Molecular Mechanism Of Interaction Between Enterovirus 71(EV71)and Host Restriction Factors

Enterovirus 71(EV71),which is the main pathogen causing hand,foot and mouth disease,has been widely concerned,and the hand,foot and mouth disease caused by it has erupted every year around the world.What is even more worrying is that EV71 can induce severe hand,foot and mouth disease.The symptoms are pulmonary edema,aseptic meningitis,etc.Even a small number of sick children develop rapidly and die.Although hand,foot and mouth disease erupts every year,therapeutic drugs for EV71 have been vacant.Therefore,in order to develop specific drugs for EV71 and even enteroviruses,the infection mechanism of enterovirus and its interaction with host restriction factors are urgently needed,and it is the only way.Based on previous studies,this study focused on the interaction of EV71 with host restriction factors.According to different host factors,it is mainly divided into two parts:The first part: the interaction and mechanism of EV71 and host restriction factor A3 G.First,we performed the same titer of EV71 virus infection in H3 cells with high expression of A3 G and Jurkat cells with little expression of A3 G,and found the antiviral effect of A3 G on EV71.Subsequently,we performed an EV71 virus infection experiment in A3 G knockdown H9 cell line and A3 G overexpressing HEK293 T cells,again verifying the antiviral effect of A3 G on EV71.Second,in the presence of A3 G,we examined changes in viral RNA and protein levels.It was found that A3 G affected the RNA replication of EV71,which in turn affected the expression of viral proteins.Finally,we studied the mechanism of A3 G anti-EV71 virus and found that the anti-EV71 activity of A3 G does not depend on its well-known cytosine deaminase activity,which indicates that A3 G has other ways to play a role in anti-intestinal virus.This is also the first time to identify the limiting effect of A3 G on enterovirus,and pointed out its potential new antiviral mechanism.Through immunoprecipitation,EMSA and other techniques,we found that A3 G binds to EV71 viral RNA and identifies key sites for A3 G binding to the 5'UTR.So,does A3 G binding alter the secondary structure of viral RNA,or does it affect other transcriptional regulators required for EV71 replication? In order to explore its mechanism of action,in the presence of A3 G,we examined the binding of some transcription factors necessary for EV71 replication and translation to EV71-5'UTR.We found that the combination of PCBP1 and EV71-5'UTR was attenuated in the presence of A3 G.Subsequently,we demonstrated the competition of A3 G for PCBP1 by RNA pull down assay and RNA IP assay,and elaborated the molecular mechanism of A3 G restriction of EV71 virus replication.In the interaction between the virus and the host,the virus has also evolved a corresponding mechanism to evade the host.So,how does the EV71 virus escape the role of the host restriction factor A3G? In studying the mechanism of A3 G against EV71,we found that the non-structural protein 2C of EV71 virus can specifically down-regulate the expression of A3 G protein.In order to explore the mechanism by which 2C protein down-regulates A3 G,we first determined that 2C did not down-regulate A3 G m RNA by RT-PCR.Secondly,it was confirmed that 2C degraded A3 G by the autophagy-lysosomal pathway by adding inhibitors such as MG132 and Baf-A1.At the same time,through immunoprecipitation and immunofluorescence techniques,we found that 2C can specifically bind to A3 G and ubiquitinate it,thereby inducing selective autophagy degradation of A3 G.In summary,we discovered the limiting role of A3 G on EV71 and systematically described the molecular mechanism of A3G-restricted EV71.At the same time,we also found that the EV71 virus breaks through the A3 G restriction mechanism,that is,the non-structural protein 2C induces A3 G for selective autophagy.The second part: the interaction between EV71 and host restriction factor SAMHD1 and its mechanism.First,we screened the restriction factors by different sensitivities of different cells infected with EV71,and found that SAMHD1 restricted the replication of EV71 virus.Furthermore,a stable cell line of SAMHD1 knockdown was constructed by RNA interference technology,and the antiviral effect of SAMHD1 was determined.Second,we compared the antiviral activity of wild-type SAMHD1 and its various active deletion mutants.As a result,it was found that the effect of SAMHD1 against EV71 virus was not related to the reported activities such as d NTPase activity,RNAse activity,and tetramerization of SAMHD1.However,it is related to the phosphorylation of SAMHD1 at position 592 and is consistent with the effect of SAMHD1 on HIV replication,that is,non-phosphorylated SAMHD1 has an antiviral effect,while phosphorylated SAMHD1 loses its antiviral activity.When we used the EV71 susceptible cell line-RD cells to construct a stable expression of SAMHD1 cell line,it was found that even if overexpressed,the expression level of SAMHD1 protein was still low,and the addition of MG132 could increase the expression of SAMHD1.This indicates the presence of a pathway that specifically degrades SAMHD1 in RD cells.By immunoprecipitation tandem mass spectrometry,we screened the E3 ubiquitin ligase,TRIM21,which ubiquitinates SAMHD1.At the same time,the site of ubiquitination of SAMHD1 was identified as the lysine at position 622 by point mutation.In EV71-infected cells,we found that the expression of TRIM21 was greatly enhanced,while SAMHD1 was degraded.To verify the relationship between the three,we constructed the SAMHD1-K622 A mutant(which could not be ubiquitinated by TRIM21)and found that the SAMHD1-K622 A mutant could not be degraded by EV71.At the same time,according to the literature,TRIM21,as a class of interferon-stimulated genes(ISG),can be activated by interferon.By establishing a stable cell line that knocks down IFNAR1,we determined that the ability of EV71 to downregulate SAMHD1 was greatly attenuated after IFNAR1 was knocked down.The above results have jointly proved that the limiting effect of EV71 in breaking SAMHD1 is to induce the expression of TRIM21 through the interferon system activated during viral replication,thereby achieving the purpose of degrading SAMHD1.In summary,this paper comprehensively studied the molecular mechanism of interaction between enterovirus EV71 and host restriction factors(A3G and SAMHD1).It provides a potential target for the development of specific new drugs for EV71 infection,and also provides a new direction for the study of the way viruses interact with the host.