Article 71 VP1 Protein Regulates Viral Replication in SH-SY5Y Cells via the mTOR Autophagy Signaling Pathway

Zi-Wei Liu 1, Zhi-Chao Zhuang 2, Rui Chen 1, Xiao-Rui Wang 1, Hai-Lu Zhang 1, Shu-Han Li 1, Zhi-Yu Wang 1 and Hong-Ling Wen 1,* 1 Key Laboratory for Infectious Disease Control and Prevention, Department of Microbiological Laboratory Technology, School of Public Health, Shandong University, 250012, ; [email protected] (Z.-W.L.); [email protected] (R.C.); [email protected] (X.-R.W.); [email protected] (H.-L.Z.); [email protected] (S.-H.L.); [email protected] (Z.-Y.W.) 2 Department of pathogenic microbiology, Tianjin Center for Disease Control and Prevention, Tianjin 300000, China; [email protected] * Correspondence: [email protected]

 Received: 14 November 2019; Accepted: 16 December 2019; Published: 20 December 2019 

Abstract: Background: Enterovirus 71 (EV71) is the main pathogen that causes severe hand, foot, and mouth disease with fatal neurological complications. However, its neurovirulence mechanism is still unclear. Candidate virulence sites were screened out at structural protein VP1, but the function of these candidate virulence sites remains unclear. Several studies have shown that autophagy is associated with viral replication. However, the relationship between VP1 and autophagy in human neurons has not been studied. Methods: A recombinant —SDLY107-VP1, obtained by replacing the VP1 full-length gene of the SDLY107 strain with the VP1 full-length gene of the attenuated strain SDJN2015-01—was constructed and tested for replication and virulence. We then tested the effect of the recombinant virus on autophagy in nerve cells. The effect of autophagy on virus replication was detected by western blot and plaque test. Finally, the changes of mTOR signaling molecules during EV71 infection and the effect of mTOR on virus replication at the RNA level were detected. Results: Viral recombination triggered virulence attenuation. The replication ability of recombinant virus SDLY107-VP1 was significantly weaker than that of the parent strain SDLY107. The SDLY107 strain could inhibit autophagic flux and led to accumulation of autophagosomes, while the SDLY107-VP1 strain could not cause autophagosome accumulation. The synthesis of EV71 RNA was inhibited by inhibiting mTOR. Conclusions: Replacement of VP1 weakened the replication ability of virulent strains and reduced the level of autophagy in nerve cells. This autophagy facilitates the replication of virulent strains in nerve cells. VP1 is an important neurovirulence determinant of EV71, which affects virus replication by regulating cell autophagy. mTOR is a key molecule in this type of autophagy.

Keywords: EV71; VP1; autophagy; virus replication

1. Introduction Enterovirus 71 (EV71), which belongs to the Picornaviridae family, is an icosahedral non-enveloped single-stranded RNA virus and is the second most important neurotropic enterovirus [1]. It causes hand, foot, and mouth disease (HFMD), which occurs mainly in young children below 4 years of age and usually presents as a self-limiting, mild febrile disease with symptoms including ulcers in the mouth, maculopapular rashes, or blister-like eruptions on the palms and soles [2]. EV71 has been increasingly associated with neurological disorders that range from aseptic meningitis with or without pulmonary edema to brain stem encephalitis and poliomyelitis-like acute flaccid paralysis, particularly

Viruses 2020, 12, 11; doi:10.3390/v12010011 www.mdpi.com/journal/viruses Viruses 2020, 12, 11 2 of 14 among infants. EV71-related hand, foot, and mouth disease outbreaks have been reported throughout the world, including in Singapore, Malaysia, Vietnam, Cambodia, China, Australia, and Japan [3–8]. Unfortunately, there are no effective prophylactic or therapeutic agents against EV71, although recent progress has been made. A large precursor protein is encoded by the RNA genome of EV71, which is then processed by viral proteases into structural (VP1, VP2, VP3, and VP4) and nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins [9]. The structural proteins constitute the viral capsid, and the nonstructural proteins participate in virus replication. Among the four structural proteins, VP1 is the most external, surface-accessible, and immunodominant protein among the capsid proteins [10]. Virus–receptor binding ability and virulence are affected by VP1 mutations, which even allow the virus to escape the host immune response [11–14]. EV71 VP1 protein activates calmodulin-dependent protein kinase II, which phosphorylates the N-terminal domain of vimentin on serine 82. Vimentin phosphorylation and rearrangement may enhance EV71 replication by playing structural roles in the formation of the replication factories [15]. In addition, human annexin II protein can bind to EV71 VP1 via VP1 amino acids 40–100, a region different from the known receptor-binding domain, thereby enhancing EV71 replication [16]. Effective cleavage of VP0 precursors is regulated by the amino acid at position 107 of VP1 during EV71 assembly. Virus growth kinetics are significantly reduced due to mutation of amino acid at position 107 of VP1 [17]. EV71 replication is inhibited by miR-2911, a honeysuckle-encoded atypical microRNA targeting the VP1 gene [18]. In conclusion, the VP1 protein plays an important role in the replication cycle of EV71. Autophagy is a degradative process that degrades protein aggregates and damages organelles. It is monitored by the conversion of the microtubule-associated protein 1A/B-light chain 3 (LC3) from its cytosolic resting state (LC3-I) to the lapidated form LC3-II. Protein p62 is considered a marker for autophagy-mediated protein degradation or autophagic flux. Many pathogens interact positively or negatively with the host autophagic pathway. The release of EV71 virions can be inhibited by inhibiting autophagy in RD cells [19]. Autophagy was induced in EV71-infected NSC-34 cells, which facilitated viral replication and non-lytic exit [20]. Mouse Schwann cell autophagy was promoted by EV71 VP1, which can up-regulate endoplasmic reticulum stress [21]. However, the relationship between VP1 and autophagy in human nerve cells has not been studied. The mammalian target of rapamycin (mTOR) is a key homeostatic regulator of cell growth, proliferation, survival, and metabolism via upregulating protein and lipid synthesis and inhibiting excessive autophagy. In mammalian cells, the phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is the primary pathway that regulates autophagy when cells are exposed to certain conditions, such as starvation, oxidative stress, infection, and tumor suppression. mTOR is closely related to autophagy induced by viral infection. Replication of the porcine epidemic diarrhea virus (PEDV) is promoted by rapamycin, an autophagy inducer that inhibits the mTOR signaling pathway [22]. Inhibition of mTOR by rapamycin promotes autophagy and viral mRNA replication, but does not affect VP1 expression during CVB3 infection [23]. Inhibition of mTOR promotes viral replication. However, the relationship between mTOR and EV71 replication is unclear. In this study, the recombinant virus SDLY107-VP1 obtained by replacing the full-length VP1 gene of the SDLY107 strain with the full-length VP1 gene of the attenuated strain SDJN2015-01 was constructed to explore the effect of VP1 on viral replication. On this basis, the relationship between VP1 and autophagy in viral replication was explored, and the relationship between EV71 replication and mTOR was further explored. This research provides new ideas for the study of the pathogenesis of EV71 and the development of antiviral drugs. Viruses 2020, 12, 11 3 of 14

2. Materials and Methods

2.1. Antibodies and Chemical Reagents The antibodies used in the study were anti-LC3B (Boston, USA, Cell Signaling Technology, Cat#2775), anti-p62 (Cambridge, UK, Abcam, 56416), anti-enterovirus 71 (Abcam 169442), anti-βactin (, China, ZSGB-BIO), anti-GAPDH (Wuhan, China, Proteintech, 60004), anti-mTOR (Cell Signaling Technology Cat#2983), anti-phospho-mTOR (Boston, USA, Cell Signaling Technology, Cat#5536), FICT-conjugated Affinipure Goat Anti-Mouse IgG (ZSGB-BIO), HPR-conjugated Affinipure Goat Anti-Rabbit IgG (Proteintech, SA-00001-2), and HPR-conjugated Affinipure Goat Anti-Mouse IgG (Proteintech, SA-00001-1). The anti-EV71 antibody used in the immunofluorescent assay was a mouse-derived polyclonal antibody made by our group [24]. Chemical reagents used in the study were rapamycin (Sigma, Germany, V900930), 3-Methyladenine (3-MA, Sigma, M9281) and chloroquine (CQ, Sigma, C6628).

