A multicellular liver organoid model for investigating hepatitis C virus infection and nonalcoholic fatty liver disease progression.

BACKGROUND AND AIMS: HCV infection can be successfully managed with antiviral therapies; however, progression to chronic liver disease states, including NAFLD, is common. There is currently no reliable in vitro model for investigating host-viral interactions underlying the link between HCV and NAFLD; although liver organoids (LOs) show promise, they currently lack nonparenchymal cells, which are key to modeling disease progression. APPROACH AND RESULTS: Here, we present a novel, multicellular LO model using a coculture system of macrophages and LOs differentiated from the same human pluripotent stem cells (PSCs). The cocultured macrophages shifted toward a Kupffer-like cell type, the liver-resident macrophages present in vivo , providing a suitable model for investigating NAFLD pathogenesis. With this multicellular Kupffer-like cell-containing LO model, we found that HCV infection led to lipid accumulation in LOs by upregulating host lipogenesis, which was more marked with macrophage coculture. Reciprocally, long-term treatment of LOs with fatty acids upregulated HCV amplification and promoted inflammation and fibrosis. Notably, in our Kupffer-like cell-containing LO model, the effects of 3 drugs for NASH that have reached phase 3 clinical trials exhibited consistent results with the clinical outcomes. CONCLUSIONS: Taken together, we introduced a multicellular LO model consisting of hepatocytes, Kupffer-like cells, and HSCs, which recapitulated host-virus intercommunication and intercellular interactions. With this novel model, we present a physiologically relevant system for the investigation of NAFLD progression in patients with HCV.

Thiophen urea derivatives as a new class of hepatitis C virus entry inhibitors.

To develop unique small-molecule inhibitors of hepatitis C virus (HCV), thiophen urea (TU) derivatives were synthesised and screened for HCV entry inhibitory activities. Among them, seven TU compounds exhibited portent anti-viral activities against genotypes 1/2 (EC50 < 30 nM) and subsequently, they were further investigated; based on the pharmacological, metabolic, pharmacokinetic, and safety profiles, J2H-1701 was selected as the optimised lead compound as an HCV entry inhibitor. J2H-1701 possesses effective multi-genotypic antiviral activity. The docking results suggested the potential interaction of J2H-1701 with the HCV E2 glycoprotein. These results suggest that J2H-1701 can be a potential candidate drug for the development of HCV entry inhibitors.

High Environmental Stability of Hepatitis B Virus and Inactivation Requirements for Chemical Biocides.

Hepatitis B virus (HBV) infection is considered a major public health problem worldwide, and a significant number of reports on nosocomial and occupational outbreaks have been reported. This systematic investigation of HBV stability and susceptibility to different antiseptics revealed that HBV infectivity was very stable, with a half-life of >22 days at 37°C. At 4°C, infectivity was barely reduced for up to 9 months. Different alcohols and commercially available hand antiseptics had a virucidal effect against HBV. We propose that very strict compliance with established hygienic guidelines should be mandatory to avoid and prevent HBV infections.

Efficient long-term amplification of hepatitis B virus isolates after infection of slow proliferating HepG2-NTCP cells.

