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1.
J Med Virol ; : e28314, 2022 Nov 15.
Article in English | MEDLINE | ID: covidwho-2115797

ABSTRACT

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent for Kaposi's sarcoma (KS), an HIV/AIDS-associated malignancy. Effective treatments against KS remain to be developed. The sugar analog 2-deoxy- d-glucose (2-DG) is an anticancer agent that is well-tolerated and safe in patients and was recently demonstrated to be a potent antiviral, including KSHV and severe acute respiratory syndrome coronavirus 2. Because 2-DG inhibits glycolysis and N-glycosylation, identifying its molecular targets is challenging. Here we compare the antiviral effect of 2-DG with 2-fluoro-deoxy- d-glucose, a glycolysis inhibitor, and 2-deoxy-fluoro- d-mannose (2-DFM), a specific N-glycosylation inhibitor. At doses similar to those clinically achievable with 2-DG, the three drugs impair KSHV replication and virion production in iSLK.219 cells via downregulation of viral structural glycoprotein expression (K8.1 and gB), being 2-DFM the most potent KSHV inhibitor. Consistently with the higher potency of 2-DFM, we found that d-mannose rescues KSHV glycoprotein synthesis and virus production, indicating that inhibition of N-glycosylation is the main antiviral target using d-mannose competition experiments. Suppression of N-glycosylation by the sugar drugs triggers ER stress. It activates the host unfolded protein response (UPR), counteracting KSHV-induced inhibition of the protein kinase R-like endoplasmic reticulum kinase branch, particularly activating transcription factor 4 and C/EBP homologous protein expression. Finally, we demonstrate that sugar analogs induce autophagy (a prosurvival mechanism) and, thus, inhibit viral replication playing a protective role against KSHV-induced cell death, further supporting their direct antiviral effect and potential therapeutic use. Our work identifies inhibition of N-glycosylation leading to ER stress and UPR as an antienveloped virus target and sugar analogs such as 2-DG and the newly identified 2-DFM as antiviral drugs.

2.
mBio ; 13(5): e0241522, 2022 10 26.
Article in English | MEDLINE | ID: covidwho-2088413

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has killed over 6 million individuals worldwide and continues to spread in countries where vaccines are not yet widely available or its citizens are hesitant to become vaccinated. Therefore, it is critical to unravel the molecular mechanisms that allow SARS-CoV-2 and other coronaviruses to infect and overtake the host machinery of human cells. Coronavirus replication triggers endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR), a key host cell pathway widely believed to be essential for viral replication. We examined the master UPR sensor IRE1α kinase/RNase and its downstream transcription factor effector XBP1s, which is processed through an IRE1α-mediated mRNA splicing event, in human lung-derived cells infected with betacoronaviruses. We found that human respiratory coronavirus OC43 (HCoV-OC43), Middle East respiratory syndrome coronavirus (MERS-CoV), and murine coronavirus (MHV) all induce ER stress and strongly trigger the kinase and RNase activities of IRE1α as well as XBP1 splicing. In contrast, SARS-CoV-2 only partially activates IRE1α through autophosphorylation, but its RNase activity fails to splice XBP1. Moreover, while IRE1α was dispensable for replication in human cells for all coronaviruses tested, it was required for maximal expression of genes associated with several key cellular functions, including the interferon signaling pathway, during SARS-CoV-2 infection. Our data suggest that SARS-CoV-2 actively inhibits the RNase of autophosphorylated IRE1α, perhaps as a strategy to eliminate detection by the host immune system. IMPORTANCE SARS-CoV-2 is the third lethal respiratory coronavirus, after MERS-CoV and SARS-CoV, to emerge this century, causing millions of deaths worldwide. Other common coronaviruses such as HCoV-OC43 cause less severe respiratory disease. Thus, it is imperative to understand the similarities and differences among these viruses in how each interacts with host cells. We focused here on the inositol-requiring enzyme 1α (IRE1α) pathway, part of the host unfolded protein response to virus-induced stress. We found that while MERS-CoV and HCoV-OC43 fully activate the IRE1α kinase and RNase activities, SARS-CoV-2 only partially activates IRE1α, promoting its kinase activity but not RNase activity. Based on IRE1α-dependent gene expression changes during infection, we propose that SARS-CoV-2 prevents IRE1α RNase activation as a strategy to limit detection by the host immune system.


