| After virus invades cells, virus-host interaction affects progression and prognosis of disease. Autophagy and apoptosis play an important role in virus-host interaction. Autophagy is a kind of highly conservative metabolism process in eukaryotic cells. Autophagy allows cells to efficiently remove unneeded or damaged intracellular protein and organelles via lysosomal degradation to maintain cell-autonomous homeostasis and promote cell survival. Virus infection can induce autophagy and autophagy in turn plays dual roles in virus infection. On the one hand, Autophagy plays an antiviral role through activating TLR,participating in virus antigen processing and presenting, sequestrating and degrading virus directly. On the other hand, a number of viruses have evolved many strategies to evade or even subvert autophagy for their benefit. They can prevent autophagosome-lysosome fusion, reshape the endomembrane system to create membrane-associated replication“factories” to favor virus replication and suppress anti-viral innate immunity.Apoptosis also plays a “double-edged sword” role in virus-host interaction. Apoptosis is an autonomic ordered programmed cell death controlled by serial genes in order to maintain homeostasis. Virus infection usually triggers activation of apoptosis. The ability of cells to activate such kind of cell suicide provides an important host defense mechanism for limiting virus replication and spread. However, viruses have developed myriad mechanisms to regulate cellular apoptosis to favor their replicating,releasing and spreading. These mechanisms include: Virus can replicate more efficiently in apoptotic cells, delay or inhibit apoptosis to promote its replication and induce apoptosis at certain stage to favor its releasing and spreading avoiding of inflammation.Although autophagy and apoptosis are completely different physiological process, there is plenty of evidence to show that they are closely related. They can regulate each other and even be transferred under certain conditions. The dynamic balance between them affects viral survival and pathogenicity. We can lay the foundation to clarify virus pathogenic mechanism and develop antiviral drugs by exploring virus-host interaction. Respiratory epithelial cells serve as the first line of host defense, it is not clear that how can they interact with Respiratory Syncytial Virus(RSV). We use RSV as a model to investigated the effect of RSV infection on autophagy of Hep2 cells and the role of autophagy in virus proliferation kinetics from the perspective of interaction between virus and host cells. And we tried to discuss possible mechanism from visual angle of mutual regulation between autophagy and apoptosis during virus infection. This study will settle foundation to clarify the interrelationship between RSV and respiratory epithelial cells and exploit new antiviral treatment targeting autophagy.Part one The effect of RSV on autophagy of respiratory epithelial cells.Objective: To explore the effect of RSV on autophagy of respiratory epithelial cells.Methods:1 The effect of RSV infection on autophagy of respiratory epithelial cells.1.1 The expression of autophagic marker LC3Ⅱof Hep2 cells infected with RSV at different Multiplicities of Infection(MOI = 1, MOI = 3, MOI= 5) for 4 h, 8 h, 12 h, 24 h, 36 h and 48 h were determined by Western blot.1.2 The expression of Atg12-Atg5 complex and autophagy-related protein Atg5, Atg7, Beclin1 of Hep2 cells infected with RSV(MOI = 5) for24 h, 36 h and 48 h were detecte by Western blot.1.3 Hep2 cells were transfected with p BABE-puro-EGFP-LC3 plasmid for 24 hours, then infected with RSV for another 24 hours and cytoplasmic EGFP-LC3 puncta was visualized by fluorescence microscopy.1.4 Autophagic vacuoles of Hep2 cells infected with RSV for 24 hours were detected by Transmission electron.2 The effect of RSV infection on autophagy flux of Hep2 cells.2.1 Expression of