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.

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.

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.

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.

Mono- and combinational drug therapies for global viral pandemic preparedness.

Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies.

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.

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.

Synergistic Interferon-Alpha-Based Combinations for Treatment of SARS-CoV-2 and Other Viral Infections.

BACKGROUND: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. METHODS: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro. RESULTS: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies. CONCLUSIONS: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

Determination of infectious hepatitis B virus particles by an end-point dilution assay identifies a novel class of inhibitors.

The quantification of infectious virus particles is fundamental to perform in vitro virology studies. To determine the number of hepatitis B virus (HBV) genome-containing particles in vitro, the genome equivalents (GEq) are measured using quantitative PCR (qPCR). However, in addition to infectious virions, HBV DNA-containing, non-infectious HBV particles are also produced in vitro, which can lead to an over-estimation of the number of infectious HBV particles when analyzed by qPCR. Here, we establish an end-point dilution assay that can precisely determine the number of infectious HBV particles. The cell-based HBV infection assay uses a 384-well plate format and enables the calculation of the 50% tissue culture infective dose (TCID50) in a semi-automated manner. Cell culture-derived HBV (HBVcc), produced by either stable HBV-replicating cells (HepAD38) or HBV-infected HepG2-NTCP cells, as well as patient-derived HBV sera were serially diluted and used to infect naïve target cells. Applying the end-point dilution assay, we infected HepG2-NTCP cells with PEG precipitated HBV derived from HepAD38-and HepG2-NTCPsec+ cell supernatants, calculated the TCID50/mL, converted to plaque-forming units (PFUs), and generated the specific infectivity (ratio of PFU/GEq). As a result, a TCID50/mL of 7.22 × 106 and 2.16 × 106, and the specific infectivity of 1/13,816 and 1/8798 were calculated for HepAD38 and HepG2-NTCPsec+ cell supernatants, respectively. The specific infectivity further increased by approximately 2-fold after removal of non-infectious "naked" particles by immunoprecipitation. Purification of HepAD38 cell supernatants by heparin columns increased the TCID50/mL and specific infectivity by 18- and 15-fold, respectively. Interestingly, non-purified patient-derived HBV sera from two individuals had a specific infectivity of 1/88 and 1/3609. After converting TCID50 to multiplicity of infection (MOI) values, we inoculated HepG2-NTCP cells with HBVcc based on GEq or MOI values and demonstrated that MOI-based infection leads to more reproducible infection rates. Furthermore, the assay was validated using serially diluted lamivudine, an HBV replication inhibitor, inhibiting HBV DNA secretion and infectious viral progeny by approx. 56- and 470-fold, respectively. Interestingly, we identified dexmedetomidine (DMM), an alpha-2 adrenergic agonist, inhibiting the secretion of infectious viral progeny by approx. 6-fold, without interfering in the secretion of HBV DNA. Taken together, we developed an assay that is suitable for the standard quantification of infectious HBV particles. We identified DMM as a novel inhibitor that exclusively interferes with the secretion of infectious HBV particles without affecting the secretion of HBV genomes. This end-point dilution assay enables the precise determination of the number of infectious HBV particles, assessment of the specific infectivity and stability of HBV particles, and identification of novel classes of HBV inhibitors.

Screening of FDA-Approved Drugs Using a MERS-CoV Clinical Isolate from South Korea Identifies Potential Therapeutic Options for COVID-19.

Therapeutic options for coronaviruses remain limited. To address this unmet medical need, we screened 5406 compounds, including United States Food and Drug Administration (FDA)-approved drugs and bioactives, for activity against a South Korean Middle East respiratory syndrome coronavirus (MERS-CoV) clinical isolate. Among 221 identified hits, 54 had therapeutic indexes (TI) greater than 6, representing effective drugs. The time-of-addition studies with selected drugs demonstrated eight and four FDA-approved drugs which acted on the early and late stages of the viral life cycle, respectively. Confirmed hits included several cardiotonic agents (TI > 100), atovaquone, an anti-malarial (TI > 34), and ciclesonide, an inhalable corticosteroid (TI > 6). Furthermore, utilizing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we tested combinations of remdesivir with selected drugs in Vero-E6 and Calu-3 cells, in lung organoids, and identified ciclesonide, nelfinavir, and camostat to be at least additive in vitro. Our results identify potential therapeutic options for MERS-CoV infections, and provide a basis to treat coronavirus disease 2019 (COVID-19) and other coronavirus-related illnesses.

Molecular basis of the interaction of the human tyrosine phosphatase PTPN3 with the hepatitis B virus core protein.

