Transcription Factor JunB Suppresses Hepatitis C Virus Replication.

We previously reported that hepatitis C virus (HCV) infection activates the reactive oxygen species (ROS)/c-Jun N-terminal kinase (JNK) signaling pathway. Activation of JNK contributes to the development of liver diseases, including metabolic disorders, steatosis, liver cirrhosis and hepatocellular carcinoma. JNK is known to have numerous target genes, including JunB, a member of activator protein-1 transcription factor family. However, the roles of JunB in the HCV life cycle and HCV-associated pathogenesis remain unclear. To clarify a physiological role of JunB in HCV infection, we investigated the phosphorylation of JunB in HCV J6/JFH1-infected Huh-7.5 cells. Immunoblot analysis revealed that HCV-induced ROS/JNK activation promoted phosphorylation of JunB. The small interfering RNA (siRNA) knockdown of JunB significantly increased the amount of intracellular HCV RNA as well as the intracellular and extracellular HCV infectivity titers. Conversely, overexpression of JunB significantly reduced the amount of intracellular HCV RNA and the intracellular and extracellular HCV infectivity titers. These results suggest that JunB plays a role in inhibiting HCV propagation. Additionally, HCV-mediated JunB activation promoted hepcidin promoter activity and hepcidin mRNA levels, a key factor in modulating iron homeostasis, suggesting that JunB is involved in HCV-induced transcriptional upregulation of hepcidin. Taken together, we propose that the HCV-induced ROS/JNK/JunB signaling pathway plays roles in inhibiting HCV replication and contributing to HCV-mediated iron metabolism disorder.

Hydrogen Peroxide Inhibits Hepatitis B Virus Replication by Downregulating HBx Levels via Siah-1-Mediated Proteasomal Degradation in Human Hepatoma Cells.

The hepatitis B virus (HBV) is constantly exposed to significant oxidative stress characterized by elevated levels of reactive oxygen species (ROS), such as H2O2, during infection in hepatocytes of patients. In this study, we demonstrated that H2O2 inhibits HBV replication in a p53-dependent fashion in human hepatoma cell lines expressing sodium taurocholate cotransporting polypeptide. Interestingly, H2O2 failed to inhibit the replication of an HBV X protein (HBx)-null HBV mutant, but this defect was successfully complemented by ectopic expression of HBx. Additionally, H2O2 upregulated p53 levels, leading to increased expression of seven in absentia homolog 1 (Siah-1) levels. Siah-1, an E3 ligase, induced the ubiquitination-dependent proteasomal degradation of HBx. The inhibitory effect of H2O2 was nearly abolished not only by treatment with a representative antioxidant, N-acetyl-L-cysteine but also by knockdown of either p53 or Siah-1 using specific short hairpin RNA, confirming the role of p53 and Siah-1 in the inhibition of HBV replication by H2O2. The present study provides insights into the mechanism that regulates HBV replication under conditions of oxidative stress in patients.

A natural variation in the RNA polymerase of severe fever with thrombocytopenia syndrome virus enhances viral replication and in vivo virulence.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne disease with high mortality in Eastern Asia. The disease is caused by the SFTS virus (SFTSV), also known as Dabie bandavirus, which has a segmented RNA genome consisting of L, M, and S segments. Previous studies have suggested differential viral virulence depending on the genotypes of SFTSV; however, the critical viral factor involved in the differential viral virulence is unknown. Here, we found a significant difference in viral replication in vitro and virulence in vivo between two Korean isolates belonging to the F and B genotypes, respectively. By generating viral reassortants using the two viral strains, we demonstrated that the L segment, which encodes viral RNA-dependent RNA polymerase (RdRp), is responsible for the enhanced viral replication and virulence. Comparison of amino acid sequences and viral replication rates revealed a point variation, E251K, on the surface of RdRp to be the most significant determinant for the enhanced viral replication rate and in vivo virulence. The effect of the variation was further confirmed using recombinant SFTSV generated by reverse genetic engineering. Therefore, our results indicate that natural variations affecting the viral replicase activity could significantly contribute to the viral virulence of SFTSV.

Mutational analysis reveals a novel role for hepatitis C virus NS5A domain I in cyclophilin-dependent genome replication.

