Identification and Characterization of Cell Lines HepG2, Hep3B217 and SNU387 as Models for Porcine Epidemic Diarrhea Coronavirus Infection.

Porcine epidemic diarrhea virus (PEDV), a member of the genera alphacoronavirus, causes acute watery diarrhea and dehydration in suckling piglets and results in enormous economic losses in the swine industry worldwide. Identification and characterization of different cell lines are not only invaluable for PEDV entry and replication studies but also important for the development of various types of biological pharmaceuticals against PEDV. In this study, we present an approach to identify suitable permissive cell lines for PEDV research. Human cell lines were screened for a high correlation coefficient with the established PEDV infection model Huh7 based on RNA-seq data from the Cancer Cell Line Encyclopedia (CCLE). Experimentally testing permissiveness towards PEDV infection, three highly permissive human cell lines, HepG2, Hep3B217, and SNU387 were identified. The replication kinetics of PEDV in HepG2, Hep3B217, and SNU387 cells were similar to that in Vero and Huh7 cells. Additionally, the transcriptomes analysis showed robust induction of transcripts associated with the innate immune in response to PEDV infection in all three cell lines, including hundreds of inflammatory cytokine and interferon genes. Moreover, the expression of inflammatory cytokines and interferons were confirmed by qPCR assay. Our findings indicate that HepG2, Hep3B217, and SNU387 are suitable cell lines for PEDV replication and innate immune response studies.

3D culture of cancer cells in alginate hydrogel beads as an effective technique for emergency cell storage and transportation in the pandemic era.

Due to the restrictions in accessing research laboratories and the challenges in providing proper storage and transportation of cells during the COVID-19 pandemic, having an effective and feasible mean to solve these challenges would be of immense help. Therefore, we developed a 3D culture setting of cancer cells using alginate beads and tested its effectiveness in different storage and transportation conditions. The viability and proliferation of cancer cells were assessed using trypan blue staining and quantitative CCK-8 kit, respectively. The developed beads allowed cancer cells survival up to 4 weeks with less frequent maintenance measures such as change of the culture media or subculture of cells. In addition, the recovery of cancer cells and proliferation pattern were significantly faster with better outcomes in the developed 3D alginate beads compared to the standard cryopreservation of cells or the 2D culture conditions. The 3D alginate beads also supported the viability of cells while the shipment at room temperature for a duration of up to 5 days with no humidity or CO2  support. Therefore, 3D culture in alginate beads can be used to store or ship biological cells with ease at room temperature with minimal preparations.

Porphyrin Molecules Decorated on Metal-Organic Frameworks for Multi-Functional Biomedical Applications.

Metal-organic frameworks (MOFs) have been widely used as porous nanomaterials for different applications ranging from industrial to biomedicals. An unpredictable one-pot method is introduced to synthesize NH2-MIL-53 assisted by high-gravity in a greener media for the first time. Then, porphyrins were deployed to adorn the surface of MOF to increase the sensitivity of the prepared nanocomposite to the genetic materials and in-situ cellular protein structures. The hydrogen bond formation between genetic domains and the porphyrin' nitrogen as well as the surface hydroxyl groups is equally probable and could be considered a milestone in chemical physics and physical chemistry for biomedical applications. In this context, the role of incorporating different forms of porphyrins, their relationship with the final surface morphology, and their drug/gene loading efficiency were investigated to provide a predictable pattern in regard to the previous works. The conceptual phenomenon was optimized to increase the interactions between the biomolecules and the substrate by reaching the limit of detection to 10 pM for the Anti-cas9 protein, 20 pM for the single-stranded DNA (ssDNA), below 10 pM for the single guide RNA (sgRNA) and also around 10 nM for recombinant SARS-CoV-2 spike antigen. Also, the MTT assay showed acceptable relative cell viability of more than 85% in most cases, even by increasing the dose of the prepared nanostructures.

Eicosanoid regulation of debris-stimulated metastasis.