BACKGROUND & AIMS: As hepatitis B virus (HBV) spreads through the infected liver it is simultaneously secreted into the blood. HBV-susceptible in vitro infection models do not efficiently amplify viral progeny or support cell-to-cell spread. We sought to establish a cell culture system for the amplification of infectious HBV from clinical specimens. METHODS: An HBV-susceptible sodium-taurocholate cotransporting polypeptide-overexpressing HepG2 cell clone (HepG2-NTCPsec+) producing high titers of infectious progeny was selected. Secreted HBV progeny were characterized by native gel electrophoresis and electron microscopy. Comparative RNA-seq transcriptomics was performed to quantify the expression of host proviral and restriction factors. Viral spread routes were evaluated using HBV entry- or replication inhibitors, visualization of viral cell-to-cell spread in reporter cells, and nearest neighbor infection determination. Amplification kinetics of HBV genotypes B-D were analyzed. RESULTS: Infected HepG2-NTCPsec+ secreted high levels of large HBV surface protein-enveloped infectious HBV progeny with typical appearance under electron microscopy. RNA-seq transcriptomics revealed that HBV does not induce significant gene expression changes in HepG2-NTCPsec+, however, transcription factors favoring HBV amplification were more strongly expressed than in less permissive HepG2-NTCPsec-. Upon inoculation with HBV-containing patient sera, rates of infected cells increased from 10% initially to 70% by viral spread to adjacent cells, and viral progeny and antigens were efficiently secreted. HepG2-NTCPsec+ supported up to 1,300-fold net amplification of HBV genomes depending on the source of virus. Viral spread and amplification were abolished by entry and replication inhibitors; viral rebound was observed after inhibitor discontinuation. CONCLUSIONS: The novel HepG2-NTCPsec+ cells efficiently support the complete HBV life cycle, long-term viral spread and amplification of HBV derived from patients or cell culture, resembling relevant features of HBV-infected patients. LAY SUMMARY: Currently available laboratory systems are unable to reproduce the dynamics of hepatitis B virus (HBV) spread through the infected liver and release into the blood. We developed a slowly dividing liver-derived cell line which multiplies infectious viral particles upon inoculation with patient- or cell culture-derived HBV. This new infection model can improve therapy by measuring, in advance, the sensitivity of a patient's HBV strain to specific antiviral drugs.

TALEN/CRISPR-mediated engineering of a promoterless anti-viral RNAi hairpin into an endogenous miRNA locus.

Successful RNAi applications depend on strategies allowing robust and persistent expression of minimal gene silencing triggers without perturbing endogenous gene expression. Here, we propose a novel avenue which is integration of a promoterless shmiRNA, i.e. a shRNA embedded in a micro-RNA (miRNA) scaffold, into an engineered genomic miRNA locus. For proof-of-concept, we used TALE or CRISPR/Cas9 nucleases to site-specifically integrate an anti-hepatitis C virus (HCV) shmiRNA into the liver-specific miR-122/hcr locus in hepatoma cells, with the aim to obtain cellular clones that are genetically protected against HCV infection. Using reporter assays, Northern blotting and qRT-PCR, we confirmed anti-HCV shmiRNA expression as well as miR-122 integrity and functionality in selected cellular progeny. Moreover, we employed a comprehensive battery of PCR, cDNA/miRNA profiling and whole genome sequencing analyses to validate targeted integration of a single shmiRNA molecule at the expected position, and to rule out deleterious effects on the genomes or transcriptomes of the engineered cells. Importantly, a subgenomic HCV replicon and a full-length reporter virus, but not a Dengue virus control, were significantly impaired in the modified cells. Our original combination of DNA engineering and RNAi expression technologies benefits numerous applications, from miRNA, genome and transgenesis research, to human gene therapy.

Suppression of Hepatitis C Virus Genome Replication and Particle Production by a Novel Diacylglycerol Acyltransferases Inhibitor.

Diacylglycerol acyltransferases (DGATs) play a critical role in the biosynthesis of endogenous triglycerides (TGs) and formation of lipid droplets (LDs) in the liver. In particular, one member of DGATs, DGAT-1 was reported to be an essential host factor for the efficient production of hepatitis C virus (HCV) particles. By utilizing our previously characterized three different groups of twelve DGAT inhibitors, we found that one of the DGAT inhibitors, a 2-((4-adamantylphenoxy) methyl)-N-(furan-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxam (10j) is a potent suppressor of both HCV genome replication and particle production. 10j was able to induce inhibition of these two critical viral functions in a mutually separate manner. Abrogation of the viral genome replication by 10j led to a significant reduction in the viral protein expression as well. Interestingly, we found that its antiviral effect did not depend on the reduction of TG biosynthesis by 10j. This suggests that the inhibitory activity of 10j against DGATs may not be directly related with its antiviral action.

Virucidal Activity of World Health Organization-Recommended Formulations Against Enveloped Viruses, Including Zika, Ebola, and Emerging Coronaviruses.