The hepatitis C virus (HCV) NS5A protein is comprised of three domains (D1-3). Previously, we observed that two alanine substitutions in D1 (V67A, P145A) abrogated replication of a genotype 2a isolate (JFH-1) sub-genomic replicon (SGR) in Huh7 cells, but this phenotype was partially restored in Huh7.5 cells. Here we demonstrate that five additional residues, surface-exposed and proximal to V67 or P145, exhibited the same phenotype. In contrast, the analogous mutants in a genotype 3a isolate (DBN3a) SGR exhibited different phenotypes in each cell line, consistent with fundamental differences in the functions of genotypes 2 and 3 NS5A. The difference between Huh7 and Huh7.5 cells was reminiscent of the observation that cyclophilin inhibitors are more potent against HCV replication in the former and suggested a role for D1 in cyclophilin dependence. Consistent with this, all JFH-1 and DBN3a mutants exhibited increased sensitivity to cyclosporin A treatment compared to wild-type. Silencing of cyclophilin A (CypA) in Huh7 cells inhibited replication of both JFH-1 and DBN3a. However, in Huh7.5 cells CypA silencing did not inhibit JFH-1 wild-type, but abrogated replication of all the JFH-1 mutants, and both DBN3a wild-type and all mutants. CypB silencing in Huh7 cells had no effect on DBN3a, but abrogated replication of JFH-1. CypB silencing in Huh7.5 cells had no effect on either SGR. Lastly, we confirmed that JFH-1 NS5A D1 interacted with CypA in vitro. These data demonstrate both a direct involvement of NS5A D1 in cyclophilin-dependent genome replication and functional differences between genotype 2 and 3 NS5A.

Functional Landscape of African Swine Fever Virus-Host and Virus-Virus Protein Interactions.

Viral replication fully relies on the host cell machinery, and physical interactions between viral and host proteins mediate key steps of the viral life cycle. Therefore, identifying virus-host protein-protein interactions (PPIs) provides insights into the molecular mechanisms governing virus infection and is crucial for designing novel antiviral strategies. In the case of the African swine fever virus (ASFV), a large DNA virus that causes a deadly panzootic disease in pigs, the limited understanding of host and viral targets hinders the development of effective vaccines and treatments. This review summarizes the current knowledge of virus-host and virus-virus PPIs by collecting and analyzing studies of individual viral proteins. We have compiled a dataset of experimentally determined host and virus protein targets, the molecular mechanisms involved, and the biological functions of the identified virus-host and virus-virus protein interactions during infection. Ultimately, this work provides a comprehensive and systematic overview of ASFV interactome, identifies knowledge gaps, and proposes future research directions.

Inhibition of Marek's Disease Virus Replication and Spread by 25-hydroxycholesterol and 27-hydroxycholesterol In Vitro.

Marek's disease virus (MDV) causes a deadly lymphoproliferative disease in chickens, resulting in huge economic losses in the poultry industry. It has been suggested that MDV suppresses the induction of type I interferons and thus escapes immune control. Cholesterol 25-hydroxylase (CH25H), a gene that encodes an enzyme that catalyses cholesterol to 25-hydroxycholesterol (25-HC), is an interferon-stimulating gene (ISG) known to exert antiviral activities. Other oxysterols, such as 27-hydroxycholesterols (27-HC), have also been shown to exert antiviral activities, and 27-HC is synthesised by the catalysis of cholesterol via the cytochrome P450 enzyme oxidase sterol 27-hydroxylase A1 (CYP27A1). At 24 h post infection (hpi), MDV stimulated a type I interferon (IFN-α) response, which was significantly reduced at 48 and 72 hpi, as detected using the luciferase assay for chicken type I IFNs. Then, using RT-PCR, we demonstrated that chicken type I IFN (IFN-α) upregulates chicken CH25H and CYP27A1 genes in chicken embryo fibroblast (CEF) cells. In parallel, our results demonstrate a moderate and transient upregulation of CH25H at 48 hpi and CYP27A1 at 72hpi in MDV-infected CEF cells. A significant reduction in MDV titer and plaque sizes was observed in CEFs treated with 25-HC or 27-HC in vitro, as demonstrated using a standard plaque assay for MDV. Taken together, our results suggest that 25-HC and 27-HC may be useful antiviral agents to control MDV replication and spread.

HMGB1 is involved in viral replication and the inflammatory response in coxsackievirus A16-infected 16HBE cells via proteomic analysis and identification.

