Enterovirus 71 Induce Cell Autophagy Involving PSGL-1

Objective:Hand-foot-and-mouth disease(HFMD)is a seasonal epidemic disease with a high mortality rate,aseptic meningitis associated with children and severe neurological complications,which is mainly enterovirus 71(EV71)infection in the confirmed cases.Therefore,in this study,we investigated the role of PSGL-1 in the regulation of autophagy in normal gastric epithelial cells infected with EV71 by the means of cell morphology and methods combined with receptor and autophagy,so as to provide the theoretical basis and important experimental basis for exploring the pathogenesis of EV71 infection,and provide new ideas for the early prevention and clinical treatment of HFMD disease.Materials and methods:1.Cell culture and virus propagation Rhabdomyosarcoma cells(RD-A)and Human gastric epithelial cells(GES-1)cells were inoculated with Dulbecco's modified Eagle's medium(DMEM)containing L-glutamine,added 1% penicillin/streptomycin(200 U/ml)and 10% fetal bovine serum(FBS)to medium.Cells were matained in a 37? incubator containing 5% CO2.GES-1cells were digested by 0.25% trypsin without EDTA at 90% confluence,and sub-cultured in the complete medium.GES-1 cells were infected with EV71(Fuyang-0805)at the indicated multiplicity of infection(MOI).After 3 hours of infection,cells were cultured in medium containing the 2% FBS after washing viruses off.The culture supernatant and cells were collected at the indicated time points of infection.Viruses were separated from the collected cells by repeated freezing and thawing for 3 times.Viral stocks were frozen at-80? and recovered at 4? before the infection,and virus titers were measured with the50% culture infective dose(TCID50)by 96-well plates covered with RD-A cells,whichaccorded to the Reed-Muench method.2.Cell treatment GES-1 were infected with EV71(6 h,12 h,24 h for MOI = 10).Also,anti-PSGL-1antibody(2 mg/ml)were inoculated with GES-1 was used to block the combination of PSGL-1 with EV71 before the infection.For autophagy detection,the cultured GES-1 were transduced with the RFP-LC3/GFP-LC3 for 24 h,and then infected with viruses.The number of FITC-LC3 puncta was assessed by using an average manual count of 5 fields.With the same magnification,the number of nuclei stained by DAPI was counted in the same regions.The number of FITC-LC3 dot/cell was evaluated as the level of autophagy.3.Real-time PCR We extracted total RNA from differently treated GES-1 cells with a TRIzol Reagent.Total RNA(2 ?g)was converted to c DNA using the Fast Quant First strand c DNA synthesis Kit.A real time quantitative RT-PCR analysis was performed with the Bestar?Sybr Green q PCR Mastermix to assess PSGL-1.Set a thermal cycling condition: 95?initial denaturation of 2 min,and then performed a 95? denaturation for 10 s,a anneal temperature 63? for 30 s and a 72? extension of 30 s for 40 cycles.Compared with GAPDH,the RNA level of PSGL-1 was expressed as fold change.4.Immunofluorescence microscopy Cells were fixed with PBS containing 4% paraformaldehyde at 4? for 20 min.The fixed cells were permeabilized using 1% Triton X-100 in PBS for 15 min at room temperature and were subsequently blocked with 1% BSA in PBS for 1 h at room temperature,and then incubated with LC3/PSGL-1-specific m Ab as the primary antibody overnight at 4?.The next day,after washing,the cells were incubated with second antibodies labeled with fluorescence.For better visualization of nuclei,cells were stained with 4',6-diamidino-2-phenylindole(DAPI).Then the cells were scrutinized under a fluorescence microscope.5.Immunoprecipitation and western blottingThe harvested cells were lysed and protein concentration was measued using a BCA assay kit.Briefly,12% SDS-PAGE was used to seperated samples and then samples were electrophoretically transferred onto a polyvinylidene difluoride(PVDF)membrane.The membranes were blocked with 5% skimmed milk for 2 h at room temperature and then incubated at 4? overnight respectively with an anti-P62,anti-LC3,anti-VP1,anti-PSGL-1and anti-?