Wild-type Rabies Virus Induces Autophagy In Human And Mouse Neuroblastoma Cell Lines

Rabies is an acute zoonotic infectious disease caused by Rabies virus(RABV) with infection of the central nervous system. Almost 100% Human and animals which infected with RABV and become symptomatic will be die. Prevention and treatment of rabies has become one of the important subjects in recent decades. At present, for rabies, prevention is the primary means and little achievement in treatment. Virus is a kind of specific cell parasitic organisms, which depends on the replication and dissemination of organisms. Therefore, it is very important to study the interaction between rabies virus and cells.Autophagy, as a self-protection mechanism of cell, highly conserved in evolution exists widely in eukaryotes, the main function of autophagy is to transport cell biological macromolecules and damaged organelles to lysosomal and digested them for reutilize. Autophagy, with apoptosis and necrosis constitute the three major cell programmed cell death mechanism. Autophagy, based on the function, has been divided into three categories: macroautophagy, microautophagy and chaperone-mediated autophagy.In recent years, there are a lot of reports about the relationship between autophagy and virus. Viruses evolved the function of evasion of host cell autophagy, or use autophagy to promote self-replication. Other members of the Rhabdoviridae: vesicular stomatitis virus(VSV), hemorrhagic septicemia virus(VHSV) and spring viraemia of carp virus(SVCV) can induce autophagy, but research of the relationship between RABV and autophagy is absent, in this paper, we will discussed at following several aspects.First, Autophagy can be induced in RABV-infected SK and NA cell. In order to determine whether the rabies virus can trigger autophagy, we selected two strains according to the very different biological characteristics: the virulent RABV GD-SH-01 and attenuated RABV HEP-Flury. We detected autophagy by western-blot, electron microscope observation and fluorescence point observation by transfecting of GFP-LC3 B plasmid in SK and NA cells, we finally determined the virulent strain GD-SH-01 can induce obvious autophagy, while attenuated RABV can not, This phenomenon concluded that the ability of RABV to induce autophagosomes is limited to the virulent RABV strain GD-SH-01. It remains to be discussed whether there is a correlation between the differences in autophagy induction and the characteristics of RABV in virulence.Second, the relationship between autophagy and apoptosis induced by RABV. As is a double-edged sword, autophagy is a kind of cellular protective mechanisms can also induce cell death. Both of autophagy and apoptosis are kind of programmed cell death, the relevant studies about the relationship between them have been reported and mainly summarized as cooperative, adversarial and support relationship. Our results showed that the RABV virulent strain GD-SH-01 can induce both autophagy and apoptosis, we next explored the relationship between autophagy and apoptosis induced by RABV by enhancing one of them and detected the other one. Our results showed that the apoptosis rate in GD-SH-01-infected cells decreased after pretreatment with rapamycin. We concluded that autophagy triggered by GD-SH-01 may act as a protective mechanism against apoptosis which served as an adverse factor induced by RABV.Third, the effect of the M gene of GD-SH-01 on autophagy. Rabies virus genome structure consists of 5 structural proteins: N, P, M, G, and L, but which gene plays the crucial role in promoting RABV-induced autophagy remains unkown. To further identify the virus constituent causing autophagy, five chimeric recombinant viruses carrying single genes of HEP-Flury instead of those of GD-SH-01 were rescued: r HEP-sh N, r HEP-sh P, r HEP-sh M, r HEP-sh G and r HEP-sh L. While the HEP-Flury virus carrying the wild-type matrix protein(M) gene of RABV triggered LC3-I/LC3-II conversion in SK and NA cells, replacement of genes of nucleoprotein(N), phosphoprotein(P) and glycoprotein(G) produced only minor autophagy. So we concluded that M gene GD-SH-01 plays a most important role in the induction of autophagy. Next, we constructed 4 plasmids which could express the fusion protein of structure protein and m RFP respectively. we found that no one single structure protein of GD-SH-01 was able to induce autophagy by transfecting the plasmids, seems to indicate that a single structure protein is not sufficient to trigger autophagy and is relevant to the biological characteristics of RABV.This paper first reveals the relationship of rabies virus and autophagy. To our knowledge, this is the first study providing evidence that autophagy is induced by RABV in the human neuroblastoma cell line(SK) and the mouse neuroblastoma cell line(NA), and the relationship between autophagy and apoptosis induced by RABV was also preliminarily clarified. M gene of RABV, as a vital role in promoting RABV-induced autophagy induced autophagy, is how to play this function, its relationship with the host cells need further exploring.