Coxsackievirus A16 (CV-A16) is still an important pathogen that causes hand, foot and mouth disease (HFMD) in young children and infants worldwide. Previous studies indicated that CV-A16 infection is usually mild or self-limiting, but it was also found that CV-A16 infection can trigger severe neurological complications and even death. However, there are currently no vaccines or antiviral compounds available to either prevent or treat CV-A16 infection. Therefore, investigation of the virus‒host interaction and identification of host proteins that play a crucial regulatory role in the pathogenesis of CV-A16 infection may provide a novel strategy to develop antiviral drugs. Here, to increase our understanding of the interaction of CV-A16 with the host cell, we analyzed changes in the proteome of 16HBE cells in response to CV-A16 using tandem mass tag (TMT) in combination with LC‒MS/MS. There were 6615 proteins quantified, and 172 proteins showed a significant alteration during CV-A16 infection. These differentially regulated proteins were involved in fundamental biological processes and signaling pathways, including metabolic processes, cytokine‒cytokine receptor interactions, B-cell receptor signaling pathways, and neuroactive ligand‒receptor interactions. Further bioinformatics analysis revealed the characteristics of the protein domains and subcellular localization of these differentially expressed proteins. Then, to validate the proteomics data, 3 randomly selected proteins exhibited consistent changes in protein expression with the TMT results using Western blotting and immunofluorescence methods. Finally, among these differentially regulated proteins, we primarily focused on HMGB1 based on its potential effects on viral replication and virus infection-induced inflammatory responses. It was demonstrated that overexpression of HMGB1 could decrease viral replication and upregulate the release of inflammatory cytokines, but deletion of HMGB1 increased viral replication and downregulated the release of inflammatory cytokines. In conclusion, the results from this study have helped further elucidate the potential molecular pathogenesis of CV-A16 based on numerous protein changes and the functions of HMGB1 Found to be involved in the processes of viral replication and inflammatory response, which may facilitate the development of new antiviral therapies as well as innovative diagnostic methods.

Diketo acid inhibitors of nsp13 of SARS-CoV-2 block viral replication.

For RNA viruses, RNA helicases have long been recognized to play critical roles during virus replication cycles, facilitating proper folding and replication of viral RNAs, therefore representing an ideal target for drug discovery. SARS-CoV-2 helicase, the non-structural protein 13 (nsp13) is a highly conserved protein among all known coronaviruses, and, at the moment, is one of the most explored viral targets to identify new possible antiviral agents. In the present study, we present six diketo acids (DKAs) as nsp13 inhibitors able to block both SARS-CoV-2 nsp13 enzymatic functions. Among them four compounds were able to inhibit viral replication in the low micromolar range, being active also on other human coronaviruses such as HCoV229E and MERS CoV. The experimental investigation of the binding mode revealed ATP-non-competitive kinetics of inhibition, not affected by substrate-displacement effect, suggesting an allosteric binding mode that was further supported by molecular modelling calculations predicting the binding into an allosteric conserved site located in the RecA2 domain.

Inhibition of salt inducible kinases reduces rhythmic HIV-1 replication and reactivation from latency.

Human immunodeficiency virus type 1 (HIV-1) causes a major burden on global health, and eradication of latent virus infection is one of the biggest challenges in the field. The circadian clock is an endogenous timing system that oscillates with a ~24 h period regulating multiple physiological processes and cellular functions, and we recently reported that the cell intrinsic clock regulates rhythmic HIV-1 replication. Salt inducible kinases (SIK) contribute to circadian regulatory networks, however, there is limited evidence for SIKs regulating HIV-1 infection. Here, we show that pharmacological inhibition of SIKs perturbed the cellular clock and reduced rhythmic HIV-1 replication in circadian synchronised cells. Further, SIK inhibitors or genetic silencing of Sik expression inhibited viral replication in primary cells and in a latency model, respectively. Overall, this study demonstrates a role for salt inducible kinases in regulating HIV-1 replication and latency reactivation, which can provide innovative routes to better understand and target latent HIV-1 infection.

Molecular insights into Spindlin1-HBx interplay and its impact on HBV transcription from cccDNA minichromosome.

