| Autophagy,an evolutionarily conserved intracellular process,is essential for tissue homeostasis and development.Autophagy and/or autophagy genes play both anti-viral and proviral roles in the virus life cycles.However,there is still no systematic research on RABV and cellular autophagy relations.This study based on the basic theory to N2a cells as the research object,explore the relationship of RABV infection and host cell autophagy.With laser confocal,western blot,drug treatment,RNA interference and co-immunoprecipitation method to explore RABV infection affect cell autophagy.Confocal microscopy detected that the changes of exogenous GFP-LC3 from the punctate to clump at 12 h,24 h,36 h,48 h with HEP-Flury or CVS-11 infection and total endogenous LC3 had significant co-localization with RABV N protein at 16 h,24 h and gathered into large lumpy.Simultaneously westernblot detection found the obvious upregulation of endogenous LC3-Ⅱ and no detectable alteration of SQSTM1 at 4 h,8 h,12 h,24 h,36 h,48 h.At the same time,we use ICR Kunming suckling mice to carry the RABV infection experiment.Westernblot detection showed that endogenous LC3-Ⅱ had significantly upregulated,but no change in SQSTM1.In addition,confocal microscopy analysis found that RABV infection caused the non-fusion of autophagic vacuoles and lysosome.To explain the effect autophagy on RABV infection,Westernblot detection showedthat autophagy activators EBSS/Rapa or inhibitors wortmannin/3-MA or shRNAtreatment respectively,autophagy enhancementto help the viral transcription,protein translation and viral release,conversely,autophagy inhibition reduced the levels.these results show RABV uses incomplete autophagy to enhangce viral replication in host cells.In order to clarify whether an incomplete autophagy response induced by RABV infection was caused by viral prortein,we analyzed theimpact of various structural proteins of RABV on autophagy.We transfected plasmids expressing viral proteins Flag-N/P/M/G/L in N2a cells,found only viral N/P protein could induce a large group of exogenous GFP-LC3 massive aggregation,and the N/P proteins could enhance the expression of overall endogenous LC3 by confocal microscopy.Westernblot futher detection found that significant upregulation of LC3-Ⅱ levels in N/P transfection groups,but SQSTM1 levels did not change significantly.Similarly,to testify whether the failure fusion of lysosomes and autophagic vacuoles was caused by viral protein,we co-transfected GFP-LC3 with Flag-N/P,confocal analysis found that RABV N/P proteins inhibited the fusion of autophagic vacuoles and lysosomes.To further examine the effect of P protein on autophagy,Western blot results found 1-222 amino acid at N end of P protein is the key area to the induction of incomplete autophagy.These results indicate that the N/P protein is the key proteins to induce incomplete autophagy.To further clarify the mechanism of signaling pathways by which RABV induced incomplete autophagy,we use genetic interference means to explore the mechanism by westernblot and confocal microscopy.The results showed that RABV infection inhibited CASP2,and activated the phosphorylation levels of AKT and MTOR,also activated the phosphorylation levels of AMPK,ERK1/2,P38.Knockdown AKT-MTOR,AMPK,MAPK pathway elements,overexpression CASP2,the results showed CASP2 is the upstream molecular elements of these pathway to mediate AMPK-MAPK/AKT-MTOR cascade signaling pathway and thus induced incomplete autophagic response.Simultaneously western blot detection found that N/P proteins could activate CASP2 mediated signaling pathway,and co-immunoprecipitation analysis found P protein interacted with BECN1.In order to further validate whether BECN1-p protein interaction induced the CASP2 dependent signaling pathway,western blot detection showed CASP2 dependent signaling pathways were significantly inhibited in BECN1-knocked down cells transfected with viral gene P or infected with RABV.These results suggest that BECN1 binding P protein mediate CASP2 dependent signaling pathway.To sum up,our comprehensive utilization of cell biology,immunology,molecular biology,biochemistry,and other means to deeply investigate the molecular mechanism of RABV infection induced incomplete autophagy.The results show that the incomplete autophagy induced by RABV infection isdue to BECN1 binding to P protein resulting in CASP2 mediated a cascade signaling pathways involved in the activation of AMPK-MAPK/AKT-MTOR.and thushelpsRABV replication.It will provide new theoretical basis to further research on RABV infection characteristics and pathogenic mechanisms. |