Subject(s)
COVID-19 , Middle East Respiratory Syndrome Coronavirus , Animals , Mice , Humans , Endoribonucleases/genetics , Endoribonucleases/metabolism , Endoplasmic Reticulum Stress/genetics , SARS-CoV-2/genetics , Inositol , Protein Serine-Threonine Kinases/genetics , Middle East Respiratory Syndrome Coronavirus/genetics , Middle East Respiratory Syndrome Coronavirus/metabolism , Ribonucleases/genetics , Transcription Factors , RNA, Messenger , Lung/metabolism , Interferons , X-Box Binding Protein 1/genetics
3.
Biochemistry (Mosc) ; 87(9): 916-931, 2022 Sep.
Article in English | MEDLINE | ID: covidwho-2038256

ABSTRACT

Endoplasmic reticulum (ER) is a multifunctional membrane-enclosed organelle. One of the major ER functions is cotranslational transport and processing of secretory, lysosomal, and transmembrane proteins. Impaired protein processing caused by disturbances in the ER homeostasis results in the ER stress. Restoration of normal ER functioning requires activation of an adaptive mechanism involving cell response to misfolded proteins, the so-called unfolded protein response (UPR). Besides controlling protein folding, UPR plays a key role in other physiological processes, in particular, differentiation of cells of connective, muscle, epithelial, and neural tissues. Cell differentiation is induced by the physiological levels of ER stress, while excessive ER stress suppresses differentiation and can result in cell death. So far, it remains unknown whether UPR activation induces cell differentiation or if UPR is initiated by the upregulated synthesis of secretory proteins during cell differentiation. Cell differentiation is an important stage in the development of multicellular organisms and is tightly controlled. Suppression or excessive activation of this process can lead to the development of various pathologies in an organism. In particular, impairments in the differentiation of connective tissue cells can result in the development of fibrosis, obesity, and osteoporosis. Recently, special attention has been paid to fibrosis as one of the major complications of COVID-19. Therefore, studying the role of UPR in the activation of cell differentiation is of both theoretical and practical interest, as it might result in the identification of molecular targets for selective regulation of cell differentiation stages and as well as the potential to modulate the mechanisms involved in the development of various pathological states.


Subject(s)
COVID-19 , Endoplasmic Reticulum Stress , Cell Differentiation , Fibrosis , Humans , Unfolded Protein Response
4.
American Journal of Cancer Research ; 12(7):3280-3293, 2022.
Article in English | EMBASE | ID: covidwho-2006849

ABSTRACT

Proteasome inhibitors are among the most potent classes of drugs in multiple myeloma treatment. One of the main challenges in myeloma therapy is acquired resistance to drugs. Several theories have been proposed to describe the mechanisms responsible for resistance to the most commonly used proteasome inhibitors bortezomib and carfilzomib. This study aimed to describe functional differences between sensitive myeloma cells (MM1S WT) and their daughter cell lines resistant to either bortezomib (MM1S/R BTZ) or carfilzomib (MM1S/R CFZ), as well as between both resistant cell lines. Bortezomib- and carfilzomib-resistant cell lines were successfully generated by continuous exposure to the drugs. When exposed to different drugs than during the resistance generation period, MM1S/R BTZ cells showed cross-resistance to carfilzomib, whereas MM1S/R CFZ cells were similarly sensitive to bortezomib as MM1S WT cells. Following proteomic profiling, unsupervised principal component analysis revealed that the MM1S/R BTZ and MM1S/R CFZ cell lines differed significantly from the MM1S WT cell line and from each other. Canonical pathway analysis showed similar pathways enriched in both comparisons - MM1S WT vs. MM1S/R CFZ and MM1S WT vs. MM1S/R BTZ. However, important differences were present in the statistical significance of particular pathways. Key alterations included the ubiquitin-proteasome system, metabolic pathways responsible for redox homeostasis and the unfolded protein response. In functional studies, both drugs continued to reduce chymotrypsin-like proteasome activity in resistant cells. However, the baseline activity of all three catalytic domains of the proteasome was higher in the resistant cells. Differences in generation of reactive oxygen species were identified in MM1S/R BTZ (decreased) and MM1S/CFZ cells (increased) in comparison to MM1S WT cells. Both baseline and drug-induced activity of the unfolded protein response were higher in resistant cells than in MM1S WT cells and included all three arms of this pathway: IRE1α/XBP1s, ATF6 and EIF2α/ATF4 (downstream effectors of PERK). In conclusion, contrary to some previous reports, resistant MM1S cells show upregulation of unfolded protein response activity, reflecting the heterogeneity of multiple myeloma and prompting further studies on the role of this pathway in resistance to proteasome inhibitors.

