Enterovirus 71 structural viral protein 1 promotes autophagy by inducing an m6A-mediated PMP22 overexpression in mouse Schwann cells

Objective Enterovirus 71 (EV71), one of the enteroviruses responsible for the hand, foot and mouth disease (HFMD), can cause severe neurologic diseases such as brainstem encephalitis and demyelination. The molecular mechanism of demyelination is still not fully understood. This study aims to investigate the mechanism of how the EV71 structural viral protein 1, VP1 can act on host cellular pathways in mouse Schwann cells. Methods An EV71 VP1-expressing vector was generated and transfected into mouse Schwann cells (MSCs). Selective mRNA methylation inhibitor (DAA) was employed to identify key members of m 6 A pathway that are targeted by VP1. To investigate the role of METTL14 and YTHDF1 in PMP22 expression, small interfering RNA against METTL14 and YTHDF1 was employed to knockdown the METTL14 and YTHDF1 expression in MSCs. Real-time PCR and Western blot analysis were performed to determine the expression of PMP22 and m 6 A modication-associated proteins. results demonstrated VP1 upregulated m 6 A pathway by targeting METTL14 and YTHDF1. The expression of PMP22 was decreased by inhibiting the expression of METTL14 and YTHDF1. The interactions between host and viral proteins are crucial to the establishment of a viral infection. previous studies have found that through interacting with VP1, the human scavenger receptor class B member 2 (SCARB2) serves as a cellular receptor and mediates the endocytosis of EV71, allowing viral entry (25). Our study investigated beyond viral adsorption and delved further into the pathogenesis of EV71-associated neurological complications by unravelling any interaction between viral proteins and downstream host pathways. Specically, we speculated that the host peripheral myelin protein PMP22 might be a potential target of EV71 during infection, suggested by a positive correlation between the EV71 structural protein VP1 and PMP22 in mouse Schwann cells. Furthermore, our study has provided a possible mechanism responsible for the interaction between VP1 and PMP22. Our results have demonstrated that VP1 is able to drive up the expression of key elements in m 6 A RNA modication, thus upregulating the methylation of PMP22 and subsequently its expression in mouse Schwann cells. Subsequently, high levels of PMP22 promote autophagy in mouse Schwann cells, leading to Schwann cell dysfunction and death, disabling them from producing intact myelin sheaths. Demyelination impairs signal propagation along the axons and causes axons to deteriorate, leading to neurological problems such as neuronal necrosis and neuronophagia (26). Therefore, our proposed mechanism behind virus-induced demyelination of peripheral nerves may provide more insights in developing effective early interventions to the neurological complications among HFMD patients. repair (33, 34). During EV71 m 6 A modication modulates through an interaction between its key component METTL3 and a viral RNA polymerase (22). In the present study, we have shown that viral structural protein VP1 is also involved in m[anipulating m 6 A modications. We have also identied two host proteins targeted by VP1, METTL14 and YTHDF1, key members of the m 6 A pathway, and we have shown that VP1 overexpression could lead to the hypermethylation of PMP22. From our results, we are able to propose a possible mechanism of how PMP22 is upregulated during EV71 infection. Further experiments are needed to illustrate a more detailed mechanism of VP1 targeting on m 6 A. Considering the role of VP1 in manipulating autophagy, m 6 A methylation and PMP22 expression, we conclude that VP1 may be a potential therapeutic target for EV71-induced demyelinating damage. However, as VP1 has been shown to have other important functions in the viral life cycle, such as in the adsorption of the viral particle, it is likely that the VP1 also interferes with other host cellular pathways. In summary, our study has not only uncovered a potential therapeutic target specically for EV71 infections, but it has also improved our understanding of host-pathogen interactions in general. In the long run, we hope that the information that we are getting from this study could aid in developing better treatments for the early intervention of EV71-related neurological diseases.