2.2. Cell Lines and Viruses Rhabdomyosarcoma (RD) cells were provided by the Shandong Center for Disease Control (Purchased from ATCC, USA) and cultured in minimum essential medium (MEM, USA, Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) at 37 ◦C in the presence of 5% CO2. Human neuroblastoma (SH-SY5Y) cells provided by the Department of Biochemistry and Molecular Biology, School of Basic Medicine, Shandong University (Purchased from ATCC, USA) were cultured in MEM medium (Gibco) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco) at 37 ◦C in the presence of 5 % CO2. The severe EV71 strain SDLY107 was isolated from a fatal case of HFMD from Linyi City, Shandong Province, China in 2011 [25]. In vivo and in vitro experiments showed that this strain has faster replication and stronger pathogenicity compared to strains isolated from patients with a mild illness [26,27]. The full-length infectious cDNA clones of the SDLY107 strain (SDLY107RV) were successfully constructed using reverse genetics technology, and no significant differences in biological characteristics were observed between wild-type SDLY107 and rescued SDLY107RV [28]. The mild EV71 strain SDJN2015-01 was isolated from a child with mild hand, foot, and mouth disease and no neurological complications. The recombinant EV71 virus SDLY107-VP1 was rescued by chimeric infectious clone pMD19T-107-VP1, which was constructed by replacing the VP1 full-length fragment of SDLY107 with the VP1 gene of SDJN2015-01. The target fragment to be replaced was amplified by PCR and ligated to the vector to obtain a recombinant plasmid containing the DNA fragment of the VP1 sequence of the SDJN2015-01 strain. The recombinant plasmid and the full-length plasmid of pMD-19T-107 preserved in the laboratory were identified by restriction endonuclease Nru I and BsiW I for restriction endonuclease digestion. The DNA fragment containing the VP1 sequence of the SDJN2015-01 strain and the DNA fragment of the remaining sequence of the SDLY107 strain except the VP1 sequence were then recovered. The recovered products were transformed after T4 connection to obtain a full-length DNA fragment of SDLY107-VP1, which was identified and subjected to in vitro transcription to obtain the RNA of SDLY107-VP1. All strains were propagated in RD cells in MEM supplemented with 1% FBS. The virus titer was routinely determined by plaque assay. The virus suspension of different dilutions was inoculated on RD monolayer cells to make the virus adsorb onto the cells, and then covered with a layer of nutrient methylcellulose culture medium. When there was no new plaque, crystal violet fuel was added. Because dead cells cannot uptake crystal violet fuel, the associated plaque will be unstained. In theory, a plaque is formed by infection of a virus particle. The plaque morphology was observed and the number of plaques was counted and multiplied with the dilution multiple to get the virus titer.

2.3. Virus Infection SH-SY5Y cells were infected with SDLY107, SDJIN2015-01, and SDLY107-VP1 at a multiplicity of infection (MOI) of 1. They were then replenished with fresh MEM containing 1% FBS, which was Viruses 2020, 12, 11 4 of 14 used for the sham group after washing with PBS. For experiments that added inhibitors, cells were pre-treated with the inhibitors (dissolved in MEM or DMSO) for 2 h before viral infection. They were then washed with PBS and infected with the virus for 1 h, and fresh MEM containing 1% FBS for the sham group.

2.4. Replication Kinetics SH-SY5Y cells and RD cells were seeded at a density of 2 105 cells per mL in 12 well plates × and, when SH-SY5Y reached 70%–80% percent confluence, cells were infected with EV71 strains at MOI = 1. The cells and culture supernatant were sampled together every 12 h post infection. The corresponding viral load at 0 h was the amount of RNA in the prepared virus suspension. Total viral RNA was extracted using an RNA extraction kit (E.Z.N.A.®Viral RNA Kit, OMEGA, Guangzhou, China) according to the manufacturer’s instructions. The replication kinetics of SDLY107, SDLY107-VP1, and SDJN2015-01 were determined by real-time quantitative PCR (qRT-PCR) as described in a previous study [29]. Experiments were performed in triplicate, with three repeats for each experiment.

2.5. Lactate Dehydrogenase (LDH) Cytotoxicity Assay SH-SY5Y cells were seeded at a density of 2 105 cells per mL in 12 well plates and, when × SH-SY5Y reached 70%–80% percent confluence, cells were infected with EV71 strains at MOI = 1. LDH activity was detected using a Cytotoxicity Assay Kit (Shanghai, China, Beyotime,) according to the manufacturer’s protocol. The rate of cell injury was calculated by LDH content as follows: Rate absorbance of treatment group absorbance of control group − of cell injury (%) = absorbance of maximum enzyme activity group absorbance of control group . Experiments were performed in triplicate, with three repeats for each experiment.−

2.6. Drugs Used to Induce or Inhibit Autophagy A measure of 3-MA (50 µM, dissolved in MEM) was used to inhibit the early stage of autophagy. CQ (20 µM, dissolved in MEM) was used to inhibit autophagosome degradation, and rapamycin (10 nM, dissolved in DMSO) was used to promote degradation of autolysosome. Two h later, cells were washed three times and infected with the three strains of the virus. MEM containing 1% FBS was used for the sham group.

2.7. Detection of Autophagosomes, Viruses, and mTOR in Cells by Immunofluorescent Assay SH-SY5Y cells were seeded on glass coverslips at 70%–80%confluency and infected with EV71. The cells were fixed in 4% paraformaldehyde for 10 min at room temperature, and blocked with 5% bovine serum albumin in 0.1% Triton X-100 (diluted in PBS) for 2 h, followed by incubation with the primary antibody overnight at 4 ◦C. After washing three times, the samples were incubated with secondary antibodies for 60 min, and observed under a fluorescent microscope after three washes. Autophagosomes were detected using the CYTO-ID® Autophagy Detection kit (New York, USA, Enzo) in strict accordance with the instructions. The images were captured using a Carl Zeiss Microscope and processed using the software provided by the manufacturer.

2.8. Detection of Autophagy-Related Protein and Virus VP1 Protein by Western Blot SH-SY5Y cells exposed to various conditions were washed twice with ice-cold PBS, then lysed with RIPA lysis buffer (Beyotime) containing 0.1% phenylmethylsulfonyl fluoride (PMSF, Beyotime), and centrifuged at 15,000 rpm for 10 min at 4 ◦C. The samples were subsequently boiled and denatured. Next, 30 µg of cell protein samples were subjected to 10%–12% SDS polyacrylamide gel electrophoresis after measuring the protein concentration using the BCA Protein Assay kit (Beyotime Institute of Biotechnology, Shanghai, China), and transferred to 0.22 µm PVDF membranes (Millipore) and then blocked with 5% non-fat milk for 1 h (room temperature). The membranes were incubated with the primary antibody at 4 ◦C overnight, and incubated with horseradish-peroxidase-conjugated Viruses 2020, 12, 11 5 of 14 secondary antibodies for 60 min followed by washing. Finally, protein bands were detected using the ultra-sensitive multi-function imager.

3. Results

3.1.Viruses Construction 2019, 11, x FOR and PEER Rescue REVIEW of Recombinant Virus SDLY107-VP1 5 of 14

WeWe compared compared thethe VP1VP1 aminoamino acid sequences sequences of of the the SDLY107 SDLY107 and and SDJN2015-01 SDJN2015-01 strains, strains, and and the the results are shown in Figure1A. There were di fferences between the 146th (V I) and 147th results are shown in Figure 1A. There were differences between the 146th (V→I) and→ 147th (V→A) (V A) amino acids of SDLY107 and SDJN2015-01. To explore the role of VP1 mutations in EV71 amino→ acids of SDLY107 and SDJN2015-01. To explore the role of VP1 mutations in EV71 replication, replication,a recombinant a recombinant virus replacing virus replacing the VP1 the full-length VP1 full-length fragment fragment was constructed was constructed and andrescued. rescued. The Theconstruction construction schematic schematic diagram diagram ofof SDLY107-VP1 SDLY107-VP1 is is shown shown in in Figure Figure 11B–D.B–D. TheThe VP1 VP1 upstream upstream and and downstreamdownstream fragments fragments of of the the SDLY107 SDLY107 strain strain and and the the VP1 VP1 fragment fragment of of the the SDJN2015-01 SDJN2015-01 strain strain were were obtainedobtained by by PCR PCR reaction reaction (Figure (Figure2A). 2A). The The recombinant recombinant DNA DNA fragments fragments were obtainedwere obtained by fusion by fusion PCR usingPCR theusing three the DNAthree fragmentsDNA fragments as templates as templates (Figure (Figure2B), and 2B), linked and linked to the pMD-19Tto the pMD-19T vector vector for blue for andblue white and spotwhite screening. spot screening. The pMD-19T The pMD-19T plasmid plasmid containing containing the recombinant the recombinant DNA fragment DNA fragment and the full-lengthand the full-length pMD-19T-107 pMD-19T-107 plasmid were plasmid identified were by identifieddouble digestion by double with restrictiondigestion with endonucleases restriction Nruendonucleases I and BsiW Nru I (Figure I and2 C).BsiW The I (Figure DNA fragments 2C). The DNA containing fragments the VP1 containing sequence the of VP1 the sequence SDJN2015-01 of the strainSDJN2015-01 were recovered strain were and recovered the remaining and the fragments remaining of thefragments VP1 sequence of the VP1 of the sequence SDLY107 of the strain SDLY107 were removed.strain were The removed.recovered products The recovered were transformed products were after T4 transformed connection, after and the T4 recombinant connection, plasmid and the pMD-19T-107-VP1recombinant plasmid was successfully pMD-19T-107-VP1 obtained was and digested successfully by restriction obtained endonuclease and digested XbaI by and restriction Hind III.endonuclease The full-length XbaI DNA and fragment Hind III. of The SDLY107-VP1 full-length was DNA recovered fragment (Figure of SDLY107-VP12D) and transcribed was recoveredin vitro to(Figure obtain 2D) the RNAand transcribed of the recombinant in vitro virusto obtain SDLY107-VP1.The the RNA of the SDLY107 recombinant strain virus and theSDLY107-VP1.The SDLY107-VP1 strainSDLY107 were strain all identical and the except SDLY107-VP1 for the VP1 strain gene. were all identical except for the VP1 gene.

