Function Of Rap1b During Internalization Process Of Avian Hepatitis E Virus Into Permissive Cells

Avian hepatitis E virus(Hepatitis E virus,HEV)is a hepatotropic virus that spreads widely in chickens,and it mainly causes Big liver and spleen syndrome(BLS)in infected chickens.The specific manifestations of the infection include hepatosplenomegaly,decreased egg production rate and a certain mortality rate,which has brought huge economic losses to the poultry breeding industry.Due to the lack of efficient in vitro virus proliferation cell lines,research on the life cycle of HEV is progressing slowly and greatly hinders the development of vaccines.In the previous study,the avian HEV truncated capsid protein ap237(aa313-549)was used as a bait protein in the GST pull-down assay to pull the potential host protein in chicken liver cells.After mass spectrometry,bioinformatics analysis and functional verification,viral infection-related membrane protein organic anion transport peptide 1A2(OATP1A2)with high expression level in the liver was identified to construct a cell line LMH-IA2 that can support the replication of avian HEV.On this basis,another membrane protein Ras-related protein 1b(Rap1b)that interacts with ap237 was screened,and found that Rap1b was significantly up-regulated in the liver of virus-infected animals.Therefore,this study explored the relationship between Rap1b and avian HEV infection for the first time,and clarified the related cascade signal pathway induced by its interaction with the avian HEV capsid protein.The obtained results of the molecular mechanism during early infection of avian HEV was preliminarily analyzed,and are as follows:1.Rap1b is associated with early infection of CaHEVFirstly,the host protein Rap1b is determined to be the interaction protein of the viral capsid protein by Co-IP,ELISA and Confocal assays.Subsequently,the transcription and post-translational expression levels of Rap1b under in vivo and in vitro were monitored after the Chinese avian HEV isolate(CaHEV)infection and showed significantly up-regulation.In addition,there was a time-dependent activation of Rap1b during the early stage of viral infection,indicating a key role in this process.Of note,the internalization of the virus is significantly inhibited when the Rap1 protein is knocked down,which further implies that Rap1b plays an important role in the entry process.2.The activation of Rap1b induced by CaHEV infection is regulated by PKA and the chaperonin Smg GDSThe activation of Rap1b is regulated by its upstream protein PKA.Phosphorylated PKA promotes the phosphorylation of Rap1b and inhibits its potential for excessive activation.Considering of the time-dependent activation manner of Rap1b,the activation of PKA was detected during the process of virus internalization.It was found that PKA phosphorylation directly participates in the activation of Rap1b and is negatively correlated with virus internalization.Interrestingly,while treatment of cells with the inhibitor H-89 and agonist Forskolin targeting PKA phosphorylation,the activation of Rap1b showed consistentency with the trend of the virus internalization efficiency.Meanwhile,the small GTP binding protein dissociation stimulating factor(Smg GDS),which promotes the activation of Rap1b by GTP loading,has also been shown to play a decisive role in the activation of Rap1b.The knockdown of Smg GDS almost offset the promotion effect of Forskolin on the activation of Rap1b induced by viral internalization stimulation,and even made the content of Rap1b-GTP significantly lower than that of untreated cells.3.Activation of Rap1b promotes membrane transfer between RIAM and Talin-1After Rap1b activation analysis,the intracellular distribution of the Rap1 interaction effector(RIAM)was preliminarily investigated,and found that the transfer of RIAM to the membrane was consistent with the activation of Rap1b induced by virus internalization.When the polyclonal antibody(anti-CaHEV p Ab)derived from CaHEV-positive chicken serum was used to effectively inhibit the internalization of the virus,the transfer of RIAM was significantly inhibited as expect.Importantly,the transfer manner of Talin-1 in the cytoplasm and membrane indicated a similarity as RIAM,and the treatment of anti-CaHEV p Ab also resulted in the blocking of Talin-1 membrane anchoring.In addition,the membrane retention of Talin-1 was also suppressed when the RIAM protein was knocked down.Further research focused on the activation of integrin α5/β1 revealed same up-regulation at the same time,and the interaction between Talin-1 and integrin α5/β1 was enhanced after stimulation with virus internalization by Co-IP assay.Subsequently,activation of integrin α5/β1 were also showed inhibition with si RNA knockdown of Talin-1and RIAM proteins,and the accumulation of capsid protein also appeared on the cell membrane,suggesting a positive regulation effect of Rap1b-RIAM-Talin-1 signal axis on the integrin α5/β1.4.Rap1b-integrin α5/β1-Cofilin cascade pathway is involved in viral internalization mediated by cell membrane rearrangementIn order to analyze the downstream signaling pathway of integrin α5/β1 related to Cofilin-mediated cell membrane rearrangement,the activation of its adaptor protein was monitored.It was found that the activation of the integrin α5/β1-CDC42/RAC1-PAK1-LIMK1 pathway regulates the activity of Cofilin by promoting the phosphorylation of FAK instead of Src.As expectation,no mater Forskolin,H-89 treated cells both declared a regulation on the activation of integrin α5/β1 same as ATN-161,an inhibitor of integrinα5/β1.Moreover,all of the downstream proteins of integrin α5/β1 were regulated,which proved the signaling pathway starting from Rap1b activates its downstream cascade via integrin α5/β1.In summary,Rap1b interacts with the avian capsid protein ap237 to promote the transfer of its downstream proteins RIAM and Talin-1 to the cell membrane,while Talin-1interacts with integrin α5/β1 and further promotes its activation,and then activates downstream signaling pathway FAK-CDC42/RAC1-PAK1-LIMK1-Cofilin.Finally,the activation of Cofilin directly participates in F-acin-mediated cytoskeletal rearrangement,and this normal cellular physiological process is utilized by CaHEV to invade the host cell.