Introduction
Hand, foot and mouth disease (HFMD) is a viral infection caused by enterovirus species A and it mainly affects children under 5 years of age. HFMD is usually self-limiting, but in the cases associated with enterovirus A71 (EV-A71 or EV71), patients may develop severe or even fatal neuro-systemic complications (1). EV71 is a highly neurotropic virus that causes severe neurological symptoms such as polio-like paralysis, spinal cord myelitis, brainstem encephalitis, etc. According to a recent study, EV71 infection accounts for at least 80% of the severe cases and 90% of deaths among HFMD patients in China (2). Autopsy in EV71-related deaths revealed neurophagy (neuronal autophagy) and demyelination (3)(4)(5), which were similar to the EV71-infected mice (6-9). However, the pathogenesis of these conditions remains unclear.
Myelin is a lipid-rich material that insulates the axons to increase the speed of the action potentials in neurons. During myelination, Schwann cells are precisely regulated to extend their membranes and wrap around axons, forming the multilayered, compact-membrane structures known as myelin sheaths (10). Myelin plays a key role in both the development of nervous system and the remyelination of damaged, demyelinated axons (10). Peripheral myelin protein 22 (PMP22) is a tetrameric transmembrane protein crucial to the development and maintenance of compact myelin, and it is expressed at high levels in the myelinating Schwann cells of the peripheral nervous system (10). A case study has reported that PMP22 is required for the normal functioning of cranial nerve motor neurons and spinal nerve sensory neurons during early development (11). Excessive accumulation of PMP22 affects protein homeostasis in cells, leading to Schwann cell dysfunction and death, as well as demyelination (12). Therefore, PMP22 needs to be tightly regulated to ensure proper myelination and therefore poses a potential therapeutic target of myelin disorders and neuron protection. Moreover, our group has previously noticed an astonishing symptomatic resemblance in patients diagnosed with PMP22 abnormality and EV71-associated neurological complications (13). We found that 80% of the EV71-associated neurological complications occurred in patients under ve years of age; early symptoms include cranial nerve malfunctions such as dysphagia and impaired eye movements, suggesting a possible link to abnormal PMP22 or aberrant myelination (13).
However, the mechanism of myelin damage in the extracranial cranial nerve in the early stage of EV71 infection has not been reported. In the early stage, our research group adopted the technical methods of Transmission Electron Microscopy (TEM), Quantitative real-time PCR (qPCR), Immuno uorescence, Western blot and autophagy marker showed that PMP22 expression and autophagy were enhanced in Mouse Schwann cells with VP1 overexpression, Inhibition of PMP22 expression eliminated autophagy enhancement, suggesting that PMP22 plays an important role in autophagy (14). Excessive accumulation of PMP22 affects the protein homeostasis in cells and leads to Schwann cell dysfunction and demyelination (15,16).
Since PMP22 seems to be one of the primary targets of EV71, it is important to understand the mechanism by which PMP22 can be affected. Studies have shown that PMP22 undergoes methylation on the RNA level and a hypermethylation can lead to the overexpression of PMP22 (17,18). N6-methyladenosine (m 6 A) is one of the most common post-transcriptional RNA modi cations in eukaryotic cells. M 6 A mainly involves three classes of proteins: 1) methylases, such as METTL3/14 and m6A-METTL-associated complex (MACOM), 2) Demethylases, such as FTO and ALKBH5, and 3) M6A methylation recognition protein, such as YTH (19,20). While m 6 A assists mRNA processing and transport, it has also been associated with the development of tumors and neurological disorders (21). Studies have also associated EV71 infection with RNA methylation and reported that the m 6 A modi cation complex modulates the replication of EV71 (22). However, it is not clear whether EV71 infection can alter the expression of key genes during m 6 A reversely. We hypothesized that EV71 infection could induce the hypermethylation of PMP22 in mouse Schwann cells (MSCs), increasing its gene dosage and resulting in the subsequent manipulation of downstream host cellular pathways.
The aim of this study was to investigate the pathogenesis of neurological complications associated to EV71 infection. We were especially interested in the development of neurophagy and demyelination in the fatal cases of EV71 infection. We speculated that the protein of our interest, the host peripheral myelin protein PMP22 was tightly linked to the autopsy-con rmed disease phenotypes. The overarching goal of this study was to uncover the correlations between EV71 infection, m 6 A methylation and PMP22 gene regulation. By understanding the mechanisms behind these molecular processes and the host-pathogen protein interactions in vitro, we hope that our study will aid in the development of a more effective HFMD treatment that can lower the risks of EV71-associated neurological complications and deaths.

