3-Deazaadenosine – Prostaglandin E2 chemical

N6-methyladenosine modiﬁcations enhance enterovirus 71 ORF translation through METTL3 cytoplasmic distribution

Min Yao, Yangchao Dong, Yuan Wang, He Liu, Hongwei Ma, Hui Zhang, Liang Zhang, Linfeng Cheng, Xin Lv, Zhikai Xu, Fanglin Zhang, Yingfeng Lei, Wei Ye
Department of Microbiology, School of Preclinical Medicine, Fourth Military Medical University, Xi’an, 710032, China

A B S T R A C T
During replication, numerous viral RNAs are modiﬁed by N6-methyladenosine (m6A), the most abundant internal RNA modiﬁcation. m6A is believed to regulate elements of RNA metabolism, such as splicing, stability, translation, secondary structure formation, and viral replication. In this study, we assessed the occurrence of m6A modiﬁcation of the EV71 genome in human cells and revealed a preferred, conserved modiﬁcation site across diverse viral strains. A single m6A modiﬁcation at the 5’ UTR-VP4 junction was shown to perform a protranslational function. Depletion of the METTL3 methyltransferase or treatment with 3-deazaadenosine signiﬁcantly reduced EV71 replication. Speciﬁcally, METTL3 colocalized with the viral dsRNA replication intermediate in the cytoplasm during EV71 infection. As a nuclear resident protein, METTL3 relies on the binding of the nuclear import protein karyopherin to its nuclear locali- zation signal (NLS) for nuclear translocation. We observed that EV71 2A and METTL3 share nuclear import proteins. The results of this study revealed an inner mechanism by which EV71 2A regulates the subcellular location of METTL3 to amplify its own gene expression, providing an increased understanding of RNA epitranscriptomics during the EV71 replication cycle.

1. Introduction
In recent years, RNA modiﬁcations have been shown to play pivotal roles in regulating various biological processes. Among these modiﬁcations, N6-methyladenosine (m6A) is one of the most prevalent and abundant modiﬁcations. m6A modiﬁcations are catalyzed by the nuclear resident methyltransferase complex “writer”, which minimally consists of the 70 kDa subunit of the N6- adenosine methyltransferase complex, methyltransferase-like 3 (METTL3), METTL14, and their chaperone Wilms’ tumor 1- associated protein (WTAP) [1]. Other proteins, such as Vir-like m6A methyltransferase associated (VIRMA), RNA-binding motif protein 15 (RBM15) and RBM15B, have also been reported to beinvolved in this process. Among these factors, METTL3 adds a methyl group to the RNA substrate and forms a heterodimer with METTL14, which contains a pseudoactive center. m6A modiﬁcations are reversible, and two proteins, fat mass and obesity-associated protein (FTO) and alkB homolog 5 (ALKBH5), can remove methyl groups from RNA and are therefore referred to as demethylases or “erasers”. m6A-modiﬁed RNA is recognized by a number of proteins containing a YT521-B homology (YTH) domain, and these proteins play a fundamental role in elements of RNA biology, such as mRNA splicing, nuclear export, stability, structural regulation, and translation.
m6A sites can exist in 50 UTRs, 3’ UTRs or within the codingregions and share a common RRACH motif (where H denotes U, A or C; and R represents A or G). Although m6A modiﬁcation during viral infection was observed in the early 1970s, the precise role of this modiﬁcation remains highly enigmatic due to insufﬁcient available methodology. However, recent research has extensively expanded our knowledge of this modiﬁcation through the development of m6A-methylated RNA immunoprecipitation (MeRIP) and MeRIP sequencing (MeRIP-Seq) techniques [2,3]. For viruses, because progeny virions are derived from host cells, it is natural to infer the role played by this ubiquitous RNA modiﬁcation in the viralreplication cycle.
