The Function And Mechanism Of Microfilament Cytoskeleton Mediated Porcine Hemagglutinating Encephalomyelitis Virus Invading Nerve Cells

Porcine hemagglutinating encephalomyelitis(PHE)is an acute and highly contagious disease caused by infection with porcine hemagglutinating encephalomyelitis virus(PHEV),which mainly affects piglets within 3 weeks of age,causing vomiting and wasting disease and/or obvious neurological symptoms.The mortality rate can reach 100%.The virus mainly attacks the nervous system.However,the mechanism of nerve damage caused by the virus is not fully elucidated.Microfilament(Fibrillar actin,F-actin)is an important component of the cytoskeleton of eukaryotic cells and serves as the first obstacle to the entry of pathogens into host cells.Additionally,the morphological structure and function of nerve cells depend on the dynamic regulation of the F-actin.Based on our previous studies,PHEV enters nerve cells through clathrin-mediated endocytosis,and this process relies on the integrity of the F-actin.Currently,Whether F-actin is involved when PHEV enters the host cell,and the regulation pattern of the actin cytoskeleton and its molecular mechanism remain unclear.Therefore,exploring the mechanism of neuronal injury induced by PHEV from the perspective of the F-actin not only helps elucidate the pathogenesis of PHEV but also provides a theoretical basis for the search for new antiviral targets.In this study,the chnages of F-actin structure in PHEV invasion of mouse neuroblastoma(Neuro-2a)cells will be analyzed by fluorescence staining,invasion kinetics based on PHEV infected neural cell model.Compared with inactivated virus and mock groups,we found that PHEV entry into N2 a cells induced the formation of filopodia and lamellipodia or stress fibers at different times.Flow cytometry results showed that F-actin polymerized rapidly after 5 min(mpi)of virus infection and then began to depolymerize at 20 mpi.These results indicate that PHEV infection induces N2 a cells actin cytoskeletal remodeling.Subsequently,the kinetic curve of the virus entering the cell was determined by indirect immunofluorescence and qRT-PCR methods.The results showed that the viral particles moved into the cells along the filopodia,and the virus entry of the cell primarily occurred after 15 mpi,suggesting that F-actin structural remodeling may be associated with viral entry.Furthermore,we treated N2 a cells with Cyto D,which binds to and cleaves F-actin and binds at the ends of the filaments to impede the polymerization of actin at that site.qRT-PCR,Western blotting,indirect immunofluorescence and virus growth kinetics were used to detect the entry and proliferation of PHEV.The results indicate that Cyto D inhibits virus entry and reduces virus titer in a dose-dependent manner.The above results indicated that the entry of the virus is closely related to the dynamic changes of the cytoskeleton caused by the virus.Cofilin is an important regulatory protein in the cytoskeletal depolymerization factor family,which can regulate its dynamic changes by cutting F-actin.Because PHEV invasion of cells led to remodeling of the actin cytoskeleton,we examined the changes in cofilin activity during the viral infection.The activation of cofilin during virus entry was detected by Western blotting and indirect immunofluorescence.The results showed that the dynamic activity of cofilin was consistent with the trend of F-actin,suggesting that it may be involved in the process of PHEV invasion.Furthermore,N2 a cells were transfected with cofilin siRNA,a wild-type expressing vector(WT),a constitutively unphosphorylated mutant cofilin(activated,S3A)or the constitutively phosphorylated mutant cofilin(inactivated,S3D),respectively,and then inoculated with PHEV.The amount of virus entry and the change of F-actin were detected by qRT-PCR,Western blotting and indirect immunofluorescence.Notably,all three constructs inhibited PHEV entry in a dose-dependent manner,similar to the results of inhibition of cofilin expression,indicating that the effective entry of PHEV into cells requires dynamic phosphorylation by cofilin rather than a certain fixed state.In addition,cofilin-specific siRNAs could dilapidate cellular stress fibers,form small protrusions on the cell surface,and PHEV can reduce the formation of rod-like structures caused by enhanced cofilin activity.These observations suggested that alteration of cofilin activity favors viral adsorption and entry into cells.At the same time,the activation