Interactions between the hepatitis B virus core protein (HBc) and host cell proteins are poorly understood, although they may be essential for the propagation of the virus and its pathogenicity. HBc has a C-terminal PDZ (PSD-95, Dlg1, ZO-1)-binding motif (PBM) that is responsible for interactions with host PDZ domain-containing proteins. In this work, we focused on the human protein tyrosine phosphatase non-receptor type 3 (PTPN3) and its interaction with HBc. We solved the crystal structure of the PDZ domain of PTPN3 in complex with the PBM of HBc, revealing a network of interactions specific to class I PDZ domains despite the presence of a C-terminal cysteine in this atypical PBM. We further showed that PTPN3 binds the HBc protein within capsids or as a homodimer. We demonstrate that overexpression of PTPN3 significantly affects HBV infection in HepG2 NTCP cells. Finally, we performed proteomics studies on both sides by pull-down assays and screening of a human PDZ domain library. We identified a pool of human PBM-containing proteins that might interact with PTPN3 in cells and that could be in competition with the HBc PBM during infection, and we also identified potential cellular partners of HBc through PDZ-PBM interactions. This study opens up many avenues of future investigations into the pathophysiology of HBV.

Immuno-informatics-based Identification of Novel Potential B Cell and T Cell Epitopes to Fight Zika Virus Infections.

BACKGROUND: Globally, the recent outbreak of Zika virus (ZIKV) in Brazil, Asia Pacific, and other countries highlighted the unmet medical needs. Currently, there are neither effective vaccines nor therapeutics available to prevent or treat ZIKV infection. OBJECTIVE: In this study, we aimed to design an epitope-based vaccine for ZIKV using an in silico approach to predict and analyze B- and T-cell epitopes. METHODS: The prediction of the most antigenic epitopes has targeted the capsid and envelope proteins as well as non-structural proteins NS5 and NS3 using immune-informatics tools PROTPARAM, CFSSP, PSIPRED, and Vaxijen v2.0. B and T-cell epitopes were predicted using ABCpred, IEDB, TepiTool, and their toxicity was evaluated using ToxinPred. The 3-dimensional epitope structures were generated by PEP-FOLD. Energy minimization was performed using Swiss- Pdb Viewer, and molecular docking was conducted using PatchDock and FireDock server. RESULTS: As a result, we predicted 307 epitopes of MHCI (major histocompatibility complex class I) and 102 epitopes of MHCII (major histocompatibility complex class II). Based on immunogenicity and antigenicity scores, we identified the four most antigenic MHC I epitopes: MVLAILAFLR (HLA-A*68:01), ETLHGTVTV (HLA-A*68:02), DENHPYRTW (HLA-B*44:02), QEGVFH TMW (HLA-B*44:03) and TASGRVIEEW (HLA-B*58:01), and MHC II epitopes: IIKKFKKDLAAMLRI (HLA-DRB3*02:02), ENSKMMLELDPPFGD (HLA-DRB3*01:01), HAET WFFDENHPYRT (HLA-DRB3*01:01), TDGVYRVMTRRLLGS (HLA-DRB1*11:01), and DGCW YGMEIRPRKEP (HLA-DRB5*01:01). CONCLUSION: This study provides novel potential B cell and T cell epitopes to fight against Zika virus infections and may prompt further development of vaccines against ZIKV and other emerging infectious diseases. However, further investigations for protective immune response by in vitro and in vivo studies to ratify immunogenicity, the safety of the predicted structure, and ultimately for the vaccine properties to prevent ZIKV infections are warranted.

Structure-guided discovery approach identifies potential lead compounds targeting Mpro of SARS-CoV-2.

The ongoing coronavirus disease 19 caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become fatal for the world with affected population crossing over 25 million in more than 217 countries, consequently declared a global pandemic by the World Health Organization. Unfortunately, neither specific prophylactic or therapeutic drugs nor vaccines are available. To address the unmet medical needs, we explored a strategy identifying new compounds targeting the main protease (Mpro) of SARS-CoV-2. Targeting the SARS-CoV-2 Mpro crystal structure (PDB ID: 6LU7) a combination of in silico screening, molecular docking, and dynamic approaches, a set of 5000 compounds of the ZINC database were screened. As a result, we identified and ranked the top 20 compounds based on the scores of ligand-interaction, their drug-likeness properties, and their predicted antiviral efficacies. The prominent drug-like and potent inhibitory compounds are 2-[2-(2-aminoacetyl) aminoacetyl] amino-3-(4-hydroxyphenyl)-propanamide (ZINC000004762511), 6'-fluoroaristeromycin (ZINC000001483267) and cyclo (L-histidyl-L-histidyl) (ZINC000005116916) scaffolds. Further in vitro and in vivo validations are required to demonstrate anti-SARS-CoV-2 activities.