Molecular interplay between host epigenetic factors and viral proteins constitutes an intriguing mechanism for sustaining hepatitis B virus (HBV) life cycle and its chronic infection. HBV encodes a regulatory protein, HBx, which activates transcription and replication of HBV genome organized as covalently closed circular (ccc) DNA minichromosome. Here we illustrate how HBx accomplishes its task by hijacking Spindlin1, an epigenetic reader comprising three consecutive Tudor domains. Our biochemical and structural studies have revealed that the highly conserved N-terminal 2-21 segment of HBx (HBx2-21) associates intimately with Tudor 3 of Spindlin1, enhancing histone H3 "K4me3-K9me3" readout by Tudors 2 and 1. Functionally, Spindlin1-HBx engagement promotes gene expression from the chromatinized cccDNA, accompanied by an epigenetic switch from an H3K9me3-enriched repressive state to an H3K4me3-marked active state, as well as a conformational switch of HBx that may occur in coordination with other HBx-binding factors, such as DDB1. Despite a proposed transrepression activity of HBx2-21, our study reveals a key role of Spindlin1 in derepressing this conserved motif, thereby promoting HBV transcription from its chromatinized genome.

Hepatitis C virus non-structural proteins modulate cellular kinases for increased cytoplasmic abundance of host factor HuR and facilitate viral replication.

Host protein HuR translocation from nucleus to cytoplasm following infection is crucial for the life cycle of several RNA viruses including hepatitis C virus (HCV), a major causative agent of hepatocellular carcinoma. HuR assists the assembly of replication-complex on the viral-3'UTR, and its depletion hampers viral replication. Although cytoplasmic HuR is crucial for HCV replication, little is known about how the virus orchestrates the mobilization of HuR into the cytoplasm from the nucleus. We show that two viral proteins, NS3 and NS5A, act co-ordinately to alter the equilibrium of the nucleo-cytoplasmic movement of HuR. NS3 activates protein kinase C (PKC)-δ, which in-turn phosphorylates HuR on S318 residue, triggering its export to the cytoplasm. NS5A inactivates AMP-activated kinase (AMPK) resulting in diminished nuclear import of HuR through blockade of AMPK-mediated phosphorylation and acetylation of importin-α1. Cytoplasmic retention or entry of HuR can be reversed by an AMPK activator or a PKC-δ inhibitor. Our findings suggest that efforts should be made to develop inhibitors of PKC-δ and activators of AMPK, either separately or in combination, to inhibit HCV infection.

Biological Roles of Hepatitis B Viral X Protein in the Viral Replication and Hepatocarcinogenesis.

Hepatitis B virus is a pathogenic virus that infects 300 million people worldwide and causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Hepatitis B virus encodes four proteins. Among them, the HBx protein plays a central role in the HBV pathogenesis. Because the HBx protein is considered to play a central role in the induction of viral replication and hepatocarcinogenesis, the regulation of its function could be a key factor in the development of new interventions against hepatitis B. In this review, HBx protein-related viral replication and hepatocarcinogenesis mechanisms are described, with a focus on the recently reported viral replication mechanisms related to degradation of the Smc5/6 protein complex. We also discuss our recent discovery of a compound that inhibits HBx protein-induced degradation of the Smc5/6 protein complex, and that exerts inhibitory effects on both viral replication and hepatocarcinogenesis. Finally, prospects for future research on the HBx protein are described.

N-dihydrogalactochitosan reduces mortality in a lethal mouse model of SARS-CoV-2.

The rapid emergence and global dissemination of SARS-CoV-2 that causes COVID-19 continues to cause an unprecedented global health burden resulting in nearly 7 million deaths. While multiple vaccine countermeasures have been approved for emergency use, additional treatments are still needed due to sluggish vaccine rollout, vaccine hesitancy, and inefficient vaccine-mediated protection. Immunoadjuvant compounds delivered intranasally can guide non-specific innate immune responses during the critical early stages of viral replication, reducing morbidity and mortality. N-dihydrogalactochitosan (GC) is a novel mucoadhesive immunostimulatory polymer of ß-0-4-linked N-acetylglucosamine that is solubilized by the conjugation of galactose glycans with current applications as a cancer immunotherapeutic. We tested GC as a potential countermeasure for COVID-19. GC was well-tolerated and did not produce histopathologic lesions in the mouse lung. GC administered intranasally before and after SARS-CoV-2 exposure diminished morbidity and mortality in humanized ACE2 receptor expressing mice by up to 75% and reduced infectious virus levels in the upper airway. Fluorescent labeling of GC shows that it is confined to the lumen or superficial mucosa of the nasal cavity, without involvement of adjacent or deeper tissues. Our findings demonstrate a new application for soluble immunoadjuvants such as GC for preventing disease associated with SARS-CoV-2 and may be particularly attractive to persons who are needle-averse.

MAVS signaling is required for preventing persistent chikungunya heart infection and chronic vascular tissue inflammation.

