Study on potential common action and mechanism of Reduning Injection against SARS, MERS and COVID-19 based on network pharmacology and molecular docking technology.

Objective To explore the potential common mechanism and active ingredients of Reduning Injection against SARS, MERS and COVID-19 through network pharmacology and molecular docking technology. Methods The TCMSP database was used to retrieve the chemical components and targets of Artemisiae Annuae Herba, Lonicerae Japonicae Flos and Gardeniae Fructus in Reduning Injection. The gene corresponding to the target was searched by UniProt database, and Cytoscape 3.8.2 was used to build a medicinal material-compound-target (gene) network. Three coronavirus-related targets were collected in the Gene Cards database with the key words of "SARS""MERS" and "COVID-19", and common target of three coronavirus infection diseases were screened out through Venny 2.1.0 database. The common targets of SARS, MERS and COVID-19 were intersected with the targets of Reduning Injection, and the common targets were selected as research targets. Protein-protein interaction (PPI) network map were constructed by Cytoscape3.8.2 software after importing the common targets into the STRING database to obtain data. R language was used to carry out GO biological function enrichment analysis and KEGG signaling pathway enrichment analysis, histograms and bubble charts were drew, and component-target-pathway network diagrams was constructed. The key compounds in the component-target-pathway network were selected for molecular docking with important target proteins, novel coronavirus (SARS-CoV-2) 3CL hydrolase, and angiotensin-converting enzyme II (ACE2). Results 31 active compounds and 207 corresponding targets were obtained from Reduning Injection. 2 453 SARS-related targets, 805 MERS-related targets, 2 571 COVID-19-related targets, and 786 targets for the three diseases. 11 common targets with Reduning Injection: HSPA5, CRP, MAPK1, HMOX1, TGFB1, HSP90AA1, TP53, DPP4, CXCL10, PLAT, PRKACA. GO function enrichment analysis revealed 995 biological processes (BP), 71 molecular functions (MF), and 31 cellular components (CC). KEGG pathway enrichment analysis screened 99 signal pathways (P < 0.05), mainly related to prostate cancer, fluid shear stress and atherosclerosis, hepatocellular carcinoma, proteoglycans in cancer, lipid and atherosclerosis, human T-cell leukemia virus 1 infection, MAPK signaling pathway, etc. The molecular docking results showed that the three core active flavonoids of quercetin, luteolin, and kaempferol in Reduning Injection had good affinity with key targets MAPK1, PRKACA, and HSP90AA1, and the combination of the three active compounds with SARS-CoV-2 3CL hydrolase and ACE2 was less than the recommended chemical drugs. Conclusion Reduning Injection has potential common effects on the three diseases of SARS, MERS and COVID-19. This effect may be related to those active compounds such as quercetin, luteolin, and kaempferol acting on targets such as MAPK1, PRKACA, HSP90AA1 to regulate multiple signal pathways and exert anti-virus, suppression of inflammatory storm, and regulation of immune function.Copyright © 2022 Drug Evaluation Research. All rights reserved.

The unfolded protein response components IRE1α and XBP1 promote human coronavirus infection.