-actin antibodies,followed by incubation with the second antibodies labeled with horseradish peroxidase(HRP)for 1 h at room temperature.Final product was viewed using chemiluminescent substrates.Cell lysate was incubated with 1 mg of m Ab overnight at4? for immunoprecipitation,then added Protein G Agarose and incubated the mixture for an additional 2 h.Western blotting was used to separated the immunoprecipitates in 12%SDS-PAGE after washing out the beads.6.Statistical analyses All data were analyzed using SPSS 13.0 and Sigmaplot 12.3.Results are presented as mean ± standard error of the mean(S.E.M.).Student's t test was used to analyze the statistical comparisons.A p-value less than 0.05 was considered statistically significant.Results:We used immunoblotting to evaluate conversion of LC3-I/LC3-II.Compared with uninfected cells,the level of LC3-II in GES-1 cells was significantly increased after 12 h of EV71 infection and continued to increase for 24 h,whereas LC3-I levels were gradually decreased.These results suggested that the cumulative increase in the formation of autophagosome with the course of infection.Furthermore,we detected the puncta formation of vacuoles labeled FITC-LC3 in uninfected cells compared with GES-1 cells infected with EV71(Fuyang-0805)at the MOI = 10 for 12 hours.The conclusion that EV71 infection indeed induced the formation of autophagosomes was proved by these experiments results.To confirm whether PSGL-1 was involved in the early infection of EV71(Fuyang-0805)entry after binding to GES-1 cells,the specific monoclonal antibodies(m Abs)PSGL-1 was used to block the receptor function.After PSGL-1 was blocked,the invasion of viruses was markedly inhibited,suggesting that virus phagocytosis was more effective by absolutely activating this receptor.We firstly sought to determine the EV71VP1 protein in infected GES-1 cells by immunoblotting.The VP1 proteins levels were notably reduced in GES-1 cells with prior-treatment with lg G isotype.Live virus titers in GES-1 cells infected virus by determined by TCID50 assay supported this result.Our results showed PSGL-1 expression in surface of GES-1 cells served as the functional receptor and binded EV71(Fuyang-0805)in the early infection.To investigate dynamics of PSGL-1 in the EV71 early infection,we examined PSGL-1 on the level of both transcription and translation.The PSGL-1 proteins in GES-1cells infected with EV71 had a significant increase compared with uninfected cells by western blotting assay.Meanwhile,we detected that RNA level increased markedly in infected cells with RT-PCR,and which confirmed that EV71 infection upregulated PSGL-1expression in human GES-1 cells.Next,we examined the distribution of PSGL-1 in cells using immunofluorescence microscopy and found that there were punctuated accumulation occurring in cytoplasm of the EV71 infected cells as compared with uninfected cells.These experimental results suggested that PSGL-1 had a significant change in the quantity and distribution along the course of EV71 infection.To test whether PSGL-1 participated in EV71 infection-induced autophagy in human GES-1 cells,we first sought to determine whether PSGL-1 involved autophagosomes formation.The immunefluorescent assay results showed that RFP-LC3 expressed in human GES-1 cells co-localize with PSGL-1.To investigate whether the association of PSGL-1with autophagosomes was a direct interaction,we tested the interaction between PSGL-1proteins and autophagy-related proteinsin infected cells.Finally,we found the interaction of PSGL-1 with SQSTM1/P62 by immunoprecipitation assay results,indicated that PSGL-1 interacts with SQSTM1 /P62,and thereby participated in EV71 infection-induced autophagy.Conclusion:EV71 infection induces autophagy in GES-1,which may be mediated by the protein interaction between PSGL-1 and P62.These results may provide a theoretical basis for us to understand the mechanism of EV71 induced autophagy and provide a new strategy for the prevention and treatment of EV71 infection.