The Effect Of Cytoskeleton In Rabies Virus Infection And Replication

Rabies is a highly contagious lethal zoonosis caused by rabies virus, which ischaracterized as the neurotropic infection. Despite the typical neurological symptoms,no significant neuronal apoptosis in brain tissue is detected under RABV naturalinfectious conditions. However, the structure of neuronal cytoskeletons whichrepresent the structural basis for maintaining normal physiological function of nervecells are changed.To study the role of neuronal cytoskeleton in the pathogenesis of Rabies,Colchicine (Col) and cytochalasin D (CytoD) were chosen and their suitableconcentration on neuroblastoma cell line SH-SY5Y were screened by cell viabilitiesand morphological changes of cytoskeleton under Immunofluorescent staining. Theeffects of cytoskeleton formation inhibitors were evaluated in earlier infection andreplication of RABV, and the dynamic of cytoskeletal associated proteins wereanalyzed, and fluorescent-fused β-actin and α-tubulin plasmids were also constructed.These will be attempted to elucidate the pathogenesis of rabies and to build therelationships between cytoskeleton and the neuronal dyfunction.The results showed that When Col were at0.065mmol/L and0.13mmol/L，respectively，and CytoD at0.25μg/mL and0.5μg/mL，no significant cell viabilityand structural changes of microtubules and actin were observed comparing with thecontrol (p>0.05). Cytoskeleton inhibitors preventing RABV earlier infection andreplication were confirmed by ELSIA、 RT-PCR、 Western blotting andImmunofluorescent staining．The inhibitory effect of Col on RABV titer was moredistinct than CytoD’s after RABV infection. The result of Western blotting shows thatthe levels of G protein and N protein were declined dramatically. Moreover, thenumber of virus-specific fluorescence was less than normal group. This went to provethat more significant role of the Col played in RABV replication than that of theCytoD. RABV infection upregulated ARPC3expression at4h.p.i, and tau and P-taudid not change significantly. However, at72h, ARPC3expression level dropped and P-tau mildly raised. Finally, pLEGFP-actin and pLEGFP-tubulin which fusedgreen-fluorescent protein were constructed and transfected into SH-SY5Ysuccessfully.Through this study, the following conclusions can be drawn:1) Suitableconcentrations of colchicine (Col) and cytochalasin (CytoD) on SH-SY5Y cells werescreened;2) CytoD had more pronounced effect in RABV infection than Col;3)Inhibitory effect of Col on RABV replication were more obvious than CytoD;4)RABV infection can stimulate ARPC3protein expression at early stage, and decreaseit at the late one;5) Successfully constructed filaments and microtubules fused with afluorescent protein plasmid and transfected them into SH-SY5Y cells.