Cancer therapy reduces tumor burden via tumor cell death ("debris"), which can accelerate tumor progression via the failure of inflammation resolution. Thus, there is an urgent need to develop treatment modalities that stimulate the clearance or resolution of inflammation-associated debris. Here, we demonstrate that chemotherapy-generated debris stimulates metastasis by up-regulating soluble epoxide hydrolase (sEH) and the prostaglandin E2 receptor 4 (EP4). Therapy-induced tumor cell debris triggers a storm of proinflammatory and proangiogenic eicosanoid-driven cytokines. Thus, targeting a single eicosanoid or cytokine is unlikely to prevent chemotherapy-induced metastasis. Pharmacological abrogation of both sEH and EP4 eicosanoid pathways prevents hepato-pancreatic tumor growth and liver metastasis by promoting macrophage phagocytosis of debris and counterregulating a protumorigenic eicosanoid and cytokine storm. Therefore, stimulating the clearance of tumor cell debris via combined sEH and EP4 inhibition is an approach to prevent debris-stimulated metastasis and tumor growth.

A dual-role of SARS-CoV-2 nucleocapsid protein in regulating innate immune response.

The recently emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of ongoing global pandemic of COVID-19, may trigger immunosuppression in the early stage and overactive immune response in the late stage of infection; However, the underlying mechanisms are not well understood. Here we demonstrated that the SARS-CoV-2 nucleocapsid (N) protein dually regulated innate immune responses, i.e., the low-dose N protein suppressed type I interferon (IFN-I) signaling and inflammatory cytokines, whereas high-dose N protein promoted IFN-I signaling and inflammatory cytokines. Mechanistically, the SARS-CoV-2 N protein dually regulated the phosphorylation and nuclear translocation of IRF3, STAT1, and STAT2. Additionally, low-dose N protein combined with TRIM25 could suppress the ubiquitination and activation of retinoic acid-inducible gene I (RIG-I). Our findings revealed a regulatory mechanism of innate immune responses by the SARS-CoV-2 N protein, which would contribute to understanding the pathogenesis of SARS-CoV-2 and other SARS-like coronaviruses, and development of more effective strategies for controlling COVID-19.

Inhibiting SARS-CoV-2 infection in vitro by suppressing its receptor, angiotensin-converting enzyme 2, via aryl-hydrocarbon receptor signal.

Since understanding molecular mechanisms of SARS-CoV-2 infection is extremely important for developing effective therapies against COVID-19, we focused on the internalization mechanism of SARS-CoV-2 via ACE2. Although cigarette smoke is generally believed to be harmful to the pathogenesis of COVID-19, cigarette smoke extract (CSE) treatments were surprisingly found to suppress the expression of ACE2 in HepG2 cells. We thus tried to clarify the mechanism of CSE effects on expression of ACE2 in mammalian cells. Because RNA-seq analysis suggested that suppressive effects on ACE2 might be inversely correlated with induction of the genes regulated by aryl hydrocarbon receptor (AHR), the AHR agonists 6-formylindolo(3,2-b)carbazole (FICZ) and omeprazole (OMP) were tested to assess whether those treatments affected ACE2 expression. Both FICZ and OMP clearly suppressed ACE2 expression in a dose-dependent manner along with inducing CYP1A1. Knock-down experiments indicated a reduction of ACE2 by FICZ treatment in an AHR-dependent manner. Finally, treatments of AHR agonists inhibited SARS-CoV-2 infection into Vero E6 cells as determined with immunoblotting analyses detecting SARS-CoV-2 specific nucleocapsid protein. We here demonstrate that treatment with AHR agonists, including FICZ, and OMP, decreases expression of ACE2 via AHR activation, resulting in suppression of SARS-CoV-2 infection in mammalian cells.

