The Inhibition Of Hepatitis E Virus By Zinc Finger Antiviral Protein ZAP

Hepatitis E virus(HEV),a kind of enterically transmitted viral hepatitis,caused serious damages to human health.However,the relationship between HEV and the host is not very clear.The zinc finger antiviral protein ZAP is a host restriction factor,which can specifically bind the ZRE region of the viral genome to cut viral genome by recruiting host enzymes,so as to achieve the antiviral effect.Virus and interferon would stimulate a significant increase of ZAP,of which belongs to the interferon stimulated gene ISG.But the relationship between ZAP and HEV has not been studied clearly.The main work of this study is to explore the effect of ZAP on HEV replication and the preliminary study of its mechanism,including 4 parts:1.HEV regulates ZAP in replication cycleZAP is a member of the interferon stimulated gene ISG.In order to explore whether HEV can regulate the expression of ZAP,serum of HEV acute infection were detected,serum of HEV acute un-infection as control.The results showed that ZAP was significantly inhibited in HEV acute infection patient,at the same time,IFN-P was also significantly inhibited.To explore the relationship between the regulation of ZAP by HEV and HEV replication cycle,we transfected HEV RNA into A549 cells and collected at different time points.The results showed that ZAP was inhibited at 4 h of HEV infected cells,however,and ZAP were significantly up-regulated by host innate immunity at 12-120 h after HEV infection.2.ZAP inhibited HEV replication effectivelyThe interaction of virus and host is a hotspot to research the pathogenic mechanism.In order to explore the effect of ZAP on HEV replication,we overexpressed ZAP in A549 cells then transfected HEV RNA.Results showed that overexpression of ZAP inhibit the replication of HEV effectively.In order to verify the effect of ZAP on HEV replication,we performed on A549 cells by knocking down ZAP then transfection HEV RNA.Results showed that ZAP knockdown promoted the replication of HEV significantly.In order to find the specific region ZRE in the HEV binding by ZAP,we constructed the pMIR-Report-HEV-5 luciferase reporter vector and pMIR-Report-HEV-3 'luciferase reporter gene vector,then co-transfected pMIR-Report-HEV-5'-luc,pMIR-Report-HEV-3'-luc and ZAP.The results showed that ZAP could inhibit pMIR-Report-HEV-5'-luc luciferase activity.3.ZAP assisted IFN-p to inhibit HEV replicationInnate immunity is the first line of defense against pathogen invasion in host cells.By identifying the virus replication intermediates dsRNA,the host activated the phosphorylation of IRF3,synthetized a large number of type I interferon,thereby eliminated pathogens invading.ZAP as a member of the ISG gene,in order to explore whether ZAP is involved in inhibition of HEV replication by IFN-?,we first transfectd A549 cells with HEV RNA,then transfected into ZAP-shRNA,poly(I:C),ZAP-shRNA+poly(I:C),respectively.The results showed that ZAP knockdown would increase HEV replication,and poly(I:C)can inhibit the replication of HEV,but knocking down ZAP could weaken the ability of poly(I:C)to stimulate the host to suppress HEV replication.4.To investigate the effect of ZAP on HEV replication in animal modelsPrevious experiments,we found that after HEV infection,ZAP showed a significant increase trend with virus replication.Over expression of ZAP can effectively inhibit the replication of HEV,knockdown of ZAP will weaken the poly(I:C)to stimulate the host to suppress HEV replication.In order to further explore whether in in vivo environment ZAP still can effectively inhibit the replication of HEV,we had the expression of ZAP in BALB/c mice,which was infected by HEV 24 h later.Results showed that experimental infection of HEV for 24 h,ZAP was up-regulated in serum and liver in mice,while overexpression of ZAP could effectively inhibit HEV replication with an enhancing inhibition effect under ZAP concentration gradient.This paper found that ZAP would be inhibited in HEV acute infection,and ZAP specifically bound to HEV 5 '-UTR to inhibit HEV replication.ZAP assisted IFN-? to inhibit HEV replication.Overexpression of ZAP showed a concentration gradient on the effective inhibition to HEV replication in BALB/c mice.