5.
Virus Res ; 320: 198897, 2022 Oct 15.
Article in English | MEDLINE | ID: covidwho-1996618

ABSTRACT

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently emerged throughout the world, resulting in more than 400 million cases and over 6 million deaths worldwide as of January 2022. Coronaviruses subvert or use certain aspects of the unfolded protein response in the endoplasmic reticulum to overcome protein translation shutdown to benefit their replication. New virions use the ER-Golgi intermediate compartment to assemble and gain transportation to the cell membrane. Extensive remodeling of the ER has been demonstrated during SARS-CoV-2 infection. In this review article, we discuss the role of the endoplasmic reticulum secretory pathway in the replication cycle of SARS-CoV-2. Currently, there is a dearth of therapeutic options for intervening with SARS-CoV-2 infection. To accelerate drug development, efforts around the globe have been focusing on repurposing drugs that have already been approved for clinical use by regulatory agencies. Targeting the ERS pathway is reasonable, as prior work has shown that SARS-CoV-2 egress is dependent on this pathway. Here we discuss the feasibility of off-patent, FDA-approved, pharmacological inhibitors of the ERS pathway to suppress the SARS-CoV-2 replication cycle, a promising approach that warrants investigation.


Subject(s)
COVID-19 , SARS-CoV-2 , Antiviral Agents/pharmacology , Antiviral Agents/therapeutic use , COVID-19/drug therapy , Endoplasmic Reticulum , Humans , Secretory Pathway
6.
J Clin Med ; 11(15)2022 Aug 05.
Article in English | MEDLINE | ID: covidwho-1979280

ABSTRACT

BACKGROUND: The different waves of SARS-CoV-2 infection have strained hospital resources and, notably, intensive care units (ICUs). Identifying patients at risk of developing a critical condition is essential to correctly refer patients to the appropriate structure and to spare limited resources. The soluble form of RAGE (sRAGE), the endoplasmic stress response and its surrogates, GRP78 and VEGF-A, may be interesting markers. METHODS: This was a prospective monocenter cohort study of adult patients admitted to the ICU for severe COVID-19 pneumonia. The plasma levels of sRAGE, GRP78 and VEGF-A were measured within the first 24 h. Patients were classified as critical if they further needed vasopressor therapy, renal replacement therapy, or invasive mechanical ventilation, or died during their ICU stay, and were otherwise classified as not critical. RESULTS: A total of 98 patients were included and 39 developed a critical condition. Critical patients presented higher sRAGE (626 [450-1043] vs. 227 [137-404] pg/mL, p < 0.0001), interleukin-6 (43 [15-112] vs. 11 [5-20] pg/mL, p < 0.0001), troponin T (17 [9-39] vs. 10 [6-18] pg/mL, p = 0.003) and NT-pro-BNP (321 [118-446] vs. 169 [63-366] pg/mL, p = 0.009) plasma levels. No difference was observed for VEGF-A and GRP78. The variables independently associated with worsening in the ICU were sRAGE (1.03 [1.01-1.05] per 10 pg/mL) and age (1.7 [1.2-2.4] per 5 years). An sRAGE value of 449.5 pg/mL predicted worsening with a sensitivity of 77% and a specificity of 80%. CONCLUSION: sRAGE may allow the identification of patients at risk of developing a critical form of COVID-19 pneumonia, and thus may be useful to correctly refer patients to the appropriate structure of care.

7.
Proc Natl Acad Sci U S A ; 119(31): e2121453119, 2022 08 02.
Article in English | MEDLINE | ID: covidwho-1960614

ABSTRACT

Human ZAP inhibits many viruses, including HIV and coronaviruses, by binding to viral RNAs to promote their degradation and/or translation suppression. However, the regulatory role of ZAP in host mRNAs is largely unknown. Two major alternatively spliced ZAP isoforms, the constitutively expressed ZAPL and the infection-inducible ZAPS, play overlapping yet different antiviral and other roles that need further characterization. We found that the splicing factors hnRNPA1/A2, PTBP1/2, and U1-snRNP inhibit ZAPS production and demonstrated the feasibility to modulate the ZAPL/S balance by splice-switching antisense oligonucleotides in human cells. Transcriptomic analysis of ZAP-isoform-specific knockout cells revealed uncharacterized host mRNAs targeted by ZAPL/S with broad cellular functions such as unfolded protein response (UPR), epithelial-mesenchymal transition (EMT), and innate immunity. We established that endogenous ZAPL and ZAPS localize to membrane compartments and cytosol, respectively, and that the differential localization correlates with their target-RNA specificity. We showed that the ZAP isoforms regulated different UPR branches under resting and stress conditions and affected cell viability during ER stress. We also provided evidence for a different function of the ZAP isoforms in EMT-related cell migration, with effects that are cell-type dependent. Overall, this study demonstrates that the competition between splicing and IPA is a potential target for the modulation of the ZAPL/S balance, and reports new cellular transcripts and processes regulated by the ZAP isoforms.