P62 of Hep2 cells infected with RSV for 24 hours and 48 hours was detected by Western blot.2.2 Hep2 cells were treated with Baf A1(10 n M) for 2 h prior to RSV infection for another 24 h, endogenous expression of LC3Ⅱ and P62 was examined by Western blot to identify if RSV can induce complete autophagy flux.3 The effect of RSV infection on the autophagy of lungs of balb / c mouse5-week old female Balb / c mice infected with RSV(5×106 PFU per mouse) intranasally were randomly divided into 8 groups(5 mice / group)which are PBS control and infected with RSV for 1 day, 2 days, 3 days, 4days, 5 days, 6 days and 7 days respectively. LC3Ⅱ expression of lungs were examined by Western blot to see if RSV can induce autophagy in vivo.Results:1 RSV infection induce autophagosome formation in Hep2 cells.1.1 The expression of LC3Ⅱ was significantly increased in Hep2 cells infected with RSV. Its expression began to increase after 24 hours and sustained to final time point of 48 hours(P<0.05).1.2 The expression of Atg12-Atg5 complex and autophagy-related protein Atg5,Atg7 and Beclin1 aslo was significantly increased compared with the mock control(P<0.05).1.3 EGFP-LC3 puncta of Hep2 cells infected with RSV was visualized by fluorescence microscopy.1.4 Transmission electron showed that double-membraned autophagicvacuoles were formed in the cytoplasm of Hep2 cells infected with RSV.2 RSV induce complete autophagy flux of Hep2 cells infected with RSV.2.1Expression of P62 of Hep2 cells infected with RSV is significantly decreased at 48 hours(P<0.05).2.2 Baf A1,a vacuolar H+-ATPase inhibitor,significantly increase the amount of LC3 and P62 in Hep2 cells infected with RSV.3 RSV can induce autophagy of mice lungs infected with RSVLC3Ⅱ expression of mice lungs infected with RSV intranasallybegan to increaseat 3 days and sustained to final time point of 7 days(P<0.05).Part two RSV induce autophagy through ROS-dependent activation of AMPK-m TOR signaling in Hep2 cells infected with RSV.Objective: To explore the role of ROS in RSV induced autophagyand signalling pathway involved.Methods:1 Intracellular reactive oxygen species(ROS) production of Hep2 cells infected with RSV for 6 h, 12 h and 24 h was detected by DCFH-DA assay.2 Expression of LC3Ⅱ in Hep2 cells pretreated with NAC(5 m M)for 2 h and followed by RSV infection for 24 h were determined by Western blot to explore the relationship between ROS and autophagy.。3 The expression of signaling molecules, including p-AMPK,p-m TOR, p-ERK and p-JNK were detected in Hep2 cells infected with RSV at different time points.4 LC3Ⅱexpression was detected in Hep2 cells pretreated with 5 μM Compoud C(AMPK inhibitor), 10 μM SP600125(JNK inhibitor) and 10μM PD98059(ERK inhibitor) respectively for 1 hour and followed by RSV infection for another 24 hours to indicate signaling pathways involved in autophagy induction.The expression of p-AMPK,p-m TOR and LC3 Ⅱ of Hep2 cellspretreated with 5 m M N-acetyl-l-cysteine for 1 hour and followed by RSV infection for another 24 hours were examined by Western blot to explore the relationship between ROS and AMPK signaling pathway. Then we can conclude main signaling pathway involved in ROS-dependent autophagy induction.Results:1 ROS level elevated in Hep2 cells infected with RSV at 6 hours,reach a peak at 12 hours and sustained to final time point of 24 hours.2 N-acetyl-l-cysteine(ROS inhibitor) can decrease RSV induced high expression of LC3Ⅱ in Hep2 cells(P<0.05). The results indicated that RSV induced autophagy through ROS dependent mechanism.3 The expression of signaling molecules p-AMPK in Hep2 cells infected with RSV began to increase at 6 hours,and sustained to final time point of 24 hours, accompanied by p-m TOR decrease(P < 0.05). The expression of signaling molecules p-ERK in Hep2 cells infected with RSV began to increased at 30 min,and sustained to final time point of 24 hours(P < 0.05). The expression of