Molecular Mechanism Of Rac1 Binding To Rabies Virus P Protein And Regulation Of Viral Infection

Rabies is a neurotropic zoonotic acute infectious disease caused by rabies virus（rabies virus,RABV）infection.Once infected,the fatality rate is almost 100%.There is no effective clinical treatment method.Its pathogenic mechanism is still unclear and needs to be further explored.Rac1 is one of the key molecules in the Rho GTPase family,which cycles between an inactive GDP-bound form and an active GTP-bound form.It also controls various cellular functions through multiple downstream signaling pathways,including the regulation of the dynamic actin,cell migration,and gene expression.Actin cytoskeleton plays an important role in the maintenance of cell morphology and participated in endocytosis and cell migration.The abnormality of the microfilament skeleton system is closely related to neurological dysfunction.Rac1 is the intermediate hub of the signaling pathway that regulates actin microfilament cytoskeleton assembly.Rac1activates downstream LIMK by phosphorylating PAK1,promotes cofilin1phosphorylation,and regulates the dynamic actin.First,si RNA was employed to knockdown Rac1 in N2a cells.After infection by RABV,RABV N protein level,m RNA level and virus titer were determined by western blot,RT-q PCR,and TCID₅₀ respectively.The results showed that the m RNA and protein level of RABV N were both decreased,and the viral titer was consistently reduced.Following transfection with constitutively active mutants of Rac1,wild-type and dominant negative mutants of Rac1,the infection level of RABV was detected.The results showed that compared with transfection of Rac1 WT,the levels of RABV infection showed an inhibitory trend after transfection of Rac1 DN,while transfection with Rac1 CA led to a promotion of viral infection.After treatment of Rac1 activity inhibitor,NSC23766,the viral infection was significantly attenuated at 0,2,4,6 and 8h after inoculation.These above results suggest that Rac1 activity was involved in the regulation of RABV infection.Second,we explored the correlation between RABV and Rac1.IP-MS analysis showed that RABV P binds to Rac1,when Flag-P protein was overexpressed in N2a cells,After co-transfection with GFP-Rac1 and Flag-P plasmids into 293T cells,IP assays were performed to identify the interaction between Rac1 and Flag-P.The colocalization of Rac1 and RABV P was observed by immunofluorescence experiment.Truncated plasmids of RABV P were constructed and overexpressed with GFP-Rac1.Western blot results showed that 82-137 amino acid at the N-terminal of P is responsible for binding to Rac1.A decrease in Rac1 activity was both detected by Glisa and Pull down assay detection,after infection with RABV and transfection with Flag-P in N2a cells.Simultaneously,the phosphorylation levels of PAK1,LIMK,and cofilin1 were all decreased.In addition,the activity of Rac1 was detected at 0,15,30,45,60,90,120min p.i and showed a dynamic trend which is increased from 0 to 45 min and declined at 60 min p.i..It was also observed that the dynamic actin filaments were disrupted after RABV infection,and there were large actin dots at the cell periphery.The results showed that Rac1 activity was enhanced and actin polymerization was accelerated when RABV begins to enter host cells,which promoted RABV infection.In later stages of RABV infection,along with RABV uncoating,P binded to the Rac1,Rac1 activity was down-regulated,Rac1-PAK1 signaling pathway was obstructed,and actin polymerization was disturbed.This study disclosed the crucial role of Rac1 during RABV infection and provided a new insight for the pathogenic mechanism of RABV.