Figure 1. Schematic diagram of SDLY107-VP1. (A) The VP1 amino acid sequence comparison results of SDLY107Figure 1. and Schematic SDJN2015-01 diagram strains of SDLY107-VP1. are shown. (B )(A The) The upstream VP1 amino and downstreamacid sequence fragments comparison of VP1 results of SDLY107of SDLY107 strain and and SDJN2015-01 VP1 of SDJN2015-01 strains are strain shown. were (B used) The as upstream templates and for fusiondownstream PCR. The fragments obtained of recombinantVP1 of SDLY107 DNA strain fragments and VP1 were of linked SDJN2015-01 to the pMD-19T strain were vector used and as identified templates by for double fusion digestion. PCR. The (Cobtained) The full-length recombinant pMD-19T-107 DNA fragments plasmid were was identifiedlinked to the by doublepMD-19T digestion, vector and it contains identified a full-length by double genedigestion. including (C) theThe untranslatedfull-length pMD-19T-107 region (UTR) plasmid of SDLY107 was strain.identified (D) by DNA double fragments digestion, containing it contains the a VP1full-length sequence gene of the including SDJN2015-01 the untranslated strain and the region remaining (UTR) DNA of SDLY107 fragments strain. without (D the) DNA VP1 fragment fragments ofcontaining the SDLY107 the strainVP1 sequence were recovered, of the SDJN2015-01 then transformed strain and after the T4 remaining connection, DNA and fragments the recombinant without plasmidthe VP1 pMD-19T-107-VP1 fragment of the SDLY107 was successfully strain were obtained. recovered, then transformed after T4 connection, and the recombinant plasmid pMD-19T-107-VP1 was successfully obtained.

Viruses 2019, 11, x FOR PEER REVIEW 6 of 14 Viruses 2020, 12, 11 6 of 14 Viruses 2019, 11, x FOR PEER REVIEW 6 of 14

Figure 2. Recombinant virus SDLY107-VP1. (A) The PCR product of the 1430–2459 bp upstream fragment of VP1 of the SDLY107 strain, the 3308–3742 bp downstream fragment, and the VP1 FigureFigure 2. 2. RecombinantRecombinant virus virus SDLY107-VP1. SDLY107-VP1. (A (A) ) The The PCR PCR product product of of the the 1430–2459 1430–2459 bp bp upstream upstream fragment of the SDJN2015-01 strain. M is DL2000 DNA Marker; Lane 1 is upstream 1430–2459bp of fragmentfragment of of VP1 VP1 of of the the SDLY107 SDLY107 strain, strain, the 3308–3742 the 3308–3742 bp downstream bp downstream fragment, fragment, and the and VP1 fragmentthe VP1 VP1 of the SDLY107 strain, and the length is about 1029 bp; Lane 2 is VP1 fragment of the SDJN2015- fragmentof the SDJN2015-01 of the SDJN2015-01 strain. M isstrain. DL2000 M is DNA DL2000 Marker; DNA Lane Marker; 1 is upstream Lane 1 is 1430–2459upstream bp1430–2459bp of VP1 of theof 01 strain, and the length is about 891 bp; Lane 3 is downstream 3308–3742 bp of VP1 of the SDLY107 VP1SDLY107 of the strain,SDLY107 and strain, the length and the is aboutlength 1029 is about bp; Lane1029 2bp; is Lane VP1 fragment2 is VP1 fragment of the SDJN2015-01 of the SDJN2015- strain, strain, and the length is approximately 434 bp. (B) The fusion products of the 1430–2459 bp upstream 01and strain, the length and the is aboutlength 891 is about bp; Lane 891 3bp; is downstreamLane 3 is downstream 3308–3742 3308–3742 bp of VP1 bp of theof VP1 SDLY107 of the strain, SDLY107 and fragment, 3308–3742 bp downstream fragment and VP1 fragment of the SDJN2015-01 strain were strain,the length and the is approximately length is approximately 434 bp. (B )434 The bp. fusion (B) The products fusion ofproducts the 1430–2459 of the 1430–2459 bp upstream bp upstream fragment, obtained. M is a DL5000 DNA Marker; Lanes 1–3 are three DNA fragment fusion PCR products, and fragment,3308–3742 3308–3742 bp downstream bp downstream fragment and fragment VP1 fragment and VP1 of thefragment SDJN2015-01 of the SDJN2015-01 strain were obtained. strain were M is the length is about 2313 bp. (C) Plasmid pMD-19T-107 and pMD-19T-VP1 double digestion enzyme obtained.a DL5000 M DNA is a DL5000 Marker; DNA Lanes Marker; 1–3 are Lanes three DNA1–3 are fragment three DNA fusion fragment PCR products, fusion PCR and products, the length and is electrophoresis. M is DL1,0000 DNA Marker; Lane 1 is the product of the pMD-19T-107 plasmid after theabout length 2313 is bp. about (C) Plasmid2313 bp. pMD-19T-107(C) Plasmid pMD-19T-107 and pMD-19T-VP1 and pMD-19T-VP1 double digestion double enzyme digestion electrophoresis. enzyme Nru I and BsiW I digestion, the fragment length is about 8.1 kb and 2.0 kb; Lane 2 is the pMD-19T- electrophoresis.M is DL1,0000 DNA M is Marker;DL1,0000 Lane DNA 1 isMarker; the product Lane of1 is the the pMD-19T-107 product of the plasmid pMD-19T-107 after Nru plasmid I and BsiW after I VP1 plasmid after Nru I and BsiW I digestion. The product fragment length is approximately 3.0 kb Nrudigestion, I and theBsiW fragment I digestion, length the is fragment about 8.1 kblength and 2.0is about kb; Lane 8.1 2kb is and the pMD-19T-VP12.0 kb; Lane 2 plasmidis the pMD-19T- after Nru and 2.0 kb. (D) Plasmid pMD-19T-107-VP1 double digestion enzyme electrophoresis. M isD DL1,0000 VP1I and plasmid BsiW I digestion.after Nru I The and product BsiW I digestion. fragment lengthThe product is approximately fragment length 3.0 kb is and approximately 2.0 kb. ( ) Plasmid 3.0 kb pMD-19T-107-VP1DNA Marker; Lanes double 1 and digestion 2 are the enzyme product electrophoresis. of the pMD-19T-107-VP1 M is DL1,0000 plasmid DNA Marker;digested Lanes with 1XbaI and and 2.0 kb. (D) Plasmid pMD-19T-107-VP1 double digestion enzyme electrophoresis. M is DL1,0000 2and are HindIII, the product and ofthe the fragment pMD-19T-107-VP1 length is about plasmid 7.4 kb digested and 2.7 withkb. XbaI and HindIII, and the fragment DNA Marker; Lanes 1 and 2 are the product of the pMD-19T-107-VP1 plasmid digested with XbaI length is about 7.4 kb and 2.7 kb. and HindIII, and the fragment length is about 7.4 kb and 2.7 kb. 3.2. Recombinant Virus Biological Characteristics 3.2. Recombinant Virus Biological Characteristics 3.2. RecombinantSH-SY5Y cells Virus and Biological RD cells Characteristics were infected with the SDLY107 strain, SDLY107-VP1 strain, and SDJN2015-01SH-SY5Y strain. cells andVirus RD replication cells were curves infected were with analyzed the SDLY107 by detecting strain, the SDLY107-VP1 total viral RNA. strain, There and SH-SY5Y cells and RD cells were infected with the SDLY107 strain, SDLY107-VP1 strain, and SDJN2015-01were no significant strain. differences Virus replication among curvesthe replication were analyzed curves byof detectingthe three strains the total of viralvirus RNA. in RD There cells SDJN2015-01 strain. Virus replication curves were analyzed by detecting the total viral RNA. There were(Figure no significant 3A). However, differences the replication among the replication power of curves the recombinant of the three strains virus SDLY107-VP1 of virus in RD cells was were no significant differences among the replication curves of the three strains of virus in RD cells (Figuresignificantly3A). However, weaker than the replicationthat of the parent power strain of the SDLY107, recombinant especially virus SDLY107-VP1 between 24 h was and significantly 84 h (Figure (Figure 3A). However, the replication power of the recombinant virus SDLY107-VP1 was weaker3B). Replacement than that of of the VP1 parent gene strain was SDLY107, concluded especially to reduce between viral 24 replication h and 84 h (Figurein neurons.3B). Replacement Changes in significantly weaker than that of the parent strain SDLY107, especially between 24 h and 84 h (Figure ofamino VP1 acids gene was146 and concluded 147 were to reducerelated viralto the replication neurovirulence in neurons. of the Changes virus. SH-SY5Y in amino cells acids were 146 then and 3B). Replacement of VP1 gene was concluded to reduce viral replication in neurons. Changes in 147infected were with related the tothree the neurovirulencestrains. The rateof ofthe cell virus. injury SH-SY5Y was determined cells were by thenLDH infected at 24 h after with infection. the three amino acids 146 and 147 were related to the neurovirulence of the virus. SH-SY5Y cells were then strains.The rate The of cell rate injury of cell of injury the recombinant was determined virus by SDLY107-VP1 LDH at 24 h after was infection.significantly The weaker rate of cellthan injury that of infected with the three strains. The rate of cell injury was determined by LDH at 24 h after infection. the recombinant parent strain virus SDLY107 SDLY107-VP1 (Figure was 3C); significantly replacement weaker of VP1 than fragments that of the attenuated parent strain the ability SDLY107 of The rate of cell injury of the recombinant virus SDLY107-VP1 was significantly weaker than that of (Figurevirulent3 C);strains replacement to cause ofcell VP1 injury. fragments attenuated the ability of virulent strains to cause cell injury. the parent strain SDLY107 (Figure 3C); replacement of VP1 fragments attenuated the ability of virulent strains to cause cell injury. B A C