CAR-NK Cells Effectively Target SARS-CoV-2-Spike-Expressing Cell Lines In Vitro.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is highly contagious and presents a significant public health issue. Current therapies used to treat coronavirus disease 2019 (COVID-19) include monoclonal antibody cocktail, convalescent plasma, antivirals, immunomodulators, and anticoagulants. The vaccines from Pfizer and Moderna have recently been authorized for emergency use, which are invaluable for the prevention of SARS-CoV-2 infection. However, their long-term side effects are not yet documented, and populations with immunocompromised conditions (e.g., organ-transplantation and immunodeficient patients) may not be able to mount an effective immune response. In addition, there are concerns that wide-scale immunity to SARS-CoV-2 may introduce immune pressure that could select for escape mutants to the existing vaccines and monoclonal antibody therapies. Emerging evidence has shown that chimeric antigen receptor (CAR)- natural killer (NK) immunotherapy has potent antitumor response in hematologic cancers with minimal adverse effects in recent studies, however, the potentials of CAR-NK cells in treating COVID-19 has not yet been fully exploited. Here, we improve upon a novel approach for the generation of CAR-NK cells for targeting SARS-CoV-2 and its various mutants. CAR-NK cells were generated using the scFv domain of S309 (henceforward, S309-CAR-NK), a SARS-CoV and SARS-CoV-2 neutralizing antibody (NAbs) that targets the highly conserved region of SARS-CoV-2 spike (S) glycoprotein and is therefore more likely to recognize different variants of SARS-CoV-2 isolates. S309-CAR-NK cells can specifically bind to pseudotyped SARS-CoV-2 virus and its D614G, N501Y, and E484K mutants. Furthermore, S309-CAR-NK cells can specifically kill target cells expressing SARS-CoV-2 S protein in vitro and show superior killing activity and cytokine production, compared to that of the recently reported CR3022-CAR-NK cells. Thus, these results pave the way for generating 'off-the-shelf' S309-CAR-NK cells for treatment in high-risk individuals as well as provide an alternative strategy for patients unresponsive to current vaccines.

SARS-CoV-2 Entry: At the Crossroads of CD147 and ACE2.

In late 2019, the betacoronavirus SARS-CoV-2 was identified as the viral agent responsible for the coronavirus disease 2019 (COVID-19) pandemic. Coronaviruses Spike proteins are responsible for their ability to interact with host membrane receptors and different proteins have been identified as SARS-CoV-2 interactors, among which Angiotensin-converting enzyme 2 (ACE2), and Basigin2/EMMPRIN/CD147 (CD147). CD147 plays an important role in human immunodeficiency virus type 1, hepatitis C virus, hepatitis B virus, Kaposi's sarcoma-associated herpesvirus, and severe acute respiratory syndrome coronavirus infections. In particular, SARS-CoV recognizes the CD147 receptor expressed on the surface of host cells by its nucleocapsid protein binding to cyclophilin A (CyPA), a ligand for CD147. However, the involvement of CD147 in SARS-CoV-2 infection is still debated. Interference with both the function (blocking antibody) and the expression (knock down) of CD147 showed that this receptor partakes in SARS-CoV-2 infection and provided additional clues on the underlying mechanism: CD147 binding to CyPA does not play a role; CD147 regulates ACE2 levels and both receptors are affected by virus infection. Altogether, these findings suggest that CD147 is involved in SARS-CoV-2 tropism and represents a possible therapeutic target to challenge COVID-19.

Discovery of a AHR pelargonidin agonist that counter-regulates Ace2 expression and attenuates ACE2-SARS-CoV-2 interaction.

The severe acute respiratory syndrome (SARS)-CoV-2 is the pathogenetic agent of Corona Virus Induced Disease (COVID)19. The virus enters the human cells after binding to the angiotensin converting enzyme (ACE)2 receptor in target tissues. ACE2 expression is induced in response to inflammation. The colon expression of ACE2 is upregulated in patients with inflammatory bowel disease (IBD), highlighting a potential risk of intestinal inflammation in promoting viral entry in the human body. Because mechanisms that regulate ACE2 expression in the intestine are poorly understood and there is a need of anti-SARS-CoV-2 therapies, we have settled to investigate whether natural flavonoids might regulate the expression of Ace2 in intestinal models of inflammation. The results of these studies demonstrated that pelargonidin activates the Aryl hydrocarbon Receptor (AHR) in vitro and reverses intestinal inflammation caused by chronic exposure to high fat diet or to the intestinal braking-barrier agent TNBS in a AhR-dependent manner. In these two models, development of colon inflammation associated with upregulation of Ace2 mRNA expression. Colon levels of Ace2 mRNA were directly correlated with Tnf-α mRNA levels. Molecular docking studies suggested that pelargonidin binds a fatty acid binding pocket on the receptor binding domain of SARS-CoV-2 Spike protein. In vitro studies demonstrated that pelargonidin significantly reduces the binding of SARS-CoV-2 Spike protein to ACE2 and reduces the SARS-CoV-2 replication in a concentration-dependent manner. In summary, we have provided evidence that a natural flavonoid might hold potential in reducing intestinal inflammation and ACE2 induction in the inflamed colon in a AhR-dependent manner.

Translational adaptation of human viruses to the tissues they infect.