The cellular processes that support human coronavirus replication and contribute to the pathogenesis of severe disease remain incompletely understood. Many viruses, including coronaviruses, cause endoplasmic reticulum (ER) stress during infection. IRE1α is a component of the cellular response to ER stress that initiates non-conventional splicing of XBP1 mRNA. Spliced XBP1 encodes a transcription factor that induces the expression of ER-related targets. Activation of the IRE1α-XBP1 pathway occurs in association with risk factors for severe human coronavirus infection. In this study, we found that the human coronaviruses HCoV-OC43 (human coronavirus OC43) and SARS-CoV-2 (severe acute respiratory syndrome coronavirus-2) both robustly activate the IRE1α-XBP1 branch of the unfolded protein response in cultured cells. Using IRE1α nuclease inhibitors and genetic knockdown of IRE1α and XBP1, we found that these host factors are required for optimal replication of both viruses. Our data suggest that IRE1α supports infection downstream of initial viral attachment and entry. In addition, we found that ER stress-inducing conditions are sufficient to enhance human coronavirus replication. Furthermore, we found markedly increased XBP1 in circulation in human patients with severe coronavirus disease 2019 (COVID-19). Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.IMPORTANCEThere is a critical need to understand the cellular processes co-opted during human coronavirus replication, with an emphasis on identifying mechanisms underlying severe disease and potential therapeutic targets. Here, we demonstrate that the host proteins IRE1α and XBP1 are required for robust infection by the human coronaviruses, SARS-CoV-2 and HCoV-OC43. IRE1α and XBP1 participate in the cellular response to ER stress and are activated during conditions that predispose to severe COVID-19. We found enhanced viral replication with exogenous IRE1α activation, and evidence that this pathway is activated in humans during severe COVID-19. Together, these results demonstrate the importance of IRE1α and XBP1 for human coronavirus infection.

Pre-Golgi Intermediate Compartment

The pre-Golgi intermediate compartment (IC), also referred to as the endoplasmic reticulum (ER)-Golgi IC (ERGIC), functions in molecular sorting and two-way trafficking in the early biosynthetic-secretory pathway. It receives newly synthesized proteins and lipids from the ER, associates with cytoskeletal filaments and binds COPI protein coats, thereby generating saccular, tubular and vesicular carriers that mediate antero- and retrograde transport at the ER-Golgi interface. Although the dynamic IC elements have traditionally been considered as transient transport intermediates, recent results showing their persistence during cell division, multifunctionality, and operation at the crossroads of Golgi-dependent and -independent transport routes provide evidence that they constitute a permanent organelle. © 2023 Elsevier Inc. All rights reserved.

Coronaviral ORF6 protein mediates inter-organelle contacts and modulates host cell lipid flux for virus production.

Lipid droplets (LDs) form inter-organelle contacts with the endoplasmic reticulum (ER) that promote their biogenesis, while LD contacts with mitochondria enhance ß-oxidation of contained fatty acids. Viruses have been shown to take advantage of lipid droplets to promote viral production, but it remains unclear whether they also modulate the interactions between LDs and other organelles. Here, we showed that coronavirus ORF6 protein targets LDs and is localized to the mitochondria-LD and ER-LD contact sites, where it regulates LD biogenesis and lipolysis. At the molecular level, we find that ORF6 inserts into the LD lipid monolayer via its two amphipathic helices. ORF6 further interacts with ER membrane proteins BAP31 and USE1 to mediate ER-LDs contact formation. Additionally, ORF6 interacts with the SAM complex in the mitochondrial outer membrane to link mitochondria to LDs. In doing so, ORF6 promotes cellular lipolysis and LD biogenesis to reprogram host cell lipid flux and facilitate viral production.

The use of alanine, free carnitine and IGFBP-1 as potential biomarkers for glycogen storage disease type I

