The Mechanism Research Of The Interaction Between Autophagy And Apoptosis In CVB3-infected Cells

Autophagy and apoptosis are important for virus infectious.On the one hand,autophagy and apoptosis play a key role in anti-viral infections by degradation of viral components,presenting viral antigens,activation of the immune response;on the other hand,the virus can also escape the protective antiviral activity and maintain their own survival and replication by inducing apoptosis and autophagy of the host cells.lt has been proved that CVB3could induce autophagy and apoptosis, but how CVB3induced the autophagosome formation increased;whether the apoptosis denpends on the caspase way and whether a relationship exists between these processes upon infection,and whether and how they influence viral replication and release are currently unknown.In this study, HeLa cells were infected with CVB3to evaluate the questions above in order to know the interaction between CVB3and cells better.In this study,we observed the autophagy level and the underlying mechanisms of autophagosome formation.In CVB3infected HeLa cells, the autophagosome formation increasd,and its level further increased in the bafilomycin-treated infected cells compared with the non-treated infected cells. These data indicating that autophagosomes had some remaining capacity to combine with lysosomes and subsequently degrade.CVB3infection-mediated autophagosome accumulation occurs by inducing autophagosome formation.To explore the machinery of autophagosome formation during CVB3infection,ERK activation was observed.We found ERK was activated.Inhibition of ERK phosphorylation with U0126suppressed CVB3-induced autophagosome accumulation.Therefore,it is apparent that CVB3infection triggers autophagy in HeLa cells through the ERK signaling pathway.To characterize the molecular mechanisms underlying increased ERK activation in CVB3infection, we observed the activity of AMPK/MEK/ERK pathway:we found AMPK and MEK were both activated,and the interaction between p-MEK and p-AMPK increased.Then we found the MEK、ERK activation decreased after giving comound C, indicating that AMPK might be an upstream regulator of ERK. Then,we found concentration of free calcium in the cytoplasm increased,but the concentration of ATP and the mitochondrial membrane potential decreased in the CVB3-infected HeLa cell than mock cells.These data indicate that AMPK/MEK/ERK signaling pathway was activated because CVB3-infected induced the injury of cytomemebrane, then the concentration of free calcium in the cytoplasm increased which injuried the mitochondrion.The mitochondrion was injuried induced the concentration of ATP decreased,then AMPK was activated.We then explored whether CVB3infection activated Ras/Raf/MEK/ERK signaling pathway:we found that Raf and Ras were both activated and the interaction between p-MEK and Rafl increased. Then we found the MEK、ERK activation decreased after giving GW5074, indicating that ERK could be activated via Ras/Raf/MEK/ERK signaling pathway.While RasGAP was cleaved at9h.p.i.,indicating that RasGAP itself is cleaved during CVB3infection,promoting the activation of a Ras-activated kinase cascade(Ras pathway) as well as the phosphorylation of specific MAPK target protein.However,the Ras/Raf/MEK/ERK and AMPK/MEK/ERK pathway signaling pathway were both still at a high activation level,the amount of autophagosome began to decreased.Thus, the expression of Atg5and beclin-1protein was explored,we found Beclin-1and Atg5were cleaved over time after CVB3infection.But their cleavage were decresed or disappeared by using Caspase inhibitors.What’s more?The level of apoptosis decreased by using these Caspase inhibitors.These observations indicate that CVB3infection induced apoptosis is caspase-dependent.Caspase cleaved Atg5and beclin-1induced the level of autophagosome decreased but apoptosis increased.To explore whether apoptosis occurs depended on the inhibition of autophagy, we observed the effect of authophagy on apoptosis.The effect of autophagy on apoptosis, however, is complicated. We observed that autophagy inhibitors decreased the level of apoptosis, while autophagy inducers increased this level at18h.p.i. This result may relate to changes in viral load because it has been well documented that CVB3proteins can directly induce apoptosis.Therefore, in order to minimize the effects of viral proteins on apoptosis, we selected a time point (3h.p.i.) exhibiting the least amount of viral protein production to conduct our further experiments. Under these conditions, the level of apoptosis increased upon inhibiting autophagy with3-MA but no change upon promoting autophagy with rapamycin, which differed from the results obtained at18h.p.i., suggesting that autophagy suppressed apoptosis after CVB3infection. The mechanism is p62mediate caspase-8activity by autophagy.What’s more?We found p62protein levels significantly decreased from9h.p.i.According to the former reports,we belived CVB3direct cleaved p62protein. Then the balance between autophagy and apoptosis was destroyed and autophagy converts to apoptosis. However, we observed that total LC3, Atg5, and Beclin-1protein as well as their mRNA levels increased from3to12h.p.i., indicating that CVB3-induced autophagy was regulated at both the transcriptional and translational levels to avoid the cells occuring apoptosis too early.To determine whether and how these CVB3-induced changes in autophagy and apoptosis influenced viral replication and release of CVB3from the cell, we treated HeLa cells with the different pharmacological agents and evaluated CVB3mRNA levels in cells and CVB3titers in the supernatant. Upon rapamycin treatment to promote autophagy, we found a dose-dependent increase in both CVB3mRNA within cells and viral titers in the supernatant, while the opposite result was observed after3-MA treatment to inhibit autophagy. To test the role for apoptosis, we treated HeLa cells with the3caspase inhibitors (Z-VAD-FMK, Z-IETD-FMK, Z-LEHD-FMK). At low concentrations, CVB3mRNA levels were a little higher, but viral titers in the supernatant decreased; upon increasing the inhibitor concentrations, both the CVB3mRNA inside the cells and titers in the supernatant decreased. Thus, these results suggest that autophagy promotes CVB3replication and eventual release while apoptosis promotes CVB3release from the cells.Above all,we belived CVB3could induce autophagy and apoptosis and it can complete its life cycle via the convert from autophagy to apoptosis, details are as following:on the one hand, RasGAP itself is cleaved by CVB3infection,promoting the activation of a Ras-activated kinase cascade(Ras pathway) as well as the phosphorylation of specific MAPK target protein;on the other hand, CVB3-infected induced the injury of cytomemebrane, then the concentration of free calcium in the cytoplasm increased which injuried the mitochondrion.The mitochondrion was injuried induced the concentration of ATP decreased,then AMPK was activated.Then ERK was activated induced autophagosome accumulation.Authophagosome is beneficial for the CVB3replication.Meanwhile,p62mediated the degradation of activate caspase-8via autophagy pathway to advoid cells occuring apoptosis too early.However,from9h.p.i.,the activated caspase-8could not be degraded because the CVB32A and3B proteins cleaved p62protein.Then the activated caspase-8increased combined the activated caspase-9cleaved Atg5and beclin-1,inhibit the formation of autophagosome.So the activated caspase-8increased again and autophagy convert to apoptosis.However, autophagy was regulated at both the transcriptional and translational levels to control the apoptosis in order to make sure that the CVB3has enough time to complete their complicate before release. These date help us to better understand the interaction between CVB3life cycle and cell,and provide new ideas for clinical treatment of related CVB3-infected diseases.