RD Cell B SH-SY5Y Cell 50 SH-SY5Y Cell A C SDLY107 8 6 SDLY107-VP1 SDJN2015-01 * * SDJN2015-01 40 * ** SDJN2015-01 RD Cell * ** SDLY107-VP1 7 SDLY107-VP1 SH-SY5Y Cell 5030 SH-SY5Y Cell SDLY107 SDLY107 4 SDLY107 8 6 SDLY107-VP1 6 SDJN2015-01 * * * SDJN2015-01 4020 * ** * ** SDJN2015-01 7 SDLY107-VP1 SDLY107-VP1 5 3010 SDLY107 2 SDLY107 4 Rate cellof injury(%) 6 * 20 4 0 7 1 0 P -01 5 10 1 5 3 20 01 Rate Rate cellof injury(%) DLY 2 S h h h h Y107-V 0h 12h 24h 36h 48h 60h 72h 0h 4h 8h 0h L DJN 12h 24h 36 48 60 72h 8 96 0 S 4 viral load(log copies/µl ofviral RNA) 10 12 SD viralload (log copies/µl of viral RNA) 7 1 0 P -01 Infection time Infection time 1 Infected strain 5 0 01 3 DLY 2 S Y107-V h h h h L 0h 12h 24h 36h 48h 60h 72h 0h 4h 8h 0h DJN 12h 24h 36 48 60 72h 8 96 S viral load(log copies/µl ofviral RNA) 10 12 SD viralload (log copies/µl of viral RNA) FigureInfection 3. time(A)The replication curves of theInfection three time strains on rhabdomyosarcomaInfected (RD) strain cells. (B) The replicationFigure 3. (A curves)The replication of the three curves strains of on the SH-SY5Y three strains cells. on (C )Therhabdomyosarcoma rate of cell injury (RD) of SH-SY5Y cells. (B) cellsThe infectedreplication with curves the three of the strains three at 24strains h. The on data SH-SY5Y represent cells. means (C)The+ SD rate from of threecell injury experiments. of SH-SY5Y* p < 0.05cells, Figure 3. (A)The replication curves of the three strains on rhabdomyosarcoma (RD) cells. (B) The ** p < 0.001 compared with the SDLY107-VP1 treated group. replication curves of the three strains on SH-SY5Y cells. (C)The rate of cell injury of SH-SY5Y cells

Viruses 2019, 11, x FOR PEER REVIEW 7 of 14

Viruses 2020, 12, 11 7 of 14 infected with the three strains at 24 h. The data represent means + SD from three experiments. *p < 0.05, **p < 0.001 compared with the SDLY107-VP1 treated group. 3.3. Autophagic Flux in SH-SY5Y Cells Is Inhibited by EV71 VP1 Protein 3.3. Autophagic Flux in SH-SY5Y Cells is Inhibited by EV71 VP1 Protein To explore the mechanism by which the VP1 gene affects the replication ability of virus in nerve cells,To autophagy, explore the which mechanism has been by proven which to the be VP1 related gene to viralaffects replication, the replication was quantitativelyability of virus analyzed. in nerve SH-SY5Ycells, autophagy, cells were which infected has with been the three proven strains. to be Cell related lysates to were viral collected replication, at 6 h, was 9 h, 12quantitatively h, and 24 h. Uninfectedanalyzed. SH-SY5Y cells served cells as were the shaminfected control. with the LC3 three and p62strains. were Cell examined lysates were by western collected blot at analysis.6 h, 9 h, SDLY10712 h, and strain 24 h. infectionUninfected resulted cells served in an increase as the sham in the control. LC3 and LC3 p62 and content, p62 were but the examined SDJN2015-01 by western strain andblot SDLY107-VP1analysis. SDLY107 strain strain did notinfection cause significantresulted in changesan increase in LC3in the and LC3 p62 and levels p62 (Figure content,4A,C,D). but the ThisSDJN2015-01 indicated strain that SDLY107and SDLY107-VP1 could inhibit strain autophagic did not cause flux significant in SH-SY5Y changes cells. in TheLC3 SDLY107-VP1and p62 levels and(Figure SDJN2015-01 4A,C,D). This strains indicated did not that have SDLY107 a significant could impactinhibit autophagic on autophagic flux fluxin SH-SY5Y in SH-SY5Y cells. cells. The FigureSDLY107-VP14B and 4E and shows SDJN2015-01 the fluorescent strains staining did not of have autophagosomes a significant impact in SH-SY5Y on autophagic cells. The numberflux in SH- of autophagosomesSY5Y cells. Figure in 4B SH-SY5Y and 4E cellsshows infected the fluorescent with SDLY107 staining strain of increasedautophagosomes with the in prolongation SH-SY5Y cells. of infectionThe number time. of However, autophagosomes the number in SH-SY5Y of autophages cells infected did not increasewith SDLY107 significantly strain afterincreased infection with with the SDLY107-VP1prolongation of and infection SDJN2015-01 time. strains.However, This the indicated number thatof autophages SDLY107 could did not lead increase to the accumulation significantly ofafter autophagosomes, infection with SDLY107-VP1 which is beneficial and SDJN2015-01 for viral replication. strains. This The replacementindicated that of SDLY107 VP1 attenuated could lead the inhibitionto the accumulation of autophagic of autophagosomes, flow by virulent strainswhich inis beneficial SH-SY5Y cells.for viral replication. The replacement of VP1 attenuated the inhibition of autophagic flow by virulent strains in SH-SY5Y cells.

Figure 4. Cont.

VirusesViruses 20202019,, 1211,, 11x FOR PEER REVIEW 8 of 14

Figure 4. (A) Western blot assay for the effect of recombinant virus SDLY107-VP1 strain, parental virus SDLY107Figure 4. strain,(A) Western and SDJN2015-01 blot assay for strain the on effect autophagy. of recombinant SH-SY5Y virus cells SDLY107-VP1 were infected withstrain, EV71. parental Cell lysatesvirus SDLY107 were collected strain, at and 6 h, SDJN2015-01 9 h, 12 h, and strain 24 h. Uninfected on autophagy. cells SH-SY5Y served as cells the sham were control. infected LC3 with and EV71. p62 wereCell lysates examined were by collected western at blot. 6 h, ( B9) h, The 12 eh,ff ectsand of24 theh. Uninfected recombinant cells virus served SDLY107-VP1 as the sham strain, control. parental LC3 virusand p62 SDLY107 were examined strain, and by western SDJN2015-01 blot. ( strainB) The oneffects autophagy of the recombinant were detected virus by SDLY107-VP1 immunofluorescent strain, staining.parental Hoechst virus SDLY107 33342 blue strain, fluorescent and dye SDJN2015-01 stains the cell strain nucleus on blue, autophagy and Cyto ID were green detected fluorescent by dyeimmunofluorescent stains autophagous staining. bodies Hoechst green 33342 during blue autophagy. fluorescent (C )dye The stains content the ofcell p62 nucleus protein blue, in SH-SY5Yand Cyto cellsID green infected fluorescent with the dye three stains viruses. autophagous (D) The bodies content green of LC3 during protein autophagy. in SH-SY5Y (C) cellsThe content infected of with p62 theprotein three in viruses.SH-SY5Y (E cells) The infected number with of autophagesthe three viruses. in SH-SY5Y (D) The cells content infected of LC3 with protein the three in SH-SY5Y viruses. Autophagouscells infected with bodies the in three each viruses. cell were (E) counted, The number 100 cellsof autophages per sample. in TheSH-SY5Y data representcells infected means with+ theSD fromthree threeviruses. experiments. Autophagous * p bodies< 0.05, in compared each cell were with thecounted, mock 100 group; cells # perp < sample.0.001, compared The data represent with the mockmeans group; + SD from ** p < three0.05, experiments. compared with *p < the 0.05, SDLY107-VP1-treated compared with the group.mock group; #p < 0.001, compared with the mock group; ** p < 0.05, compared with the SDLY107-VP1-treated group. 3.4. VP1 Protein Affects Virus Replication Associated with Cell Autophagy