Background: Glycogen Storage Disease Ia (GSDIa) and Ib (GSDIb) are inborn errors of carbohydrate metabolism due to a deficiency of glucose-6-phosphatase (G6Pase) or glucose-6-phosphate translocase (G6PT), respectively. Consuming prescribed amounts of uncooked cornstarch (UCCS) to prevent hypoglycemia is the standard of care for GSDIa and GSDIb. Patients followed in our GSD Program are admitted to the hospital annually for evaluation of their metabolic control by measuring glucose and lactate levels and revising treatment regimens accordingly. Lack of bed space due to the COVID-19 pandemic has created a need for alternate markers of metabolic control as lactate measurements are unreliable in the outpatient setting. This research aims to identify alternative biomarkers to show degree of metabolic control in individuals with GSDI. Method(s): A retrospective chart review was conducted on 45 adults and children with GSDI using data from January 1, 2014 toMay 6, 2021. Plasma alanine and free carnitine levels were compared with laboratory reference ranges. Results from the three tests were not available on every subject. Plasma alanine was evaluated on 24 subjects (16-GSDIa, 8-GSDIb) and free carnitine was evaluated on 25 subjects (17-GSDIa, 8-GSDIb). Result(s): Alanine levels in subjects with GSDIa ranged from 378 to 786 umol/L, while alanine levels in subjects with GSDIb ranged from 254 to 506 umol/L (reference range = 103-528 umol/L). Free carnitine levels ranged from26 to 72 umol/L in subjects with GSDIa and from 44 to 90 umol/L in subjects with GSDIb (reference range = 19-55 umol/L). Conclusion(s): Our analysis showed that plasma alanine and free carnitine have potential to be used as biomarkers of metabolic control. For plasma alanine, there seemed to be differences between subjects with GSDIa and GSDIb, as the majority of subjects with GSDIa had elevations in plasma alanine, while subjects with GSDIb did not. Elevated plasma alanine levels indicate lactic acidosis. For GSDIb, we hypothesize that there may be some type of G6Pase enzyme activity that occurs outside of the endoplasmic reticulum. When looking at both groups, free carnitine levels were mostly elevated. This indicates that there could be inhibition of fatty acid oxidation.Copyright © 2022 Elsevier Inc. All rights reserved.

Fangchinoline Exerts Anticancer Effects on Colorectal Cancer Cells by Evoking Cell Apoptosis via Endoplasmic Reticulum Stress.

We studied the anti-tumor effect of fangchinoline (FAN) against human colorectal cancer cell lines CCL-244 and SW480 and analyzed the mechanism of FAN action. The cell viability and apoptosis were assessed by MTT test and Annexin V-PI staining; caspase-3 activity was measured by Western blotting. The expression of endoplasmic reticulum stress-related proteins was assessed by real-time PCR, Western blotting, and gene transfection. It was found that FAN inhibited cell growth and induced apoptosis in human colorectal cancer cell lines CCL-244 and SW480 in a dose-dependent manner. The caspase-3 inhibitor Ac-DEVD-CHO could reverse the inhibitory effect of FAN. Moreover, FAN significantly increased the expression of endoplasmic reticulum stress-related proteins p-PERK, p-eIF2α, ATF4, and CHOP in CCL-244 and SW480 cells. In addition, endoplasmic reticulum stress inhibitor 4-phenylbutyric acid or CHOP knockdown could prevent FAN-induced apoptosis. Thus, FAN induced apoptosis of human colorectal cancer through activation of endoplasmic reticulum stress.

Is There any Alternative Receptor for SARS-CoV-2?

Angiotensin-converting enzyme II (ACE2) in association with type II transmembrane serine protease (TMPRSS2) is considered the main receptor of SARS-CoV-2. However, considering the clinical complications of COVID-19 in different organs, there is no strong association between the abundance of ACE2/TMPRSS2 co-expression and clinical features of the disease and the severity of complications. Since SARS-CoV-2 affects certain organs that lack or have low expression of ACE2/TMPRSS2, it may be possible that the virus employs other receptors for colonization and entry. Based on recent studies, glucose-regulated protein 78 (GRP78) can be a potential alternative receptor for SARS-CoV-2 entry. In this letter, supporting evidence proposed GRP78 as an alternative receptor in SARS-CoV-2 infection.

