Ubiquitin-activating-enzyme Inhibitor PYR41 Reduces EV71 Replication Involving Autophagy

Objective:Hand foot and mouth disease?HFMD?is an infectious disease with high mortality,good incidence of children and easy to be accompanied by aseptic meningitis and serious neurological complications.In recent years,the incidence and mortality of HFMD in class C infectious diseases are the highest in China.The data showed that in these large number of confirmed severe cases,enterovirus 71 infection is the main cause of children's hand foot and mouth disease.At present,hand foot and mouth disease has become a emerging infectious disease that has a potential threat to global health.Many studies have shown that the host factors play a key role in the pathogenesis of EV71^[1-4].Given the wide range of role of the ubiquitin proteasome system in cell metabolism,cell proliferation,cell cycle regulation and apoptosis,so our experiment starts from ubiquitination and autophagy,which aims to study the ubiquitination change after viral infection and the regulation interplay between ubiquitination and autophagy.Other than,it also lays the foundation for further exploring the effect of this regulation on virus replication and the antiviral effect of the body.Methods:1.Cell cultureRD-A cells were inoculated in the MEM complete medium containing 10%fetal bovine serum and 100U penicillin,100mg/ml streptomycin at 37?incubator containing5%CO₂.The fresh cell culture medium was replaced every 2 days,and 0.25%trypsin was used to digest the RD-A cells when they were at 80%confluence.2.Titration detection of EV71 virus-TCID50According to"Hand Foot and Mouth disease Prevention and Control Guidelines?2009Edition?"and the Reed-Muench method.RD-A cells were seeded in 96 well plates with concentration of 5×10⁴/ml,per hole 100?l,when they were attached,the collected infection supernatant to be measured was 10-fold serial diluted,added to the 96 well culture plate with 8 holes of each dilution,each hole 100?l,at the same time,with dilute solution as a negative control.After 5 days,all the holes were observed and recorded?Cytopathic Effect,CPE?,according to the formula to calculate the titer of virus Kaposi's.3.Virus propagationThe TCID50 of the virus was measured before this experiment.The number of Multiplicity of Infection?MOI?was determined according to the amount of virus and the number of cells.Counting cells,adjusting the concentration and planking.RD-A cells were infected with EV71 at the indicated MOI or time.At the indicated infection time,the cultural supernatant and cells were collected for titer detection and VP1quantification.4.Plasmid transfectionThe logarithmic phase cells were seeded in 6-well plates,1×10⁵/ml,the cell fusion degree reached 80%,it was changed into fresh medium containing serum not containing antibiotics.The plasmid pPLK/GFP-Atg5-shRNA?2.5?g/hole?and the transfection reagent lipo6000^TM?5?l/hole?were used without serum or antibiotics medium configuration,static 5min for good mixing.Static 20min again,respectively added to the corresponding hole with 250?l/hole system,under the condition of 37?and 5%CO₂,after 6h,it was changed into complete medium.The appropriate dose of EV71 virus was added at the indicated time.Group situation:blank control group?uninfected group,12h postinfection?,negative control group pPLK/GFP-shRNA?uninfected group,12h postinfection?,interference group pPLK/GFP-Atg5-shRNA?uninfected group,12h postinfection?.5.Detection of cell activity of PYR41 by CCK-8The logarithmic phase cells were counted,the density was 1×10⁵/ml,and 100?l were planked per hole on 96 hole plates.After the cells adhered,the RD-A cells were treated with 0.0,0.1,1.0,5.0 and 10.0?mol/L PYR41 respectively,with 5 holes in each concentration.After 12h,the cell activity was detected by CCK-8.10?l CCK-8 solution?5mg/ml?was added 2h before termination,and the absorbance?OD?value of each pore was measured at 450 nm,and the mean OD value of each pore was calculated.The cell viability rate of the control group was regarded as 100%.The cell viability rates of each group were calculated,and the cell viability rate=?ODtreatment/ODcontrol?×100%.6.Detection of autophagy related proteins and viral protein VP1 by Western blotThe logarithmic phase cells were seeded in 6-well plates,1×10⁵/ml,when the cellconfluence reached 80%,adding PYR41?5?M?pretreatment for 12h and then infected with EV71?MOI=1?,and set up no drug treatment group and the no-infection control group,after 6h and 12h of EV71 infection,the cells were collected,following with the total protein extraction reagent and protease inhibitor PMSF for cell lysis on ice.Then centrifuged at the condition of 4?,15min,13800rpm,quantified by BCA method and adjusted the concentration of protein.Finally added 4×loading buffer to obtain protein samples by boiling at 100?for 5min.The whole protein of the samples was seperated by 12%SDS-PAGE electrophoresis and transferred to the NC membrane,then the membranes was blocked with 5%skimmed milk powder for 2h at room temperature,after that,they were incubated respectedly with anti-LC3-I/II?1:1000?,anti-P62?1:2000?,anti-VP1?1:1000?,anti-beta-actin?1:2000?at 4?overnight.After TBST washing three times?for 5min each time?,following by adding HRP-labeled the second antibodies?1:5000?and incubating at room temperature for 2h.Then washed three times with TBST again,finally added ECL chemiluminescence chromogenic substrate and exposured in Bio-Rad Gel Imager.7.Detection of viral VP1 mRNA by fluorescence quantitative PCRIn accordance with the method 6,RD-A cells were pretreated by PYR41?5?M?for 12h,and then exposed to virus for 6h and 12h,respectively.Cells were collected to extract total RNA with TRIzol Reagent.RT-PCR was performed by converting the total RNA to cDNA according to the method of cDNA the first-chain fast generation kit.Then