3.4. VP1We treatedProtein cellsAffects with Virus 3-MA Replication 50 µM, CQAssociated 20 µM, andwith rapamycin10Cell Autophagy nM, and the levels of p62 and LC3 in cells were measured after 24 h. The 3-MA caused an increase in intracellular p62 levels and a decrease We treated cells with 3-MA 50 μM, CQ 20 μM, and rapamycin10 nM, and the levels of p62 and in LC3 levels (Figure5A,B). CQ led to an increase in intracellular p62 and LC3 levels (Figure5C,D). LC3 in cells were measured after 24 h. The 3-MA caused an increase in intracellular p62 levels and a However, rapamycin caused a decrease in intracellular p62 and LC3 levels (Figure5E,F). Our results decrease in LC3 levels (Figure 5A,B). CQ led to an increase in intracellular p62 and LC3 levels (Figure indicated that 3-MA can inhibit the formation of autophagosomes, CQ can inhibit autophagic flux and 5C,D). However, rapamycin caused a decrease in intracellular p62 and LC3 levels (Figure 5E,F). Our lead to the accumulation of autophagosomes, while rapamycin can promote anautophagic flux and results indicated that 3-MA can inhibit the formation of autophagosomes, CQ can inhibit autophagic accelerate the degradation of autophagosomes. In order to explore the relationship between autophagy flux and lead to the accumulation of autophagosomes, while rapamycin can promote anautophagic and viral replication, SH-SY5Y cells were treated with 3-MA, CQ, and rapamycin for 2 h and then flux and accelerate the degradation of autophagosomes. In order to explore the relationship between infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. Cell lysates were harvested after autophagy and viral replication, SH-SY5Y cells were treated with 3-MA, CQ, and rapamycin for 2 h 24h. LC3, p62, and capsid protein VP1 levels were detected by western blot. The culture supernatant and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. Cell lysates were was collected after 24 h and subjected to plaque assay to determine the viral titer. After infection with harvested after 24h. LC3, p62, and capsid protein VP1 levels were detected by western blot. The SDLY107 and SDLY107-VP1, the virus content in cell and culture supernatants decreased, and the culture supernatant was collected after 24 h and subjected to plaque assay to determine the viral titer. overall replication level of virus decreased in the rapamycin-treated group. In the 3-MA-treated group, After infection with SDLY107 and SDLY107-VP1, the virus content in cell and culture supernatants the virus content in cells did not change significantly, but the virus content in culture supernatant decreased, and the overall replication level of virus decreased in the rapamycin-treated group. In the decreased, and the overall replication level of the virus decreased. However, in the CQ-treated group, 3-MA-treated group, the virus content in cells did not change significantly, but the virus content in the virus content in cells and culture supernatant increased and the overall replication level of virus culture supernatant decreased, and the overall replication level of the virus decreased. However, in increased (Figure6A,B). Unfortunately, due to the weak replication ability of SDJN2015-01 in nerve the CQ-treated group, the virus content in cells and culture supernatant increased and the overall cells, no obvious VP1 protein band was detected by western blotting except in the CQ treatment group, replication level of virus increased (Figure 6A,B). Unfortunately, due to the weak replication ability but the change of virus titer in the culture supernatant was the same as that of the other two viruses. of SDJN2015-01 in nerve cells, no obvious VP1 protein band was detected by western blotting except The results showed that the accumulation of autophagosomes is beneficial to virus replication in in the CQ treatment group, but the change of virus titer in the culture supernatant was the same as SH-SY5Y cells. The replication ability of the SDLY107 strain was stronger than that of the replacement that of the other two viruses. The results showed that the accumulation of autophagosomes is strain SDLY107-VP1 because the SDLY107 strain can lead to the accumulation of autophagosomes. The beneficial to virus replication in SH-SY5Y cells. The replication ability of the SDLY107 strain was replacement of VP1 results in the loss of ability of virulent strain to inhibit autolysosome degradation. stronger than that of the replacement strain SDLY107-VP1 because the SDLY107 strain can lead to the accumulation of autophagosomes. The replacement of VP1 results in the loss of ability of virulent strain to inhibit autolysosome degradation.

Viruses 2019, 11, x FOR PEER REVIEW 9 of 14

Viruses 2020, 12, 11 9 of 14 Viruses 2019, 11, x FOR PEER REVIEW 9 of 14

Figure 5. Cells were treated with 3-MA 50 μM, CQ 20 μM, and rapamycin 10 nM. After 2 h, cells were washed three times, and the levels of p62 and LC3 in the cells were measured after 24 h. (A) Western blot detects the effect of 3-MA on autophagy in SH-SY5Y cells. (B) Changes in intracellular p62 and LC3 levels in SH-SY5Y cells after 3-MA treatment of cells. (C) Western blot detects the effect of CQ on autophagy in SH-SY5Y cells. (D) Changes in intracellular p62 and LC3 levels in SH-SY5Y cells after CQ treatment of cells. (E) Western blot detects the effect of rapamycin on autophagy in SH-SY5Y cells. (F) Changes in intracellular p62 and LC3 levels in SH-SY5Y cells after rapamycin treatment of cells. The data represent means + SD from three experiments, **p < 0.001, compared with the control group.

3.5. Autophagy Affects the Survival of Nerve Cells Infected with EV71 SH-SY5Y cells were treated with 3-MA, CQ, and rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. The rate of cell injury was detected by LDH at 24 Figureh after 5.infection.Cells were After treated infection with 3-MA with 50 threeµM, CQ strains, 20 µM, the and rate rapamycin of cell injury 10 nM. was After higher 2 h, cells in were the CQ- Figure 5. Cells were treated with 3-MA 50 μM, CQ 20 μM, and rapamycin 10 nM. After 2 h, cells were treatedwashed group three than times, in the and controlthe levels group, of p62 andwhereas LC3 in the the effect cells were of the measured rapamycin after 24 treatment h. (A) Western was the washed three times, and the levels of p62 and LC3 in the cells were measured after 24 h. (A) Western opposite.blot detectsThe cell the injury effect ofrate 3-MA of the on autophagy3-MA treatment in SH-SY5Y group cells. was (B not) Changes significantly in intracellular different p62 from and that blot detects the effect of 3-MA on autophagy in SH-SY5Y cells. (B) Changes in intracellular p62 and of theLC3 control levels group in SH-SY5Y (Figure cells 6C). after This 3-MA suggests treatment that of cells.the accumulation (C) Western blot of detectsautophagosomes the effect of CQincreased on LC3 levels in SH-SY5Y cells after 3-MA treatment of cells. (C) Western blot detects the effect of CQ on autophagy in SH-SY5Y cells. (D) Changes in intracellular p62 and LC3 levels in SH-SY5Y cells after the cellautophagy injury rate.in SH-SY5Y The difference cells. (D) Changes of cell injuryin intracellular ability betweenp62 and LC3 the levels SDLY107 in SH-SY5Y and SDLY107-VP1 cells after CQ treatment of cells. (E) Western blot detects the effect of rapamycin on autophagy in SH-SY5Y cells. strainsCQ in treatment SH-SY5Y of cells cells. was (E) Western related blotnot detectsonly to the viral effect replication of rapamycin but on also autophagy to autophagy; in SH-SY5Y the SDLY107 cells. (F) Changes in intracellular p62 and LC3 levels in SH-SY5Y cells after rapamycin treatment of cells. strain( Fand) Changes the SDLY107-VP1 in intracellular strain p62 and were LC3 identical levels in except SH-SY5Y for cells the afterVP1 rapamycingene. Therefore, treatment VP1 of regulatescells. The data represent means + SD from three experiments, ** p < 0.001, compared with the control group. viral replicationThe data represent and cell means injury + SD by from affecting three experiments,the accumulation **p < 0.001, of autophages. compared with the control group. A B C 3.5. Autophagy Affects the Survival of Nerve Cells Infected with EV71 SH-SY5Y cells were treated with 3-MA, CQ, and rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. The rate of cell injury was detected by LDH at 24 h after infection. After infection with three strains, the rate of cell injury was higher in the CQ- treated group than in the control group, whereas the effect of the rapamycin treatment was the

1 1 1 P 01 opposite. 7The cell injury0 rate of the 3-MA treatment group was not significantly differentV 5- from that 0 P - 1 1 -V 5 Y 7 1 07- L 0 0 D 1 2 SDLY107 S Y N L J of the controlD groupD (Figure 6C). This suggests that the accumulation of autophagosomesSDLY1 SDJN20 increased S S the cell injury rate. The difference of cell injury ability between the SDLY107 and SDLY107-VP1 strainsFigure in SH-SY5Y 6. (A) Intracellular cells was related VP1 protein not only content. to viral SH-SY5Y replication cells but were also treated to autophagy; with 3-MA, the CQ, SDLY107 or Figure 6. (A) Intracellular VP1 protein content. SH-SY5Y cells were treated with 3-MA, CQ, or strainrapamycin and the SDLY107-VP1 for 2 h and then infectedstrain were with identical SDLY107, SDLY107-VP1,except for the and VP1 SDJN2015-01, gene. Therefore, respectively. VP1 regulates Cell rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. viral lysatesreplication were harvestedand cell injury after 24 by h. affecting LC3, p62, the and accumulation capsid protein of VP1 autophages. levels were detected by western Cell lysates were harvested after 24 h. LC3, p62, and capsid protein VP1 levels were detected by blot. (B) Virus titer of culture supernatant. SH-SY5Y cells were treated with 3-MA, CQ, or rapamycin Awestern blot. (B) Virus titer of culture B supernatant. SH-SY5Y cells were treatedC with 3-MA, CQ, or for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. After 24 h, the rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. cell culture supernatant was harvested and the culture supernatant titer was measured by plaque assay. After 24 h, the cell culture supernatant was harvested and the culture supernatant titer was measured (C)Effect of autophagy on cell injury. SH-SY5Y cells were treated with 3-MA, CQ, or rapamycin for 2 by plaque assay. (C) Effect of autophagy on cell injury. SH-SY5Y cells were treated with 3-MA, CQ, h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. After 24 h, LDH or rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. activity was assayed using the Cytotoxicity Assay Kit according to the manufacturer’s protocol. Rapa

stands for rapamycin. The data represent means +SD from three experiments. * p < 0.05, compared