Coagulopathies after vaccination against SARS-CoV-2: The sole solution might lead to another problem

The common reported adverse impacts of COVID-19 vaccination include the injection site's local reaction followed by various non-specific flu-like symptoms. Nevertheless, uncommon cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) following viral vector vaccines (ChAdOx1 nCoV-19 vaccine, Ad26.COV2 vaccine) have been reported. This literature review was performed using PubMed and Google Scholar databases using appropriate keywords and their combinations: SARS-CoV-2, adenovirus, spike protein, thrombosis, thrombocytopenia, vaccine-induced immune thrombotic thrombocytopenia (VITT), NF-kappaB, adenoviral vector, platelet factor 4 (PF4), COVID-19 Vaccine, AstraZeneca COVID vaccine, ChAdOx1 nCoV-19 COVID vaccine, AZD1222 COVID vaccine, coagulopathy. The s and titles of each article were assessed by authors for screening and inclusion English reports about post-vaccine CVST and VITT in humans were also collected. Some SARS-CoV-2 vaccines based on viral vector, mRNA, or inactivated SARS-CoV-2 virus have been accepted and are being pragmatic global. Nevertheless, the recent augmented statistics of normally very infrequent types of thrombosis associated with thrombocytopenia have been stated, predominantly in the context of the adenoviral vector vaccine ChAdOx1 nCoV-19 from Astra Zeneca. The numerical prevalence of these side effects seems to associate with this particular vaccine type, i.e., adenoviral vector-based vaccines, but the meticulous molecular mechanisms are still not clear. The present review summarizes the latest data and hypotheses for molecular and cellular mechanisms into one integrated hypothesis demonstrating that coagulopathies, including thromboses, thrombocytopenia, and other associated side effects, are correlated to an interaction of the two components in the COVID-19 vaccine.Copyright © 2022, Iranian Pediatric Hematology and Oncology Society. All rights reserved.

Cell surface GRP78 functions as an alternative virus entry receptor on monocytes during SARS-CoV- 2 infection

Background: It has been widely acknowledged that severe acute respiratory syndrome coronavirus 2 (SARS-CoV- 2) infects host cells via the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) entry mechanism. However, ACE2 and TMPRSS2 cannot explain the Toll-like receptor driven response of monocytes since there is no ACE2 expressed on monocytes, suggesting alternative receptor(s) on these cells. Here, we report cell surface glucose-regulated protein 78 (csGRP78) which is abundantly expressed on monocytes to function as an alternative receptor for SARS-CoV- 2 internalization. Method(s): Blood from COVID-19 patients and healthy donors were collected for csGRP78 and monocyte activation marker as well as cytokine concentration. In vitro SPR, GST pull-down and Co-IP assay were used to determine interaction between SARS-CoV- 2 spike protein and GRP78. Cytokine mixture of IL-1beta, IL-6, TNF and IFN-gamma were used to stimulated csGRP78 upregulation on human monocytic cell line THP-1. GRP78-overexpressing- THP- 1 was also established. pseudo-typed virus expressing spike protein was used to infect mock or GRP78 over-expressing THP-1 cells. Result(s): Our results show that csGRP78 is upregulated on the monocyte of COVID-19 patients. Moreover, in vitro cell culture experiments revealed that stimulation of wtTHP-1 and GRP78 over-expressing THP-1 with the relevant cytokines IL-1beta, IL-6, TNF and IFN-gamma induces similar csGRP78 and activation marker upregulation on cell surface as found on patients' monocytes. In vitro spike protein and GRP78 interaction tests, confirmed direct binding of spike protein and GRP78. Finally, pseudo-typed virus infection assay showed that virus entered GRP78 over-expressing THP-1 cells but not control THP-1 cells. Conclusion(s): Our results demonstrate that csGRP78 acts as a potential functional receptor for SARS-CoV- 2 spike protein and mediates ACE2 independent SARS-CoV- 2 entry into monocytes. These findings provide insight into role of monocytes in the pathophysiology of COVID-19, and suggest a new therapeutic target candidate for anti-SARS- CoV- 2 treatment.