In recent decades, m6A-modiﬁed RNA has been observed in both DNA virus- and RNA virus-infected cells and maintains different functionalities [4e6]. This modiﬁcation is even detectable within the genomes of cytoplasm-replicating viruses, such as hepatitis C virus (HCV) [7], Zika virus (ZIKV) [8], and recently, enterovirus 71 (EV71) [9].
The roles of m6A modiﬁcation in the viral life cycle can be subdivided into three types. First, by inhibiting viral replication at different stages, such as HCV, m6A modiﬁcation inhibits HCV egress without inﬂuencing viral protein translation [7,8]. Second, m6A modiﬁcation enhances viral replication, as observed for HIV, simian virus 40 (SV40), EV71 [9e13] and lytic Kaposi’s sarcoma-associated herpesvirus (KSHV) infections [14]. Third, m6A modiﬁcation can perform dual functions, having either enhancing or inhibitory functions, during viral replication in different cell lines or at different positions, as observed for KSHV and HBV [15,16]. Furthermore, the reader protein YTHDF was recently shown to inhibit HIV replication [17]. However, the mechanism by which this modiﬁcation is manipulated by viruses is largely unknown. Addi- tionally, because m6A modiﬁcation participates in regulating type I interferon responses, an increasingly dynamic role of m6A modiﬁ- cation in regulating viral replication has emerged [18e20].
EV71 is the pathogen responsible for the severe symptoms ofhand, foot and mouth disease (HFMD) and other fatal neurological diseases worldwide [21] and belongs to the family Picornaviridae. As a picornavirus, EV71 is nonenveloped and has a single-stranded, positive-sense RNA genome encoding a single polyprotein precur- sor that is subsequently processed into four structural (VP1, VP2, VP3, and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins via the 2A and 3C proteinases [22].
In this study, we identiﬁed an EV71 strain isolated from Xi’an, China with a genome that is m6A-modiﬁed in rhabdomyosarcoma (RD) cells and located the modiﬁcation positions. Meanwhile, METTL3-mediated m6A modiﬁcation enhanced EV71 open reading frame (ORF) translation. Furthermore, the key writer protein METTL3 share the same nuclear import protein karyopherins with nonstructural protein 2A which may contributes to partial METTL3 cytoplasmic distribution. In summary, our results indicate a role for m6A modiﬁcation in enhancing EV71 ORF translation and elucidate the mechanism by which the virus hijacks resident nuclear METTL3 for its own beneﬁt.

2. Results
2.1. N6-methyladenosine modiﬁcations in the human cell-derived EV71 genome
Studying the inﬂuence of RNA modiﬁcations, especially the most abundant modiﬁcation, m6A, on the viral replication cycle is a rapidly evolving research area. We focused on the most prevalent pathogen of HFMD, EV71. Since MeRIP-Seq data from previous research were collected in a monkey cell line [9], we wanted to determine whether these m6A modiﬁcation sites are conserved in human cells. Thus, MeRIP-Seq was performed using EV71-infected RD cells. The locations of m6A enrichment (Fig. 1A) closely matched the locations in a previously proposed MeRIP-Seq map [9], with most of the m6A modiﬁcation sites being located in the VP4, VP1, and 2A region, although slight differences between our results and those previously reported were observed. In contrast to our results, Hao et al. [9] observed signiﬁcant enrichment around the start of the VP3 coding area. Two other m6A enrichments in the 3A and 3C region were not detected in EV71 cultured in Vero cells. Other enrichment peaks, such as those at the 5’ UTR-VP4 junction and at the end of the VP3, VP1, 2C and 3D coding regions correspondedbody. This similarity suggests a conserved m6A modiﬁcation pref- erence for EV71 independent of where the strain was isolated from or which primate cells were used for growth. These differences could be attributed to differences in multiple factors, such as the experimental procedure, the nucleic acids of various strains, cell origins, or even the passage number of the cultured virus since isolation. This question awaits further investigation of the m6A modiﬁcation map, especially with regard to clinical isolates with a low number of passages.