The World Health Organization (WHO) published 2 alcohol-based formulations to be used in healthcare settings and for outbreak-associated infections, but inactivation efficacies of these products have not been determined against (re-)emerging viruses. In this study, we evaluated the virucidal activity of these WHO products in a comparative analysis. Zika virus (ZIKV), Ebola virus (EBOV), severe acute respiratory syndrome coronavirus (SARS-CoV), and Middle East respiratory syndrome coronavirus (MERS-CoV) as (re-)emerging viral pathogens and other enveloped viruses could be efficiently inactivated by both WHO formulations, implicating their use in healthcare systems and viral outbreak situations.

DDX60L Is an Interferon-Stimulated Gene Product Restricting Hepatitis C Virus Replication in Cell Culture.

UNLABELLED: All major types of interferon (IFN) efficiently inhibit hepatitis C virus (HCV) replication in vitro and in vivo. Remarkably, HCV replication is not sensitive to IFN-γ in the hepatoma cell line Huh6, despite an intact signaling pathway. We performed transcriptome analyses between Huh6 and Huh-7 cells to identify effector genes of the IFN-γ response and thereby identified the DExD/H box helicase DEAD box polypeptide 60-like (DDX60L) as a restriction factor of HCV replication. DDX60L and its homolog DEAD box polypeptide 60 (DDX60) were both induced upon viral infection and IFN treatment in primary human hepatocytes. However, exclusively DDX60L knockdown increased HCV replication in Huh-7 cells and rescued HCV replication from type II IFN as well as type I and III IFN treatment, suggesting that DDX60L is an important effector protein of the innate immune response against HCV. In contrast, we found no impact of DDX60L on replication of hepatitis A virus. DDX60L protein was detectable only upon strong ectopic overexpression, displayed a broad cytoplasmic distribution, but caused cytopathic effects under these conditions. DDX60L knockdown did not alter interferon-stimulated gene (ISG) induction after IFN treatment but inhibited HCV replication upon ectopic expression, suggesting that it is a direct effector of the innate immune response. It most likely inhibits viral RNA replication, since we found neither impact of DDX60L on translation or stability of HCV subgenomic replicons nor additional impact on assembly of infectious virus. Similar to DDX60, DDX60L had a moderate impact on RIG-I dependent activation of innate immunity, suggesting additional functions in the sensing of viral RNA. IMPORTANCE: Interferons induce a plethora of interferon-stimulated genes (ISGs), which are our first line of defense against viral infections. In addition, IFNs have been used in antiviral therapy, in particular against the human pathogen hepatitis C virus (HCV); still, their mechanism of action is not well understood, since diverse, overlapping sets of antagonistic effector ISGs target viruses with different biologies. Our work identifies DDX60L as a novel factor that inhibits replication of HCV. DDX60L expression is regulated similarly to that of its homolog DDX60, but our data suggest that it has distinct functions, since we found no contribution of DDX60 in combatting HCV replication. The identification of novel components of the innate immune response contributes to a comprehensive understanding of the complex mechanisms governing antiviral defense.

DDX3 DEAD-box RNA helicase is a host factor that restricts hepatitis B virus replication at the transcriptional level.

UNLABELLED: DDX3 is a member of the DEAD-box RNA helicase family, involved in mRNA metabolism, including transcription, splicing, and translation. We previously identified DDX3 as a hepatitis B virus (HBV) polymerase (Pol) binding protein, and by using a transient transfection, we found that DDX3 inhibits HBV replication at the posttranscriptional level, perhaps following encapsidation. To determine the exact mechanism of the inhibition, we here employed a diverse HBV experimental system. Inconsistently, we found that DDX3-mediated inhibition occurs at the level of transcription. By using tetracycline-inducible HBV-producing cells, we observed that lentivirus-mediated DDX3 expression led to a reduced level of HBV RNAs. Importantly, knockdown of DDX3 by short hairpin RNA resulted in augmentation of HBV RNAs in two distinct HBV replication systems: (i) tetracycline-inducible HBV-producing cells and (ii) constitutive HBV-producing HepG2.2.15 cells. Moreover, DDX3 knockdown in HBV-susceptible HepG2-NTCP cells, where covalently closed circular DNA (cccDNA) serves as the template for viral transcription, resulted in increased HBV RNAs, validating that transcription regulation by DDX3 occurs on a physiological template. Overall, our results demonstrate that DDX3 represents an intrinsic host antiviral factor that restricts HBV transcription. IMPORTANCE: Upon entry into host cells, viruses encounter host factors that restrict viral infection. During evolution, viruses have acquired the ability to subvert cellular factors that adversely affect their replication. Such host factors include TRIM5α and APOBEC3G, which were discovered in retroviruses. The discovery of host restriction factors provided deeper insight into the innate immune response and viral pathogenesis, leading to better understanding of host-virus interactions. In contrast to the case with retroviruses, little is known about host factors that restrict hepatitis B virus (HBV), a virus distantly related to retroviruses. DDX3 DEAD box RNA helicase is best characterized as an RNA helicase involved in RNA metabolism, such as RNA processing and translation. Here, we show that DDX3 inhibits HBV infection at the level of viral transcription.