Subject(s)
Epithelial-Mesenchymal Transition , RNA, Messenger , RNA, Viral , RNA-Binding Proteins , Unfolded Protein Response , Epithelial-Mesenchymal Transition/genetics , Heterogeneous Nuclear Ribonucleoprotein A1/metabolism , Heterogeneous-Nuclear Ribonucleoproteins/genetics , Heterogeneous-Nuclear Ribonucleoproteins/metabolism , Humans , Polypyrimidine Tract-Binding Protein/genetics , Polypyrimidine Tract-Binding Protein/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA, Messenger/metabolism , RNA, Viral/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Ribonucleoproteins, Small Nuclear/metabolism
8.
Alcoholism: Clinical and Experimental Research ; 46:142A, 2022.
Article in English | EMBASE | ID: covidwho-1937893

ABSTRACT

As the delta and omicron SARS-CoV-2 variants spread across the world, more tools to fight off serious infection have been developed. COVID antiviral drugs that can be taken orally at home could cut serious illness and reduce the risk of hospitalization and death. However, significant population of people consume alcohol before the infection and use of the antiviral drugs, which could potentiate side effects of the drugs on the liver. We investigated the role of alcohol in anti-Covid drug-induced stress responses in live cells. METHODS: HepG2 cells or primary mouse hepatocytes (PMH) were pre-treated with alcohol (50 mMlow dose or 100 mMhigh dose) for 6-24 hours and then treated with the newly developed oral anti-Covid drugs: nirmatrelvir, ritonavir, molnupiravir, and remdesivir at 10- 30 lg/ml for 6-24 hours. Unfolded protein response (UPR)/ER stress molecular markers (e.g. IRE1 GRP78, PERK, Xbp1 and CHOP), Golgi stress response (GSR) markers of GCP60, HSP47 and TFE3, and STAT3 were measured after the treatments. Cell death was assessed through double staining the liver cells with Syntox Green and Hoesche's Blue. RESULTS: ER stress response as indicated by IRE1, Xbp1 and CHOP was insignificant or mild in either HepG2 or PMH treated individually with alcohol at the low dose, nirmatrelvir, ritonavir, molnupiravir, or remdesivir. Alcohol or remdesivir induced moderate GSR based on mRNA increase of GCP60, HSP47 and TFE3, which was accompanied with apparent Golgi fragmentation in either HepG2 or PMH. Cell death rates in HepG2 treated with alcohol, nirmatrelvir, ritonavir, molnupiravir, or remdesivir individually were less than 5%. Pre-exposure to alcohol combined with subsequent treatment with nirmatrelvir, ritonavir molnupiravir, or remdesivir significantly increased both ER stress and GSR markers and expression of phosphorylated STAT3 (p-STAT3). Most significantly, cell death rates in HepG2 or PMH were increased by 2- to 5-fold by pre-alcohol exposure plus ritonavir, nirmatrelvir, molnupiravir, or remdesivir. The organelle stress markers, p-STAT3 and cell death were all further increased in alcoholand anti-Covid drug-treated HepG2 or primary mouse hepatocytes that were pre-infected with the lentiviruses that were pseudotyped with the SARS-CoV-2 spike protein. CONCLUSION: Our results indicate that pre-exposure to alcohol potentiates the liver cells to anti-Covid-19 drugs induced stress responses and cell death.

9.
Redox Biol ; 54: 102388, 2022 08.
Article in English | MEDLINE | ID: covidwho-1907715

ABSTRACT

The replication machinery of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is closely associated with the endoplasmic reticulum (ER) in host cells. Activation of the unfolded protein response (UPR) is a strategy hijacked by coronavirus to facilitate its replication and suppress host innate immunity. Here, we have found that SARS-CoV-2 ORF8 protein accumulates in the ER and escapes the degradation system by forming mixed disulfide complexes with ER oxidoreductases. ORF8 induces the activation of three UPR pathways through targeting key UPR components, remodels ER morphology and accelerates protein trafficking. Moreover, small molecule reducing agents release ORF8 from the mixed disulfide complexes and facilitate its degradation, therefore mitigate ER stress. Our study reveals a unique mechanism by which SARS-CoV-2 ORF8 escapes degradation by host cells and regulates ER reshaping. Targeting ORF8-involved mixed disulfide complexes could be a new strategy to alleviate SARS-CoV-2 induced ER stress and related diseases.