Intracellular "in silico microscopes"-fully 3D spatial Hepatitis C virus replication model simulations

Virus pandemics and endemics cause enormous pain and economic, political, and social costs and turmoil. While the Covid19 pandemics induced obvious damages, the "silent" Hepatitis C virus (HCV) infection induced liver damages are the main reason for liver transplantations. HCV-generated virus genome replication factories are housed within virus-induced intracellular structures termed membranous webs (MW) which are derived from the Endoplasmatic Reticulum (ER). Up to now, very advanced experimental data such as highly spatially resolved fuorescence and electron-tomography data often do not enter computational HCV viral RNA (vRNA) cycle models. Based upon difusion-reaction partial differential equation (PDE) models, we are developing fully 3D resolved "in silico microscopes" to mirror in vitro / in vivo experiments of the intracellular vRNA cycle dynamics. Our first models described the major components (vRNA, non-structural viral proteins-NSPs-and a host factor). The next steps incorporated additional parameters: Different aggregate states of vRNA and NSPs, and population dynamics inspired difusion and reaction co-Effcients instead of multilinear ones. Our work in progress framework presently is merging effects restricted to 2D manifold surface grids (e.g. ER surface, NSP difusion) with others occurring in 3D volume meshes (e.g. cytosol, host factor supply). We estimate and incorporate realistic parameters such as NSP difusion constants. The simulations are performed upon experimental data based reconstructed cell geometries and help understanding the relation of form and function of virus replication. In the long run, our framework might help to facilitate the systematic development of Effcient direct antiviral agents and vaccines.

Cell surface GRP78 functions as an alternative virus entry receptor on monocytes during SARS-CoV-2 infection

It has been widely acknowledged that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infects host cells via the angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) entry mechanism. However, ACE2 and TMPRSS2 cannot explain the Toll-like receptor driven response of monocytes since there is no ACE2 expressed on monocytes, suggesting alternative receptor(s) on these cells. Here, we report cell surface glucose-regulated protein 78 (csGRP78) which is abundantly expressed on monocytes to function as an alternative receptor for SARS-CoV-2 internalization. Our results show that csGRP78 is upregulated on the monocyte of COVID-19 patients. Moreover, in vitro cell culture experiments revealed that GRP78 over-expressing THP-1 cells and stimulation of wtTHP-1 cells with the relevant cytokines IL-1beta, IL-6, TNF and IFN-gamma induces similar csGRP78 and activation marker upregulation on cell surface as found on patients' monocytes. In vitro spike protein and GRP78 interaction tests (SPR assay, GST-pull down and Co-IP), confirmed direct binding of spike protein and GRP78. Finally, pseudo-typed virus expressing spike protein was used to infect mock or GRP78 over-expressing THP-1 cells. We found that pseudo-typed virus entered GRP78 over-expressing THP-1 cells but not control THP-1 cells. Our results demonstrate that csGRP78 acts as a potential functional receptor for SARS-CoV-2 spike protein and mediates ACE2 independent SARS-CoV-2 entry into monocytes. These findings provide insight into role of monocytes in the pathophysiology of COVID-19, and suggest a new therapeutic target candidate for anti-SARS-CoV2 treatment.

Advance in human coronaviruses research of host interactions.

2019-novel coronavirus (2019-nCoV) is a highly pathogenic human CoV that first emerged in Wuhan in 2019. 2019-nCoV has a zoonotic origin and poses a major threat to public health. However, little is known about the viral factors contributing to the high virulence of 2019-nCoV. Many animal viruses, including CoVs, encode proteins that interfere with host gene expression, including those involved in antiviral immune responses, and these viral proteins are often major virulence factors. Human coronaviruses (HCoVs) are known respiratory pathogens associated with a range of respiratory infection. In the past 17 years, the onset of 2019-nCoV, severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) have thrust HCoVs into spotlight of the research community due to their high pathogenicity in humans. The recent study of HCoVs-host interactions has contributed extensively to our understanding of infection pathogenesis of 2019-nCoV. This review discuss various host physiopathologic mechanism, such as apoptosis, innate immunity, endoplasmic reticulum (ER) stress response, mitogen-activated protein kinase (MAPK) pathway and nuclear factor kappa B (NF-kappaB) pathway that may be modulated by HCoVs and provides evidence for the intensive investigate of 2019-nCoV infection.Copyright © 2020 by the Chinese Medical Association.

