Rabies Virus Changes The Expression Of Microtubule And Actin Associated Proteins

Rabies is an acute and fatal viral infection of the mammalian‘s central nervous system. At present, it is included among the leading zoonotic diseases of the world. Rabies infected animals show horrifying clinical signs and s ymptoms that gradually lead to progressive and inevitable death. Recently, 50,000 to 59,000 deaths have been reported mainly in Asia and Africa. Among several wild and domestic reservoirs of rabies virus, dog is still considered as the principal source par ticularly in Asia and Africa. The virus travels from peripheral nerves to the central nervous system(axon to cell body) as route of traveling, where it causes apoptosis, neuronal dysfunction, oxidative stress, dysfunctional mitochondria and inhibits innate immune response. It is a cunning virus that uses evasive strategies to manipulate the defense mechanism of host cel s.In vitro and in vivo experiments using different strains of rabies virus have shown several degenerative changes in neuronal processes, dendrites and axons that are closely related to the pathological alterations in the neuronal cytoskeleton. In addition to structural support, dynamic actin & microtubule s also regulate neuronal growth, versatile intracellular framework for guided transport, trafficking and other mechanical and signaling processes. However, actin- microtubule cytoskeletons not only function individually or mutually but, their dynamic functions are purely regulated by their corresponding binding proteins. These associated or binding partners of cytoskeleton are vital in maintaining overall neuronal architecture and dendritic spine morphology in particular. These are the major proteins that regulate fundamental actin- microtubule based cellular events and cross-talks in neuronal cells. Dendritic spines are the highly motile spot of dendritic network that are abundant in actin and constitute major postsynaptic sites for excitatory synaptic transmission thus, regulating overall physiological activity of the central nervous system.Rabies virus follows clathrin- mediated endocytosis for the internalization within the host cells, and later follows specific GTPases for early(rab5, EEA1) and late endosomes(rab7, Lamp1). The endocytic process and initial transport kinetics of IV rabies virus are dependent on actin and microtubule. Therefore, the present study was designed to investigate the essential actin- microtubule based binding proteins that essentially regulate dynamic actin and microtubule polarized ends to maintain branched dendritic network and spines. Moreover, early and late endosomal GTPases were also evaluated that could colocalize rabies virus in order to authenticate the earlier events and mechanical route of the virus inside host cells.During the first part of the research, cortical neurons were processed through immunofluorescence using specific marker antibodies for early and late endosomes in order to observe their colocalization with rabies virus in neurons. The RNA interference and western blot were aimed to analyze the down-regulation effect of rab5 and rab7 on rabies virus infection in neuroblastoma cells line. In the second part, gene expressions of different actin and microtubule associated or related proteins were evaluated using quantitative real time PCR under fixed strain of rabies virus. Similarly, CVS and MRV strains of RV were employed for the analysis of relative gene expressions and protein contents of EB3(microtubule plus end binding protein) and p140cap(actin binding protein) using quantitative real time PCR and western blot respectively. Furthermore, distribution of EB3 in immuno-stained fixed neurons was also studied. In the last part, MRV strain was intracerebrally inoculated in mice to analyze the antigen distribution and pathological lesions in different parts of mice brain using immunohistochemistry and histopathology respectively. These experiments were processed to observe the effect of two viral strains on the neuronal architecture by disturbing the cytoskeleton related proteins. The whole data were subjected to SPSS and ANOVA for the determination of significance between control and experimental groups, and relative analysis of different gene expressions was obtained using comparative CT method(2-ΔΔCT method).The results showed that nucleoprotein o f rabies virus successfully colocalized with the markers for early(rab5, EEA1) and late(rab7, LAMP1) endosomes in fixed neurons. RNA interference of rab5 and rab7 considerably reduced the rabies virus infection in neuroblastoma cell lines. The western blot test also verified that the down-regulation of rab5 and rab7 inhibited nucleoprotein expression of rabies virus. Further, rabies virus titers were significantly decreased in the early rabies virus infection. Both strains of rabies virus significantly do wn-regulated EB3 and p140 cap gene expression and decreased their corresponding proteins contents in western blot analysis. However, the fixed strain of rabies virus largely reduced protein contents of EB3 as compared to p140 cap. Uneven distribution of EB3 along with fractured microtubules could possibly indicate the degeneration of actin- microtubule cytoskeleton under these two strains of rabies virus. Different expression levels of microtubule related proteins(Tesk1, EB3, Limk1), actin binding proteins(Profilin, P140 cap, Vasp, Fascin1 and Ena) and synapse related proteins(Rb12, Tppp, GIT2) were down-regulated in quantitative real time PCR. On the other hand, up-regulated levels of gene expressions were also seen for microtubule related proteins(Tesk2, Katnal), actin binding proteins(Cofilin, Gelsolin) and synapse related proteins(Ppp3ca, Ssh1). Interestingly, these findings demonstrated that the expression levels of all the actin- microtubule associated proteins were not negatively affected. The immunohistochemistry showed minute distribution of viral antigens in various sections of mice brain, while cerebrum came to be the area of choice for viral antigens. Histopathology showed dumble-shaped neurons and vacuolated changes in the neuronal cel s of the cerebrum.The results enable us to discuss the initial transport dynamics with respect to early and late endosmes. The conclusions about colocalization give us the clue to elucidate the retrograde transport of rabies virus using fluorescence based rabies virus vector. According to previous findings between rab5 and microtubules, their intricate relationship is a little bit more appealing. The down-regulation of microtubule and actin associated proteins have revealed that rabies virus negatively influence the structural integrity of the actin- microtubule cytoskeleton that is mandatory for the cellular morphology. The lesions and degenerative changes in paraffin-embedded sections help us to differentiate the pathogenesis of different viral strains.