Viruses need to hijack the translational machinery of the host cell for a productive infection to happen. However, given the dynamic landscape of tRNA pools among tissues, it is unclear whether different viruses infecting different tissues have adapted their codon usage toward their tropism. Here, we collect the coding sequences of 502 human-infecting viruses and determine that tropism explains changes in codon usage. Using the tRNA abundances across 23 human tissues from The Cancer Genome Atlas (TCGA), we build an in silico model of translational efficiency that validates the correspondence of the viral codon usage with the translational machinery of their tropism. For instance, we detect that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is specifically adapted to the upper respiratory tract and alveoli. Furthermore, this correspondence is specifically defined in early viral proteins. The observed tissue-specific translational efficiency could be useful for the development of antiviral therapies and vaccines.

Flow cytometry multiplexed method for the detection of neutralizing human antibodies to the native SARS-CoV-2 spike protein.

A correct identification of seropositive individuals for the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection is of paramount relevance to assess the degree of protection of a human population to present and future outbreaks of the COVID-19 pandemic. We describe here a sensitive and quantitative flow cytometry method using the cytometer-friendly non-adherent Jurkat T-cell line that stably expresses the full-length native spike "S" protein of SARS-CoV-2 and a truncated form of the human EGFR that serves a normalizing role. S protein and huEGFRt coding sequences are separated by a T2A self-cleaving sequence, allowing to accurately quantify the presence of anti-S immunoglobulins by calculating a score based on the ratio of fluorescence intensities obtained by double-staining with the test sera and anti-EGFR. The method allows to detect immune individuals regardless of the result of other serological tests or even repeated PCR monitoring. As examples of its use, we show that as much as 28% of the personnel working at the CBMSO in Madrid is already immune. Additionally, we show that anti-S antibodies with protective neutralizing activity are long-lasting and can be detected in sera 8 months after infection.

A Ligand Selection Strategy Identifies Chemical Probes Targeting the Proteases of SARS-CoV-2.

Activity-based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS-CoV-2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases.

Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.

CETSA MS Profiling for a Comparative Assessment of FDA-Approved Antivirals Repurposed for COVID-19 Therapy Identifies TRIP13 as a Remdesivir Off-Target.

The reuse of preexisting small molecules for a novel emerging disease threat is a rapid measure to discover unknown applications for previously validated therapies. A pertinent and recent example where such a strategy could be employed is in the fight against coronavirus disease 2019 (COVID-19). Therapies designed or discovered to target viral proteins also have off-target effects on the host proteome when employed in a complex physiological environment. This study aims to assess these host cell targets for a panel of FDA-approved antiviral compounds including remdesivir, using the cellular thermal shift assay (CETSA) coupled with mass spectrometry (CETSA MS) in noninfected cells. CETSA MS is a powerful method to delineate direct and indirect interactions between small molecules and protein targets in intact cells. Biologically active compounds can induce changes in thermal stability, in their primary binding partners, and in proteins that in turn interact with the direct targets. Such engagement of host targets by antiviral drugs may contribute to the clinical effect against the virus but can also constitute a liability. We present here a comparative study of CETSA molecular target engagement fingerprints of antiviral drugs to better understand the link between off-targets and efficacy.

GB-2 inhibits ACE2 and TMPRSS2 expression: In vivo and in vitro studies.

After the first case of Coronavirus disease 2019 (COVID-19) was reported in Wuhan, COVID-19 has rapidly spread to almost all parts of world. Angiotensin converting enzyme 2 (ACE2) receptor can bind to spike protein of SARS-CoV-2. Then, the spike protein of SARS-CoV-2 can be cleaved and activated by transmembrane protease, serine 2 (TMPRSS2) of the host cells for SARS-CoV-2 infection. Therefore, ACE2 and TMPRSS2 are potential antiviral targets for treatment of prevention of SARS-CoV-2 infection. In this study, we discovered that 10-250 µg/mL of GB-2, from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit ACE2 mRNA expression and ACE2 and TMPRSS2 protein expression in HepG2 and 293 T cells without cytotoxicity. GB-2 treatment could decrease ACE2 and TMPRSS2 expression level of lung tissue and kidney tissue without adverse effects, including nephrotoxicity and hepatotoxicity, in animal model. In the compositions of GB-2, we discovered that 50 µg/mL of theaflavin could inhibit protein expression of ACE2 and TMPRSS2. Theaflavin could inhibit the mRNA expression of ACE2. In conclusion, our results suggest that GB-2 and theaflavin could act as potential compounds for ACE2 and TMPRSS2 inhibitors in the further clinical study.