of the cofilin upstream kinase LIMK during PHEV infection and its effect on viral entry were examined.The results showed that LIMK activity also showed dynamic changes,and the trend was consistent with the trend of cofilin activity.Decreasing endogenous LIMK expression in cells also inhibits viral entry,indicating that LIMK acts as a cofilin upstream kinase to regulate the invasion of PHEV cells.Cytoskeletal signaling molecules are usually mediated by G-protein coupled receptors,lectins and receptor tyrosine kinases(RTKs)in the regulation of cofilin activity.To further investigate the molecular mechanism of F-actin-mediated PHEV invasion of N2 a cells,specific chemical inhibitors of the above various signal molecules are used to block the signaling pathway mediated.The effects of different signaling molecules on cofilin activity and PHEV invasion were detected by qRT-PCR,Western blotting,GST-pull down and indirect immunofluorescence.The results showed that RTKs were not involved in PHEV entry;integrin α5β1 not only accumulated when PHEV is attached,but also promoted activation of downstream kinases FAK and PI3 K / Akt signaling pathways;Src was not involved in PHEV entry,indicating that PHEV regulates F-actin rearrangement through the FAK signaling pathway that is independent of c-Src interaction;Rac 1 and Cdc 42 GTPases were downstream Rho GTPases of the integrin α5β1-FAK pathway,and both Rac 1 and Cdc 42 GTPases affect the rearrangement of F-actin and cofilin activity by regulating downstream PAK 1 molecules;Rho A was the downstream target protein for the integrin α5β1-PI3 K / Akt pathway and affects the rearrangement of F-actin and cofilin activity by regulating downstream ROCK kinase.We determined that cofilin is a key factor required for PHEV infection.PHEV entry required a two-stage process of rapid polymerization and depolymerization of actin,and PHEV triggered bidirectional regulation of cofilin protein activity.Viral binding induced phosphorylation of cofilin by the FAK-Rac 1 / Cdc 42-PAK 1-LIMK and PI3 K / Akt-Rho A-ROCK-LIMK signaling pathways downstream of the integrin α5β1 signaling molecule for actin polymerization and efficient entry.Subsequent viral penetration activated cofilin,resulting in the fragmentation of existing actin filaments to promote viral trafficking.Integrin α5β1 signaling molecule is a key factor for PHEV to enter N2 a cells.To determine if integrin α5β1 has similar functions in vivo,this study established a mouse infection model to detect activation of this signaling molecule and downstream pathways.The results showed that the expression of integrin α5β1 was increased during viral infection in mice,consistented with the results at the cellular level;the FAK-Rac 1 / Cdc 42-PAK 1-LIMK pathway was activated,while the PI3 K / Akt-Rho A-ROCK-LIMK pathway was not fully activated,suggesting that integrin α5β1-FAK-Rac 1 / Cdc 42-PAK 1-LIMK-cofilin may also play a role in viral infection in vivo.BALB/c mice were injected intravenously with ATN-161 to reduce integrin α5β1 expression in the brain.Whether ATN-161 has antiviral effect in vivo is detected by qRT-PCR,Western blotting,GST-pull down,indirect immunofluorescence,H&E staining.The results showed that ATN-161 treatment delayed the onset of mice,reduced symptoms,decreased weight loss,and significantly prolonged survival.H&E staining showed that the neuronal cells of ATN-161 treated mice tend to be normal,and there were no typical non-suppurative encephalitis symptoms at 5 dpi.In addition,viral proliferation and integrin α5β1-FAK signaling pathway were significantly inhibited after intravenous injection of ATN-161.These experiments confirmed that integrin α5β1-FAK-Rac 1 / Cdc 42-PAK 1-LIMK-cofilin can promote PHEV proliferation in vivo;and integrin α5β1 inhibitor ATN-161 has a certain antiviral effect in vivo.In conclusion,this experiment for the first time elucidated the specific mechanism of F-actin cytoskeletal remodeling during PHEV entry into N2 a cells and confirmed that cofilin activity is a molecular switch between F-actin remodeling and viral entry,which could provide a theoretical basis for a better understanding of the pathogenesis of PHEV-induced neurological symptoms.In addition,the integrin α5β1 specific inhibitor ATN-161 has a certain antiviral effect in vivo,indicating that cofilin and upstream signaling molecules are viable targets for development of therapeutic antiviral strategies,which could provide theoretical support for the development of new antiviral drugs.