| The incidence of hand, foot, and mouth disease (HFMD) caused by enterovirus71(EV71) is still very high in recent years. To date, neither effective vaccine norantiviral treatment is available for EV71infection. Interferons (IFN), asabroad-spectrum antiviral drugs, have been successfully in the treatment of a varietyof viral infection diseases. High concentration IFN can inhibit EV71and EV71couldantagonize IFN response. This work is aimed to explore how EV71antagonize theantiviral activity of IFN.Five EV71strains were used in this work, including Anhui2007, VR1432,VR1775, GDV103and GDV102. Three viral genomes (Anhui\VR1432\GDV103)were sequenced. The accession number is KC954662、KC954664and KC954663. Thephylogenetic analysis based on the complete sequence showed that Anhui andGDV103strain belongs to C4sub-genotype and VR1432strain belongs to C2sub-genotype. Vero E6and MRC-5cells were pretreated with IFN-ω or IFN-α2bbefore Anhui, VR1432, GDV103and GDV102infection, further the antiviralactivities of IFN-ω and IFN-α were assayed using the cytopathic effect method bycrystal violet staining. The results showed that IFN-ω displayed better antiviralactivity than IFN-α2b and EV71was more sensitive to IFN in vero E6than MRC-5cell. The EC50of IFN-ω in Anhui, VR1432, GDV103and GDV102-infected cells is931.5IU/ml,1810.7IU/ml,385.8IU/ml and40.6IU/ml. Subsequently, the copynumber of GDV103and VR1432was determined by quantitative real-time PCR(qRT-PCR) when vero E6was pretreated with IFN-ω or IFN-α2b, the resultsindiciated that the number of viral copies decreased sharply with interferonconcentration increased.In order to explore the mechanism of the interaction of EV71and interferon,MRC-5and RD cells were infected with GDV103and VR1432before stimulatedwith IFN-α2b. To assess the effect of EV71infection on type I IFN response, themRNA and protein level of IFN-β was investigated at different time points postinfection, VSV infection as positive control. RT-PCR and WB results suggested thatEV71infection in MRC-5cells suppressed the induction of IFN-β. The MRC-5cells were mock or GDV103infected at MOI of10for10h and then cells were stimulatedwith IFN-α2b (1000U/ml) for another1h. PCRArray detection of gene expression inIFN signal pathway showed that EV71inhibited ISGs expression. Taken together,these results revealed that EV71facilitates its survival and replication not only byinhibiting the endogenous IFN production but also bleach the exogenous IFNtreatment.Type I interferon can activate the downstream antiviral protein gene through theclassic JAK-STAT signaling pathway. Each stage of the JAK-STAT signaling pathwaycan be disrupted by viral proteins. As STAT1and STAT2are key players in IFNsignaling pathway, we first checked if EV71infection altered the phosphorylation ofSTAT1and STAT2. Western Blot analysis showed that there was no significantchange in p-STAT1，p-STAT2expression after viral infection at low MOI in bothMRC-5and RD cells. But when infected with EV71at a high MOI, thephosphorylated STAT1and STAT2were significantly reduced in the EV71infectedcells after IFN-α2b treatment. To examine the translocation of ISGF3inEV71-infected cells, we transfected vero E6cells with pEGFP-C1-STAT1andsubsequently infected the cells with VR1432. Nuclear and cytoplasmic of the cellswere fractioned and Western Blot indicated that EV71infection blocked nucleartranslocation of the ISGF3(STAT1/STAT2/IRF9). Since EV71infection inhibited thephosphorylation of STAT1and STAT2, we further wanted to know if IFNARinfluenced the phosphorylation of STAT1and STAT2. Western Blot and FACSanalysis showed that neither GDV103nor VR1432infection significantly altered theIFNAR1protein levels in MRC-5cells and treatment with IFN-α2b (1000U/ml)didn’t reduce the expression of surface IFNAR1on MRC-5cells. Further weexamined expression and activation of the Jak kinases that link receptor activation toSTAT protein phosphorylation. GDV103and VR1432markedly downregulated Jak110h p.i., which correlated with loss of detectable phosphorylated Jak1and Tyk2.However viral infection did not signifcantly affect Tyk2expression.Further assay showed that the downregulated Jak1protein did not result frommRNA level of Jak1and the downregulation of Jak1was proteasome-independent. Ithas reported that EV71can facilitate their replication and proliferation throughautophagy. We want to know whether the reduction of Jak1was caused by autophagy.MRC-5cells were pretreated with3-MA, the autophagy inhibitor, infected with EV71and harvested to measure Jak1level. The results indicated that treatment with3-MA didn’t reverse the reduction of Jak1in EV71-infected cells. Further assay also showedthat the lysosome may mediate the reduction of Jak1.IFN-induced JAK-STAT signaling can be inhibited at different levels by viralprotein and cellular factors. There are some cellular key regulators, capable ofnegatively regulating JAK-STAT signaling, include suppressor of cytokine signaling,(SOCS), protein inhibitor of activated STATs (PIAS), protein tyrosine phosphatases(PTP). But our results showed that none of the proteins mediated the inhibition ofIFN-α-induced STAT1and STAT2phosphorylation in EV71-infected cells. Toidentify the potential viral proteins2A and3C which may function as antagonists toIFN signaling, we constructed pcDNA3.1(+)-2A and pcDNA3.1(+)-3C plasmids.Transiently transfection with the two plasmids and Western Blot showed that neither2A nor3C was an antagonist of IFN signaling.In our work, we provided the evidence that the downregulation of Janus tyrosinekinase (Jak1) induced by EV71inhibited IFN’s anti-viral pathway while IFNAR1didnot significantly changed after EV71infection. Also we found that the inhibition ofSTAT1and STAT2phosphorylation was not mediated by phosphatases (PTP) andSOCS1or SOCS3. Downregulation of Jak1reduced the phosphorylation of Jak1andTyk2, next to phosphorylation of STAT1and STAT2and the downregulation of Jak1was proteasome-independent but may lysosome-dependent. The protein2A and3C ofEV71were not responsible for the downregulation of Jak1and phosphoraletd STAT1and STAT2. |
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