Molecular Mechanism Of Autophagy In SSN-1 Cells Induced By The Infection Of Snakehead Fish Vesiculovirus

Rhabdoviruses are single-stranded,negative-sense,enveloped RNA viruses.Viral particles are rod-like or bullet-shaped.The viral nucleocapsid consist of genomic RNA,nucleoprotein?N?,RNA-dependent RNA polymerase?L?and phosphoprotein?P?.In addition,the nucleocapsid is surrounded by matrix protein?M?and glycoprotein?G?.The Rhabdoviridae consists of more than 185 different viruses isolated from various hosts,including plants,insects,fish and mammals,and cause serious diseases of the infected hosts.More than twenty species of fish rhabdoviruses have been reported.They can cause great economic loss in aquaculture.Snakehead fish vesiculovirus?SHVV?was isolated from the diseased hybrid snakehead fish.It causes serious disease of snakehead fish and leads to great economic losses in snakehead fish culture.However,there is no high effective strategy to fight against SHVV infection.Understanding viral pathogenicity and the mechanism of host defense against virus will pave a new way for development of effective preventional strategy against SHVV infection.Autophagy is the process that eukaryotic cells transport the macromolecular substances in cells or damage organelles to the lysosomes with vesicle structure,and then these cargos would be digested in lysosome.The most important function of this process is to maintain cellular homeostasis.Autophagy plays multiple roles in the process of pathogens invasion.For example,autophagy can clean up the viral protein,or transport the viral particles to the lysosome for degradation,and inhibits viral replication.On the other hand,viruses can utilize autophagy to escape the removal effect of host immune system,this will facilitate viral multiplication.In this study,we focused our studies on SHVV infection and autophagy,the main results are follows.1.Microtubule-associated protein 1 light chain 3-beta?LC3B?gene cloning,bioinformatics analysis and antibody preparationLC3B is the main marker for autophagy.According to the transcriptome database of SSN-1 cells infected with SHVV,LC3 B gene was amplified by PCR.The length of this gene was 378 bp encoding 125 amino acids.The predicted theoretical molecular weight of LC3 B was 14.8 kDa with theoretical isoelectric point of 5.23.The homology of LC3 B gene from 10 animal species was analyzed,the results showed that snakehead LC3 B was closed to the LC3 Bs from grass carp and common herring with 93% identity.The plasmid pET-32a-LC3 B encoding his-tag-fused LC3 B protein was constructed.pET-32a-LC3 B was pressed in E.coli and his-tag-LC3 B was purified.The purified LC3 B was used for antibody generation.The polyclonal rabbit antibody against LC3 B was obtained.2.Viral particles observed under transmission electronic microscopeAfter infection with SHVV for 24 h,SSN-1 cells were subjected to routine sample preparation,and the cell sections were observed under transmission electronic microscope.Typical bullet-shaped particles of rhabdovirus which size of 70×120 nm were observed.3.SHVV infection induced autophagy in SSN-1 cells.There are three main assays for autophagy confirmation.They are observation by transmission electronic microscopy?TEM?,laser scanning confocal microscopy,and Western blot.SSN-1 cells infected with or without SHVV were subjected to the above mentioned assays.The results showed that typical membrane vesicles were observed only in SHVV infected samples by TEM.The gathered dots of YFP-SSN-LC3 B were revelaled only in SHVV infected samples by immunofluorescence microscopy.The conversion of LC3-I to LC3-II was examined only in SHVV infected samples by Western blot.Taken together,autophagy was activated in SSN-1 cells infected by SHVV,but not in non-infected SSN-1 cells.4.Activated autophagy inhibited SHVV multiplication in SSN-1 cellsCommonly,rapamycin is an activator of autophagy,while 3-MA is an inhibitor of autophagy.To study the effect of autophagy on SHVV multiplication,SSN-1 cells were incubated with rapamycin or 3-MA,so as to induce or inhibit autophagy in SSN-1 cells.Thereafter,the treated cells were infected with SHVV.The levels of SHVV-N mRNA and protein,viral titer(TCID₅₀)in the supernatant were measured.The results showed that autophagy inhibited SHVV multiplication in SSN-1 cells.5.The mechanism underlying SHVV inducing autophagy in SSN-1 cellsWhen the ultraviolet-inactivated SHVV were incubated with SSN-1cells,they could induce autophagy in SSN-1 cells indicating that the induced autophagy was not dependent on SHVV replication.To identify the viral proteins which could induce autophagy,SHHV structural protein genes were cloned into PcDNA3.1 plamsids which were used to tansfect SSN-1 cells,respectively.The results showed that glycoprotein?G?alone,but not other viral proteins,could activate the autophagy in SSN-1 cells.To further elucidate the mechanism underlying the autophagy activation of G protein,a number of peptides derived from G protein were synthesized and used for the incubation of SSN-1 cells.The results showed that p340 located in the receptor-binding domain of glycoprotein could induce autophagy of SSN-1.