Subject(s)
Disulfides , Endoplasmic Reticulum , SARS-CoV-2 , Viral Proteins , COVID-19 , Disulfides/metabolism , Endoplasmic Reticulum/metabolism , Humans , Oxidoreductases/metabolism , Viral Proteins/metabolism
10.
Vet Microbiol ; 271: 109494, 2022 Aug.
Article in English | MEDLINE | ID: covidwho-1886124

ABSTRACT

Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that has the potential for cross-species infection. Many viruses have been reported to induce endoplasmic reticulum stress (ERS) and activate the unfolded protein response (UPR). To date, little is known about whether and, if so, how the UPR is activated by PDCoV infection. Here, we investigated the activation state of UPR pathways and their effects on viral replication during PDCoV infection. We found that PDCoV infection induced ERS and activated all three known UPR pathways (inositol-requiring enzyme 1 [IRE1], activating transcription factor 6 [ATF6], and PKR-like ER kinase [PERK]), as demonstrated by IRE1-mediated XBP1 mRNA cleavage and increased mRNA expression of XBP1s, ATF4, CHOP, GADD34, GRP78, and GRP94, as well as phosphorylated eIF2α expression. Through pharmacologic treatment, RNA interference, and overexpression experiments, we confirmed the negative role of the PERK-eIF2α pathway and the positive regulatory role of the ATF6 pathway, but found no obvious effect of IRE1 pathway, on PDCoV replication. Taken together, our results characterize, for the first time, the state of the ERS response during PDCoV infection and identify the PERK and ATF6 pathways as potential antiviral targets.


Subject(s)
Protein Serine-Threonine Kinases , Unfolded Protein Response , Animals , Deltacoronavirus , Endoplasmic Reticulum Stress , Eukaryotic Initiation Factor-2/metabolism , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/metabolism , Swine , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
11.
Journal of Virology ; 96(1):1-23, 2022.
Article in English | A9H | ID: covidwho-1647636

ABSTRACT

The replication of coronaviruses, including severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERSCoV), 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 demonstrate 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 eIF2a phosphorylation kinase, interferon (IFN)-induced double-stranded RNA-dependent protein kinase (PKR), was also activated and acted cooperatively with PERK to decrease PHEV replication. Furthermore, we demonstrate that the PERK/PKR-eIF2a pathways negatively regulated PHEV replication by attenuating global protein translation. Phosphorylated eIF2a also promoted the formation of stress granules (SGs), 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 eIF2a) for antiviral drugs. [ FROM AUTHOR] Copyright of Journal of Virology is the property of American Society for Microbiology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full . (Copyright applies to all s.)

12.
Front Aging Neurosci ; 13: 767493, 2021.
Article in English | MEDLINE | ID: covidwho-1526773

ABSTRACT

Abnormal accumulation of misfolded proteins in the endoplasmic reticulum and their aggregation causes inflammation and endoplasmic reticulum stress. This promotes accumulation of toxic proteins in the body tissues especially brain leading to manifestation of neurodegenerative diseases. The studies suggest that deregulation of proteostasis, particularly aberrant unfolded protein response (UPR) signaling, may be a common morbific process in the development of neurodegeneration. Curcumin, the mixture of low molecular weight polyphenolic compounds from turmeric, Curcuma longa has shown promising response to prevents many diseases including current global severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and neurodegenerative disorders. The UPR which correlates positively with neurodegenerative disorders were found affected by curcumin. In this review, we examine the evidence from many model systems illustrating how curcumin interacts with UPR and slows down the development of various neurodegenerative disorders (ND), e.g., Alzheimer's and Parkinson's diseases. The recent global increase in ND patients indicates that researchers and practitioners will need to develop a new pharmacological drug or treatment to manage and cure these neurodegenerative diseases.

13.
mBio ; 12(4): e0157221, 2021 08 31.
Article in English | MEDLINE | ID: covidwho-1349194