Identification of type I and type II interferon-induced effectors controlling hepatitis C virus replication.

UNLABELLED: Persistent infection with hepatitis C virus (HCV) can lead to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. All current therapies of hepatitis C include interferon-alpha (IFN-α). Moreover, IFN-gamma (IFN-γ), the only type II IFN, strongly inhibits HCV replication in vitro and is the primary mediator of HCV-specific antiviral T-cell responses. However, for both cytokines the precise set of effector protein(s) responsible for replication inhibition is not known. The aim of this study was the identification of IFN-α and IFN-γ stimulated genes (ISGs) responsible for controlling HCV replication. We devised an RNA interference (RNAi)-based "gain of function" screen and identified, in addition to known ISGs earlier reported to suppress HCV replication, several new ones with proven antiviral activity. These include IFIT3 (IFN-induced protein with tetratricopeptide repeats 3), TRIM14 (tripartite motif containing 14), PLSCR1 (phospholipid scramblase 1), and NOS2 (nitric oxide synthase 2, inducible). All ISGs identified in this study were up-regulated both by IFN-α and IFN-γ, demonstrating a substantial overlap of HCV-specific effectors induced by either cytokine. Nevertheless, some ISGs were more specific for IFN-α or IFN-γ, which was most pronounced in case of PLSCR1 and NOS2 that were identified as main effectors of IFN-γ-mediated anti-HCV activity. Combinatorial knockdowns of ISGs suggest additive or synergistic effects demonstrating that with either IFN, inhibition of HCV replication is caused by the combined action of multiple ISGs. CONCLUSION: Our study identifies a number of novel ISGs contributing to the suppression of HCV replication by type I and type II IFN. We demonstrate a substantial overlap of antiviral programs triggered by either cytokine and show that suppression of HCV replication is mediated by the concerted action of multiple effectors.

Identification of a series of 1,3,4-trisubstituted pyrazoles as novel hepatitis C virus entry inhibitors.

In this report we describe the identification of novel pyrazole analogs as potent hepatitis C virus (HCV) entry inhibitor. The pyrazoles were identified by our phenotypic high-throughput screening using infectious HCV. A series of pyrazole derivatives was synthesized and evaluated for inhibitory activity against HCV in the infectious cell culture system. Through evaluation of selected compounds we observed that the pyrazoles did not interfere with HCV RNA replication but with viral entry as shown by experiments with HCV replicons and HCV pseudo particles, respectively.

Identification of Zika virus NS2B-NS3 protease and NS5 polymerase inhibitors by structure-based virtual screening of FDA-approved drugs.

Zika virus (ZIKV) is a mosquito-borne human flavivirus responsible that causing emergency outbreaks in Brazil. ZIKV is suspected of causing Guillain-Barre syndrome in adults and microcephaly. The NS2B-NS3 protease and NS5 RNA-dependent RNA polymerase (RdRp), central to ZIKV multiplication, have been identified as attractive molecular targets for drugs. We performed a structure-based virtual screening of 2,659 FDA-approved small molecule drugs in the DrugBank database using AutoDock Vina in PyRx v0.8. Accordingly, 15 potential drugs were selected as ZIKV inhibitors because of their high values (binding affinity - binding energy) and we analyzed the molecular interactions between the active site amino acids and the compounds. Among these drugs, tamsulosin was found to interact most efficiently with NS2B/NS3 protease, as indicated by the lowest binding energy value (-8.27 kJ/mol), the highest binding affinity (-5.7 Kcal/mol), and formed H-bonds with amino acid residues TYRB130, SERB135, TYRB150. Furthermore, biotin was found to interact most efficiently with NS5 RdRp with a binding energy of -150.624 kJ/mol, a binding affinity of -5.6 Kcal/mol, and formed H-bonds with the amino acid residues ASPA665 and ASPA540. In vitro, in vivo, and clinical studies are needed to demonstrate anti-ZIKV safety and the efficacy of these FDA-approved drug candidates.Communicated by Ramaswamy H. Sarma.

