Under conditions of cell stress such as nutrient deprivation or as a result of the accumulation of damaged organelles and misfolded proteins a lysosomal pathway is induced which degrades proteins as well organelles independent of the proteasomal pathway. The core machinery of this pathway -termed autophagy from the Greek auto-, "self" and phagein, "to eat” (literally “selfeating”) - was discovered in genetic screens of yeast for genes required for survival under nutrient starvation and since been shown to be conserved in mammalian and plant cells alike, providing metabolic substrates in the absence of external sources as well as contributing to cell survival by degrading non-functional organelles. As discussed in previous posts, autophagy also contributes to the degradation of viral proteins, the presentation of viral antigens to the immune system via the MHC Class II system, as well as contributing to the release of virions and the formation of replication centers. In the context of viral infection therefore the induction of autophagy can have both pro- as well as antiviral effects. Indeed, a number of viral proteins such as the coronaviral nsp-6 or PLP2 proteins both induce the formation of autophagosomes or autophagosome-like structures whilst preventing the fusion of these structures with the lysosome whilst the genome of other viruses encode for protein(s) which might prevent the fusion of autophagosomes whilst not inducing the formation of autophagosomes. In addition to inducing the degradation of viral proteins and/or viral RNA, the induction of autophagy can also induce cell death, which in contrast to the induction apoptosis is (primarily) independent of the induction of caspases and morphologically different from apoptosis. In general, the induction of autophagy induced cell death by cellular stress such as DNA damage, mitochondrial damage, or the accumulation of misfolded proteins can be the result of defective autophagy. Insufficient autophagy characterized by a decrease of autophagy may induce both cell death and also contribute to the immortalization of cells by failure of clearance of micronuclei thus contributing to the accumulation reassembled chromosomes. Excessive autophagy in contrast, induces lipid depletion of the ER and/or nuclear membrane, and inducing a process termed autosis characterized by ER fragmentation, which may be distinct from “classical” autophagy induced cell death, the latter being induced by the release of Cathepsin-D and subsequent activation of caspases.
|Induction of different pathways due to dysregulated autophagy|
In contrast to autosis, which may or may not involve the induction of ER stress response, the release of Cathepsin-D increases the release of Cytochrome C and subsequent activates Caspase-3/-7 thus inducing the depolarization of the mitochondrial membrane, linking defective autophagy to Caspase-dependent apoptosis. Indeed, the overexpression of Cathepsin-D in human malignant glioblastoma M059J cells induces autophagy and thus prevents apoptosis. The connection between autophagy and apoptosis however is quite complex, highlighted by results obtained from U87 human glioblastoma cells treated with arsenic trioxide, in which the inhibition of autophagy using 3-MA protects cells from arsenite toxicity whilst high concentrations of Bafilomycin-A1 did not, indicating that the accumulation of mature lysosomes induces apoptosis probably due to the release of Cathepsin-D, similar to treatment of U87, LN229, LN308, U118 and U251 glioblastoma cell lines with Chloroquine, and that low concentrations of Bafilomycin-A1 favors the downregulation of Cathepsin-B and Cathepsin-L thus protecting cells from apoptosis.
In the case of Enterovirus-71 infected human rhabdomyosarcoma (RD-A) cells, the increase of Cathepsin-D at 12 hrs p.i. has been postulated to induce caspase-dependent apoptosis, whereas at earlier time points the inhibition of the fusion of the autophagosome with the lysosome via UVRAG and Bax prevents apoptosis; interestingly, the application of both Z-VAD-FMK and Z-DEVD-FMK inhibitors not only prevents apoptosis but also promotes autophagic flux as measured by the degradation of p62/SQSTM-1, indicating that both processes are competing. In contrast to EV-71 infected RD-A cells, Chloroquine treatment of the aforementioned glioblastoma cell lines however induces caspase independent cell death, indicating that the outcome might be cell line specific. It should be noted however that the involvement of caspases in autophagy related cell death might be secondary and that caspase activation might only be partial.