Because the N6-methyladenosine modiﬁcation is executed by a methyltransferase complex “writer”, especially METTL3, we con- structed a reporter plasmid retaining the terminal 50 UTR-VP4 and 30 UTR regions among which other ORF areas substituted with the NanoLuc luciferase reporter gene, and a modiﬁed S1 aptamer (S1m) tag was added at the end of the 3’ UTR [23e25]. The S1m aptamer is speciﬁcally binds to streptavidin. This simulated viral RNA was used as bait to enrich the putative RNA-binding protein (RBP), and the eluted samples was blotted with the antibody against METTL3. As shown in Fig. 1B, an apparent band was observed in the eluted RBP, suggesting that METTL3 directly binds to the EV71 RNA genome.

2.2. N6-methyladenosine methylation promotes EV71 gene expression
As the EV71 RNA genome derived from human cells is m6A modiﬁed, an interesting question is how the m6A modiﬁcation af- fects EV71 replication. Previous study showed that m6A modiﬁca- tion can enhance mRNA translation. To test this possibility, the compound 3-deazaadenosine (3-DAA) was used. Because 3-DAA can inhibit the formation of S-adenosyl methionine (SAM) from S-adenosyl homocysteine (SAH) without affecting mRNA capping [26], it can serve as a nonspeciﬁc inhibitor of m6A addition. With the addition of 3-DAA into the culture medium, the overall m6A modiﬁcation level of cellular RNA was reduced in the 3-DAA- treated groups (Fig. 2A). Consequently, the EV71 ORF translation rate was markedly reduced (Fig. 2B) without an appreciable change in cell viability at the concentrations used (Figure S1). Thus, m6A modiﬁcation could enhance EV71 ORF translation, consistent with the results of a previous study [9].
Because the primary methyltransferase, METTL3, binds to theEV71 RNA genome, we subsequently assessed the role of METTL3 in EV71 genomic RNA translation. We ﬁrst selected two shRNA clones against METTL3 and evaluated their gene knockdown efﬁciency. As clone 1 exhibited higher efﬁciency (Figure S2), an A549 cell line stably transfected with this clone was generated through puro- mycin selection. In the A549 cells with stable knockdown of METTL3, EV71 ORF translation was lower than that observed in parental A549 cells (Fig. 2C), suggesting that METTL3-executed m6A modiﬁcation promotes EV71 ORF translation.
To further verify the inﬂuence of the m6A modiﬁcation on EV71 ORF translation, the previously described reporter plasmid was transfected into A549 cells with or without stable METTL3 knock- down. The performance of this reporter was evaluated for different transfection ratios, and the group with the highest luciferase ac- tivity level was used in subsequent experiments (data not shown). As expected, the luciferase activity level was higher in parental A549 cells than in the METTL3 knockdown cells (Fig. 2D), indicating that METTL3 could enhance the EV71 reporter translation.
Moreover, because the RRACH motif is the key domain allowing METTL3 to identify and modify RNA, a control EV71 reporter plasmid, RRACHmut, was derived by substituting the putative RRACH sequence with RRUCH within the 50 UTR-VP4 junction (Figure S3). Then, the luciferase activity levels of the NanoLuc luciferase reporter for the wild-type 50 UTR-VP4 and RRACHmutreporter plasmids were measured. A signiﬁcant decrease was observed in the reporter level for the RRACHmut group (Fig. 1H), suggesting that the m6A modiﬁcation within the 5’ UTR-VP4 junction plays a protranslational role during the EV71 lifecycle.

2.3. METTL3 is localized in the cytoplasm during EV71 infection
As METTL3 is predominantly located in the nucleus under normal conditions, how does this protein contribute to the repli- cation of cytoplasmic resident enterovirus? Using confocal micro- scopy, we observed that METTL3 colocalized with VP1, a structural protein of EV71 (Fig. 3A). Moreover, the dsRNA replication inter- mediate during virus lifecycle also colocalized with METTL3 in the cytosol (Fig. 3A), indicating that METTL3 localized in the cytoplasm during EV71 infection. Next, we performed cytoplasmic and nu- clear fractionation. As shown in Fig. 3B, appreciable amounts of cytosolic METTL3 were detected in the EV71-infected group compared to that observed in the mock-treated group, and the nuclear resident METTL3 level was also reduced. Taken together, these results suggest that METTL3 is localized in the cytoplasm during EV71 infection, which modiﬁes viral RNA and promotes viral translation, and may serve as part of the EV71 replication complex.