Materials And Methods
Sampling and EV71 isolation EV71 was isolated from clinical specimens including throat and anal swabs and stools of a patient with HFMD due to EV71 infection, provided by the Center for Disease Control and Prevention of Guangdong Province (Guangzhou, China). The patient was diagnosed at Zhongshan School of Medicine, Sun Yat-sen University (Guangzhou, China). The study was approved by the ethics committee of the Guangzhou Medical University, Guangzhou Women and Children's Medical Center, IRB, Wuhan Institute of Virology, CAS (no. 2017122501, 2017 − 396 and WIVA07201904). All procedures performed involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Written informed consent was obtained from the legal guardians of the patient from whom EV71 was isolated for research purposes.
Trypan blue staining to detect cell viability was 90%, and the cell concentration was adjusted to 5×10 7 /ml for experiments.
Gene silencing by siRNA Genes of interest are targeted and silenced by siRNA. METTL14 and YTHDF1 siRNA (minic) are ordered from Santa Cruz Biotechnology., Inc. (Cat. #sc-42037, Santa Cruz, CA, USA) ( Table 1

Quantitative real-time PCR (qPCR)
The total RNA of the sample was extracted using TRE-Trizol (Invitrogen,Cat. #15596-026). The design of uorescence quantitative PCR primers is shown in Table 2. Complementary DNA (cDNA) was obtained by the reverse transcription of mRNA, which was performed in strict accordance with the instructions of TaKaRa PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa Bio Inc., Cat. # D6210A). The preparation and reaction parameters of the uorescent quantitative PCR reaction System were performed according to the instructions of the TaKaRa SYBR® Premix Ex Taq™ II (Perfect Real Time) kit (TaKaRa Bio Inc., Cat. # RR039B). Ampli cation and fusion curves of Real Time PCR were performed using ABI PRISM® 7500 Sequence Detection System (REAL-TIME PCR System). Fluorescence quantitative RT-PCR data was analysis using the SDSShell Software1.6 software, and the 2 − Δ Δ Ct method to calculate.

Statistical analysis
All experiments were repeated at least three times. data are expressed as mean ± standard error (SE).
Statistical signi cance was assessed by the Student's t test or one-way ANOVA with the Least Signi cance Difference (LSD) post hoc test, using the SPSS 16.0 statistical software (SPSS, IBM). P < 0.05 indicates statistical signi cance.

Results
Key elements involved in m 6 A are upregulated on both RNA and protein levels by VP1 As mentioned, PMP22 undergoes post-transcriptional methylation (17,23). Previous studies have shown that hypermethylation results in higher level of PMP22 (17,18), which may increase the risk of tumorigenesis and nervous system disease (21). Therefore, to understand the pathogenesis of EV71 infection, it is important to explore whether VP1 overexpression would lead to hypermethylation. To achieve this goal, we examined the expression of key genes involved in m 6 A modi cation in VP1-transfected mouse Schwann cells. qRT-PCR results showed that the gene expression levels of METTL3/14 and YTHDF1/2/3 were signi cantly increased. (Supplementary Table 1 To identify the proteins that are actively involved in m 6 A modi cation, EV71-VP1-overexpressing mouse Schwann cells were treated with an mRNA methylation inhibitor DAA (Fig. 3) (24). RT-PCR results showed that the gene expression of METTL 3/14 and YTHDF 1/2 decreased signi cantly after DAA treatment, even when VP1 is overexpressed (Supplementary Table 3, Fig. 3). These results further veri ed that METTL 3/14 and YTHDF 1/2 were key members of the methylation pathway that are affected by VP1.
Next, we focused on the two key elements of m 6 A to see whether inactivation of METTL14 or YTHDF1 alone could rescue the increase in PMP22 mediated by VP1. In the presence of VP1, the level of PMP22 increased in comparison to un-transfected and negative controls (Fig. 4). When expressions of METTL14 and YTHDF1 were suppressed by the targeting siRNA respectively, the level of PMP22 decreased, counteracting the effect of VP1 (Fig. 4).