Generally, autosis is less well defined and might be in fact a subtype of autophagy induced cell death. In cells undergoing autosis, the ER is highly fragmented and focal ballooning of the perinuclear space can be observed. Whilst being independent of caspase activation, autosis is induced by excessive autophagy experimentally triggered by the expression of an autophagy inducing peptide, Tat-Beclin, and dependent on the activity of the Na+/K+ ATPase. So far, no viral protein or virus has been demonstrated to interfere with autosis but it seems conceivable that the treatment of HIV, Chikungunya Virus, or West Nile Virus infected cells with Tat-Beclin might trigger autosis.
|Tat-Beclin might induce autosis in cells expressing HIV-Nef or degradation of Nef|
Epstein Barr Virus (EBV) LMP-1 and LMP-2: induction of autophagy via different pathways
During the latent phase of the replication cycle, the EBV latent membrane protein 1 (LMP-1) oncogene has been demonstrated to induce the unfolded protein response (UPR)/ ER stress response pathway via the N terminal six membrane spanning domain by activating all three branches of the UPR (PERK, ATF6, and IRE-1) in a sequential manner. First, LMP-1 induces the expression and activation of ATF4 via phosphorylation of eIF2α, thus inducing not only the expression of LMP-1 but also inducing the expression of autophagy related genes, preventing the induction of apoptosis due to prolonged induction of PERK. Activation of IRE-1 promotes the accumulation of spliced XBP1, inducing the formation of EDEMosomes via increasing the expression of EDEM-1 and EDEM-2 as well as the activating Beclin-1 by JNK mediated phosphorylation of Bcl-2 whilst inhibiting autophagy via CHOP. Cleavage of ATF6 generates ATF6α, inducing the ERAD pathway and activating CHOP. Translocation of ATF6α from the ER/Golgi into the nucleus increases the expression of EBV genes, thus promoting expression of viral genes. An increased level of phosphorylated eIF2α however inhibits general translation, lowering LMP-1 levels, allowing the cycle to begin anew.
High levels of autophagy induced via the UPR, thus promoting cell survival, also degrade LMP-1. Indeed, transfection of EBV infected B-lymphocytes with siBeclin-1 not only inhibits LMP-1 induced autophagy but also increases LMP-1 levels and induces apoptosis. LMP-1 induced autophagy therefore has a pro-survival rather than an pro-apoptotic effect, at least in the context of viral infection. In addition to autophagy induced as a result of ER stress, LMP-1 might induce the formation of autophagosomes via induction of PI3K.
The expression of another EBV latent membrane protein, LMP-2A, also induces autophagy and prevents apoptosis/anoikis in human nonmalignant breast epithelial MCF10A cells. In this case, blocking the formation of autophagosomes with 3-MA or the degradation of the autolysosome with Chloroquine not only prevents autophagy but also induces the cleavage of Caspase-3. Conversely, the expression of LMP-2 in the absence of inhibitors promotes the formation of autophagosomes in a Atg5 and Atg7 dependent manner probably involving the K11-ubiquitiylation of Beclin-1 by NEDD4 E3 ubiquitin ligases such as Itch or RNF5 as well as cleavage of ATG4B cysteine protease and subsequent lipidation of LC3-I (converting LC3-I to LC3-II) via the PY domain. LMP-2A therefore might facilitate the lipidation of LC3-I -and thus the formation of the mature autophagosome- by ATG4B mediated cleavage of LC3-I. In addition, LMP-2A forms a complex with ATG5 and ATG12, implying that LMP-2A induces the formation of the phagophore by sequestering Beclin-1, leading to the recruitment of Vps34, UVRAG, AMBRA1, ATG14L, p150 and/or Bif-1 independent of ULK-1 as well as promoting the conversion of LC3-I to LC3-II.
|Regulation of autophagy in cells expressing both LMP-1 and LMP2A: model|
In the context of viral infection both LMP-1 and LMP-2A co-localize to lipid rafts, indicating that lipid rafts are the membrane source for the autophagic vesicles induced by both proteins, similar to HTLV-1 Tax. Since the expression of LMP-1 induces the ER stress response, it might be possible that LMP-2A increases the clearance of LMP-1 by enhancing LMP-1 induced autophagy. It remains to be seen if the combined expression of both LMP-1 and LMP-2A in the absence of other viral proteins such as EBNA-1 induces autophagic cell death or autosis due to lipid depletion. Also it remains to be seen if the induction of autophagy by LMP-1 and/or LMP-2A interferes with immune sensing pathways, including STING mediated signaling by clearing mitochondria bound to the ER via MAM. Moreover, the induction of autophagy by EBV has been demonstrated to decrease viral replication and autophagic flux is inhibited in EBV infected cells entering the lytic cycle. Since both LMP-1 and LMP-2A are expressed during latency, LMP-1/LMP-2A induced autophagy might be essential to maintain latency. If the experimental inhibition of LMP-1 and/or LMP-2A induced autophagy is however sufficient to abolish latency remains to be seen.
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