Coronavirus-induced ER stress response and its involvement in regulation of coronavirus-host interactions

Abstract

Coronavirus replication is structurally and functionally associated with the endoplasmic reticulum (ER), a major site of protein synthesis, folding, modification and sorting in the eukaryotic cells. Disturbance of ER homeostasis may occur under various physiological or pathological conditions. In response to the ER stress, signaling pathways of the unfolded protein response (UPR) are activated. UPR is mediated by three ER transmembrane sensors, namely the PKR-like ER protein kinase (PERK), the inositol-requiring protein 1 (IRE1) and the activating transcriptional factor 6 (ATF6). UPR facilitates adaptation to ER stress by reversible translation attenuation, enhancement of ER protein folding capacity and activation of ER-associated degradation (ERAD). In cells under prolonged and irremediable ER stress, UPR can also trigger apoptotic cell death. Accumulating evidence has shown that coronavirus infection causes ER stress and induces UPR in the infected cells. UPR is closely associated with a number of major signaling pathways, including autophagy, apoptosis, the mitogen-activated protein (MAP) kinase pathways, innate immunity and pro-inflammatory response. Therefore, studies on the UPR are pivotal in elucidating the complicated issue of coronavirus-host interaction. In this paper, we present the up-to-date knowledge on coronavirus-induced UPR and discuss its potential involvement in regulation of innate immunity and apoptosis.

Keywords: Apoptosis; Coronavirus; ER stress; Innate immune response; Pro-inflammatory cytokines; Unfolded protein response.

Fig. 1

Signaling pathways of three branches of UPR and modulations by coronavirus infection. (A) Coronavirus replication causes ER stress by three major mechanisms. The ER stress sensors PERK, IRE1, and ATF6 are activated and trigger UPR in an attempt to counter ER stress. (B) The signaling pathway of integrated stress response and coronavirus intervention. Infection with MHV-A59, SARS-CoV, and IBV has been shown to cause eIF2α phosphorylation, which is most likely mediated by PKR and/or PERK. The phosphorylated eIF2α sequesters eIF2B, inhibits recycling of GTP-bound eIF2α and leads to translation attenuation. (C) The IRE1 signaling pathway and coronavirus intervention. IRE1 mediates splicing of XBP1, which induces UPR genes such as ERdj4 and p58^IPK. IRE1 can also recruit TRAF2 and activate JNK-mediated apoptosis. The over-expression of spike proteins of IBV and MHV-A59 has been shown to activate IRE1, whereas the SARS-CoV E protein inhibits XBP1 splicing. (D) The ATF6 signaling pathway and coronavirus intervention. ATF6 protein is cleaved by S1P and S2P under ER stress. The released fragment (ATF6f) translocates to the nucleus and induces UPR genes. Transfection of SARS-CoV accessory protein 8ab or infection with IBV or MHV-A59 has been shown to induce ATF6 cleavage.

Fig. 2

Involvement of UPR in coronavirus-induced apoptosis. Under prolonged ER stress, phosphorylation of eIF2α by PKR/PERK up-regulates CHOP, which has been shown to suppress the pro-survival kinase ERK and the anti-apoptotic mitochondrial protein Bcl-2. IRE1 has been shown to protect IBV-infected cells from apoptosis, by converting the pro-apoptotic unspliced XBP1 to the anti-apoptotic spliced form (XBP1s). Also, in IBV-infected cells, IRE1 activates the pro-survival kinase AKT and suppresses the pro-apoptotic kinase JNK. ATF6 potentially facilitates apoptosis by induction of CHOP and XBP1u. ER-stress induced apoptosis has also been associated with cleavage of caspase 12 (Casp 12) in mouse or caspase 4 in human, although their involvement during coronavirus infection is unknown. Pro-apoptotic factors are shown in red boxes, while pro-survival proteins in blue boxes.

Fig. 3

Over-expression of CHOP, but not its N-terminal deletion mutant, promotes IBV-induced apoptosis. (A) H1299 cells were transfected with different amount of FLAG-tag CHOP plasmid or empty vector. At 24 h post transfection, cells were infected with IBV or incubated with mock lysate for 22 h. Cell lysates were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane for western blotting using antibodies against FLAG-tag, IBV N protein and the apoptosis marker poly (ADP-ribose) polymerase (PARP). The percentages of PARP cleavage (intensity of cleavage band [Cl] divided by total intensities of full-length [FL] and cleavage bands) were determined and indicated below. The β-tubulin protein was used as loading control. The presented blot is one representative blot from three independent experiments. (B) Schematic diagram showing the known functional domains of CHOP and the two N-terminal deletion mutants (ΔN36 and ΔN70) used in (C). Amino acid 10–18 have been shown to interact with TRIB3. Serine 79 and 82 are phosphorylated by p38, while Leucine 134 and 141 are responsible for DNA binding of the bZIP domain. (C) H1299 cells were transfected with FLAG-tag wild type CHOP or N-terminal deletion mutants. At 24 h post transfection, cells were infected with IBV or incubated with mock lysate. Cell lysates were harvest and subjected to SDS-PAGE and western blot analysis as in (A). Percentage of PARP cleavage is determined as in (A) and indicated below. The β-actin protein was used as loading control. The presented blot is one representative blot from three independent experiments.

Fig. 4

Involvement of UPR in innate immune response during coronavirus infection. The PKR/PERK mediated eIF2α phosphorylation leads to translation attenuation and lower level of IκBα synthesis. On the other hand, IRE1 recruits TRAF2 and activates IKK to phosphorylate IκBα, promoting its ubiquitination and degradation. The outcome is a lower protein level of IκBα, releasing NF-κB for activation of type-I interferons and/or cytokines. The PERK branch also activates NF-κB by up-regulation of CHOP, which forms heterodimer with C/EBPβ and prevent it from activating PPARγ that suppresses NF-κB activation. The MAP kinases p38 and JNK activate AP-1 and promote cytokine production. Under ER stress, JNK is phosphorylated by IRE1/TRAF2 complex, while ATF3 has been shown to induce DUSP1 that de-phosphorylates p38. Several proteins, such as GADD34 or the spliced form of XBP1, have been shown to cross-talk with the innate immune signaling. Refer to text for detailed description.

Fig. 5

The cross-talks between three UPR branches and temporal control of UPR during coronavirus infection, using IBV as an example. At the early stage (1–8 h) of infection, the PERK/PKR triggers translational block by eIF2α phosphorylation. The activation of ATF4 and its downstream signaling leads to translation recovery and accumulation of CHOP. The ATF6 and IRE1 branches possibly activate at a much later time of IBV infection (12–16 h). Enhanced ER folding and activation of ERAD may promote adaptation to the ER stress and cell survival. Finally, prolonged ER stress due to continuous IBV replication and budding led to the dead phase of UPR, characterized by caspase cleavage and other apoptosis mediated cell demolitions.