2.4. Interaction between 2A and karyopherin alpha contributes to the cytoplasmic distribution of METTL3
To determine the potential mechanisms on promoting the cytoplasmic shuttling of METTL3 during EV71 infection, we ana- lysed the nonstructural proteins of EV71 using POSRT II, an algo- rithm based on the k-nearest-neighbor classiﬁer [27]. EV71 2A also contains an NLS, and this motif appears to be conserved acrosspicornaviruses (Figure S4). The 2A proteinase is a virulence deter- minant of EV71 due to its versatile function in modulating cellular processes [22]. To investigate the possible function of 2A in METTL3 nucleocytoplasmic shuttling, we ﬁrstly generated a proteinase- function-deﬁcient 2A mutant by mutating cysteine 110 to alanine (C110A). This mutant can be expressed and detected by western blotting independently of WT 2A (Figure S5). When 2A/C110A was transfected into cells, cytosolic METTL3 levels were slightly elevated, but the difference between the groups was minimal. Then, METTL3 and transfected 2A were immunostained. As shown in Fig. 4B, the 2A/C110A mutant was predominantly localized in the nucleus, whereas the low level cytosolic 2A/C110A was localized with cytosolic METTL3. Moreover, two other mutants, RRKH-AAAA and NLVWEDSSRDLLV-A, exhibited different localization patterns (Fig. 4B, S4,S6), indicating that the additional signals are also required.
Next, we sought to determine the mechanism by which 2Acontributes to the cytosolic distribution of METTL3. A previous study showed that METTL3 harbors two different NLSs that are important for its nuclear localization [28]. During nuclear trans- location, the translated protein was either transported by kar- yopherin beta alone or by coupled karyopherin alpha and beta subunits. We hypothesized that the 2A-induced partial cytoplasmic localization of METTL3 may occur due to the sharing of theDis hypothesis, we cotransfected cells with 2A/C110A and different karyopherin alpha subunits. The IP results showed that kar- yopherin alpha subunits 1, 2, 6, and 7 interacted with 2A/C110A or METTL3 (Fig. 4C), indicating that 2A may competitively interact with METTL3 for the shared karyopherin alpha subunit. Moreover, co-IP results indicated that there is an interaction between METTL3 and 2A/C110A (Fig. 4D). To determine whether this 2A/C110A-induced distribution of METTL3 in the cytosol contributes to EV71 genomic RNA m6A modiﬁcation and translational enhancement, we cotransfected cells with the EV71 reporter and either 2A/C110A or the vector. As shown in Figure S7C, 2A/C110A signiﬁcantly increased NanoLuc activity, indicating that 2A/C110A could induce the cytosolic distribution of METTL3 and subsequently promote EV71 translation.
A previous study showed that the EV71 nonstructural protein 3D is responsible for the cytoplasmic distribution of METTL3 due to the SUMOylation and ubiquitination of the latter protein [9]. In addition to 3D and 2A, we also evaluated interactions between other nonstructural proteins of EV71 and METTL3 during infection [all clones were veriﬁed by western blotting (Fig. S7A)] and observed that other than 3D and 2A, 2C also induced the partial cytosolic localization of METTL3 (Fig. S7B). Furthermore, 2C, 3AB, 3C/C147A and 3D all enhanced EV71 reporter-driven NanoLuc translation with different efﬁciencies (Fig. S7C), indicating more functional roles of other nonstructural proteins in regulating m6A modiﬁcation machinery.