signaling molecules p-JNK in Hep2 cells infected with RSV began to increased at 30min(P<0.05).4 SP600125 and PD98059 could not inhibit LC3Ⅱhigh expression in Hep2 cells induced by RSV(P<0.05), while Compound C could reverse p-m TOR low expression and LC3Ⅱhigh expression in Hep2 cells induced by RSV(P < 0.05), The results indicated that AMPK-m TOR signaling pathways played a major role in RSV induced autophagy.N-acetyl-l-cysteine could reverse p-AMPK high expression and p-m TOR low expression in Hep2 cells induced by RSV(P<0.05), inhibit LC3Ⅱhigh expression in Hep2 cells induced by RSV(P < 0.05). The results indicated that ROS production served as an upstream signal to activate AMPK-m TOR signaling pathways to induce autophagy in Hep2 cells infected with RSV.Part three Autophagy promote RSV replication through affecting RSV-induced cellular apoptosisObjective: To explore the effect of autophagy of Hep2 induced by RSV on virus proliferation kinetics and its possible mechanism.Methods:1 The effect of autophagy of Hep2 induced by RSV on virus proliferation kinetics.1.1 LC3Ⅱexpression of Hep2 cells pretreated with rapamycin(autophagy inducer), 3-MA or wortmannin(autophagy inhibitor) in different concentrations respectively for 2 hours prior to RSV infection for another 24 hours were detected by Western blot to determine applied concentration. Hep2 cells expressing EGFP-LC3 were pretreated with optimal concentration of rapamycin, 3-MA or wortmannin for 2 hours and followed by RSV infection for another 24 hours, EGFP-LC3 puncta was visualized by fluorescence microscopy to verify the efficiency of the autopahgy inducer and inhibitors.1.2 Hep2 cells were pretreated with 100 n M rapamycin, 1 m M 3-MA or 100 n M wortmannin respectively for 2 h and followed by RSV infection for 24 h or 48 h. Viral gene relative expression were detected by fluorescence quantitative PCR. RSV F protein expressions were examined by Western blot. extracellular virus titer in the supermatant of cells were detected. We evaluate the effect of autophagy induction and inhibition on virus proliferation dynamics from above three aspects.1.3 We generated Atg5, Atg7 and Beclin1 stable knockdown Hep2 cells infected with lentivirus that express sh RNA targeting corresponding gene respectively. Expression of Atg5, Atg7 and Beclin1 of stable knockdown cells copmared with Hep2 and Hep2-sh-luciferase cells was to detected by Western blot to validate the interference effect.LC3Ⅱexpression of above cells infected with RSV for 24 h was performed by Western blot to further identify the efficiency of inhibiting autopahgy.1.4 Hep2-sh-Atg5, Hep2-sh-Atg7 and Hep2-sh-Beclin1 were infected with RSV for 24 h or 48 h. Viral gene relative expression were detected by fluorescence quantitative PCR. RSV F protein expressions were examinedby Western blot. extracellular virus titer in the supermatant of cells were detected at indicated time point. We evaluate the effect of autophagy inhibition by knockdowning autophagy related gene on virus proliferation dynamics from above three aspects.2 Autophagy promote RSV replication through affecting RSV-induced cellular apoptosis.2.1 Expression of full length casepase 3, cleaved-casepase 3, PARP,cleaved-PARP of Hep2 cells pretreated with 3-MA for 2 h prior to RSV infection for another 48 h were performed by Western blot to clarify the effect of autophagy inhibition by using 3-MA on RSV induced cell apoptosis. Apoptosis rate of cells was determined by flow cytometry assay using Annexin V-PE and 7-AAD staining.2.2 Expression of full length casepase 3, cleaved-casepase 3, PARP,cleaved-PARP of Hep2-sh-Atg5 or Hep2-sh-Beclin1 Hep2 cells infected with RSV compared with Hep2-sh-luciferase were examined by Western blot to clarify the effect of autophagy inhibition on RSV induced cell apoptosis.2.3 Hep2 cells were transfected with si RNAs targeting Atg5, Beclin1 and and non-specific control si RNA for 24 h