ABSTRACT

Tissue- and cell-specific expression patterns are highly variable within and across individuals, leading to altered host responses after acute virus infection. Unraveling key tissue-specific response patterns provides novel opportunities for defining fundamental mechanisms of virus-host interaction in disease and the identification of critical tissue-specific networks for disease intervention in the lung. Currently, there are no approved therapeutics for Middle East respiratory syndrome coronavirus (MERS-CoV) patients, and little is understood about how lung cell types contribute to disease outcomes. MERS-CoV replicates equivalently in primary human lung microvascular endothelial cells (MVE) and fibroblasts (FB) and to equivalent peak titers but with slower replication kinetics in human airway epithelial cell cultures (HAE). However, only infected MVE demonstrate observable virus-induced cytopathic effect. To explore mechanisms leading to reduced MVE viability, donor-matched human lung MVE, HAE, and FB were infected, and their transcriptomes, proteomes, and lipidomes were monitored over time. Validated functional enrichment analysis demonstrated that MERS-CoV-infected MVE were dying via an unfolded protein response (UPR)-mediated apoptosis. Pharmacologic manipulation of the UPR in MERS-CoV-infected primary lung cells reduced viral titers and in male mice improved respiratory function with accompanying reductions in weight loss, pathological signatures of acute lung injury, and times to recovery. Systems biology analysis and validation studies of global kinetic transcript, protein, and lipid data sets confirmed that inhibition of host stress pathways that are differentially regulated following MERS-CoV infection of different tissue types can alleviate symptom progression to end-stage lung disease commonly seen following emerging coronavirus outbreaks. IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe atypical pneumonia in infected individuals, but the underlying mechanisms of pathogenesis remain unknown. While much has been learned from the few reported autopsy cases, an in-depth understanding of the cells targeted by MERS-CoV in the human lung and their relative contribution to disease outcomes is needed. The host response in MERS-CoV-infected primary human lung microvascular endothelial (MVE) cells and fibroblasts (FB) was evaluated over time by analyzing total RNA, proteins, and lipids to determine the cellular pathways modulated postinfection. Findings revealed that MERS-CoV-infected MVE cells die via apoptotic mechanisms downstream of the unfolded protein response (UPR). Interruption of enzymatic processes within the UPR in MERS-CoV-infected male mice reduced disease symptoms, virus-induced lung injury, and time to recovery. These data suggest that the UPR plays an important role in MERS-CoV infection and may represent a host target for therapeutic intervention.


Subject(s)
Acute Lung Injury/pathology , Apoptosis/physiology , Coronavirus Infections/pathology , Unfolded Protein Response/physiology , Acute Lung Injury/virology , Animals , Cell Line , Endothelial Cells/metabolism , Endothelial Cells/virology , Female , Fibroblasts/metabolism , Fibroblasts/virology , Humans , Male , Mice , Middle East Respiratory Syndrome Coronavirus/immunology
14.
Vaccines (Basel) ; 8(3)2020 Jul 20.
Article in English | MEDLINE | ID: covidwho-1389560

ABSTRACT

The efficacy of SARS-CoV-2 nucleic acid-based vaccines may be limited by proteolysis of the translated product due to anomalous protein folding. This may be the case for vaccines employing linear SARS-CoV-2 B-cell epitopes identified in previous studies since most of them participate in secondary structure formation. In contrast, we have employed a consensus of predictors for epitopic zones plus a structural filter for identifying 20 unstructured B-cell epitope-containing loops (uBCELs) in S, M, and N proteins. Phylogenetic comparison suggests epitope switching with respect to SARS-CoV in some of the identified uBCELs. Such events may be associated with the reported lack of serum cross-protection between the 2003 and 2019 pandemic strains. Incipient variability within a sample of 1639 SARS-CoV-2 isolates was also detected for 10 uBCELs which could cause vaccine failure. Intermediate stages of the putative epitope switch events were observed in bat coronaviruses in which additive mutational processes possibly facilitating evasion of the bat immune system appear to have taken place prior to transfer to humans. While there was some overlap between uBCELs and previously validated SARS-CoV B-cell epitopes, multiple uBCELs had not been identified in prior studies. Overall, these uBCELs may facilitate the development of biomedical products for SARS-CoV-2.

15.
Cell Stress Chaperones ; 26(5): 859-868, 2021 09.
Article in English | MEDLINE | ID: covidwho-1353732

ABSTRACT

Vaccinations are widely credited with reducing death rates from COVID-19, but the underlying host-viral mechanisms/interactions for morbidity and mortality of SARS-CoV-2 infection remain poorly understood. Acute respiratory distress syndrome (ARDS) describes the severe lung injury, which is pathologically associated with alveolar damage, inflammation, non-cardiogenic edema, and hyaline membrane formation. Because proteostatic pathways play central roles in cellular protection, immune modulation, protein degradation, and tissue repair, we examined the pathological features for the unfolded protein response (UPR) using the surrogate biomarker glucose-regulated protein 78 (GRP78) and co-receptor for SARS-CoV-2. At autopsy, immunostaining of COVID-19 lungs showed highly elevated expression of GRP78 in both pneumocytes and macrophages compared with that of non-COVID control lungs. GRP78 expression was detected in both SARS-CoV-2-infected and un-infected pneumocytes as determined by multiplexed immunostaining for nucleocapsid protein. In macrophages, immunohistochemical staining for GRP78 from deceased COVID-19 patients was increased but overlapped with GRP78 expression taken from surgical resections of non-COVID-19 controls. In contrast, the robust in situ GRP78 immunostaining of pneumocytes from COVID-19 autopsies exhibited no overlap and was independent of age, race/ethnicity, and gender compared with that from non-COVID-19 controls. Our findings bring new insights for stress-response pathways involving the proteostatic network implicated for host resilience and suggest that targeting of GRP78 expression with existing therapeutics might afford an alternative therapeutic strategy to modulate host-viral interactions during SARS-CoV-2 infections.