MARCH8 Inhibits Ebola Virus Glycoprotein, Human Immunodeficiency Virus Type 1 Envelope Glycoprotein, and Avian Influenza Virus H5N1 Hemagglutinin Maturation.

Membrane-associated RING-CH-type 8 (MARCH8) strongly blocks human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) incorporation into virions by downregulating its cell surface expression, but the mechanism is still unclear. We now report that MARCH8 also blocks the Ebola virus (EBOV) glycoprotein (GP) incorporation via surface downregulation. To understand how these viral fusion proteins are downregulated, we investigated the effects of MARCH8 on EBOV GP maturation and externalization via the conventional secretion pathway. MARCH8 interacted with EBOV GP and furin when detected by immunoprecipitation and retained the GP/furin complex in the Golgi when their location was tracked by a bimolecular fluorescence complementation (BiFC) assay. MARCH8 did not reduce the GP expression or affect the GP modification by high-mannose N-glycans in the endoplasmic reticulum (ER), but it inhibited the formation of complex N-glycans on the GP in the Golgi. Additionally, the GP O-glycosylation and furin-mediated proteolytic cleavage were also inhibited. Moreover, we identified a novel furin cleavage site on EBOV GP and found that only those fully glycosylated GPs were processed by furin and incorporated into virions. Furthermore, the GP shedding and secretion were all blocked by MARCH8. MARCH8 also blocked the furin-mediated cleavage of HIV-1 Env (gp160) and the highly pathogenic avian influenza virus H5N1 hemagglutinin (HA). We conclude that MARCH8 has a very broad antiviral activity by prohibiting different viral fusion proteins from glycosylation and proteolytic cleavage in the Golgi, which inhibits their transport from the Golgi to the plasma membrane and incorporation into virions.IMPORTANCE Enveloped viruses express three classes of fusion proteins that are required for their entry into host cells via mediating virus and cell membrane fusion. Class I fusion proteins are produced from influenza viruses, retroviruses, Ebola viruses, and coronaviruses. They are first synthesized as a type I transmembrane polypeptide precursor that is subsequently glycosylated and oligomerized. Most of these precursors are cleaved en route to the plasma membrane by a cellular protease furin in the late secretory pathway, generating the trimeric N-terminal receptor-binding and C-terminal fusion subunits. Here, we show that a cellular protein, MARCH8, specifically inhibits the furin-mediated cleavage of EBOV GP, HIV-1 Env, and H5N1 HA. Further analyses uncovered that MARCH8 blocked the EBOV GP glycosylation in the Golgi and inhibited its transport from the Golgi to the plasma membrane. Thus, MARCH8 has a very broad antiviral activity by specifically inactivating different viral fusion proteins.

The cholesterol metabolite 27-hydroxycholesterol inhibits SARS-CoV-2 and is markedly decreased in COVID-19 patients.

There is an urgent need to identify antivirals against the coronavirus SARS-CoV-2 in the current COVID-19 pandemic and to contain future similar emergencies early on. Specific side-chain cholesterol oxidation products of the oxysterols family have been shown to inhibit a large variety of both enveloped and non-enveloped human viral pathogens. Here we report on the in vitro inhibitory activity of the redox active oxysterol 27-hydroxycholesterol against SARS-CoV-2 and against one of the common cold agents HCoV-OC43 human coronavirus without significant cytotoxicity. Interestingly, physiological serum levels of 27-hydroxycholesterol in SARS-CoV-2 positive subjects were significantly decreased compared to the matched control group, reaching a marked 50% reduction in severe COVID-19 cases. Moreover, no correlation at all was observed between 24-hydroxycholesterol and 25-hydroxycholesterol serum levels and the severity of the disease. Opposite to that of 27-hydroxycholesterol was the behaviour of two recognized markers of redox imbalance, i.e. 7-ketocholesterol and 7ß-hydroxycholesterol, whose serum levels were significantly increased especially in severe COVID-19. The exogenous administration of 27-hydroxycholesterol may represent in the near future a valid antiviral strategy in the worsening of diseases caused by present and emerging coronaviruses.