A new study led by researchers from the University of Chicago that involved medical scientists from the University of Pennsylvania has found that the SARS-CoV-2 coronavirus is different from all other known Betacoronaviruses in that it only partially activates the IRE1α/XBP1 endoplasmic reticulum stress pathway.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China in late 2019. It was the third lethal zoonotic coronavirus to emerge into humans, after SARS-CoV (2002) and Middle East respiratory syndrome coronavirus (MERS-CoV) (2012), each of which has been associated with acute lung injury and hypoxemic respiratory failure.
While coronaviruses are divided into four genera (alpha, beta, gamma, and delta) (1, 2), all three of the lethal human coronaviruses are betacoronaviruses, albeit from different subgenera (Fig. 1).
SARS-CoV and SARS-CoV-2 are sarbecoviruses, while MERS-CoV is a merbecovirus. Other human CoVs, including HCoV-OC43 (OC43) and HCoV-HKU1 (HKU-1), are embecoviruses, as is the model murine coronavirus mouse hepatitis virus (MHV). All CoVs have similar genome structures and replication cycles, and the human CoVs as well as some MHV strains exhibit tropism for the epithelia of the respiratory tract, the portal of entry. They replicate their RNAs and produce subgenomic mRNAs by conserved mechanisms and encode homologous structural as well as replicase proteins.
Despite the similarities among all coronaviruses, each subgenus expresses distinct accessory proteins that may confer differences in host-virus interactions. Indeed, we have previously found that SARS-CoV-2, MERS-CoV, and MHV all induce somewhat different levels of activation and/or antagonism of interferon (IFN) signaling and other double-stranded RNA (dsRNA) induced antiviral innate responses (3–11).
One key pathway involved in the virus-induced host response is the endoplasmic reticulum (ER) stress response that regulates protein homeostasis (referred to as proteostasis) in this organelle. One-third of all eukaryotic proteins, including most that are inserted into membranes or secreted, are synthesized through co-translational translocation into the ER lumen.
Likewise, viral membrane-associated proteins are translated and processed in association with the ER (12, 13). Once in the ER, these polypeptides undergo stringent quality control monitoring to ensure that they are properly processed and folded. If the capacity to fold proteins is unable to keep up with demand, misfolded proteins will accumulate in the ER lumen – a condition referred to as “ER stress.”
The presence of misfolded proteins in the ER is sensed by three transmembrane sentinel proteins – activating transcription factor 6 (ATF6), PKR-like ER kinase (PERK), and inositol-requiring enzyme (IRE)1α – which trigger an intracellular signaling pathway called the unfolded protein response (UPR).
In an effort to restore proteostasis, activation of these sensors induces transcription factors that turn on genes encoding chaperones, oxidoreductases, and ER-associated decay (ERAD) components (14). The UPR also inhibits cap-dependent translation, thus decreasing the load on the ER and giving it extra time to fold proteins already in production (15, 16). If successful, these adaptive UPR programs restore ER homeostasis.
The most ancient UPR pathway is controlled by IRE1α—an ER transmembrane bifunctional kinase/endoribonuclease (RNase) that employs autophosphorylation to control its catalytic RNase function (17, 18). In response to ER stress, IRE1α undergoes autophosphorylation and dimerization to allosterically activate its RNase domain to excise a 26-nucleotide (nt) nonconventional intron in XBP1 mRNA; religation of spliced XBP1 shifts the open reading frame, and its translation produces the homeostatic transcription factor XBP1s (s = spliced) (19, 20).
Once synthesized, XBP1s upregulates genes that expand the ER and its protein folding machinery (21). IRE1α can additionally lead to apoptosis and inflammation via JUN N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling (22). Prolonged ER stress can induce regulated IRE1-dependent decay (RIDD), promoting the cleavage of additional targets beyond XBP1 mRNA, such as secretory protein and ER-localized mRNAs (23).
In the short term, RIDD may promote adaptation through further reducing translation and the protein burden on the ER. However, prolonged RIDD leads to the depletion of vital ER resident enzymes and structural components to exacerbate ER stress and hasten cell death (17, 24).
There is a large body of evidence that viral replication in mammalian cells can trigger ER stress and UPR activation in infected cells (25), and numerous studies report that the UPR is activated upon infection of host cells by coronavirus family members (12, 13, 26–31).
Coronaviruses induce stress in the ER in several ways. First, conserved replicase-encoded, nonstructural proteins nsp3, nsp4, and nps6 are embedded into the ER membrane and, along with unknown host factors, promote membrane curvature to form double membrane vesicles (DMVs), the site of viral replication/transcription centers (RTC) (32).
In addition to remodeling the ER, coronaviruses further condition infected cells by shifting translation away from host mRNAs and instead to viral mRNAs. Translation of viral mRNAs causes the ER to be flooded with heavily glycosylated viral structural proteins (e.g., spike [S], membrane [M], and envelope [E]), challenging the organelle’s folding capacity and overall integrity.
Indeed, overexpression of coronavirus spike proteins (33) as well as several sarbecovirus accessory proteins (28, 34), has been reported to induce ER stress, although overexpression itself may cause stress irrespective of the proteins. Finally, cell membranes are depleted as enveloped virus particles are assembled into new virions in the ER-Golgi intermediate compartment before budding from the infected cell (1).
Thus, coronaviruses as well as other enveloped viruses promote a massive ER expansion and modification necessary to replicate their genomes, transcribe mRNAs, and finally, to process and package their protein products into viral particles.
We have compared the activation status and requirement of the IRE1α/XBP1 arm of the UPR in well-characterized human lung epithelial cell lines and in induced pluripotent stem cell (iPSC)-derived type II alveolar (iAT2) cells, following infection with four betacoronaviruses representing three distinct subgenera.
We find that infection with MERS-CoV, OC43, and MHV leads to phosphorylation of IRE1α and the consequent production of spliced XBP1 (XBP1s) transcription factor. Surprisingly, while we observed phosphorylation of IRE1α in SARS-CoV-2 infected cells, there was a notable absence of XBP1s, suggesting that SARS-CoV-2 inhibits downstream signaling of the IRE1α/XBP1 arm of the UPR. In addition, we report reduced SARS-CoV-2-induced interferon signaling gene expression in the absence of IRE1α.