| Background:Enterovirus D68(EV-D68)is a respiratory viral pathogen that causes severe respiratory diseases and neurologic manifestations.Since the 2014 outbreak in the US,EV-D68 has been reported to be associated to the development of acute flaccid myelitis(AFM)which causes worldwide concern.However,there are currently no approved antiviral agents or vaccines for EV-D68.Zinc ions inhibit viral replication through impairing polyprotein processing,inhibiting genome transcription and replication,interfering with viral-cell membrane fusion and facilitating viral uncoating.And zinc ions have been co-treated with antiviral therapies clinically,including the treatment on HIV,chronic HCV and viral warts and so on.Zinc ions exert the antiviral activity against many RNA viruses and DNA viruses,including coronavirus,HSV,HCV,HPV.However,EV-D68 is so different from those viruses in aspects of the nucleic acid type,the ways of infection and transmission,with or without the viral envelope and so on.There is no related report about the anti-viral effect of zinc ions on enterovirus D68.The replication of virus depends on the resources of host cells.Viruses have evolved variety of mechanisms to manipulate the cellular environment of infected host cells to satisfied the needs of viral replication.One mechanism is the regulation of the cell cycle by viruses,many of which arrest the cell cycle at a phase when it facilitates the replication.It has been reported that zinc ions regulate cell cycle by reacts with many checkpoints of cell cycle.Therefore,we tried to study whether zinc ions exert anti-viral effect of EV-D68 by regulating cell cycles.We used the m RNA sequencing to identify the genes which have impact on the ability of infection of EV-D68.In this study,we evaluate the anti-viral effect of zinc ions on enterovirus D68 and pursue the possible mechanism.Methods:1.The effect of zinc ions on the replication of EV-D68 in vitro and in vivo(1)Cytopathic effects(CPEs): The zinc ion group and control group were infected with EV-D68 Fermon strain(MOI=0.1)for 2 hours.Zinc ions(0.1 m M)were added after the infection.The CPEs was observed at 48 hpi and photographed.(2)Effect on viral progeny production: The zinc ion group and control group were infected with EV-D68(MOI=0.1).At different time points: 0 hpi,6 hpi,8 hpi,10 hpi,12 hpi,20 hpi,24 hpi or 48 hpi,the supernatant was collected for virus titer assay and western blot to test the kinetics of released VP1 expression.(3)Effect on cellular VP1 accumulation: The zinc ions group and control group were infected with EV-D68(MOI=0.1).At 0 hpi,6 hpi,8 hpi,10 hpi,12 hpi,20 hpi,24 hpi or 48 hpi,the cells were collected for western blot to test the kinetics of cellular VP1 accumulation.(4)Effect on the 2014 circulating strains of EV-D68: The zinc ions group and control group were infected with EV-D68 2014 US circulating strains: MO and KY(MOI=0.1).At 48 hpi,the cellular VP1 accumulation and viral progeny production were tested.The CPEs was observed.(5)The anti-viral effect of various zinc salts on EV-D68: The EV-D68 infected cells were treated with zinc sulfate,zinc acetate and zinc chloride at the same concentration.At 48 hpi,the cellular VP1 accumulation and viral progeny production were tested.The CPEs was observed.(6)The anti-viral effect of zinc ions in other two EV-D68 permissive cell lines:A549 and Hela were infected with EV-D68(MOI=0.1).At 48 hpi,the cellular VP1 accumulation and viral progeny production were tested.(7)The anti-viral effect of zinc ionophore(PDTC)on EV-D68: The EV-D68 infected cells were treated with zinc ions(0.0005 m M),PDTC,combination of PDTC and zinc ions(0.0005 m M).At 48 hpi,the cellular VP1 accumulation and viral progeny production were tested.The CPEs was observed.We also conducted the experiments with or without serum to test the virus titer.