cDNA was the template for qRT-PCR amplification.The qRT-PCR primers were designed and synthesized by Shanghai Sangon Industrial Co.Ltd.The PCR reaction conditions:pre-denaturation 60s at 95?;95?15s,60?15s,72?60s,40 cycles.The Bio-Rad fluorescence quantitative PCR instrument,Cq values were measured by three independent experiments.Compared with reference gene GAPDH,the RNA level of the target gene was expressed as fold changes by Prism 5.8.Detection of intracellular EV71 virus number by immunofluorescenceLogarithmic phase cells were seeded in 24 well plates,1×10⁵/ml/hole.When the cellconfluence reached 80%,which were pretreated by PYR41?5?M?for 12h and infected with EV71 for 3h,6h,12h.At the indicated time point,the cells were washed with PBS,respectively fixed with 4%paraformaldehyde and throughed with 0.3%TritonX-100.After washing,closed with the immunofluorescence sealing liquid?5%BSA?at room temperature for 30 min.Then adding anti-EV71?1:200?for binding to EV71 virus at 4?,overnight.After washing with PBS for 3 times,incubated FITC-labeled anti-Rabbit second antibody?1:200?at room temperature for 30 min.Finally washing with PBS again;allowing DAPI nuclear staining for 5 minutes,after which,watched and photographed under fluorescence microscope and then compared the changes of the number of virus.9.Statistical analysisAll data were expressed as ???±s,SPSS19.0 and Sigmaplot 12.3 was used for statisticalanalysis.The mean values between groups were compared with t test.The percentage and rate comparison were analyzed by?² test,with P<0.05 as the statistical significance.Results:1.EV71 infection increased ubiquitination level in RD-A cells.Immunoblots showed ubiquitinated proteins was significantly increased in a time-dependent pattern and reached a higher level at post infection 12h?P<0.05?.At the same time,with the increase of infection dose of EV71,ubiquitinated protein increased gradually with a higher level in MOI=5?P<0.05?.2.Inhibition of ubiquitination reduces the amount of intracellular virus.The RD-A cells were pretreated with PYR41 and exposed to EV71 for 12h.Compared with no drug but only EV71 group,VP1 in the group PYR41+EV71 was significantly lower?P<0.01?.Fluorescence quantitative PCR analysis also found that post infection12h,VP1 mRNA level in the group PYR41+EV71 was significantly lower compared with only EV71 group?P<0.01?.Immunofluorescence was used to observe the EV71distribution with FITC-labeled by fluorescence microscope.It was also found that the number of fluorescent spots in the group with PYR41 was significantly lower than that in the group without PYR41 but only EV71?P<0.05?.3.Inhibition of ubiquitination inhibited the terminal autophagy induced by EV71.Our previous study found that EV71 could induce autophagy in a time-and dose-dependent pattern.This study showed that PYR41 obviously converted LC3-I to LC3-II,especially at 12h?P<0.01?.And in the infective group with PYR41 pretreatment,LC3-II was significantly higher than that in the single infectious group?P<0.01?.At the same time,it's estimated that the effects of PYR41 on the increase of LC3-II may be similar to CQ:inhibition of the terminal autophagy.To prove it,we conducted P62protein detection.It found that P62 decreased after infection,but the trend of P62degradation was inhibited by adding PYR41?P<0.05?.4.Autophagic molecule Atg5 was ubiquitined degradation during EV71 infection.After EV71 infection,the intracellular Atg5 decreased and significantly decreased post infection 12h?P<0.01?.With the addition of PYR41,the decrease of Atg5 was significantly inhibited compared with only EV71 infection group?P<0.05?.It shows that Atg5 has been degraded by ubiquitination,which can be inhibited by PYR41.It is suggested that Atg5 depends on ubiquitination to be degraded during EV71 infection.5.PYR41 reduced the amount of intracellular virus involving autophagy.It also showed that VP1 in the PYR41 group changed with the role of 3-MA or CQ?P<0.01?.At the same time,it's suggested that the effects of PYR41 on autophagy and virus may be similar to CQ:inhibition of the terminal autophagy increased virus release,resulted in intracellular virus reduced.To prove it,the effect of PYR41 on the extracellular titer was detected by TCID50.It was found that the extracellular virus titer increased significantly in the infective group with PYR41 pretreatment?P<0.05?.6.The decrease of the key autophagic protein Atg5 is beneficial to viral replication.In our experiments,EV71 infection enhances the ubiquitinated degradation of Atg5,which may affect autophagy and the amount of intracellular viruses.After transfection of plasmid pPLK/GFP-Atg5-shRNA,the intracellular viral protein VP1 increased?P<0.05?.It is suggested that the reduction of Atg5 may be beneficial to the replication of virus or the reduction of its release.In other word,PYR41 may be beneficial to the early autophagy by inhibiting the degradation of Atg5 and reduce the number of intracellular viruses.More validation goes for our previous results.Conclusion:Ubiquitin Activase inhibitor PYR41 inhibited ubiquitination and reduced the number of intracellular viruses.And its antiviral effect was related to its autophagy effect:by inhibiting the late autophagy,resulted that the release of virus was increased and the number of intracellular viruses was reduced.At the same time,PYR41 can reduce the amount of intracellular viruses by inhibiting the ubiquitination of autophagic protein Atg5and starting early autophagy.It is concluded that ubiquitination induced by EV71infection was conducive to viral replication,which may be related to autophagy induced by EV71.These results provided an experimental basis for us to better understand the mechanism of autophagy induced by EV71 and also set up a new strategy for preventing and treating EV71 infection from the perspective of ubiquitination.