1 1 1 P 01 7 0 V 5- with the0 EV71-treatedP - group; # p < 0.05, compared with the DMSO-treated group. 1 1 -V 5 Y 7 1 07- L 0 0 D 1 2 SDLY107 S Y N L J D D SDLY1 SDJN20 3.5. AutophagyS AffectsS the Survival of Nerve Cells Infected with EV71

SH-SY5Y cells were treated with 3-MA, CQ, and rapamycin for 2 h and then infected with SDLY107, Figure 6. (A) Intracellular VP1 protein content. SH-SY5Y cells were treated with 3-MA, CQ, or SDLY107-VP1, and SDJN2015-01, respectively. The rate of cell injury was detected by LDH at 24 h after rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively. infection. After infection with three strains, the rate of cell injury was higher in the CQ-treated group Cell lysates were harvested after 24 h. LC3, p62, and capsid protein VP1 levels were detected by than in the control group, whereas the effect of the rapamycin treatment was the opposite. The cell western blot. (B) Virus titer of culture supernatant. SH-SY5Y cells were treated with 3-MA, CQ, or injuryrapamycin rate of the for 3-MA 2 h and treatment then infected group with was SDLY107, not significantly SDLY107-VP1, different and fromSDJN2015-01, that of the respectively. control group (FigureAfter6C). 24 This h, the suggests cell culture that supernatant the accumulation was harvested of autophagosomes and the culture increasedsupernatant the titer cell was injury measured rate. The by plaque assay. (C) Effect of autophagy on cell injury. SH-SY5Y cells were treated with 3-MA, CQ, or rapamycin for 2 h and then infected with SDLY107, SDLY107-VP1, and SDJN2015-01, respectively.

Viruses 2020, 12, 11 10 of 14

diVirusesfference 2019 of, 11 cell, x FOR injury PEER ability REVIEW between the SDLY107 and SDLY107-VP1 strains in SH-SY5Y 10cells of 14 was related not only to viral replication but also to autophagy; the SDLY107 strain and the SDLY107-VP1 strain wereAfter 24 identical h, LDH activity except was for assayed the VP1 using gene. the Therefore, Cytotoxicity VP1 Assay regulates Kit according viral to replication the manufacturer’s and cell injury by affectingprotocol. the Rapa accumulation stands for rapamycin. of autophages. The data represent means +SD from three experiments. *p < 0.05, compared with the EV71-treated group; # p < 0.05, compared with the DMSO-treated group. 3.6. mTOR Is a Key Molecule Affecting EV71 Replication in Nerve Cells 3.6. mTOR is a Key Molecule Affecting EV71 Replication in Nerve Cells mTOR signaling is generally involved in regulating cell survival, cell growth, cell metabolism, proteinmTOR synthesis, signaling and is autophagy, generally involved as well as in homeostasis regulating cell [30 survival,]. SH-SY5Y cell cellsgrowth, were cell infected metabolism, with the threeprotein strains, synthesis, cell lysates and autophagy, were harvested, as well as and homeostasis mTOR/p-mTOR [30]. SH-SY5Y was detected cells were by infected western with blot. the The SDLY107three strains, strain cell significantly lysates were reduced harvested, p-mTOR and content;mTOR/p-mTOR however, was no similardetected results by western were observedblot. The for SDLY107 strain significantly reduced p-mTOR content; however, no similar results were observed the other two strains (Figure7A). VP1 replacement attenuated the ability of virulent strains to inhibit for the other two strains (Figure 7A). VP1 replacement attenuated the ability of virulent strains to mTOR phosphorylation, which is beneficial to autophagy. This autophagy facilitates the replication of inhibit mTOR phosphorylation, which is beneficial to autophagy. This autophagy facilitates the virulent strains in nerve cells. VP1 affects virus replication via autophagy related to mTOR signaling replication of virulent strains in nerve cells. VP1 affects virus replication via autophagy related to molecules. We selected the SDLY107 strain to infect SH-SY5Y cells, and found by immunofluorescence mTOR signaling molecules. We selected the SDLY107 strain to infect SH-SY5Y cells, and found by testimmunofluorescence that EV71 virus and test mTORthat EV71 can virus co-locate and mTOR (Figure can7B). co-locate This also (Figure indicates 7B). This that also there indicates could be interactionthat there could between be interaction the EV71 virusbetween and the mTOR EV71 virus molecule. and mTOR Subsequently, molecule. SH-SY5YSubsequently, cells SH-SY5Y pre-treated withcells rapamycin pre-treated (10 with nM) rapamycin were infected (10 nM) with were SDLY107, infected and with the SDLY107, viral capsid and protein the viral VP1 capsid was protein examined byVP1 western was examined blot and intracellularby western blot viruses and wereintracellular detected viruses by immunofluorescent were detected by immunofluorescent assay. The amount of totalassay. RNA The and amount RNA of in total the cellRNA culture and RNA supernatant in the cell was culture detected supernatant by real-time was detected fluorescent by quantitativereal-time PCR.fluorescent Inhibition quantitative of mTOR PCR. by rapamycin, Inhibition of which mTOR can by rapamycin, promote the which degradation can promote of autolysosomes, the degradation can restrainof autolysosomes, viral RNA synthesiscan restrain and viral block RNA viral synthesis protein and VP1 block expression viral protein caused VP1 by expression the SDLY107 caused infection by (Figurethe SDLY1077C–E). infection (Figure 7C–E).

Figure 7. (A) SH-SY5Y cells were infected with the three strains. Cell lysates were collected at 24 h. Figure 7. (A) SH-SY5Y cells were infected with the three strains. Cell lysates were collected at 24 h. mTOR and p-mTOR levels were detected by western blot. (B) EV71 and mTOR were detected by mTOR and p-mTOR levels were detected by western blot. (B) EV71 and mTOR were detected by co- co-localization immunofluorescence at 24 h after infection. FICT stained EV71 green, TRITC stained localization immunofluorescence at 24 h after infection. FICT stained EV71 green, TRITC stained mTOR red, and DAPI stained nucleus blue. (C) SH-SY5Y cells were seeded on a six well plate and mTOR red, and DAPI stained nucleus blue. (C) SH-SY5Y cells were seeded on a six well plate and exposedexposed to to rapamycin rapamycin or or not not before before being being infected infected withwith EV71.EV71. TheThe viralviral VP1 protein was examined by 5 western blot after 24 h. (D) SH-SY5Y cells were inoculated into a 12 well plate with about 2 10 cells5 by western blot after 24 h. (D) SH-SY5Y cells were inoculated into a 12 well plate with about× 2 × 10 percells well per and well exposed and exposed to rapamycin to rapamycin or not or before not before being being infected infected with with EV71. EV71. Immunofluorescent Immunofluorescent assay wasassay used was to used detect to the detect intracellular the intracellular virus content virus content after 24 after h. (E 24) SH-SY5Y h. (E) SH-SY5Y cells were cells inoculated were inoculated onto a 12 wellonto plate a 12 andwell exposedplate and to exposed rapamycin to rapamycin or not before or not being before infected being infected with EV71, with EV71, and the and amount the amount of viral RNAof viral was RNA measured was measured by real-time by real-time fluorescent fluorescent quantitative quantitative PCR after PCR 24 h. after Rapa 24 standsh. Rapa for stands rapamycin. for Therapamycin. data represent The data means represent+ SD means fromthree + SD experiments,from three experiments, ** p < 0.001, **p compared < 0.001, compared with the with rapamycin the treatmentrapamycin group. treatment group.

