| Rabies is an acute and fatal central nervous system(CNS)infectious disease cause by rabies virus(RABV)which belongs to family Rhabdovirede.According to the World Health Organization(WHO)’s report there are 59,000 human death worldwide annually,and most of these occur in developing countries in Asia and Africa.At present,China has the second highest number of human rabies cases,just behind India.Human has experienced a long history to struggle with RABV.Although great progress have been made,the exact pathogenic mechanisms underlying RABV is still limited,especially on the interaction between RABV and organism/ host cells,and on host protein profiles alteration and their associated biological functions after RABV infection.And at present,studies about the divergent pathogenicity of different virulence RABV are focus on the difference in sequences or structures of viral proteins,but rarely focus on revealing the whole profile of different host effectors upon RABV infection.Analyzing virus-host interactions can play an instructive effect to help researchers explore the pathogenesis of pathogens.Recently,liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification(i TRAQ)labeling approach(i TRAQ LC-MS/MS),combined with bioinformation analysis of differently expressed proteins(DEPs)in expression patterns,functional cluster and associated biological pathways and networks,was wildly used to identify whole changings of host protein profile based on its high-throughput,high-sensitivity and quantitative accuracy.These provide a powerful method for filtrating host factors that associated with virus infection and host defense.Therefore,understanding the host protein profiles of mouse brains infected with RABV at pan-proteomic level is essential to explain the pathogenesis of RABV.Autophagy,a highly evolutionary conserved cellular catabolic process,can degradate excess or damaged cellular organelles and protein aggregateds by sequestrating ‘cargoes’ in autophagosome and fusing with lysosomes to degratate internal contents by resident lysosomal hydrolases.Autophagy is an important process that maintains cellular homeostasis and respond to stress such as starvation,nutrition deficiency,oxidative stress or infection.Several studies have demonstrated that viral infection,such as vesicu Lar stomatitis virus(VSV)and classical swine fever virus(CSFV),can induce autophagy.Contrarily,autophagy can affect viral infection through virious pathways.For instance,VSV-induced autophagy acts as an antiviral mechanism by delivering viral nucleic acids to endosome resident TLRs for IFN induction.In the other hand,viruses have evolved diverse countermeasures adopted by viruses to exploit and subvert autophagy.For example,dengue fever virus(DENV)–induced autophagic degradation of lipid dropletsresults in mobilization of triglycerides to allow ATP produce which play an pro-viral effect.Therefore,the understanding of interaction between autophagy and RABV infection can provide information of RABV pathogenesis.In this study,we employed i TRAQ LC-MS/MS approach for the systematic analysis of global host proteins in response upon challenge virus standard(CVS)-11 infection and conduct a comparative analysis between mouse brains upon pathogenic CVS-11 strain infection and attenuated SRV9 strain infection.And based on the results obtained above,we explore the relationship of RABV infection and autophagy which was significantly targeted in above proteomic analysis.This study is mainly divided into four parts:Part 1: Proteome profile of mouse brains infected with RABV CVS-11 strain.Dynamic analysis of brains infected with RABV were analyzed at 1,4 and 7 dpi.2,781 non-redundant host proteins were identified with 104 DEPs.At 1 dpi,23 proteins were up-regulated and 17 were down-regulated.At 4 dpi,16 proteins were up-regulated and 17 were down-regulated,while at 7dpi,40 proteins were up-regulated and 16 were down-regulated.Bioinformation analysis shows that DEPs were mainly associated with cytoskeleton,immune response,response to stimulate,signal transduction and energy metabolism.Strikingly,several members of interferon-inducible GTPases were significantly up-regulated,including IGRM1,GBP2,IIGP1,IGTP,and many reports have demonstrated these four interferon-inducible GTPases were associated with autophagy pathway.Therefore,we mainly focus on autophagy during RABV infection in following study.Part 2: Comparative proteome analysis of mouse brains infected with different virulence RABV.To filtrate host factors that response to different virulence RABV,kinetic comparative proteomic analysis of mouse brains infected with pathogenic strain CVS-11 and attenuated strain SRV9 were performed using i TRAQ LC-MS/MS proteome method.Using the protein profile of mock-infected mouse brains as background,90 or 227 non-redundant DEPsvs.m were identified in pathogenic CVS-11-or attenuated SRV9-infected mouse brains,respectively,indicating that attenuated SRV9 strain induced more in-depth global dysregulation.Because the major objective of this section was to determine which factors influence the pathogenicity of different strains of RABV,we focuse on proteins that exhibit different expression levels between the two infected groups and subjected these DEPsvs.s(which using global protein profile of SRV9-infected mouse brains as background)to bioinformatics analysis.Pathway analysis showed that autophagy and autophagy-associated pathways,such as free radical scavenging and NRF-mediated oxidative stress were highly targeted in CVS-11-vs.SRV9-infected mouse brains.The overview of biological information shows that autophagy was located in the center of this diagram and act as a connecting linker for upstream effectors and downstream sensors.In summary,this comparative proteome provide a strong evidence that autophagy probably involve in RABV infection.Part 3: The detect of autophagic activity upon RABV infection.Proteome in part 1and 2 both indicated autophagy probably partcipate in RABV infection,therefore in this part we validated thisphenomenon using mouse neuroblastoma(NA)cells as an in vitro model.We evaluated the autophagy activity upon RABV infection by monitoring the number of autophagosomes using electroon microscopy,fluorescrnce microscopy,western blot,and monitoring autophagic flux by detecting p62 expression levels.The results show that autophagic activity was changed during RABV infection with dissimilar capacities between different virulent strains.Part 4: The influence of autophagy on RABV replication and pathogenicity.Appropriate concentration of autophagic regulators were determined by detecting cell viability and RABV internalization.Subsequently,viral titer and genomic RNA were detected when regulated autophagic activities using pharmacological or genetic methods.The results indicate that RABV replication was independent of autophagy.Moreover,in vivo study shows that autophagy inducer increased RABV mortality in the absence of the in?uence on viral replication. |
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