they verified that m6A modification controlled the expression of ORF50 in iSLK.219 cells, and YTHDF2 deletion resulted in transcriptional defect in ORF50. All findings suggested that YTHDF2 may act as a proviral role in DNA and RNA viruses. These observations make clear that YTHDC1 and YTHDF2 play an important role in ORF50 function and suggest that regulation of ORF50 expression through m6A methylation could be a new direction to explore in the future.
Hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family. HBV infection is the leading cause of chronic hepatitis and has the risk of developing cirrhosis and hepatocellular carcinoma (54). Recently, researchers have demonstrated that the life cycle of HBV virus is regulated by m6A modification, which affects the expression of HBV-related proteins and the reverse transcription of pre-genomic RNA (pgRNA) (55). Imam et al corroborated that m6A modification of HBV transcript negatively regulated the expression of HBV protein and the half-life of pgRNA. Using siRNA to knock down METTL3 and METTL14 increased the level of protein expression of HBc and HBs and the half-life of pgRNA as the same with knockdown YTHDF2 and YTHDF3, while depletion of FTO and ALKBH5 appeared opposite outcome.
Besides HBV, Gokhale et al affirmed that the Hepatitis C virus (HCV) genome also existed m6A modification, and thus explored the mechanism about how m6A modification regulated HCV replication (56). Knockdown of METTL3 and METTL14 in host cells enhanced the viral titer of HCV, while viral titer decreased after knocking down FTO. They claimed that YTHDF protein played a negative regulatory role in HCV replication and then they detected the binding of YTHDF to viral RNA by RIP technology. These proteins competed with the HCV core protein for binding to the region of the Env gene to inhibit the packaging of viral RNA. Taken all data together, suggested that maintaining at a normal level of m6A-related proteins may provide potential therapeutic strategies for controlling these viral pathogens.
Chronic infection with HBV and HCV is the most important causes of Hepatocellular Carcinoma (HCC) (57). Not only HBV and HCV are regulated by m6A modification, but also increasing evidence has shown that m6A and regulators are critical for the development of HCC. Recently, two studies revealed that m6A modification was associated with HCC progression, mainly focusing on writers METTL3 and METTL14, respectively. One of the researchers discovered that METTL3, increasing in HCC, positively facilitated HCC cell proliferation, migration and colony formation in vitro and enhanced HCC tumorigenicity and lung metastasis in vivo(58). This article identified suppressors of cytokine signaling 2 (SOCS2) reported as an essential tumor suppressor in different cancer types, as a direct downstream target of METTL3-mediated m6A modification; after knockdown of METTL3, SOCS2 mRNA was diminished. And they demonstrated that METTL3 mediated SOCS2 abnormal relied on m6A “reader” protein YTHDF2 and also showed that lower expression of SOCS2 significantly related to poor overall survival and disease-free survival of HCC patients.
The latter reported that METTL14, which was significantly down-regulated in HCC, was mainly responsible for HCC (59). As a difference to Chen’s discovery (58), no significant difference was observed in the expression of METTL3, Wilms tumor 1–associated protein, KIAA1429, and ALKBH5 in HCC. METTL14 was associated with frequent recurrence and poorer survival; and abnormal levels of METTL14 mRNA were related to tumor differentiation, tumor stage, tumor encapsulation and microsatellite and microvascular invasion. Subsequently, they concluded that microRNA126 (miR126) was regulated in an m6A-dependent manner as a downstream target of METTL14. Overexpression of METTL14 contributed to pri-miR126 process to mature miR126 by enhancing the recognition and binding of the microprocessor protein DGCR8 to pri-miRNA. Collectively, these studies all spread that aberrant level of m6A modification played a pivotal role in HCC mediating different mechanisms on different m6A related proteins.
Deeper understanding m6A related to HBV, HCV, HCC would contribute to the treatment of these diseases. These two observations authenticated that METTL3 and SOCS2, METTL14 and miR126 were involved in HCC development by regulating m6A levels. Thereby, SOCS2 and miR126 could be a candidate target for the treatment of liver cancer.
Human papillomavirus (HPV), which belongs to the genus papillomavirus of the papillomavirus family, is a small DNA virus with a genome of approximately 8 kb. Cervical cancer (CC) that is the fourth largest cancer killer for woman is one of the most common gynecological malignancies in the world (60, 61). Although it has been made in significant advances in cancer detection and treatment over the past few decades, the 5-year survival rate remains low. Thus, it is urgent to determine the detail molecular mechanisms of CC development, to explore innovative strategies.