Viruses 2020, 12, 11 11 of 14

4. Discussion A number of virulence determinants for EV71 have been reported, which may influence virus assembly, attachment, replication, or cell tropism. However, the mechanism by which VP1 affects viral replication and virulence has not been elucidated. In this study, a recombinant virus with the VP1 fragment replaced was successfully constructed. The SDLY107 strain and the SDLY107-VP1 strain were identical except for the VP1 gene. Replacement of the VP1 fragment altered viral replication. Increased replication capacities of EV71 strains have previously been associated with increased disease severity in animal models, and positive correlations have been found between virus titers in the respiratory tract and disease severity in humans [29,31,32]. Therefore, the VP1 protein is closely related to the virulence and pathogenicity of the virus. A variety of viruses can manipulate autophagy to assist their replication [33]. Autophagy is induced in host cells by viruses, including human cytomegalovirus, hepatitis C virus, herpes simplex virus-1, CVB3, influenza A virus, HIV-I, and EV71 [34–40]. EV71 replication induced the formation of autophagosomes and increased disease severity and viral titer [41]. Virus-containing autophagic vacuoles were isolated from the culture supernatant which suggests that autophagy contributed to the release and spread of the virus [20]. Our research demonstrated that SDLY107could inhibit autophagic flux in SH-SY5Y cells and lead to the accumulation of autophagosomes. However, replacement of VP1 directly led to decreased autophagosome accumulation and reduced viral replication. The accumulation of autophagosomes is beneficial to virus replication. In this study, the viral titer and the rate of cell injury were decreased when autophagic flux was promoted by rapamycin (Figure6). Host defense is another important role of autophagy in addition to maintaining cell homeostasis. Autophagy improves host defense mechanisms through several biological functions [42–44]. The role of autophagy for host defense against viruses strongly depends on the type of infection and involves several mechanisms, especially those involved in autophagosome generation and maturation. RNA viruses seem to stabilize autophagosomes by preventing their degradation. The human immunodeficiency virus type-1 (HIV) blocks autophagosome maturation in infected macrophages [45]. The degradation of autophagosomes and the elimination of the invading pathogens are accelerated when autophagy induced by a virus is promoted. Therefore, the virus titer and cell injury rate were reduced after treatment with rapamycin, but the opposite result was obtained when autophagosome degradation was inhibited (Figure6). Although EV71 infection can induce autophagy in vivo or in vitro, its exact mechanism is unclear. The PI3K/Akt/mTOR pathway plays a vital role in the regulation of autophagy, and suppressing mTOR activity leads to the initiation of autophagy and the formation of autophagosomes [46]. This study demonstrated that VP1 replacement attenuates the ability of virulent strains to inhibit mTOR phosphorylation, which is beneficial to autophagy. The formation and accumulation of autophagosomes are beneficial to the replication of the virulent strains in nerve cells. However, inhibition of mTOR activation not only leads to the initiation of autophagy and the formation of autophagosomes, but also promotes the degradation of autolysosomes. The cell injury rate, intracellular viral VP1 protein content, and culture supernatant titer were reduced after cells were treated with rapamycin, which could promote the degradation of autolysosomes. The synthesis of viral RNA was inhibited by rapamycin. In summary, the study demonstrated that replacement of EV71 VP1 resulted in decreased replication of EV71 in SH-SY5Y cells by affecting cell autophagy via the mTOR pathway. The synthesis of viral RNA was inhibited by rapamycin, which inhibits mTOR and promotes the degradation of autolysosomes. The study provides new insights into the molecular mechanism of EV71 and EV71-related HFMD.

Author Contributions: Conceived and designed the experiments: H.-L.W., Z.-W.L. and Z.-C.Z., Collected samples: R.C., X.-R.W., H.-L.Z. and S.-H.L., Performed the experiments: Z.-W.L., Z.-C.Z., Analyzed the data: Z.-W.L., Wrote the paper: Z.-W.L., Supervision: H.-L.W. and Z.-Y.W., Funding acquisition: H.-L.W. All authors have read and agreed to the published version of the manuscript. Viruses 2020, 12, 11 12 of 14

Funding: This research was funded by the National Natural Science Foundation of China, grant number 81371833 and the Natural Science Foundation of Shandong Province, grantnumber ZR2018MH035. Acknowledgments: We thank the support of the National Natural Fundation of China (81371833) and the Natural Fundation of Shandong Province (ZR2018MH035). Thanks to Edward C. Mignot, Shandong University, for linguistic advice. Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

References

1. Ayukekbong, J.A.; Bergstrom, T. Polio will go, acute flaccid paralysis will stay. Lancet 2014, 383, 2209–2210. [CrossRef] 2. Nassef, C.; Ziemer, C.; Morrell, D.S. Hand-foot-and-mouth disease: A new look at a classic viral rash. Curr. Opin. Pediatr. 2015, 27, 486–491. [CrossRef][PubMed] 3. Wu, Y.; Yeo, A.; Phoon, M.C.; Tan, E.L.; Poh, C.L.; Quak, S.H.; Chow, V.T. The largest outbreak of hand; foot and mouth disease in Singapore in 2008: The role of enterovirus 71 and coxsackievirus A strains. Int. J. Infect. Dis. 2010, 14, 1076–1081. [CrossRef][PubMed] 4. Chan, L.G.; Parashar, U.D.; Lye, M.S.; Ong, F.G.; Zaki, S.R.; Alexander, J.P.; Ho, K.K.; Han, L.L.; Pallansch, M.A.; Suleiman, A.B.; et al. Deaths of children during an outbreak of hand, foot, and mouth disease in sarawak, malaysia: Clinical and pathological characteristics of the disease. For the Outbreak Study Group. Clin. Infect. Dis. 2000, 31, 678–683. [CrossRef][PubMed] 5. Van Tu, P.; Thao, N.T.T.; Perera, D.; Truong, K.H.; Tien, N.T.K.; Thuong, T.C.; How, O.M.; Cardosa, M.J.; McMinn, P.C. Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005. Emerg. Infect. Dis. 2007, 13, 1733–1741. [CrossRef][PubMed] 6. Zou, X.N.; Zhang, X.Z.; Wang, B.; Qiu, Y.T. Etiologic and epidemiologic analysis of hand, foot, and mouth disease in Guangzhou city: A review of 4,753 cases. Braz. J. Infect. Dis. 2012, 16, 457–465. [CrossRef] [PubMed] 7. Gilbert, G.L.; Dickson, K.E.; Waters, M.J.; Kennett, M.L.; Land, S.A.; Sneddon, M. Outbreak of enterovirus 71 infection in Victoria, Australia, with a high incidence of neurologic involvement. Pediatr. Infect. Dis. J. 1988, 7, 484–488. [CrossRef] 8. Ishimaru, Y.; Nakano, S.; Yamaoka, K.; Takami, S. Outbreaks of hand, foot, and mouth disease by enterovirus 71. High incidence of complication disorders of central nervous system. Arch. Dis. Child. 1980, 55, 583–588. [CrossRef] 9. Ng, Q.; He, F.; Kwang, J. Recent Progress towards Novel EV71 Anti-Therapeutics and Vaccines. Viruses 2015, 7, 6441–6457. [CrossRef] 10. Kim, Y.I.; Song, J.H.; Kwon, B.E.; Kim, H.N.; Seo, M.D.; Park, K.; Lee, S.; Yeo, S.G.; Kweon, M.N.; Ko, H.J.; et al. Pros and cons of VP1-specific maternal IgG for the protection of Enterovirus 71 infection. Vaccine 2015, 33, 6604–6610. [CrossRef] 11. Nishimura, Y.; Lee, H.; Hafenstein, S.; Kataoka, C.; Wakita, T.; Bergelson, J.M.; Shimizu, H. Enterovirus 71 binding to PSGL-1 on leukocytes: VP1-145 acts as a molecular switch to control receptor interaction. PLoS Pathog. 2013, 9, 1003511. [CrossRef][PubMed] 12. Wang, W.; Duo, J.; Liu, J.; Ma, C.; Zhang, L.; Wei, Q.; Qin, C. A mouse muscle-adapted enterovirus 71 strain with increased virulence in mice. Microbes Infect. 2011, 13, 862–870. [CrossRef][PubMed] 13. Mizuta, K.; Aoki, Y.; Suto, A.; Ootani, K.; Katsushima, N.; Itagaki, T.; Ohmi, A.; Okamoto, M.; Nishimura, H.; Matsuzaki, Y.; et al. Cross-antigenicity among EV71 strains from different genogroups isolated in Yamagata, Japan, between 1990 and 2007. Vaccine 2009, 27, 3153–3158. [CrossRef][PubMed] 14. Huang, S.W.; Hsu, Y.W.; Smith, D.J.; Kiang, D.; Tsai, H.P.; Lin, K.H.; Wang, S.M.; Liu, C.C.; Su, I.J.; Wang, J.R. Reemergence of enterovirus 71 in 2008 in : Dynamics of genetic and antigenic evolution from 1998 to 2008. J. Clin. Microbiol. 2009, 47, 3653–3662. [CrossRef] 15. Haolong, C.; Du, N.; Hongchao, T.; Yang, Y.; Wei, Z.; Hua, Z.; Wenliang, Z.; Lei, S.; Po, T. Enterovirus 71 VP1 activates calmodulin-dependent protein kinase II and results in the rearrangement of vimentin in human astrocyte cells. PLoS ONE 2013, 8, 73900. [CrossRef] Viruses 2020, 12, 11 13 of 14