Design of a multi-epitope Zika virus vaccine candidate - an in-silico study.

Zika virus (ZIKV), an RNA virus, rapidly spreads Aedes mosquito-borne sickness. Currently, there are neither effective vaccines nor therapeutics available to prevent or treat ZIKV infection. In this study, to address these unmet medical needs, we aimed to design B- and T-cell candidate multi-epitope-based subunit against ZIKV using an in silico approach. In this study we applied immunoinformatics, molecular docking, and dynamic simulation assessments targeting the most immunogenic proteins; the capsid (C), envelope (E) proteins and the non-stuctural protein (NS1), described in our previous study, and which predicted immunodominant B and T cell epitopes. The final non-allergenic and highly antigenic multi-epitope was constituted of immunogenic screened-epitopes (3 CTL and 3 HTL) and the ß-defensin as an adjuvant that have been linked using EAAAK, AAY, and GPGPG linkers, respectively. The final construct containing 143 amino acids was characterized for its allergenicity, antigenicity, and physiochemical properties; and found to be safe and immunogenic with a good prediction of solubility. The existence of IFN-γ epitopes asserts the capacity to trigger strong immune responses. Subsequently, the molecular docking among vaccine and immune receptors (TLR2/TLR4) was revealed with a good binding affinity with and stable molecular interactions. Molecular dynamics simulation confirmed the stability of the complexes. Finally, the construct was subjected to in silico cloning demonstrating the efficiently of its expression in E.coli. However, this study needs the experimental validation to demonstrate vaccine safety and efficacy.Communicated by Ramaswamy H. Sarma.

Mouse hepatic cells support assembly of infectious hepatitis C virus particles.

BACKGROUND & AIMS: Hepatitis C virus (HCV) has a high propensity to establish persistence; better understanding of this process requires the development of a fully permissive and immunocompetent small animal model. Mouse cells can be engineered to express the human orthologs of the entry molecules CD81 and occludin to allow entry of HCV. However, RNA replication is poor in mouse cells, and it is not clear whether they support assembly and release of infectious HCV particles. We used a trans-complementation-based system to demonstrate HCV assembly competence of mouse liver cell lines. METHODS: A panel of 3 mouse hepatoma cell lines that contain a stable subgenomic HCV replicon was used for ectopic expression of the HCV structural proteins, p7, nonstructural protein 2, and/or apolipoprotein E (apoE). Assembly and release of infectious HCV particles was determined by measuring viral RNA, proteins, and infectivity of virus released into the culture supernatant. RESULTS: Mouse replicon cells released low amounts of HCV particles, but ectopic expression of apoE increased release of infectious HCV to levels observed in the human hepatoma cell line Huh7.5. Thus, apoE is the limiting factor for assembly of HCV in mouse hepatoma cells but probably not in primary mouse hepatocytes. Products of all 3 human alleles of apoE and mouse apoE support HCV assembly with comparable efficiency. Mouse and human cell-derived HCV particles have similar biophysical properties, dependency on entry factors, and levels of association with apoE. CONCLUSIONS: Mouse hepatic cells permit HCV assembly and might be developed to create an immunocompetent and fully permissive mouse model of HCV infection.

Discovery and characterization of a novel 7-aminopyrazolo[1,5-a]pyrimidine analog as a potent hepatitis C virus inhibitor.