A recent study revealed that m6A methylation played an important role in cervical cancer. They demonstrated that m6A played a negative role in cell proliferation and CC development (62). Compared with adjacent non-cancerous tissues, the m6A level reduced significantly in cervical cancer tissues. Moreover, for patients with cervical cancer, disease-free survival (DFS) and overall survival (OS) was significantly higher in the high m6A level group than the low m6A level group, indicating that the level of m6A methylation could be a prognostic marker. Cervical cancer cell proliferation was suppressed in vitro and cervical cancer development was inhibited in vivo, this relayed on knocking-down erasers (FTO and ALKBH5) or overexpressing writers (METTL3 and METTL14). Interestingly, Zhou et al. asserted that FTO overexpression led to resistance of chemo-radiotherapy on cervical squamous cell carcinoma (CSCC), the major type of cervical cancer (63). And FTO-induced upregulation of β-catenin via mRNA demethylation and subsequently activation of excision repair cross-complementation group 1 (ERCC1) contributed to this resistance. They also highlighted that high expression level of FTO had a poor prognosis. These findings of m6A role in response to the treatment of cervical cancer would guide us to seek optimal treatment. But there is still no report to demonstrate the relation between HPV and m6A methylation, so it’s a large spare waiting to explore.
Zika virus (ZIKV), which leads to Zika virus disease (ZVD)-a type of acute infectious disease, was firstly isolated from the serum of apyrexial rhesus monkey caged in the canopy of Zika Forest (64). In 2007, it had previously outbreak in Yap Island in the Western Pacific (65), and then caused sporadic disease in French Polynesia in 2013 (66), more recent outbreaks occurred in Brazil and arrived at the epidemic peaked, next spread rapidly in South America (67). Currently, there are still no specific antiviral drugs; therefore, it is urgent to explore innovative means to aim at ZIKA infected mechanism.
To date, there has been one research involved in the existence of ZIKV to m6A modification. Lichinchi et al confirmed that ZIKV viral RNA was methylated and they identified twelve discrete m6A peaks spanning the full length of ZIKV RNA among which most were present in the region encoding the NS5 region and the 30 UTR region (68). Like other virus, ZIKV RNA adenosines were also modified by host writers and erasers; perturbation of ZIKV m6A affected ZIKV replication efficiency and viral titer. Knockdown of writers (METTL3 and METTL14) significantly increased ZIKV production, the viral titer, ZIKV RNA levels in cell supernatants and expression of ZIKV envelope protein, while the silencing of erasers (ALKBH5 and FTO) had the opposite effect. Besides, they reported that reader (YTHDF proteins) bound to ZIKV RNA could regulate replication of ZIKV as well. Especially, YTHDF2 had extremely greatest effect on ZIKV replication, RNA expression and stability of viral RNA as compared to YTHDF1 and YTHDF3. Generally, these results elaborated a new mechanism to understand the connection between host and virus by the relationship between m6A associated enzymes and ZIKV; it could provide an innovative direction to treat disease related to ZIKV infection by regulating the expression of writers, erasers and readers in the host.
Simian Virus 40 (SV40), a polyomavirus of the rhesus macaque, is a double-stranded DNA virus which is a potent DNA tumor virus reported to induce human primary brain tumors, malignant mesotheliomas, bone cancers, and non-Hodgkin’s lymphoma (69, 70). In 1979, it was reported that late SV40 16S and 19S mRNAs contain several m6A residues (32). But there was no report about where these m6A residues precisely located in and their functional significance remained unclear. A recent evaluation by Tsai et al identified and precisely mapped m6A peaks on the SV40 including eleven peaks in late transcripts, and two in the SV40 early region using photo-crosslinking-assisted m6A sequencing (PA-m6A-seq) and photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) (32). Meanwhile, they demonstrated that overexpression of YTHDF2 significantly enhanced replication of SV40, while mutation inhibited SV40 replication as well as SV40 structural gene expression after using gene editing to inactivate m6A reader YTHDF2 or writer METTL3 in the permissive cell line BSC40. In total, their investigation clearly implicated that m6A modification played a positive role in the regulation of the SV40 life cycle.
Epstein–Barr virus (EBV), a herpesvirus, is an oncogenic virus isolated and identified from a Burkitt’s lymphoma patient in 1964 (71). As one of the most common human viruses, EBV causes infectious mononucleosis and is associated with certain forms of lymphoma and there are about 200,000 cases of cancer associated with this virus, leading to 140,000 deaths each year (72). Common antiviral therapies can suppress active viral replication, but to date no existing EBV vaccine or treatment can effectively eradicate latent infection. Therefore, it’s urgent to explore a novel cure for many prevalent viral diseases.
