Mechanism Of Enterovirus A71 Evading OAS3/RNase L Antiviral Pathway

Hand-foot-mouth-disease（HFMD）is one of the most important public health problems in the world in recent years.As the main pathogen of hand-foot-mouth-disease（HFMD）,it is of great significance and practical value to study the mechanism of interaction between enterovirus and host.It is worth noting that Enterovirus A71（EVA71）is the main pathogen causing severe hand-foot-mouth disease（HFMD）,causing fatal nervous system diseases and myocardial inflammatory heart disease.Severe hand,foot and mouth disease（HFMD）and its complications pose a serious threat to the health of children worldwide,but until now,there is still a lack of specific drugs for EVA71 infection.Therefore,it is urgent to pay attention to the replication mechanism of EVA71 and other enteroviruses as well as the interaction between EVA71 and other enteroviruses and the host,so as to provide more data basis for the clinical treatment of hand-foot-mouth disease.The 2’,5’Oligoadenylate Synthetase（OAS）protein family was one of the earliest discovered interferon stimulated Genes（ISGs）,and was the first line of defense against viral infection.It induces and activates downstream RNA enzyme L（RNase L）to shear and degrade viral RNA.The OAS/RNase L-coupled antiviral pathway has been verified to have antagonistic effects against a variety of viruses.OAS3 is the main protein in the OAS protein family that activates RNase L activity,so OAS3 protein was selected as the target protein in this study to explore the interaction mechanism between OAS3/RNase L pathway and EVA71.In this study,we first verified the mechanism of non-specific Interferon（IFN）response induced by EVA71 infection.After HEK293T cells were infected with EVA71,the m RNA levels of IFN-β,IFN-γand IL-29 belonging to type Ⅰ interferon,type II interferon,and type Ⅲ interferon were widely up-regulated.After HEK293T cells were pretreated with these three IFNs and then infected with EVA71,IFN-βtreatment showed less Cytopathic Effect（CPE）,and IFN-βcould strongly up-regulate endogenous OAS3 m RNA levels.In addition,silencing of OAS3 protein in HEK293T cells significantly reduced the inhibitory effect of IFN-βon EVA71 replication.These results suggest that EVA71 infection induces the non-specific immune response of IFN-β/OAS3,and that OAS3 plays an important role in IFN-β-mediated inhibition of EVA71 replication.Then,the key Signal factor Of OAS3 protein expression induced by interferon was verified.Signal transduction and Activator of Transcription1（STAT1）or STAT3 KD（Knock Down）HEK293T cells was pretreated with IFN-βor EVA71,respectively.and the expression of endogenous STAT1,p-STAT1,STAT3,p-STAT3,and OAS3 proteins was detected.IFN-βand EVA71 significantly reduced the induction and upregulation of OAS3 protein,while STAT3 knockdown did not affect the expression of OAS3,suggesting that STAT1 is essential for the synthesis of OAS3protein induced by IFN-βand EVA71.Next,the interaction between EVA71 and OAS3/RNase L pathway was discussed.The results showed that silencing of OAS3 protein promoted EVA71replication,while overexpression of OAS3 protein inhibited EVA71 replication,and OAS3 protein inhibited EVA71 replication in an RNase L-dependent manner.The activation of RNase L was related to the activity of OAS3 protein.The four inactivated mutants D816A,D818A,D888A and K950A,which were related to the activity of OAS3 protein synthesis 2-5A,not only lost their antagonism against EVA71,but also lost the activation ability of RNase L enzyme digestion activity.Although EVA71 could up-regulate the expression of OAS3 protein to activate RNase L,EVA71 could not induce the expression of RNase L.In addition,EVA71 was found to up-regulate OAS3 protein expression in the early stage of infection,but down-regulate OAS3 protein expression in the late stage of infection.This phenomenon suggests that EVA71 may down-regulate the expression of OAS3protein through some unknown mechanism,thereby escaping the antiviral effect of OAS3/RNase L pathway.In order to verify the escape mechanism of EVA71,seven non-structural proteins of EVA71（2A-2C,3A-3D）were co-transfected withOAS3 protein respectively in this study,and the results showed that EVA71 3C^pro mediated the down-regulation of OAS3 protein expression in a dose-dependent manner.And both the addition of Rupintrivir,an inhibitor of the enzyme activity of EVA71 3C^pro,and the construction of an inactivated mutant of EVA71 3C^pro（H40G,E71A,and C147G）restored the down-regulation of OAS3 protein by EVA71 3C^pro.In addition,anti-HA antibody was used to detect the whole protein of the samples in the Western Blot experiment,and it was found that compared with the chromogenic bands of the cell samples transfected withOAS3 protein alone,the co-transfected samples of OAS3 and EVA71 3C^pro had an additional small protein band of about 20 k Da at the bottom of the membrane.This suggests that EVA71 3C^pro May cleave OAS3 protein through protease activity,thus escaping the antiviral effect of OAS3/RNase L pathway.On this basis,the cleavage sites of EVA71 3C^pro in OAS3 sequence were verified.According to the size of small protein cleavage bands and sequence analysis,four potential cleavage sites（Q726-A727,Q832-G833,Q883-S884 and Q982-G983）were identified.By constructing an inactivated mutant,Q982-G983 in OAS3 protein was identified as an important cleavage site for EVA71 3C^pro.In addition,the immunofluorescence co-localization experiment showed that compared with wild-type OAS3 protein,the binding ability of EVA71 3C^pro to OAS3 protein Q982A mutant was relatively weak,further verifying that Q982-G983 site played an important role in the enzyme digestion activity of EVA71 3C^pro.Finally,the present study investigated whether the OAS3/RNase L pathway has a broad inhibitory effect on enterovirus.The results show that the OAS3/RNase L pathway can inhibit Enterovirus A71（EVA71）,Coxsackievirus A16（CVA16）and Coxsackievirus B3（CVB3）,the three most common enteroviruses showed significant inhibitory effects.However,there were significant differences between CVA16 and the other two viruses in the replication level and the regulation level of OAS3 protein under the inhibition of OAS3/RNase L pathway.The reason for these differences was that CVA16 had different ability to antagonize OAS3/RNase L antiviral pathway from the other two viruses.Compared with 3C^pro of EVA71 and CVB3,CVA16 3C^proshowed weaker shear ability to OAS3 protein.Sequence analysis revealed three distinct amino acid sites（I36,V86 and I157）in CVA16 3C^pro,which were different from EVA71 3C^pro and CVB3 3C^pro.The amino acids corresponding to these three sites of EVA71 3C^pro were replaced by site-directed mutagenesis technology,namely,three EVA71 3C^pro mutants V36I,I86V and V157I were constructed.The results showed that the V36I and I86V mutants of EVA71 3C^pro completely lost the ability to shear OAS3,indicating that V36 and I86 were important amino acids affecting the activity of EVA71 3C^pro.These results suggest that the differentiation and evolution of different enterovirus 3C^pro enzymes is related to the escape of the virus from the OAS3/RNase L system.In conclusion,OAS3 protein specifically induced by IFN-βis coupled with the downstream RNase L protein in the antiviral pathway,which has a broad inhibitory effect on EVA71,CVA16 and CVB3,three common enteroviruses,and EVA71transcriptional induction of OAS3 protein via IFN-β/STAT1.However,EVA71 has evolved a mechanism to escape the antiviral effect of the OAS3/RNase L pathway by splicing the OAS3 protein using its own 3C^pro.This study provides a new idea for the clinical treatment of EVA71 infection and the development of specific antiviral drugs.