The Role Of CDC42 In Hepatitis E Virus Infection In Host Cells

Hepatitis E Virus(HEV),a potential zoonotic pathogen,causes acute viral hepatitis in humans worldwide and belongs to the family Hepeviridae,while a subset of HEV species,avian HEV,causes hepatitis-splenomegaly syndrome in chickens.Due to lack of efficient cell culture system,there are few reports on the host proteins interacting with HEV and being involved in viral infection.Previous pull-down assay combining mass spectrometry indicated that cell division control protein 42(CDC42),a member belonging to the Rho GTPase family,was pulled down by avian HEV capsid protein.Here,the study mainly focused on the“the role of CDC42 in HEV infection”,containing the following two parts:Part ? CDC42 and its downstream signaling are associated with avian HEV(a HEV)infection1.Identification the interaction between CDC42 and a HEV truncated capsid protein ap237To identify the interaction between CDC42 and the truncated avian HEV capsid protein ap237(aa 368-606),the CDC42 eukaryotic and prokaryotic expression vectors were firstly constructed,and the specific interaction between ap237 and exogenous and endogenous CDC42 was identified in HEK 293T cells and LMH^OATP1A2cells by co-immunoprecipitation(Co-IP)and pull-down assay,respectively.Then,the prokaryotic expressed and purified CDC42 was confirmed that it could directly bind to ap237 in vitro by Co-IP and ELISA.Besides it,colocalization of CDC42 and ap237 signals was also observed in HEK 293T cells and LMH^OATP1A2cells.Taken together,these results showed that avian HEV capsid protein ap237 and CDC42 directly interacted with each other in vitro and in vivo.2.The expression level and activity of CDC42 is positively correlated with a HEV Infection.Previous reports have demonstrated that ap237 can mimic natural HEV virions to attach and enter host cells.Here,it was detected that the amount of GTP-CDC42 and total CDC42 in cells gradually increased with the entry of ap237/a HEV in the ap237 and a HEV entry assays.And CDC42 levels in the a HEV-positive LMH^OATP1A2cells and chicken livers was also up-regulated.the positive correlation between the GTP-CDC42 and total CDC42levels and amount of ap237/a HEV was observed.Through the CDC42 in vitro activation experiment,it was found that the interaction can increase the amount of active guanosine triphosphate binding CDC42 state(GTP-CDC42).Subsequent the results of CDC42overexpression,siRNA interference and inhibitor treatment experiments showed that the expression level and activity of CDC42 was positively correlated with the ap237 entry and a HEV infection.In conclusion,these results indicated that a HEV infection was positively correlated with the expression level and activity of CDC42.3.CDC42-MRCK signaling participated in CaHEV InfectionThere are three CDC42 downstream signaling pathways,including p21-activated kinase 1(PAK1)signaling,MRCK(a CDC42-binding protein)signaling and neural Wiskott-Aldrich syndrome protein(N-WASP)signaling.To confirm which above signaling pathway involved in a HEV,the six inhibitors of Rho GTPases were used,and the results showed that once the activities of RAC1,myosin light chain kinase(MYLK)and non-myosin IIA(NMIIA)were inhibited,a HEV infection was significantly decreased,which was also comfirmed by RNA interfering and pull-down assay.Altogether,CDC42-MRCK signaling pathway that participated in a HEV infection.Part ? CDC42 signaling pathways participated mammalian HEV infectionIt has been reported that the existing HEV virions have two different forms,namely non-enveloped form(neHEV)and quasi-enveloped form(eHEV),which are thought to use different mechanisms to invade host cells.Here,we further confirmed the direct interaction between CDC42 and mammalian capsid protein using Co-IP and ELISA.The CDC42activation assay was then performed,and the result showed that the interaction also promoted the active GTP-CDC42 levels.With cell entry of neHEV,up-regulations of GTP-CDC42 and total CDC42 were observed in HepG2/C3A cells.Subsequent CDC42signaling pathways analysis found that CDC42-MRCK and CDC42-(N-)WASP signaling pathways participated neHEV infection,while CDC42-PAK1 and CDC42-(N-)WASP signaling pathways participated eHEV infection.In summary,this study revealed that CDC42 directly interacts with HEV capsid proteis,with a positive correlation observed between HEV infectivity and CDC42expression level and activity.This interaction between CDC42 and HEV capsid protein is mediated by direct binding of GTP to CDC42 such that increases in number of GTP-CDC42 complexes leads to increases in number of HEV virions.Specifically,avian and mammalian HEV were all shown that utilize CDC42 signaling pathways,although different pathways were triggered based on virus species and particle form;the CDC42-MRCK-NMIIA pathway was hijacked by non-enveloped HEV virions,the CDC42-(N-)WASP-Arp2/3 pathway was hijacked by all virion forms of HEV,and the CDC42-PAK1-NMIIA/Cofilin pathway was hijacked by quasi-enveloped HEV.These results elucidate the molecule mechanisms of CDC42 in HEV internalization and will be valuable for guiding design of therapeutics to combat HEV and other virues.