Taking together, our data reveal a potentially conserved set of m6A epitranscriptomic sites in the EV71 genome that are inde- pendent of cell line and viral strain. Furthermore, the 2A protein may play an important role in regulating METTL3 nucleocytoplas- mic shuttling to enhance EV71 ORF translation. Based on these results, we propose a model for m6A modiﬁcation-induced EV71ORF translation enhancement by 2A. Independent of its original proposed functions in nuclear pore rupture and host translation machinery shutoff, 2A could also competitively interact with METTL3 for nuclear importin protein binding, contributing to EV71 RNA m6A modiﬁcation and translational enhancement (Graphical abstract).

3. Discussion
Despite the prevalence of m6A modiﬁcations in cellular and viral mRNAs, the strategies used for m6A modiﬁcation are largely un- known, especially in virology. In this study, we showed that METTL3, the host N6-methyladenosine methyltransferase, is regu- lated by a nonstructural EV71 protein, expanding our knowledge of viral epitranscriptomics [4]. EV71 is the most prevalent causative pathogen of HFMD in mainland China and is also responsible for neurological complications, such as encephalitis, aseptic meningitis and acute ﬂaccid paralysis [29].
In this study, we showed that the RNA of the EV71 Xi’an isolate was m6A modiﬁed and shared a very similar m6A modiﬁcation pattern with the RNA of a previously reported XF strain, with a slight difference observed in the enrichment peak location [9]. For some sites located within the regulatory noncoding region that render mutagenesis difﬁcult without affecting normal function, we then generated a reporter retaining only one m6A modiﬁcationpeak at the 50 UTR-VP4 junction and used this reporter for down- stream evaluation. This m6A modiﬁcation appeared to preserve a protranslational function, consistent with previous results showing that EV71 replication is decreased in METTL3 knockdown A549 cells compared to that observed in parental cells. In addition, the universal methylation inhibitor 3-DAA diminished EV71 ORF translation.
m6A methylation and demethylation are dynamic processes in normal cell function, and METTL3 is predominantly localized in the nucleus. However, cytoplasmic METTL3 was observed and found to perform multiple functions, such as enhancing translation in hu- man cancer cells through interaction with the translation initiation machinery [30]. The genomes of other cytoplasm-replicating vi- ruses, such as ﬂaviviruses, are also m6A-modiﬁed; thus, METTL3, along with other molecules of the writer complex, must be present in the cytosol somehow. Both our group and other group have observed the cytoplasmic localization of METTL3 during EV71 infection, and these results explain why EV71 RNA replicated in the cytosol is N6 methylated. Although the mechanism by which this modiﬁcation enhances EV71 ORF translation requires further investigation, the intrinsic mechanism may be attributed in part to the abundance of METTL3, which has been shown to promote translation by Lin et al. [30]. As a picornavirus, EV71 utilizes an internal ribosome entry site (IRES)-driven mRNA translationmechanism. A previous study indicated that m6A modiﬁcation canfacilitate eIF4F-independent mRNA translation [31]; thus, m6A modiﬁcation of EV71 RNA may also use this mechanism. Moreover, recentstudies have indicated that m6A modiﬁcation can regulate the host interferon system [18,19], m6A-induced enhancement of EV71 replication may also contribute to dysfunction of interferon system.
As the nucleus and cytoplasm are separated by a lipid bilayer membrane, nucleocytoplasmic protein transport primarily occurs across the nuclear envelope via nuclear pore complexes (NPCs). NPCs are large complexes comprising nearly 30 different compo- nents named nucleoporins (Nups). Large proteins (typically larger than 40 kDa) require special sequences recognized by transport factors, called importins (karyopherin) and exportins, to be trans- ported into or out of the nucleus [32]. One example is Ebola virus VP24, a potent interferon antagonist that can target karyopherin alpha 5 to selectively compete with the nuclear import of phos- phorylated signal transducer and activator of transcription 1 (STAT1) [33].