Apoptosis, Autophagy, and Unfolded Protein Response and Cerebellar Development

Development is an evolutionary process that is tightly regulated in mammalian species. Several different cascades are involved in various stages of development. Among these mechanisms, apoptosis, autophagy, and unfolded protein response play critical roles in regulating development by affecting cell fate. All of these pathways are involved in the regulation of cell numbers via determining the life and death cycles of the cells. In this chapter, we first explain the brief mechanisms that are involved in the regulation of apoptosis, autophagy, and unfolded protein response, and later, we briefly describe how these mechanisms play roles in general development. We next address the critical role of these pathways in cerebellar development regulation and how they will aid in our knowledge of the processes behind neurodevelopmental disorders. Additionally, we summarize the present findings on neurological symptoms and disorders related to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and their linkage to autophagy pathways in the cerebellum. © 2023, The Author(s), under exclusive license to Springer Nature Switzerland AG.

In Silico Modeling of COVID-19 Pandemic Course Differentiation Using the FOD Model

Background: The strange and still unclear scenarios of COVID-19 pandemic development have raised the question about the reason for the observed essential state and personal differences con-cerning the expansion and severity of the infection process. Some custom activities are taken into consid-eration in an attempt to explain the phenomenon. Alcohol in the diet is suggested in this paper as the possible factor which could explain the observed differentiation. It easily penetrates cells modifying their natural internal environment, and independently influences tissues as the toxic agent being the source of acetyl aldehyde. Objective(s): The process in which the cell seems to be the most sensitive to altered environmental conditions is the protein folding;in particular, its portion occurring in the endoplasmic reticulum where freshly synthesized polypeptides fold and then are introduced to the cell membrane influencing its property and in particular its fluidity, which is the critical parameter deciding the virus penetration into the cell. Method(s): The application of a mathematical model, fuzzy oil drop model FOD, expressing the influence of the environment on the protein folding process shows the mechanism of this influence. Result(s): The differences between statistical assessment of epidemy in Europe and the Far East, which may be correlated with alcohol consumption, suggest the influence of diet on the status of epidemy in these regions. Conclusion(s): The protein folding seems to be the process most sensitive to environmental conditions in the cell. The different diet customs, including the use of alcohol, may disturb the folding process, lower-ing as the result the number of proteins needed for cell membrane stability, thus increasing its fluidity and the cell susceptibility to virus penetration. Observations presented in this paper are based on the initial period of pandemic development and have not been intentionally modified to prevent the influence of additional factors, like government activities or virus mutations.Copyright © 2022 Bentham Science Publishers.

Identification of selected earthworm species of Northeast India using DNA barcoding

Aim: This study aimed at the species identification of selected indigenous earthworms of Manipur and Assam, Northeast India along with an exotic species using morpho-anatomical study and DNA barcoding. Methodology: Indigenous species of earthworms were collected from Imphal and Jorhat, North-eastern part of India. The exotic species of earthworm were collected from Indian Council of Agricultural Research Complex, Manipur. The samples were collected by digging and hand sorting method. Identification of samples was done by both conventional and molecular methods. Molecular characterization was accomplished through PCR amplification of the mitochondrial cytochrome oxidase I (COI) genes. Automatic sequencing reactions were performed for the amplified PCR products on ABI3100 Genetic Analyser (Applied Biosystems). Result(s): Out of five specimens (EM1, EM2, EM4, EG5 and EM6) examined through morpho-anatomical studies, three were identified to species level while the other two were identified to their genus level only. Out of EM1 and EM2 specimens in the genus Perionyx as per the morpho-anatomical studies, DNA barcoding could deduce the EM2 specimen up to the species level as P. excavatus. The exotic EM6 specimen morphologically identified as Eisenia fetida showed 99% COI gene sequence similarity with both E. fetida and E. andrei but its sequence divergence with E. andrei was less than 1%, so, it belonged to E. andrei. Interpretation(s): This study shows the reliability of clubbing DNA barcoding experiments with classical taxonomy in supplementing and strengthening the traditional taxonomy for accurate identification of earthworms.Copyright © Triveni Enterprises, Lucknow (India)

