The Unfolded Protein Response and Autophagy on the Crossroads of Coronaviruses Infections

Abstract

The ability to sense and adequately respond to variable environmental conditions is central for cellular and organismal homeostasis. Eukaryotic cells are equipped with highly conserved stress-response mechanisms that support cellular function when homeostasis is compromised, promoting survival. Two such mechanisms - the unfolded protein response (UPR) and autophagy - are involved in the cellular response to perturbations in the endoplasmic reticulum, in calcium homeostasis, in cellular energy or redox status. Each of them operates through conserved signaling pathways to promote cellular adaptations that include re-programming transcription of genes and translation of new proteins and degradation of cellular components. In addition to their specific functions, it is becoming increasingly clear that these pathways intersect in many ways in different contexts of cellular stress. Viral infections are a major cause of cellular stress as many cellular functions are coopted to support viral replication. Both UPR and autophagy are induced upon infection with many different viruses with varying outcomes - in some instances controlling infection while in others supporting viral replication and infection. The role of UPR and autophagy in response to coronavirus infection has been a matter of debate in the last decade. It has been suggested that CoV exploit components of autophagy machinery and UPR to generate double-membrane vesicles where it establishes its replicative niche and to control the balance between cell death and survival during infection. Even though the molecular mechanisms are not fully elucidated, it is clear that UPR and autophagy are intimately associated during CoV infections. The current SARS-CoV-2 pandemic has brought renewed interest to this topic as several drugs known to modulate autophagy - including chloroquine, niclosamide, valinomycin, and spermine - were proposed as therapeutic options. Their efficacy is still debatable, highlighting the need to better understand the molecular interactions between CoV, UPR and autophagy.

Keywords: autophagy; coronavirus; host-pathogen interaction; integrated stress response; unfolded protein response.

Figure 1

The three branches of the unfolded protein response (UPR). When misfolded proteins accumulate in the endoplasmic reticulum (ER) lumen, chaperone binding immunoglobulin protein (BiP/GRP78) is detached from the luminal domains of the three ER sensors to which it was bound, allowing PKR-like ER protein kinase (PERK) and inositol-requiring protein-1 (IRE1) to form homodimers and activating transcriptional factor-6 (ATF6) to transit to the Golgi. (1) PERK phosphorylates the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), resulting in a global reduction of protein synthesis while still maintaining translation of a few key proteins, such as activating transcription factor 4 (ATF4), which induces expression of genes involved in redox homeostasis, amino acid metabolism, protein synthesis, autophagy and apoptosis, such as the transcription factor C/EBP homologous protein (CHOP). Protein synthesis is restored when eIF2α is dephosphorylated by protein phosphatase 1 (PP1) regulatory subunit GADD34, which is also induced by ATF4 when ER stress is resolved. (2) IRE1 autophosphorylates to switch on its RNase activity, inducing a process known as regulated IRE1-dependent decay (RIDD), in which IRE1 cleaves and leads to the selective degradation of a small set of mRNAs or miRNAs. (3) IRE1 also excises a short 26-nucleotide intron from the mRNA encoding transcription factor X-box-binding protein 1 (XBP1), generating the spliced Xbp1 mRNA, which is ultimately translated into the transcription factor XBP1s that, like ATF4, upregulates genes involved in multiple cell signaling pathways, such as ER-associated protein degradation (ERAD). (4) Full length ATF6 translocates from the ER to the Golgi, where it is cleaved by site-1 protease (S1P) and site-2 protease (S2P). This releases a cytosolic fragment which then transits to the nucleus, transcription factor ATF6p50, which drives a transcriptional program to reestablish homeostasis.

Figure 2

Unfolded protein response (UPR), autophagy and coronavirus (CoV) infections. This figure represents data obtained by using different cell models infected by CoV viruses mouse hepatites virus (MHV), infectious bronchitis virus (IBV), SARS-CoV or SARS-CoV-2. CoV infection begins when the viral S protein attaches to its complementary host receptor, allowing the virus to enter the host cells by endocytosis or direct fusion of the viral envelop with the host membrane. The process of SARS-CoV2 virus entry and uncoating (1) downregulates AMPK and reduces phosphorylation of ATG14, disrupting the autophagy flux, while simultaneously inhibiting mTORC1, which promotes autophagy. Once inside the cell, the viral positive-sense RNA is translated (2) and the virus induces massive rearrangement of the intracellular membrane network to generate double membrane vesicles (DMVs) (3). CoV-induced DMVs may originate from late endosomes, autophagosomes or vesicles from the early secretory pathway. MHV-induced autophagy was impaired in cells lacking ATG5, which displayed a deranged morphology of the membranes and decreased viral yield. Nonlipidated LC3 extensively colocalizes with DMV protein markers and downregulation of LC3 protects cells from CoV infection as result of defects in DMVs biogenesis. CoV replication occurs within DMVs and transmembrane structural proteins (S, M and E) are synthesized in the ER (4). Non-structural protein NSP6 from IBV, MHV and SARS-CoV are localized in the ER and participate in autophagosome formation via omegasomes, which could be used for DMV formation. Finally, new viral particles are transported to the ER-Golgi intermediate compartment (ERGIC) for assembly (5) and exported through secretory pathway in smooth-wall vesicles, which ultimately fuse with the plasma membrane to release the mature virus (6). A high demand for membranes for DMV formation and virion exocytosis contributes to ER stress. MHV activates PERK/eIF2α and IRE1 and target genes from these two branches are detected upon infection. Cleavage of ATF6 can also be observed but no target genes are detected subsequently.