(8)The anti-viral effect of zinc ions in EV-D68 infected mouse model: 2-days mouse were injected intracranially with EV-D68.The zinc ions were treated to the mouse since 1 dpi.The viral loads in muscle,lung and spinal cord were evaluated at 2,3,4 and 6 dpi.The HE staining and IHC staining are carried out.2.Study on the anti-viral mechanism of zinc ions against EV-D68(1)Prophylactic effect of zinc ions against EV-D68: The cells were 24 h pre-treated with or without zinc ions,then infected with EV-D68(MOI=0.1)at 4 ℃ and 37 ℃ separately.At 2 hpi,the RNA level of VP1 was detected by q RT-PCR.Effect of zinc ions on the attachment and entry of EV-D68: The cells were infected with EV-D68(MOI=0.1)with or without zinc treatment during the infection process at4 ℃ and 37 ℃ separately.At 2 hpi,the RNA level of VP1 was detected by q RT-PCR.(2)Effect on the regulation of G1 synchronization cells by zinc ions: The cells were pre-treated with serum free culture medium for 24 h which induces G1 synchronization.Then the zinc ions group and control group were infected with EVD68(MOI=0.1).The serum was re-added to the medium after the infection.At 24 hpi,the percentage of cells in different phases were detected by flow cytometry,and the RNA level of CDK4,CDK6 and Cyclin D was tested.(3)Relation between the regulation of cell cycle of zinc ions and its anti-viral activity: the cells are cultured with or without the serum then infected with EV-D68(MOI=0.1).At 24 hpi,the percentage of cells in different phases were detected by flow cytometry,and the RNA level of cellular VP1 and released VP1 were tested.(4)Effect on the re-entry into cell cycle of S and G2 synchronization cells after EV-D68 infection: Thymidine or nocodazole were treated to the cells 24 h prior to the infection to induce S or G2 synchronization.Then thymidine and nocodazole were removed.The zinc ions group and control group were infected with EV-D68(MOI=0.1),then cultured in the DMEM containing 10% FBS.At 24 hpi,the percentage of cells in different phases were detected by flow cytometry.(5)Effect on the expression of the CDK/Cyclin complex: The zinc ions group and control group were infected with EV-D68(MOI=0.1).At 24 hpi,the cells were collected for western blot to test the expression of CDK1,Cyclin B1,Cyclin D and VP1;and for q RT-PCR to detect the RNA levels of CDK1,Cyclin B1,CDK4,CDK6,Cyclin D and VP1.The VP1 protein expression from supernatant were also tested.(6)Effect of zinc chelator(TPEN)on cell cycle regulated by zinc ions: The EVD68 infected cells were treated with zinc ions,TPEN,combination of TEN and zinc ions.At 24 hpi,the distribution of cell cycles was tested by flow cytometry.3.The identification of genes related to the infectious ability of EV-D68: The zinc ions group and control group were infected with EV-D68 Fermon strain(MOI=0.1)for 2 hours.At 24 hpi,the cells were collected for m RNA sequencing.We conducted X the analysis of differentially expressed genes,GO enrichment analysis,establishment of PPI network and identification of the top 10 HUB genes relates to viral replication and cell cycle regulation.By carried out the knockdown or overexpressed of target genes in 293 T cell line,we tested the impact on the infectious ability of EV-D68.Results:1.The effect of zinc ions on the replication of EV-D68 in vitro and in vivo(1)Zinc ions alleviate the CPEs induced by EV-D68.(2)Zinc ions decrease the production of progeny viruses: At different time points:8 hpi,10 hpi,12 hpi,20 hpi,24 hpi,the expression of VP1 in supernatant of zinc group was decreased when compare with the control group.At 48 hpi,both the expression of VP1 in the supernatant and the virus titer of zinc group are decreased.(3)Zinc ions exert anti-viral effect at the early replication cycles: At 8 hpi,10 hpi and 12 hpi,the expression of cellular VP1 decreased in zinc group.After multiple cycles of replication(20 hpi and 24 hpi),the difference of cellular VP1 between zinc group and control group diminished.(4)Zinc ions exert anti-viral effect on EV-D68 circulating strains: The CPE induced by MO and KY was alleviated by zinc ions.Compare with the control group,the expression of released VP1 and cellular VP1 in zinc group were decreased.(5)Various zinc salts exert anti-viral effect on EV-D68: Zinc sulfate,zinc acetate and zinc chloride decreased the CPE,the virus titer and the expression of VP1 in the supernatant of zinc group.