The Endosomes Involving Rabies Virus Infection In Neuron

Rabies virus(RABV) is a highly neurotropic virus belonging to Lyssavirus genus of the Rhabdoviridae family, which follows clathrin-mediated endocytosis and p H depend fusion for invading the neuronal cells. RABV invades the peripheral nervous system at neuromuscular junctions and undergos axonal retrograde transport arriving neuronal soma and subsequently the CNS. Although an interaction between RV P and the dynein light chain mediating the virus retrograde axonal transport had proved, removal of the LC8 binding domain from RABV P does not prevent the virus spreading into the CNS, implying that the P protein is not essential for the virus transportation. Double-labled RNP and G of RABV showed the virus might be transported as intact virions. The endosomal system is a major membrane-sorting apparatus for cargos transportation initiated from endocytosis. The Rab family of proteins is a member of the Ras superfamily of monomeric G proteins, and regulate many steps of membrane traffic, including vesicle formation, vesicle movement along cytoskeleton networks, and membrane fusion. Rab5 and Rab7 are key molecular markers destined to early and late endosomes, play critical roles in endosomal sorting, maturity and targeting for cargos. Recent study have shown that intracellular transport of some enveloped viruses, such as influenza virus(IV), vesicular stomatitis virus(VSV), Adenovirus(ADV) were regulated by Rab5 and Rab7. However, roles of the Rab5 and Rab7 in RABV have not been determined yet.In this work, we prepared the embryonic rat primary cortical neurons and infected by RABV, then the early endosomes marker EEA1, Rab5 and late endosomal marker Rab7, LAMP1, were used to define types of endosomes co-localized with RABV by immunofluorescent staining. The distribution of virus particles in RABV infectied neuron were confirmed via ultratin sections and immunogold labeling of Rab5 and Rab7. N protein and TCID50 of RABV under downregulation of Rab5 or Rab7 by si RNA were detected, and recombined fusion expression vectors of RFP-Rab5 and RFP-Rab7 were also constituted.Results showed that RABV were co-localized whith both eraly and late endosomal markers in neuron, and RABV particals were observed in the membrane organelles which are labled by Rab5 and Rab7 immunogold signals via immunomicroscopy in ultrathin sections, these proved rabies virus infection involving in early and late endosomes. Co-localization of RABV with either of Rab5 and Rab7 in the cytoplasm of N2 a and SH-SY5 Y cells were confirmed, N protein expression and RABV titers were significantly decreased after Rab5 or Rab7 inhibition, suggesting that Rab5 and Rab7 closely associated with the early infection of rabies virus. RABV dispersed mostly round the cytoplasm membrane in Rab5 si RNA transfected cells were also observed, however, RABV prefered dispersively in cytoplasm when Rab7 were inhibited. All these demonstrated that Rab5 and Rab7 were crucial for endosomal sorting and transport. Recombined proteins of RFP-tagged to Rab5 and Rab7 were successfully expressed in neurons by electrotransfection.Through this study, we get the following conclusions: 1. RABV infection in neuron requires both early and late endosomes. 2. Rabies virus is regulated by Rab5 and Rab7 during the early stage of viral life cycle, these two Rabs protein play essential roles in the rabies virus endocytosis and subsequent sorting and targeting movement. 3. Successfully imported light-emitting plasmid of marker protein into neuron by electric transfection, which serve for the kinetic study of rabies virus retrograde transport in neuron later.