The PERK/PKR-eIF2α Pathway Negatively Regulates Porcine Hemagglutinating Encephalomyelitis Virus Replication by Attenuating Global Protein Translation and Facilitating Stress Granule Formation

Abstract

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is closely associated with the endoplasmic reticulum (ER) of infected cells. The unfolded protein response (UPR), which is mediated by ER stress (ERS), is a typical outcome in coronavirus-infected cells and is closely associated with the characteristics of coronaviruses. However, the interaction between virus-induced ERS and coronavirus replication is poorly understood. Here, we demonstrated that infection with the betacoronavirus porcine hemagglutinating encephalomyelitis virus (PHEV) induced ERS and triggered all three branches of the UPR signaling pathway both in vitro and in vivo. In addition, ERS suppressed PHEV replication in mouse neuro-2a (N2a) cells primarily by activating the protein kinase R-like ER kinase (PERK)-eukaryotic initiation factor 2α (eIF2α) axis of the UPR. Moreover, another eIF2α phosphorylation kinase, IFN-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrated that the PERK/PKR-eIF2α pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2α also promoted the formation of stress granule (SG), which in turn repressed PHEV replication. In summary, our study presents a vital aspect of the host innate response to invading pathogens and reveals attractive host targets (e.g., PERK, PKR and eIF2α) for antiviral drugs. IMPORTANCE Coronavirus diseases are caused by different coronaviruses of importance in humans and animals, and specific treatments are extremely limited. ERS, which can activate the UPR to modulate viral replication and the host innate response, is a frequent occurrence in coronavirus-infected cells. PHEV, a neurotropic β-coronavirus, causes nerve cell damage, which accounts for the high mortality rates in suckling piglets. However, it remains incompletely understood whether the highly developed ER in nerve cells plays an antiviral role in ERS and how ERS regulates viral proliferation. In this study, we found that PHEV infection induced ERS and activated the UPR both in vitro and in vivo and that the activated PERK/PKR-eIF2α axis inhibited PHEV replication through attenuating global protein translation and promoting SG formation. A better understanding of coronavirus-induced ERS and UPR activation may reveal the pathogenic mechanism of coronavirus and facilitate the development of new treatment strategies for these diseases.

Induction and modulation of the unfolded protein response during porcine deltacoronavirus infection

“…It is likely that the antibodies used in our experiments did not efficiently recognize the amino-terminus of porcine ATF6. Similar cases have been described in previous reports ( Echavarria-Consuegra et al, 2021 , Shi et al, 2022 ).…”

“…For example, Porcine epidemic diarrhea virus (PEDV) and TGEV infection induce ERS and promote GRP78 protein expression ( Sun et al, 2021 , Xue et al, 2018 ). Porcine hemagglutinating encephalomyelitis virus (PHEV) infection activates all three UPR arms and increases GRP78 expression at both the mRNA and protein levels ( Shi et al, 2022 ). PRRSV targets the UPR master regulator GRP78 for degradation despite inducing an increase in its mRNA levels in a time-dependent manner ( Gao et al, 2019 ).…”

Study on the Anti-Adenovirus Mechanism of Sargassum fusiforme

“…The endoplasmic reticulum (ER) is an essential organelle in eukaryotes that is mainly responsible for protein assembly, modification, folding, calcium storage, and lipid synthesis ( Metcalf et al., 2020 ). Studies have shown that when viruses interact with cells, large amounts of viral proteins accumulate in the ER lumen, which perturbs the homeostasis and function of the ER, triggering the UPR ( Prasad and Greber, 2021 ; Shaban et al., 2021 ; Shi et al., 2021 ). Regulation of the ERS on UPR signals is mainly dependent on three pathways (ATF6, PERK, and IRE1).…”

PHEV infection: A promising model of betacoronavirus-associated neurological and olfactory dysfunction

“…The TEM analysis was performed as previously described [ 32 ]. Briefly, PHEV-infected mouse brain samples were collected, fixed with 2.5% glutaraldehyde for 24 h, and then postfixed with 1% osmium tetroxide for 2 h. After dehydration using a graded ethanol series, the specimens were embedded in Renlam resin.…”

“…Neurodegenerative changes, such as axonal dysplasia, unstable dendritic spine formation, and irregular swelling and disconnection in neurites, are linked to the Ulk1-TrkA-NGF-Rab5 signaling pathway [29,30]. Furthermore, PHEV-induced neurodegeneration is related to lysosome dysfunction and endoplasmic reticulum (ER) stress [31][32][33], similar to the pathogenesis of human neurodegenerative diseases such as Parkinson's disease, frontotemporal degeneration and neuronal lipofuscinosis, indicating that PHEV might be a useful model virus to study the mechanisms of human neurodegenerative disease [33].…”