(6)Zinc ions exert anti-viral effect on EV-D68 in A549 and Hela: zinc ions decreased the virus titer and the expression of released VP1 in A549 and Hela cell lines.(7)The antiviral effect of zinc ions could be enhanced by zinc ionophore(PDTC):At 0.0005 m M,zinc ions have no anti-viral effect against EV-D68.The combination of PDTC and 0.0005 m M zinc alleviated the CPE,the cellular VP1 accumulation and the virus titer compare with the control group.The virus titer of PDTC treatment in DMEM containing 10% FBS decreased compare with that in serum free DMEM.(8)Zinc ions inhibit the viral infection in neonatal mice: the RNA level of VP1 decreased in lung,spinal cord and skeletal muscle of zinc-treatment group.According to the IHC result,viral antigens in lung,spinal cord and skeletal muscle are decreased in zinc-treatment group.No pathological change has been found between the groups.2.Study on the anti-viral mechanism of zinc ions against EV-D68 infection.(1)Zinc ions exert prophylactic effect against EV-D68: At 4℃ and 37 ℃,the RNA level of VP1 decreased in zinc group when compare with the control group.Zinc ions have no impact on the entry and attachment of EV-D68: At 4℃ and 37℃,there is no difference of the RNA level of VP1 between zinc group and the control group.(2)Zinc ions promote the exit from G1 phase: The RNA level of CDK4,CDK6 and Cyclin D in the control group decreased when compare with the negative control group.Meanwhile,the RNA level of CDK4,CDK6 and Cyclin D increased by zinc ions when compare with the control group.The percentage of cells in G1 phase decreased by zinc ions when compare with the control group.(3)Zinc ions exert the anti-viral effect through the reverse of G1 arrest induced by EV-D68: In both serum-free system and 10% serum system,the percentage of cells in G1 phase in zinc group reduced,while percentage of cells in G2 phase in zinc group went up.The RNA level of VP1 in both cells and supernatant decreased in zinc group.(4)Zinc ions induce G2 arrest: Compare with the thymidine group,the percentage of cells in S phase in zinc group reduced,the percentage of cells in G2 phase in zinc group went up.Compare with the nocodazole group,the percentage of cells in G2 phase in zinc group increased,the percentage of cells in G1 phase in zinc group decreased.(5)Zinc ions inhibit the post-translational expression of CDK1/Cyclin B1:Compare with the control group,the RNA level of CDK4,CDK6 and Cyclin D went up in zinc group;the expression of Cyclin D increased,the expression of Cyclin B1 and CDK1 decreased in zinc group.The expression of released VP1 reduced in zinc group,no difference in the expression of cellular VP1 was shown.(6)TPEN reverse the G2 arrest induced by zinc ions: Compare with the control group,the percentage of cells in G1 phase in zinc group reduced while in G2 phase increased.Compare with zinc group,the percentage of cells in G1 phase in TPEN group went up while in G2 phase went down.3.E2F1 and EGR1 are the targeted genes related with the infectious ability of EV-D68: EV-D68 infected E2F1 knock-down 293 T cell line,the cellular VP1 expression and virus titer went up;EV-D68 infected E2F1 over-expressed 293 T cell line,the cellular VP1 expression and virus titer went down;EV-D68 infected EGR1over-expressed 293 T cell line,the cellular VP1 expression and virus titer went up.Conclusion:1.Zinc ions exert anti-viral effect on both prototype and circulating strains of EVD68 in vitro.2.Zinc ions exert prophylactic effect on cells against EV-D68 infection by incubation ahead of time.3.Zinc ions exert anti-viral effect on EV-D68 infected neonatal mice.4.Zinc ions reverse the G1 arrest induced by EV-D68 results in the reduction of viral replication.Zinc ions induce G2 arrest by promoting the entry into G2 and prevent the exit from G2.Zinc ions induce G2 arrest might through inhibiting the posttranslational expression of CDK1/Cyclin B1 complex.5.E2F1 and EGR1 might be the targeted genes in host cells which relates to the infectious ability of EV-D68.Knock-down E2F1 and over-express EGR1 facilitate the replication of EV-D68;over-express E2F1 inhibits the replication of EV-D68. |
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