SARS-CoV-2 ORF8 Protein Induces Endoplasmic Reticulum Stress-like Responses and Facilitates Virus Replication by Triggering Calnexin: an Unbiased Study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the worldwide coronavirus disease 2019 (COVID-19) pandemic. The novel SARS-CoV-2 ORF8 protein is not highly homologous with known proteins, including accessory proteins of other coronaviruses. ORF8 contains a 15-amino-acid signal peptide in the N terminus that localizes the mature protein to the endoplasmic reticulum. Oligomannose-type glycosylation has been identified at the N78 site. Here, the unbiased molecular functions of ORF8 are also demonstrated. Via an immunoglobulin-like fold in a glycan-independent manner, both exogenous and endogenous ORF8 interacts with human calnexin and HSPA5. The key ORF8-binding sites of Calnexin and HSPA5 are indicated on the globular domain and the core substrate-binding domain, respectively. ORF8 induces species-dependent endoplasmic reticulum stress-like responses in human cells exclusively via the IRE1 branch, including intensive HSPA5 and PDIA4 upregulation, with increases in other stress-responding effectors, including CHOP, EDEM and DERL3. ORF8 overexpression facilitates SARS-CoV-2 replication. Both stress-like responses and viral replication induced by ORF8 have been shown to result from triggering the Calnexin switch. Thus, ORF8 serves as a key unique virulence gene of SARS-CoV-2, potentially contributing to COVID-19-specific and/or human-specific pathogenesis. IMPORTANCE Although SARS-CoV-2 is basically regarded as a homolog of SARS-CoV, with their genomic structure and the majority of their genes being highly homologous, the ORF8 genes of SARS-CoV and SARS-CoV-2 are distinct. The SARS-CoV-2 ORF8 protein also shows little homology with other viral or host proteins and is thus regarded as a novel special virulence gene of SARS-CoV-2. The molecular function of ORF8 has not been clearly known until now. Our results reveal the unbiased molecular characteristics of the SARS-CoV-2 ORF8 protein and demonstrate that it induces rapidly generated but highly controllable endoplasmic reticulum stress-like responses and facilitates virus replication by triggering Calnexin in human but not mouse cells, providing an explanation for the superficially known in vivo virulence discrepancy of ORF8 between SARS-CoV-2-infected patients and mouse.

Leukocyte surface expression of the endoplasmic reticulum chaperone GRP78 is increased in severe COVID-19.

Hyperinflammation present in individuals with severe COVID-19 has been associated with an exacerbated cytokine production and hyperactivated immune cells. Endoplasmic reticulum stress leading to the unfolded protein response has been recently reported as an active player in inducing inflammatory responses. Once unfolded protein response is activated, GRP78, an endoplasmic reticulum-resident chaperone, is translocated to the cell surface (sGRP78), where it is considered a cell stress marker; however, its presence has not been evaluated in immune cells during disease. Here we assessed the presence of sGRP78 on different cell subsets in blood samples from severe or convalescent COVID-19 patients. The frequency of CD45+sGRP78+ cells was higher in patients with the disease compared to convalescent patients. The latter showed similar frequencies to healthy controls. In patients with COVID-19, the lymphoid compartment showed the highest presence of sGRP78+ cells versus the myeloid compartment. CCL2, TNF-α, C-reactive protein, and international normalized ratio measurements showed a positive correlation with the frequency of CD45+sGRP78+ cells. Finally, gene expression microarray data showed that activated T and B cells increased the expression of GRP78, and peripheral blood mononuclear cells from healthy donors acquired sGRP78 upon activation with ionomycin and PMA. Thus, our data highlight the association of sGRP78 on immune cells in patients with severe COVID-19.