and followed by RSV infection for another 48 h. Apoptosis rate was determined by flow cytometry assay using Annexin V-FITC and PI staining.2.4 Hep2 cells were pretreated with autophagy inhibitor 3-MA for 2 h and followed by RSV infection for another 48 h during which Z-VAD-FMK was added after virus adsorption(2 h). Expression of RSV F protein were examined by Western blot and extracellular virus titer in the supermatant of cells were detected to explore if Z-VAD-FMK can reverse the effect of 3-MA on virus replication.2.5 Hep2-sh-Atg5 and Hep2-sh-Beclin1 were infected with RSV for48 h during which Z-VAD-FMK was added after virus adsorption for 2 h.The expression of RSV F protein were examined by Western blot and extracellular virus titer in the supermatant of cells were detected todetermine if Z-VAD-FMK can reverse the effect of autophagy inhibition by knockdowning autophagy-related gene on virus replication.Results:1 Autophagy favored virus replication.1.1 A serious of experiments were done to determine the optimum concentration of rapamycin, 3-MA and wortmannin and verify their effect:Autophagy inducer rapamycin was 100 n M; Autophagy inhibitor3-MA was 1m M; Autophagy inhibitor wortmannin was 100 n M.1.2 Rapamycin(100 n M) can significantly increase viral gene M, N, L,G, F, M2 and NS1 expression level, RSV F protein expression and extracelluar viral titers(P < 0.05), indicating that it can promote virus replication. The results are completely opposite if we treat cells with 3-MA or wortmannin(P<0.05).1.3 We generated Atg5, Atg7 and Beclin1 stable knockdown Hep2 cells in which Atg5, Atg7 and Beclin1 expression identified by Western blot were significantly decreased(P < 0.05) and LC3Ⅱ expression of above cells infected with RSV was significantly decreased compared with control cells Hep2-sh-luciferase. It indicated that knockdown Atg5, Atg7 and Beclin1 expression can inhibit autophagy.1.4 Knockdowning Atg5, Atg7, Beclin1 expression of Hep2 cells infected with RSV can inhibit virus replication.Compared with Hep2-sh-luciferase cells, knockdowning Atg5, Atg7,Beclin1 expression of Hep2 cells infected with RSV significantly decrease viral gene M, N, L, G, F, M2 and NS1 expression level, RSV F protein expression and extracelluar viral titers(P<0.05).2 Inhibiting autophagy can significantly increase RSV induced cell apoptosis.2.1 Copmared to RSV infection alone, 3-MA treatment can significantly increase the expression of cleaved-casepase 3 and cleaved-PARP of Hep2 cells infected RSV and the rate of apoptosis of Hep2 cells performed by flow cytometry assay using Annexin V-PE and7-AAD staining(P<0.05).2.2 Compared to Hep2-sh-luciferase cells, Expression of cleaved-casepase 3 and cleaved-PARP of Hep2-sh-Atg5 or Hep2-sh-Beclin1 Hep2 cells infected with RSV increased significantly(P<0.05).2.3 Copmared to negative control si RNA transfected cells,knockdowning Atg5 and Beclin1 expression using si RNA significantly increased the rate of apoptosis of Hep2 cells infected with RSV examined by flow cytometry assay using Annexin V-FITC and PI staining(P<0.05).3 Autopahgy favored viral replication by preventing cell apoptosis.3.1 RSV F protein expression and extracelluar viral titers of Hep2 cells pretreated with 3-MA prior to RSV infection can be enhanced by blocking apoptosis using pan-casepase inhibitor Z-VAD-FMK.3.2 RSV F protein expression and extracelluar viral titers of Hep2-sh-Atg5 and Hep2-sh-Beclin 1 cells infected with RSV can be enhanced by blocking apoptosis using pan-casepase inhibitor Z-VAD-FMK.Conclusions:1 RSV infection induced complete autophagy flux in Hep2 cells.2 RSV infection enhanced autophagy of mice lungs.3 RSV infection induced autophagy through ROS-dependent activation of AMPK-m TOR pathway in Hep2 cells.4 Autopahgy induced by RSV infection favored viral replication in Hep2 cells by preventing cell apoptosis. |
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