We describe a novel 7-aminopyrazolo[1,5-a]pyrimidine (7-APP) derivative as a potent hepatitis C virus (HCV) inhibitor. A series of 7-APPs was synthesized and evaluated for inhibitory activity against HCV in different cell culture systems. The synthesis and preliminary structure-activity relationship study of 7-APP are reported.

Optimization of BRET saturation assays for robust and sensitive cytosolic protein-protein interaction studies.

Bioluminescence resonance energy transfer (BRET) saturation is a method of studying protein-protein interaction (PPI) upon quantification of the dependence of the BRET signal on the acceptor/donor (A:D) expression ratio. In this study, using the very bright Nluc/YFP BRET pair acquired respectively with microplate reader and automated confocal microscopy, we significantly improved BRET saturation assay by extending A:D expression detection range and normalizing A:D expression with a new BRET-free probe. We next found that upon using variable instead of fixed amount of donor molecules co-expressed with increasing acceptor concentrations, BRET saturation assay robustness can be further improved when studying cytosolic protein, although the relative amounts of dimers (BRETmax) and the relative dimer affinity (BRET50) remain similar. Altogether, we show that our method can be applied to many PPI networks, involving the NF-κB pathway, high-affinity nanobody, rabies virus-host interactions, mTOR complex and JAK/STAT signaling. Altogether our approach paves the way for robust PPI validation and characterization in living cells.

Drug repurposing through virtual screening and in vitro validation identifies tigecycline as a novel putative HCV polymerase inhibitor.

The repurposing of marketed drugs for new indications is an elegant strategy to quickly and cost-efficiently address unmet medical needs. The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has been shown to be a valid drug target. We performed structure-based virtual screening to assess the off-label utilization of existing drugs as novel HCV inhibitors. The virtual screen showed that tigecycline could potentially dock with high affinity to the palm site of the HCV RdRp. In vitro validation showed that tigecycline had therapeutic indexes (CC50/EC50) greater than 13 and 6.5 against infectious HCV and subgenomic HCV replicons, respectively. Furthermore, tigecycline displayed synergistic activity with sofosbuvir and daclatasvir against HCV. In silico screening identified tigecycline as a putative inhibitor of HCV RdRp, which was validated in vitro and demonstrated synergistic effects in combination with first-line anti-HCV therapies.

Selected ginsenosides interfere efficiently with hepatitis B virus mRNA expression levels and suppress viral surface antigen secretion.

Ginsenosides are a class of natural steroid glycosides and triterpene saponins found in Panax ginseng. After screening of a commercial ginsenoside compound library for low cellular cytotoxicity and the ability to mediate efficient reductions in hepatitis B virus (HBV) mRNA expression levels in HepG2.2.15 cells, three ginsenosides (Rg6, Rh4, and Rb3) are selected. Thereafter, using the same cellular model, all three ginsenosides are shown to mediate efficient, selective inhibition of HBV mRNA expression levels, and also interfere with the secretion of both HBV particles and hepatitis B surface antigen (HBsAg). Drug combination studies are performed in both HepG2.2.15 and HBV-infected HepG2-NTCPsec+ cell models with the selected ginsenosides and lamivudine (LMV), a nucleoside analogue used to treat chronic hepatitis B (CHB) infections. These studies, involving RT-qPCR and ELISA, suggest that Rh4/LMV combinations in particular act synergistically to inhibit the secretion of HBV particles and HBsAg. Therefore, on the assumption that appropriate in vivo data are in future agreement, Rh4, in particular, might be used in combination with nucleoside/nucleotide analogues (NUCs) to devise an effective, cost-efficient combination therapy for the treatment of patients with CHB infections.

Nanobodies: an unexplored opportunity to combat COVID-19.

Coronavirus disease 2019 (COVID-19) is a highly contagious disease caused by severe acute respiratory coronavirus 2 (SARS-CoV-2). This virus is capable of human-to-human transmission, and is spreading rapidly round the globe, with markedly high fatality rates. Unfortunately, there are neither vaccines nor specific therapies available to combat it, and the developments of such approaches depend on pursuing multiple avenues in biomedical science. Accordingly, in this paper we highlight one such avenue-nanobodies-for potential utility in therapeutic and diagnostic interventions to combat COVID-19.Communicated by Ramaswamy H. Sarma.