Currently, only one research has revealed the association of m6A “writers” with EBV, mainly focusing on METTL14 (73). METTL14, dramatically increasing in EBV latently infected cells and down-regulating during EBV lytic infection, can facilitate cells growth and colony formation in EBV transformed cells and enhance EBV tumorigenicity and growth in vivo. The stability of the latent genes (including EBNA1, EBNA3C, and LMP1) and lytic genes (including BRLF1, gp350, and BMRF1) can be downregulated via METTL14. Meanwhile, EBNA3C was not only a downstream target of METTL14, but the antigen of EBV was responsible for up-regulation and stability of METTL14. EBV latent antigens are the major contributors to EBV-associated malignancies and low expression of EBV latent antigens will bring about attenuation of EBV-mediated tumorigenesis. Therefore, targeting METTL14 may be a key strategy for controlling EBV-associated cancer.
Viruses showing above are deeply bad to human health, while there still are other viruses existing m6A in their RNA including Rous sarcoma virus (RSV) (74), vesicular stomatitis virus (VSV) (75), adenoviruses (34) that are less pathogenic to the human body. Advance in the understanding of human pathogenic virus has been remarkable in recent years, but treatment methods have changed little in the past decades. For this reason, it is necessary to focus on the development of new treatments that may replace or follow standard therapy.
With emerging of posttranscriptional modification of RNAs, especially methylation of RNAs, it has become a hot topic in recent years due to their key role in regulating gene expression, cell behaviors, and physiological conditions in many species, including humans. Among more than 100 kinds of different chemical modifications, m6A is the most abundant modification which is discovered initially in the poly(A) RNA fractions and has been predicted to be functional in mRNA processing (4). m6A is dynamic and reversible that installed by “writers” and removed by “erasers”. The sites in which methylated on RNA would be bond with m6A recognition proteins- “readers”. Emerging evidence has shown that aberrant expression of proteins related m6A modification is associated with the progress of cancer, such as acute myeloid leukemia (76), lung cancer (77), hepatocellular carcinoma (58, 59) and so on. Particularly, m6A has been detected in viral mRNA and in the RNA of retroviruses as well, it can influence the expression of viral gene and viral life cycle, which could provide a new method for the treatment of many malignant virus-related diseases.
The development of corresponding targeted small molecule inhibitors for writers, erasers and readers in m6A modifications could be a potential therapeutic approach for antiviral therapy. Recent studies have shown that the use of writer inhibitors could have an impact on viral replication and also affect tumor development (1, 58, 78). Maybe inhibition of the occurrence of m6A may have an anti-tumor effect.
Increasing studies have shown that m6A plays a very significant role in human pathogenic viruses, but potential challenges still exist. First, the specific mechanism concerning related proteins of m6A modification interacting with viral RNA is largely unknown. Second, if the m6A modification-related regulatory genes and proteins can be used as prognostic and diagnostic markers for some viral diseases, their specificity and targeting need to be further explored, and whether there is interference with normal cells remains to be further verified. Third, many groups have revealed that substances similar to inhibitors could be used to block m6A-modified abnormalities to regulate related diseases, but there is still lack of a large number of clinical applications, and the corresponding effects are largely unknown. Taken together, all of these issues should be further explored clearly.
This study was supported by the National Natural Science Foundation of China (No. 81271692), the Ocean Antithrombotic Fibrinolytic Enzyme Gene Bank of Taiwan Strait (No. 2014FJPT08), the Science and Technology Innovation Public Technology Service Platform of Function of Drugs and Food (No. 3502Z20141015), Education Department of Fujian Province (JA14020) and Subsidized Project for Postgraduates’ Innovative Fund in Scientific Research of Huaqiao University (No. 17013071028).
m6A
N6-methyladenosine
ribosomal RNA
transfer RNA
5’untranslated region
3’untranslated region
methyltransferase-like 3
methyltransferase-like 14
Wilms’ tumor 1-associating protein
methyltransferase-like 16
RNA binding motif protein 15
methyltransferase
alkB homologue 5
fat mass and obesity-associated proteins
N6,2′-O-dimethyladenosine
the insulin-like growth factor 2 mRNA-binding proteins
Human immunodeficiency virus I
acquired immunodeficiency syndrome
methylated RNA immunoprecipitation sequening
Rev response element
Enterovirus 71
RNA-dependent RNA polymerase
Influenza A virus
Kaposi’s sarcoma-associated herpesvirus
Kaposi’s sarcoma
primary effusion lymphoma
multicentric Castleman’s disease
replication transcription activator
Open reading frame 50
3-deazaadenosine
Hepatitis B virus
Hepatitis C virus
Hepatocellular Carcinoma
suppressors of cytokine signaling 2
microRNA126
Human papillomavirus
Cervical cancer
disease-free survival
overall survival
cervical squamous cell carcinoma
excision repair cross-complementation group 1
Zika virus
Zika virus disease
Simian Virus 40
photo-crosslinking-assisted m6A sequencing
photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation
Rous sarcoma virus
vesicular stomatitis virus
Epstein–Barr virus