ISKNV Mediated Rac1-PAK1-LIMK-Cofilin Signaling Pathway Regulates F-Actin Rearrangement To Promote Self-Replication And Proliferation Proliferation

Infectious spleen and kidney necrosis virus（ISKNV）infects various kinds of fish.It is one of the main pathogens that cause morbidity of Siniperca siniperca（Siniperca siniperca）and endangers the healthy development of Siniperca culturing industry.F-actin is an important component of cytoskeleton,which plays an important role in virus adsorption,invasion,assembly and release.Some studies have found that F-actin plays an important role in late ISKNV infection.What is the relationship between F-actin and ISKNV replication and proliferation?What are the signaling pathways involved in the regulation of F-actin?How do they regulate ISKNV replication and proliferation?With the above questions in mind,we carried out the following research:1.The relationship between F-actin and ISKNV replication and proliferationPerca ISKNV infected with Chinese brain cell line（CPB）.RT-q PCR detected the copy number changes of perca ISKNV at different time points.The results showed that72 h after infection of Perca ISKNV was the peak of viral replication.Indirect immunofluorescence staining of F-actin cells during this period showed that F-actin contracted and converged in the lesion site of the virus compared with control cells,indicating that the replication and proliferation of ISKNV would lead to the rearrangement of F-actin.Cytochalasin D and Cucurbitacin E were used to pretreat cells with F-actin inhibitor Cytochalasin D and Cucurbitacin E.The effects of Cytochalasin D and Cucurbitacin E on ISKNV replication and proliferation were detected by RT-q PCR,Western blot and TCID₅₀.The results showed that after inhibiting F-actin rearrangement,the lesion cells,viral genome copy number and viral protein expression of ISKNV were down-regulated in a dose-dependent manner,and the virus titer was decreased.These results indicated that ISKNV induced F-actin remodeling to promote viral replication and proliferation.2.The relationship between Rho A-Rock 1 and Rac1-PAK1 signaling pathways and ISKNV replication and proliferationF-actin is a highly dynamic structure regulated by members of the Rho GTPases family by transmitting membrane receptor signals.Rho A and Rac1 are the main members of Rho-GTPase family.In order to investigate whether Rho A,Rac1 and their downstream signaling pathways were involved in ISKNV infection,Rho A,Rock 1,Rac1 and PAK1 were detected by RT-q PCR and Western blot after 72 h infection.The m RNA levels of Rho A,Rock 1,Rac1 and PAK1 were up-regulated after 72 h infection by RT-q PCR.Western blot results showed that the protein expressions of Rho A,Rock 1,Rac1 and PAK1 were significantly up-regulated and the phosphorylation of PAK1 was significantly up-regulated after 72 h infection,indicating that both Rho A-Rock 1 and Rac1-pak1 signaling pathways were involved in ISKNV replication and proliferation.In order to further elucidate the relationship between Rho A-Rock 1 and Rac1-PAK1 pathways and ISKNV replication and proliferation,inhibitors CCG-1423,Thiazovivin,Ehop-016 and IPA-3 were used to inhibit Rho A-Rock 1 and Rac1-PAK1pathways,respectively.RT-q PCR showed that the copy number of ISKNV genomic DNA was reduced by 2.4 times after inhibiting Rho A-Rock1 pathway,and the cytopathic changes caused by ISKNV were reduced by 2.3 times.These results indicated that the inhibition of Rho A-Rock1 pathway could inhibit the replication and proliferation of ISKNV.However,after inhibiting Rac1-PAK1 pathway,ISKNV genomic DNA copy number decreased by 4.3 fold,ISKNV-induced cytopathia decreased by 5.8 fold,and ISKNV-MCP protein expression was down-regulated by 3.4fold.These results indicate that inhibition of Rac1-PAK1 pathway can significantly inhibit the replication and proliferation of ISKNV.Fluorescence staining of cells treated with inhibitors Ehop-016 and IPA-3 showed that inhibitors inhibited virus-induced F-actin rearrangement,indicating that Rac1-PAK1 signaling pathway regulates F-actin rearrangement in ISKNV replication and proliferation.In conclusion,inhibition of Rho A-rock 1 and Rac1-PAK1 pathway can inhibit ISKNV replication and proliferation,but Rac1-Pak1 pathway has a more significant effect on ISKNV replication and proliferation.Therefore,Rac1-PAK1 pathway is mainly used as the research object in this follow-up experiment.3.Relationship between LIMK-Cofilin signaling pathway and ISKNV replication and proliferationLIMK,a member of the Serine/threonine protein kinase family,is a downstream kinase of PAK1.RT-q PCR and Western blot were used to detect the activation of LIMK in the replication and proliferation of ISKNV and its effect on the replication and proliferation of the virus.The results showed that the expression of LIMK m RNA,LIMK protein and phosphorylated LIMK protein were significantly up-regulated after72 h of ISKNV infection,and the activity of LIMK was significantly up-regulated,which was consistent with the change trend of PAK1.After si RNA knockdown endogenous LIMK gene in cells,RT-q PCR and Western blot were used to detect changes in ISKNV infection volume.The results showed that the replication and proliferation of ISKNV virus was significantly inhibited,indicating that Is KNV-mediated LIMK regulated the replication and proliferation of the virus.LIMK is activated by dephosphorylation of its downstream kinase Cofilin.RT-q PCR and Western blot were used to detect the activation of Cofilin after ISKNV infection.The results showed that 72 h after ISKNV infection,the expression of Cofilin m RNA,Cofilin protein and phosphorylated Cofilin protein were significantly up-regulated.The activity of Cofilin was significantly increased.CPB cells were infected with ISKNV after Cofilin knockdown by si RNA,and the amount of ISKNV virus was detected by RT-q PCR and Western blot.The results showed that the replication and proliferation of ISKNV was significantly inhibited.The above results indicated that ISKNV activated LIMK to promote self-replication and proliferation.The change trend of Rac1-PAK1 was consistent with that of the downstream LIMK-Cofilin pathway.In order to verify whether Rac1-PAK1 promoted ISKNV replication and proliferation by reshaping F-actin through its downstream LIMK-Cofilin pathway.Ehop-016 was used to inhibit Rac1 activation,and phosphorylated PAK1protein was down-regulated in a concentration-dependent manner,suggesting that PAK1was regulated by upstream Rac1.After Ehop-016 and IPA-3 inhibited Rac1-PAK1signaling pathway,Western blot detected changes in the activities of LIMK and Cofilin.The results showed that phosphorylated LIMK was significantly down-regulated,and phosphorylated Cofilin was significantly up-regulated.The results indicated that inhibition of Rac1-PAK1 signaling pathway could inhibit LIMK-Cofilin activation induced by ISKNV.These results indicate that Rac1-PAK1 regulation promotes ISKNV replication and proliferation through the downstream LIMK-Cofilin pathway.In conclusion,ISKNV infection can activate Rac1-PAK1-LIMK-Cofilin signaling pathway,and inhibit Rac1-PAK1-LIMK-Cofilin signaling pathway can inhibit F-actin remodeling and ISKNV replication and proliferation.ISKNV infection activates the Rac1-PAK1-LIMK-Cofilin signaling pathway to promote self-replication and proliferation.