Chikungunya virus (CHIKV) infection has been associated with severe cardiac manifestations, yet, how CHIKV infection leads to heart disease remains unknown. Here, we leveraged both mouse models and human primary cardiac cells to define the mechanisms of CHIKV heart infection. Using an immunocompetent mouse model of CHIKV infection as well as human primary cardiac cells, we demonstrate that CHIKV directly infects and actively replicates in cardiac fibroblasts. In immunocompetent mice, CHIKV is cleared from cardiac tissue without significant damage through the induction of a local type I interferon response from both infected and non-infected cardiac cells. Using mice deficient in major innate immunity signaling components, we found that signaling through the mitochondrial antiviral-signaling protein (MAVS) is required for viral clearance from the heart. In the absence of MAVS signaling, persistent infection leads to focal myocarditis and vasculitis of the large vessels attached to the base of the heart. Large vessel vasculitis was observed for up to 60 days post infection, suggesting CHIKV can lead to vascular inflammation and potential long-lasting cardiovascular complications. This study provides a model of CHIKV cardiac infection and mechanistic insight into CHIKV-induced heart disease, underscoring the importance of monitoring cardiac function in patients with CHIKV infections.

Identification of an Oligostilbene, Vaticanol B, from Dryobalanops aromatica Leaves as an Antiviral Compound against the Hepatitis C Virus.

Chronic hepatitis C virus (HCV) infection can lead to liver cirrhosis and hepatocellular carcinoma. Although current medications using direct-acting antivirals (DAAs) are highly effective and well-tolerated for treating patients with chronic HCV, high prices and the existence of DAA-resistant variants hamper treatment. There is thus a need for easily accessible antivirals with different mechanisms of action. During the screening of Indonesian medicinal plants for anti-HCV activity, we found that a crude extract of Dryobalanops aromatica leaves possessed strong antiviral activity against HCV. Bioassay-guided purification identified an oligostilbene, vaticanol B, as an active compound responsible for the anti-HCV activity. Vaticanol B inhibited HCV infection in a dose-dependent manner with 50% effective and cytotoxic concentrations of 3.6 and 559.5 µg/mL, respectively (Selectivity Index: 155.4). A time-of-addition study revealed that the infectivity of HCV virions was largely lost upon vaticanol B pretreatment. Also, the addition of vaticanol B following viral entry slightly but significantly suppressed HCV replication and HCV protein expression in HCV-infected and a subgenomic HCV replicon cells. Thus, the results clearly demonstrated that vaticanol B acted mainly on the viral entry step, while acting weakly on the post-entry step as well. Furthermore, co-treatment of the HCV NS5A inhibitor daclatasvir with vaticanol B increased the anti-HCV effect. Collectively, the present study has identified a plant-derived oligostilbene, vaticanol B, as a novel anti-HCV compound.

Susceptibility of perivenous macrophages to PRRSV-1 subtype 1 LV and PRRSV-1 subtype 3 Lena using a new vein explant model.

Vessel pathology such as increased permeability and blue discoloration is frequently observed with highly pathogenic PRRSV strains. However, data concerning the viral replication in the environment of blood vessels are absent. In the present study, ex vivo models with swine ear and hind leg vein explants were established to study the interaction of PRRSV-1 subtype 1 reference strain LV and highly pathogenic subtype 3 strain Lena with perivenous macrophages. The replication characteristics of these two strains were compared in vein explants by immunofluorescence analysis. The explants maintained a good viability during 48 hours of in vitro culture. We found that CD163-positive macrophages were mainly present around the veins and their number gradually decreased with increasing distance from the veins and longer incubation time. More CD163+Sn- cells than CD163+Sn+ cells (6.6 times more) were observed in the vein explants. The Lena strain demonstrated a higher replication level than the LV strain, with approximately 1.4-fold more infected cells in the surrounding areas of the ear vein and 1.1-fold more infected cells in the leg vein explants at 48 hours post inoculation. In both LV and Lena inoculated vein explants, most infected cells were identified as CD163+Sn+ (> 94%). In this study, an ex vivo vein model was successfully established, and our findings will contribute to a better understanding of the vein pathology during viral infections (e.g., PRRS, classical and African swine fever).

Inhibition of Hepatitis B Virus (HBV) replication and antigen expression by Brucea javanica (L.) Merr. oil emulsion.

