Rabies Virus Infection Downregulates Expression Of Microtubule-associated Protein EB3and P140cap

Rabies is a fatal and contagious zoonotic disease of the central nervous system,caused by RNA virus of genus lyssavirus. The attacking animals have obviousneurological symptoms such ashydrophobia, paroxysmal manic,salivation and so on.It was observed that the beading and/or swelling of dendrites and axons which wereclosely correlated with changes in the structure of the neuronal cytoskeleton in RABVinfected adult rat brain. As the important ingredient of cytoskeleton, the stability ofmicrotubule determines the cells’ normal function directly. For studying therelationship between neuron microtubules and pathogenicity of RABV, we discussedthe the relationship of RABV infection and MTs associated proteins which affectedthe MTs stability through detecting the changes of their transcription, expression andintracellular distribution based on the observation about neuron MTs changes causedby RABV infection. These studies will potentially provide evidences to furtherexplore the role in neurodegenerative disease caused by RABV infection.In this study, we infected suckling mice with RABV fixed strain (CVS) byintracerebral inoculation for viral amplification and tested viral titer. Secondly, wetested9genes which are related with the stability of MTs, including EB3, p140cap,Rbl2, Tppp, Vasp, Fascin1, Tesk2, GIT2and Ena via real-time PCR. Obviouschanged genes among these were selected for further study. Thirdly, the mRNA level,protein levels and intracellular distribution of target genes were investigated via realtime PCR, western-blotting and immune fluorescent stain to determine their changesin rat primary neuron infected by CVS.The activity of downstream protein of targetgene in CVS infected neuron was detected then. Lastly,based on the abnormality oftarget gene expression in neuron infected with CVS，we investigated the changes ofthe mRNA level,protein levels and intracellular distribution of target genes in neuroninfected with RABV street strain(MRV) to study the mechanism of neuro dysfunctioncaused by MRV.The results showed that EB3, p140cap, Rbl2, Tppp, Vasp, Fascin1, Tesk2andEna among9selected MTs related genes have abnormal transcription via qPCR,andthen we chosed two related genes changing significantly,including EB3and p140cap,and Rac1which is downstream protein of p140cap.First the dynamic changes of the CVS infected neuron EB3transcription and translation in different time wereinvestigated via qPCR and western blot.Results showed that after1hour of CVSinfection, EB3mRNA and protein contents were significantly lower as compared withthe control group. As infection time extended, the amount of EB3mRNA and proteingot reduced, and the difference with the control group was more apparent. IF resultshowed that neuronal soma has a small amount of virus specificity spots, EB3presented a few continuously spot-like distribution in neurite after48h of CVSinfection. There were more virus spots on neuron soma and neurite, and irregularspot-like EB3stainsin soma and neurite is after CVS infecting96h,while neuronwithout CVS infection own dot-like EB3distribution. Dynamic testing on p140capfound that after CVS infecting1h, p140cap’s transcription and translation level hadwere markable decline. And then Rac1activity was detected and found that CVSinhibited the activity of Rac1. For neurons, MRV infection could inhibit theexpression of EB3and influence its distribution, but p140cap expression level wasinhibited unremarkably after96h infection.After MRV infection after96h, EB3appeared sand-like even distribution on neurite.Through this study, the following conclusions can be drawn:1) CVS infectionaffected EB3(↓), p140cap (↓), Rbl2(↓), Tppp (↓), GIT2(↓), Fascin1(↓), TESK (↑)and Ena (↓) and other abnormal gene transcription, inhibited the expression of EB3and p140cap and changed EB3position in neurons, and then affected the stability ofneuronal MTs. CVS infection may affect MFs’ rearrangement through EB3-p140capsignaling pathway when CVS infection inhibited Rac1activity possibly.2)RABVStreet strain MRV could inhibit EB3expression and change its subcellular localization,but inhibited expression of p140cap in conspicuously.