However, the cytoplasmic translocation of METTL3 remains to be addressed. The results of previous investigations performed with rhinovirus and poliovirus (PV) showed that 2A can shut off cellular translation via eIF4G cleavage, thereby preventing the formation of the eIF4F complex in initiating host mRNA translation [34], disrupting the nucleopore by degrading components of the nuclear pore complex [35,36], and manipulating the innate im- mune pathway for efﬁcient replication and serving as a virulence determinant of EV71 [37e40]. Moreover, the coxsackievirus B3 (CVB3) 2A proteinase promotes encephalomyocarditis virus (EMCV) replication [41]. Because structure of 2A is largely conserved among enteroviruses [42,43], these proteins may share similar functions. Previous studies have shown that during the early stage of infection, the 2A proteins of PV and EMCV [44,45], members of the Picornaviridae family genera Picornavirus and Cardiovirus, respectively, can localize in the nucleus and inhibit cellular mRNA transcription. A subsequent study identiﬁed an NLS within EMCV 2A through mutation analysis [46]. In addition, the deletion of positions 133-147 can localize CVB3 2A to the nucleus [41]. Using PSORT II [27], we identiﬁed an NLS within EV71 2A that is conserved across picornaviruses. Additional analysis using the NetNES 1.1 serverpredicted a hydrophobic leucine-rich NES that isalso conserved (Figure S4) [47].
As noted for PV, we observed the nuclear localization of 2A soon after EV71 infection. Using a similar truncation strategy and site- directed mutagenesis, we mapped the key domain for EV71 2A nuclear localization. EMCV proteins 2A and 3BCD can localize to the nucleus and inhibit cellular mRNA transcription but not rRNA transcription [45], although whether this function is conserved in EV71 requires further investigation. This ﬁnding may explain why 2A is predominantly present in the cytosol during the late stages of infection. However, the predicted NES pattern was not identical to that of CVB3, although these proteins share the same prominent NLS. In addition, a previous study indicated that EV71 induces karyopherin alpha 1 degradation via caspase-3 signaling to prevent STAT1 nuclear translocation [48]. EV71 2A and 3D can antagonize antiviral activity by targeting either mitochondrial antiviral signaling protein (MAVS) or other components [37,39]. Moreover, similar to 2A, METTL3 appears to be transported into the nucleus by karyopherin alpha 1, 2, 6 and 7. Thus, 2A may play a pivotal role in regulating the subcellular localization of METTL3 and, hence, viral replication.
Finally, membranous replication factories can be induced bypicornaviruses and other positive-strand RNA viruses during infection [49,50]. Both 2B and 2C contain a transmembrane domain, which in 2B can form the so-called viroporin to regulate the permeability and level of Ca2þ between inner membrane or- ganelles [51e53], whereas in 2C this domain can induce inner membrane reorganization and is a key component of the viral replication factory [54]. 3A/3AB stabilizes 3D to form the replica- tion complex, and the N terminus is also important for ER-Golgi apparatus transport [55]. Thus, the colocalization of 2C and dsRNA with METTL3 together with the 3A-induced decrease in the ﬂuorescence intensity of nuclear METTL3 indicate that METTL3 may serve as a host factor for efﬁcient viral replication. In addition, we observed a signiﬁcant decrease in luciferase activity in the 2B- cotransfected group (Figure S7B). As a small hydrophobic ion channel protein, 2B was shown to induce cell apoptosis through BCL2-associated X (BAX) recruitment and activation [56], whichmay contribute to the observed reduction in the NanoLuc levels.
Collectively, we explored m6A modiﬁcation sites of the EV71 genome cultured in RD cells and revealed a preferred, conserved modiﬁcation site across diverse virus strains. We conﬁrmed that during EV71 infection, 2A could competitively interact with METTL3 for nuclear importin protein binding, contributing to METTL3 cytoplasmic location and EV71 RNA m6A modiﬁcation, hence, translation enhancement. This work revealed an inner mechanism by which EV71 2A regulates the subcellular location of METTL3 to amplify its own gene expression, augmenting knowl- edge of RNA epitranscriptomics in the EV71 replication cycle.

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