Discussion
The interactions between host and viral proteins are crucial to the establishment of a viral infection. For instance, previous studies have found that through interacting with VP1, the human scavenger receptor class B member 2 (SCARB2) serves as a cellular receptor and mediates the endocytosis of EV71, allowing viral entry (25). Our study investigated beyond viral adsorption and delved further into the pathogenesis of EV71-associated neurological complications by unravelling any interaction between viral proteins and downstream host pathways. Speci cally, we speculated that the host peripheral myelin protein PMP22 might be a potential target of EV71 during infection, suggested by a positive correlation between the EV71 structural protein VP1 and PMP22 in mouse Schwann cells. Furthermore, our study has provided a possible mechanism responsible for the interaction between VP1 and PMP22. Our results have demonstrated that VP1 is able to drive up the expression of key elements in m 6 A RNA modi cation, thus upregulating the methylation of PMP22 and subsequently its expression in mouse Schwann cells. Subsequently, high levels of PMP22 promote autophagy in mouse Schwann cells, leading to Schwann cell dysfunction and death, disabling them from producing intact myelin sheaths. Demyelination impairs signal propagation along the axons and causes axons to deteriorate, leading to neurological problems such as neuronal necrosis and neuronophagia (26). Therefore, our proposed mechanism behind virus-induced demyelination of peripheral nerves may provide more insights in developing effective early interventions to the neurological complications among HFMD patients.
EV71 carries four structural proteins: VP1, VP2, VP3, and VP4. VP1 homodimers are the main component of the icosahedral viral capsid, which contributes to the pathogenicity and stability of EV71 and allows it to survive in the gastrointestinal tract (27,28). Our previous study found that VP1 overexpression induced autophagy in Schwann cells in mice. The autophagy pathway is most likely to be activated through the interaction between VP1 and a host protein, PMP22. Peripheral myelin protein PMP22 is an essential player in regulation of the autophagy pathway in Schwann cells. When PMP22 expression was suppressed by a targeting small interfering RNA, autophagy was downregulated, and mouse Schwann cells can maintain their integrity (14,29). The adverse effect of PMP22 accumulation has been described in other studies, where excessive PMP22 affects protein homeostasis in cells (12). However, whether autophagy plays a role in the pathogenesis of neurological complication remains a question. Autophagy plays dual roles in the nervous system. Excessive autophagy may be protective in chronic neurodegenerative diseases but detrimental in acute neural damages (30). EV71 can cause neuronal autophagy (4,31), while autophagy inhibition intervention can reduce viral infections, neurological symptoms and reduce mortality (32). Nevertheless, autophagy and PMP22 may still pose as interesting therapeutic targets, as their regulations could be tightly linked to the progression of EV71 infection. Further studies are needed to fully understand the effect of autophagy on EV71 infection as well as how it might be ne-tuned.
As PMP22 and autophagy may be closely related to the neurological disease phenotype during complications after EV71 infection, we were curious about the driving force of the PMP22 upregulation.
Studies have also shown that the expression of PMP22 is sensitive to gene doses; a slight increase in the methylation of PMP22 on the RNA level causes PMP22 to overexpress (17,18). N6-methyladenosine (m 6 A) modi cation has been associated to myelination, as well as EV71 replication in the host (22). Speci cally, m 6 A is involved in chromatin modi cation and the epigenetic controls of myelin sheath regeneration and axonal repair (33,34). During EV71 infection, m 6 A modi cation complex modulates viral replication through an interaction between its key component METTL3 and a viral RNA polymerase (22). In the present study, we have shown that viral structural protein VP1 is also involved in m[anipulating m 6 A modi cations. We have also identi ed two host proteins targeted by VP1, METTL14 and YTHDF1, key members of the m 6 A pathway, and we have shown that VP1 overexpression could lead to the hypermethylation of PMP22. From our results, we are able to propose a possible mechanism of how PMP22 is upregulated during EV71 infection. Further experiments are needed to illustrate a more detailed mechanism of VP1 targeting on m 6 A.
Considering the role of VP1 in manipulating autophagy, m 6 A methylation and PMP22 expression, we conclude that VP1 may be a potential therapeutic target for EV71-induced demyelinating damage. However, as VP1 has been shown to have other important functions in the viral life cycle, such as in the adsorption of the viral particle, it is likely that the VP1 also interferes with other host cellular pathways. In summary, our study has not only uncovered a potential therapeutic target speci cally for EV71 infections, but it has also improved our understanding of host-pathogen interactions in general. In the long run, we hope that the information that we are getting from this study could aid in developing better treatments for the early intervention of EV71-related neurological diseases.