Preliminary Study On The Molecular Mechanism Of Actin Cytoskeleton Rearrangement Regulating RABV Infection

Rabies is caused by rabies virus(rabies virus,RABV)infection.It is a highly lethal zoonotic disease characterized by damage to the nervous system.The fatality rate is almost 100%.Because the pathogenic mechanism is still uncleare,it has actually become a hot spot in rabies research.The polymerization or decomposition of actin cytoskeleton plays an important role in the maintenance of cell morphology,material transportation and virus infection,and is regulated by the Rho GTPase signaling pathway.Rac1,Cdc42 and Rho A,as Rho GTPase family members,involve in the cell entry,gene expression,virus latency and budding of a variety of viruses.Rho GTPase activates downstream effector proteins,such as WASP,Rock,PAKs,LIMK,which interact with actin-binding proteins(including Profilin,Cofilin,etc.)to form a complecated signal pathway network that regulates the dynamic actin.We first explored the effect of disrupting dynamic actin filaments on RABV infection.MTT assay was emplyed to confirm the appropriate concentration of Latrunculin A and Jasplakinolide on N2a cells.Subsequently,after inhibitor treatment on N2a and infection with RABV,RABV N protein level,m RNA level and virus titer were determined by western blot,RT-q PCR,TCID₅₀respectively.The structure of microfilament cytoskeleton was observed by immunofluorescence.The results showed that the m RNA and protein level of RABV N was decreased.The viral titer is consistantly reduced.And F-actin polymerization was significantly disrupted.These results suggested that dynamic actin regulated RABV infection.Secondly,we explored whether Rac1,Cdc42 and Rho A regulated RABV infection by effecting actin cytoskeleton rearrangement.The RT-q PCR data showed that m RNA level of Rac1 increased slightly and Cdc42 and Rho A had no significant change.Following transfecttion with wild-type and dominant negative mutants of Rac1,Cdc42 and Rho A,m RNA levels of the RABV N gene was detected at 3 h p.i.and 24 h p.i..RABV infection was impaired when activity of Rac1 or Cdc42attenuated at a certain degree,while Rho A showed no significant effect.The results revealed that Rac1 and Cdc42 involved in actin rearrangement caused by RABV infection.To further explore the effect of Rac1 and Cdc42 activity on RABV infection,we treat N2a cells with Rac1 inhibitor NSC23766 and Cdc42 inhibitor ML141.MTT assay was emplyed to confirm the appropriate concentration of drugs.Subsequently,after inhibitors treatment on N2a and infection with RABV,RABV N protein level,m RNA level and virus titer were determined by western blot,RT-q PCR,TCID₅₀.The results showed that by inhibiting the activity of Rac1 and Cdc42,RABV infection showed a decreasing trend in protein level,m RNA level and virus titer.Finally,we explored the function of PAK1,downstream molecule of Rac1/Cdc42-PAK1 signaling pathway,in RABV infection.MTT assay was emplyed to confirm the appropriate concentration of PAK1 inhibitor IPA-3 on N2a cells.after inhibitor treatment on N2a and infection with RABV,RABV N protein level,m RNA level and virus titer were determined by western blot,RT-q PCR,TCID₅₀.The results demonstrated that the m RNA and protein level of RABV N was decreased in a dose-dependent manner,and the viral titers were also reduced consistantly.The phosphalation of LIMK1 and Cofilin1 were subsequently detected at 0,15,30,45,60,90,120 min p.i.After RABV infection,the phosphalation levels of LIMK1 and Cofilin1 increased during 30 min and decreased at 45 min p.i.These above results showed that Rac1/Cdc42-PAK1 signaling pathway effected actin rearrangement and regulated RABV infection.Our study constructed primary associations of Rho-GTP pathways with RABV infection.However,more work need to be done.