Introduction: The seeds of Brucea javanica (L.) Merr. (BJ) have been traditionally used to treat various types of cancers for many years in China. In this study, we systematically investigated a BJ oil emulsion (BJOE) produced from BJ seeds with the purpose of evaluating its antiviral effect against hepatitis B virus (HBV). Methods: HepG2.215 (a wild-type HBV cell line), HepG2, and Huh7, transfected with wildtype (WT) or lamivudine-resistance mutant (LMV-MT) HBV replicon plasmids, were treated with different doses of BJOE and then used for pharmacodynamic evaluation. Cell viability was determined using CCK8 assay. The levels of HBsAg/HBeAg in cell cultured supernatant, HBcAg in cell lysis solution, and HBV DNA in both were evaluated. Results: BJOE at ≤5 mg/ml was nontoxic to carcinoma cell lines, but could significantly inhibit WT/LMV-MT HBV replication and HBs/e/c antigen expression in a dose-dependent manner by upregulating interleukin-6 (IL-6), demonstrating that it possesses moderate anti-HBV activity. As one of the major components of BJOE, bruceine B was found to play a dominant role in IL-6 induction and HBV inhibition. Discussion: Our results demonstrated that BJOE suppressed HBV replication by stimulating IL-6, indicating that it has promising clinical therapeutic potential for both WT and LMV-MT HBV.

Comparison of genome replication fidelity between SARS-CoV-2 and influenza A virus in cell culture.

Since the emergence of COVID-19, several SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) variants have emerged and spread widely. These variants are produced through replication errors of the viral genome by viral RNA-dependent RNA polymerase (RdRp). Seasonal epidemics of influenza are also known to occur because of new variants of influenza A virus (IAV), which are generated by the introduction of mutations by viral RdRp with low fidelity. Variants with different antigenicities appear because of mutations in envelope glycoproteins. In this study, we calculated and compared the mutation rates in genome replication of IAV and SARS-CoV-2. Average mutation rates per passage were 9.01 × 10-5 and 3.76 × 10-6 substitutions/site for IAV and SARS-CoV-2, respectively. The mutation rate of SARS-CoV-2 was 23.9-fold lower than that of IAV because of the proofreading activity of the SARS-CoV-2 RdRp complex. Our data could be useful in establishing effective countermeasures against COVID-19.

SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication.

COVID-19, caused by SARS-CoV-2, is a respiratory disease associated with inflammation and endotheliitis. Mechanisms underling inflammatory processes are unclear, but angiotensin converting enzyme 2 (ACE2), the receptor which binds the spike protein of SARS-CoV-2 may be important. Here we investigated whether spike protein binding to ACE2 induces inflammation in endothelial cells and determined the role of ACE2 in this process. Human endothelial cells were exposed to SARS-CoV-2 spike protein, S1 subunit (rS1p) and pro-inflammatory signaling and inflammatory mediators assessed. ACE2 was modulated pharmacologically and by siRNA. Endothelial cells were also exposed to SARS-CoV-2. rSP1 increased production of IL-6, MCP-1, ICAM-1 and PAI-1, and induced NFkB activation via ACE2 in endothelial cells. rS1p increased microparticle formation, a functional marker of endothelial injury. ACE2 interacting proteins involved in inflammation and RNA biology were identified in rS1p-treated cells. Neither ACE2 expression nor ACE2 enzymatic function were affected by rSP1. Endothelial cells exposed to SARS-CoV-2 virus did not exhibit viral replication. We demonstrate that rSP1 induces endothelial inflammation via ACE2 through processes that are independent of ACE2 enzymatic activity and viral replication. We define a novel role for ACE2 in COVID-19- associated endotheliitis.

The hope and hype of ellagic acid and urolithins as ligands of SARS-CoV-2 Nsp5 and inhibitors of viral replication.

Non-structural protein 5 (Nsp5) is a cysteine protease that plays a key role in SARS-CoV-2 replication, suppressing host protein synthesis and promoting immune evasion. The investigation of natural products as a potential strategy for Nsp5 inhibition is gaining attention as a means of developing antiviral agents. In this work, we have investigated the physicochemical properties and structure-activity relationships of ellagic acid and its gut metabolites, urolithins A-D, as ligands of Nsp5. Results allow us to identify urolithin D as promising ligand of Nsp5, with a dissociation constant in the nanomolar range of potency. Although urolithin D is able to bind to the catalytic cleft of Nsp5, the appraisal of its viral replication inhibition against SARS-CoV-2 in Vero E6 assay highlights a lack of activity. While these results are discussed in the framework of the available literature reporting conflicting data on polyphenol antiviral activity, they provide new clues for natural products as potential viral protease inhibitors.