Subject(s)
Alveolar Epithelial Cells/metabolism , COVID-19/metabolism , Endoplasmic Reticulum Stress , Heat-Shock Proteins/analysis , Receptors, Coronavirus/analysis , SARS-CoV-2/pathogenicity , Adult , Aged , Aged, 80 and over , Alveolar Epithelial Cells/pathology , Alveolar Epithelial Cells/virology , Autopsy , COVID-19/mortality , COVID-19/pathology , COVID-19/virology , Case-Control Studies , Female , Host-Pathogen Interactions , Humans , Macrophages, Alveolar/metabolism , Macrophages, Alveolar/virology , Male , Middle Aged , Proteostasis , Up-Regulation , Young Adult
16.
Mol Cell Proteomics ; 20: 100120, 2021.
Article in English | MEDLINE | ID: covidwho-1284342

ABSTRACT

Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold-like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, nonstructural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV; and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) data set for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance, nuclear protein import for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.2 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.


Subject(s)
Activating Transcription Factor 6/metabolism , Coronavirus Papain-Like Proteases/metabolism , Host-Pathogen Interactions/physiology , SARS-CoV-2/pathogenicity , Coronavirus 229E, Human/metabolism , Coronavirus 229E, Human/pathogenicity , Coronavirus OC43, Human/metabolism , Coronavirus OC43, Human/pathogenicity , Coronavirus Papain-Like Proteases/genetics , Endoplasmic Reticulum-Associated Degradation , HEK293 Cells , Humans , Middle East Respiratory Syndrome Coronavirus/metabolism , Middle East Respiratory Syndrome Coronavirus/pathogenicity , Protein Interaction Maps , SARS-CoV-2/metabolism , Unfolded Protein Response , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism
17.
Int J Mol Sci ; 22(11)2021 Jun 01.
Article in English | MEDLINE | ID: covidwho-1259507

ABSTRACT

The COVID-19 pandemic is caused by the 2019-nCoV/SARS-CoV-2 virus. This severe acute respiratory syndrome is currently a global health emergency and needs much effort to generate an urgent practical treatment to reduce COVID-19 complications and mortality in humans. Viral infection activates various cellular responses in infected cells, including cellular stress responses such as unfolded protein response (UPR) and autophagy, following the inhibition of mTOR. Both UPR and autophagy mechanisms are involved in cellular and tissue homeostasis, apoptosis, innate immunity modulation, and clearance of pathogens such as viral particles. However, during an evolutionary arms race, viruses gain the ability to subvert autophagy and UPR for their benefit. SARS-CoV-2 can enter host cells through binding to cell surface receptors, including angiotensin-converting enzyme 2 (ACE2) and neuropilin-1 (NRP1). ACE2 blockage increases autophagy through mTOR inhibition, leading to gastrointestinal complications during SARS-CoV-2 virus infection. NRP1 is also regulated by the mTOR pathway. An increased NRP1 can enhance the susceptibility of immune system dendritic cells (DCs) to SARS-CoV-2 and induce cytokine storm, which is related to high COVID-19 mortality. Therefore, signaling pathways such as mTOR, UPR, and autophagy may be potential therapeutic targets for COVID-19. Hence, extensive investigations are required to confirm these potentials. Since there is currently no specific treatment for COVID-19 infection, we sought to review and discuss the important roles of autophagy, UPR, and mTOR mechanisms in the regulation of cellular responses to coronavirus infection to help identify new antiviral modalities against SARS-CoV-2 virus.