16. Yang, S.L.; Chou, Y.T.; Wu, C.N.; Ho, M.S. Annexin II binds to capsid protein VP1 of enterovirus 71 and enhances viral infectivity. J. Virol. 2011, 85, 11809–11820. [CrossRef] 17. Zhang, Y.X.; Huang, Y.M.; Li, Q.J.; Li, X.Y.; Zhou, Y.D.; Guo, F.; Zhou, J.M.; Cen, S. A highly conserved amino acid in VP1 regulates maturation of enterovirus 71. PLoS Pathog. 2017, 13, 1006625. [CrossRef] 18. Li, X.; Huang, Y.; Sun, M.; Ji, H.; Dou, H.; Hu, J.; Yan, Y.; Wang, X.; Chen, L. Honeysuckle-encoded microRNA2911 inhibits Enterovirus 71 replication via targeting VP1 gene. Antiviral Res. 2018, 152, 117–123. [CrossRef] 19. Xi, X.; Zhang, X.; Wang, B.; Wang, T.; Wang, J.; Huang, H.; Wang, J.; Jin, Q.; Zhao, Z. The interplays between autophagy and apoptosis induced by enterovirus 71. PLoS ONE 2013, 8, 56966. [CrossRef] 20. Too, I.H.; Yeo, H.; Sessions, O.M.; Yan, B.; Libau, E.A.; Howe, J.L.; Lim, Z.Q.; Suku-Maran, S.; Ong, W.Y.; Chua, K.B.; et al. Enterovirus 71 infection of motor neuron-like NSC-34 cells undergoes a non-lytic exit pathway. Sci. Rep. 2016, 6, 36983. [CrossRef] 21. Li, P.; Yang, S.; Hu, D.; Wei, D.; Lu, J.; Zheng, H.; Nie, S.; Liu, G.; Yang, H. Enterovirus 71 VP1 promotes mouse Schwann cell autophagy via ER stressmediated PMP22 upregulation. Int. J. Mol. Med. 2019, 44, 759–767. [CrossRef][PubMed] 22. Guo, X.; Zhang, M.; Zhang, X.; Tan, X.; Guo, H.; Zeng, W.; Yan, G.; Memon, A.M.; Li, Z.; Zhu, Y.; et al. Porcine Epidemic Diarrhea Virus Induces Autophagy to Benefit Its Replication. Viruses 2017, 9, 53. [CrossRef] [PubMed] 23. Chang, H.; Li, X.; Cai, Q.; Li, C.; Tian, L.; Chen, J.; Xing, X.; Gan, Y.; Ouyang, W.; Yang, Z.; et al. The PI3K/Akt/mTOR pathway is involved in CVB3-induced autophagy of HeLa cells. Int. J. Mol. Med. 2017, 40, 182–192. [CrossRef][PubMed] 24. Qiao, Q.; Li, J.; Sun, C.X.; Ma, Y.W.; Zhuang, Z.C.; Sun, L.L.; Zhao, L.; Wang, Z.Y.; Wen, H.L. Construction and identification of a chimeric cDNA infectious clone of enterovirus 71 strain. Chin. J. Microbiol. Immunol. 2015, 35, 87–93. [CrossRef] 25. Wen, H.L.; Si, L.Y.; Yuan, X.J.; Hao, S.B.; Gao, F.; Chu, F.L.; Sun, C.X.; Wang, Z.Y. Complete genome sequencing and analysis of six enterovirus 71 strains with different clinical phenotypes. Virol. J. 2013, 10, 115. [CrossRef] 26. Li, C.; Qiao, Q.; Hao, S.B.; Dong, Z.; Zhao, L.; Ji, J.; Wang, Z.Y.; Wen, H.L. Nonstructural protein 2A modulates replication and virulence of enterovirus 71. Virus Res. 2018, 244, 262–269. [CrossRef] 27. Dong, Z.; Liu, Z.W.; Chen, R.; Wen, X.J.; Ji, J.; Zheng, X.X.; Zhao, L.; Wang, Z.Y.; Wen, H.L. The untranslated regions of EV-A71 contribute to its pathogenicity and virulence. Virus Res. 2019, 263, 55–63. [CrossRef] 28. Ma, Y.W.; Hao, S.B.; Sun, L.L.; Li, J.; Qiao, Q.; Gao, F.; Zhao, L.; Yu, X.J.; Wang, Z.Y.; Wen, H.L.; et al. Construction and characterization of infectious cDNA clones of enterovirus 71 (EV71). Virol. Sin. 2015, 30, 305–308. [CrossRef] 29. Sun, L.L.; Wang, J.K.; Cui, X.Q.; Hao, S.B.; Li, J.; Zhao, L.; Yuan, X.J.; Wen, H.L.; Yu, X.J.; Wang, Z.Y.; et al. Association of viral replication capacity with the pathogenicity of enterovirus 71. Virus Res. 2014, 189, 1–7. [CrossRef] 30. Watanabe, R.; Wei, L.; Huang, J. mTOR signaling, function, novel inhibitors, and therapeutic targets. J. Nucl. Med. 2011, 52, 497–500. [CrossRef] 31. Bruning, A.H.L.; Thomas, X.V.; van der Linden, L.; Wildenbeest, J.G.; Minnaar, R.P.; Jansen, R.R.; de Jong, M.D.; Sterk, P.J.; van der Schee, M.P.; Wolthers, K.C.; et al. Clinical, virological and epidemiological characteristics of infections in early childhood: A comparison between non-hospitalised and hospitalised children. J. Clin. Virol. 2015, 73, 120–126. [CrossRef][PubMed] 32. Jansen, R.R.; Schinkel, J.; Dek, I.; Koekkoek, S.M.; Visser, C.E.; de Jong, M.D.; Molenkamp, R.; Pajkrt, D. Quantitation of respiratory viruses in relation to clinical course in children with acute respiratory tract infections. Pediatr. Infect. Dis. J. 2010, 29, 82–84. [CrossRef][PubMed] 33. Kemball, C.C.; Alirezaei, M.; Flynn, C.T.; Wood, M.R.; Harkins, S.; Kiosses, W.B.; Whitton, J.L. Coxsackievirus infection induces autophagy-like vesicles and megaphagosomes in pancreatic acinar cells in vivo. J. Virol. 2010, 84, 12110–12124. [CrossRef][PubMed] 34. Mouna, L.; Hernandez, E.; Bonte, D.; Brost, R.; Amazit, L.; Delgui, L.R.; Brune, W.; Geballe, A.P.; Beau, I.; Esclatine, A.; et al. Analysis of the role of autophagy inhibition by two complementary human cytomegalovirus BECN1/Beclin 1-binding proteins. Autophagy 2016, 12, 327–342. [CrossRef] 35. Ke PY, Chen SS: Autophagy in hepatitis C virus-host interactions: Potential roles and therapeutic targets for liver-associated diseases. World J. Gastroenterol. 2014, 20, 5773–5793. [CrossRef] Viruses 2020, 12, 11 14 of 14

36. Talloczy, Z.; Jiang, W.; Virgin HWt Leib, D.A.; Scheuner, D.; Kaufman, R.J.; Eskelinen, E.L.; Levine, B. Regulation of starvation- and virus-induced autophagy by the eIF2alpha kinase signaling pathway. Proc. Natl. Acad. Sci. USA 2002, 99, 190–195. [CrossRef] 37. Wong, J.; Zhang, J.; Si, X.; Gao, G.; Mao, I.; McManus, B.M.; Luo, H. Autophagosome supports coxsackievirus B3 replication in host cells. J. Virol. 2008, 82, 9143–9153. [CrossRef] 38. Zhou, Z.; Jiang, X.; Liu, D.; Fan, Z.; Hu, X.; Yan, J.; Wang, M.; Gao, G.F. Autophagy is involved in influenza A virus replication. Autophagy 2009, 5, 321–328. [CrossRef] 39. Espert, L.; Denizot, M.; Grimaldi, M.; Robert-Hebmann, V.; Gay, B.; Varbanov, M.; Codogno, P.; Biard-Piechaczyk, M. Autophagy is involved in T cell death after binding of HIV-1 envelope proteins to CXCR4. J. Clin. Investig. 2006, 116, 2161–2172. [CrossRef] 40. Fu, Y.; Xu, W.; Chen, D.; Feng, C.; Zhang, L.; Wang, X.; Lv, X.; Zheng, N.; Jin, Y.; Wu, Z. Enterovirus 71 induces autophagy by regulating has-miR-30a expression to promote viral replication. Antiviral Res. 2015, 124, 43–53. [CrossRef] 41. Lee, Y.R.; Wang, P.S.; Wang, J.R.; Liu, H.S. Enterovirus 71-induced autophagy increases viral replication and pathogenesis in a suckling mouse model. J. Biomed. Sci. 2014, 21, 80. [CrossRef][PubMed] 42. Henault, J.; Martinez, J.; Riggs, J.M.; Tian, J.; Mehta, P.; Clarke, L.; Sasai, M.; Latz, E.; Brinkmann, M.M.; Iwasaki, A.; et al. Noncanonical autophagy is required for type I interferon secretion in response to DNA-immune complexes. Immunity 2012, 37, 986–997. [CrossRef][PubMed] 43. Paludan, C.; Schmid, D.; Landthaler, M.; Vockerodt, M.; Kube, D.; Tuschl, T.; Munz, C. Endogenous MHC class II processing of a viral nuclear antigen after autophagy. Science 2005, 307, 593–596. [CrossRef][PubMed] 44. Buffen, K.; Oosting, M.; Quintin, J.; Ng, A.; Kleinnijenhuis, J.; Kumar, V.; van de Vosse, E.; Wijmenga, C.; van Crevel, R.; Oosterwijk, E.; et al. Autophagy controls BCG-induced trained immunity and the response to intravesical BCG therapy for bladder cancer. PLoS Pathog. 2014, 10, 1004485. [CrossRef][PubMed] 45. Kyei, G.B.; Dinkins, C.; Davis, A.S.; Roberts, E.; Singh, S.B.; Dong, C.; Wu, L.; Kominami, E.; Ueno, T.; Yamamoto, A.; et al. Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages. J. Cell. Biol. 2009, 186, 255–268. [CrossRef][PubMed] 46. Heras-Sandoval, D.; Perez-Rojas, J.M.; Hernandez-Damian, J.; Pedraza-Chaverri, J. The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal. 2014, 26, 2694–2701. [CrossRef][PubMed]

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