Subject(s)
Autophagy , COVID-19/pathology , Neuropilin-1/metabolism , Unfolded Protein Response , Antiviral Agents/pharmacology , Autophagy/drug effects , COVID-19/virology , Humans , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , SARS-CoV-2/isolation & purification , SARS-CoV-2/metabolism , Signal Transduction/drug effects , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/metabolism
18.
Int J Mol Sci ; 22(11)2021 May 26.
Article in English | MEDLINE | ID: covidwho-1244042

ABSTRACT

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and IL-6. Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this study, we investigated the roles of the integrated stress response (ISR) and activator protein-1 (AP-1) family proteins in regulating coronavirus-induced IL-8 and IL-6 upregulation. The mRNA expression of IL-8 and IL-6 was significantly induced in cells infected with infectious bronchitis virus (IBV), a gammacoronavirus, and porcine epidemic diarrhea virus, an alphacoronavirus. Overexpression of a constitutively active phosphomimetic mutant of eukaryotic translation initiation factor 2α (eIF2α), chemical inhibition of its dephosphorylation, or overexpression of its upstream double-stranded RNA-dependent protein kinase (PKR) significantly enhanced IL-8 mRNA expression in IBV-infected cells. Overexpression of the AP-1 protein cJUN or its upstream kinase also increased the IBV-induced IL-8 mRNA expression, which was synergistically enhanced by overexpression of cFOS. Taken together, this study demonstrated the important regulatory roles of ISR and AP-1 proteins in IL-8 production during coronavirus infection, highlighting the complex interactions between cellular stress pathways and the innate immune response.


Subject(s)
Coronavirus Infections/metabolism , Endoplasmic Reticulum Stress/genetics , Eukaryotic Initiation Factor-2/metabolism , Interleukin-8/metabolism , Unfolded Protein Response/genetics , Alphacoronavirus/metabolism , Alphacoronavirus/pathogenicity , Animals , Cell Line , Chlorocebus aethiops , Coronavirus Infections/genetics , Gammacoronavirus/metabolism , Gammacoronavirus/pathogenicity , Gene Expression Regulation , Humans , Immunity, Innate , Infectious bronchitis virus/metabolism , Infectious bronchitis virus/pathogenicity , Interleukin-8/genetics , Phosphorylation , Porcine epidemic diarrhea virus/metabolism , Porcine epidemic diarrhea virus/pathogenicity , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , Signal Transduction/genetics , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolism , Up-Regulation , Vero Cells , eIF-2 Kinase/genetics , eIF-2 Kinase/metabolism
19.
Front Cell Infect Microbiol ; 11: 668034, 2021.
Article in English | MEDLINE | ID: covidwho-1231324

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.


Subject(s)
COVID-19 , Autophagy , Endoplasmic Reticulum/metabolism , Endoplasmic Reticulum Stress , Humans , SARS-CoV-2 , Unfolded Protein Response
20.
Br Med Bull ; 137(1): 13-27, 2021 03 25.
Article in English | MEDLINE | ID: covidwho-1054267

ABSTRACT

BACKGROUND: Many drugs approved for other indications can control the growth of tumor cells and limit adverse events (AE). DATA SOURCES: Literature searches with keywords 'repurposing and cancer' books, websites: https://clinicaltrials.gov/, for drug structures: https://pubchem.ncbi.nlm.nih.gov/. AREAS OF AGREEMENT: Introducing approved drugs, such as those developed to treat diabetes (Metformin) or inflammation (Thalidomide), identified to have cytostatic activity, can enhance chemotherapy or even replace more cytotoxic drugs. Also, anti-inflammatory compounds, cytokines and inhibitors of proteolysis can be used to control the side effects of chemo- and immuno-therapies or as second-line treatments for tumors resistant to kinase inhibitors (KI). Drugs specifically developed for cancer therapy, such as interferons (IFN), the tyrosine KI abivertinib TKI (tyrosine kinase inhibitor) and interleukin-6 (IL-6) receptor inhibitors, may help control symptoms of Covid-19. AREAS OF CONTROVERSY: Better knowledge of mechanisms of drug activities is essential for repurposing. Chemotherapies induce ER stress and enhance mutation rates and chromosome alterations, leading to resistance that cannot always be related to mutations in the target gene. Metformin, thalidomide and cytokines (IFN, tumor necrosis factor (TNF), interleukin-2 (IL-2) and others) have pleiomorphic activities, some of which can enhance tumorigenesis. The small and fragile patient pools available for clinical trials can cloud the data on the usefulness of cotreatments. GROWING POINTS: Better understanding of drug metabolism and mechanisms should aid in repurposing drugs for primary, adjuvant and adjunct treatments. AREAS TIMELY FOR DEVELOPING RESEARCH: Optimizing drug combinations, reducing cytotoxicity of chemotherapeutics and controlling associated inflammation.


Subject(s)
COVID-19/drug therapy , Drug Repositioning , Neoplasms/drug therapy , Humans
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