Identification and Tracking of Antiviral Drug Combinations.

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.

Advanced Therapeutics, Vaccinations, and Precision Medicine in the Treatment and Management of Chronic Hepatitis B Viral Infections; Where Are We and Where Are We Going?

The management of chronic hepatitis B virus (CHB) infection is an area of massive unmet clinical need worldwide. In spite of the development of powerful nucleoside/nucleotide analogue (NUC) drugs, and the widespread use of immune stimulators such as interferon-alpha (IFNα) or PEGylated interferon-alpha (PEG-IFNα), substantial improvements in CHB standards of care are still required. We believe that the future for CHB treatment now rests with advanced therapeutics, vaccination, and precision medicine, if all are to bring under control this most resilient of virus infections. In spite of a plethora of active drug treatments, anti-viral vaccinations and diagnostic techniques, the management of CHB infection remains unresolved. The reason for this is the very complexity of the virus replication cycle itself, giving rise to multiple potential targets for therapeutic intervention some of which remain very intractable indeed. Our review is focused on discussing the potential impact that advanced therapeutics, vaccinations and precision medicine could have on the future management of CHB infection. We demonstrate that advanced therapeutic approaches for the treatment of CHB, in the form of gene and immune therapies, together with modern vaccination strategies, are now emerging rapidly to tackle the limitations of current therapeutic approaches to CHB treatment in clinic. In addition, precision medicine approaches are now gathering pace too, starting with personalized medicine. On the basis of this, we argue that the time has now come to accelerate the design and creation of precision therapeutic approaches (PTAs) for CHB treatment that are based on advanced diagnostic tools and nanomedicine, and which could maximize CHB disease detection, treatment, and monitoring in ways that could genuinely eliminate CHB infection altogether.

A Simple and Cost-Effective DNA Preparation Method Suitable for High-Throughput PCR Quantification of Hepatitis B Virus Genomes.

Hepatitis B virus (HBV) is a para-retrovirus that reverse transcribes its pregenomic RNA into relaxed circular DNA inside viral nucleocapsids. The number of HBV genomes produced in vitro is typically quantified using commercial silica-membrane-based nucleic acid purification kits to isolate total DNA followed by HBV-specific quantitative PCR (qPCR). However, despite the convenience of commercial kits, this procedure is costly and time-consuming due to multiple centrifugation steps, which produce unnecessary waste. Here, we report a rapid, cost-effective, and environmentally friendly total DNA preparation method. The assay is based on the simple incubation of detergent and proteinase K with cells or cell-free supernatants to permeabilize cells and disrupt viral particles. After heat inactivation and subsequent centrifugation to clear the lysates, DNA samples are directly subjected to qPCR to quantify HBV genomes. As a proof of concept, the assay was developed in 12-well plates to assess intra- and extracellular HBV genome equivalents (GEqs) of stably viral-replicating cell lines (e.g., HepAD38) and HBV-infected HepG2-NTCP cells, both treated with lamivudine (LMV), an HBV replication inhibitor. Viral DNA was also prepared from the serum of patients chronically infected with HBV. To validate the assay, a representative commercial DNA isolation kit was used side-by-side to isolate intra- and extracellular HBV DNA. Both methods yielded comparable amounts of HBV GEqs with comparable LMV 50% efficient concentration (EC50) values. The assay was subsequently adapted to 96- and 384-well microtiter plates using HepAD38 cells. The EC50 values were comparable to those obtained in 12-well plates. In addition, the calculated coefficient of variation, Z' values, and assay window demonstrated high reproducibility and quality. We devised a novel, robust, reproducible, high-throughput microtiter plate DNA preparation method suitable for quantifying HBV GEqs by qPCR analysis. This strategy enables rapid and convenient quantitative analysis of multiple viral DNA samples in parallel to investigate intracellular HBV replication and the secretion of DNA-containing viral particles.

Benzimidazole Derivatives as Novel Zika Virus Inhibitors.

We have synthesized 50 benzimidazole (BMZ) derivatives with 1,2-phenylenediamines and aromatic aldehydes under mild oxidation conditions by using inexpensive, nontoxic inorganic salt sodium metabisulfite in a one-pot condensation reaction and screened their ability to interfere with Zika virus (ZIKV) infection utilizing a cell-based phenotypic assay. Seven BMZs inhibited an African ZIKV strain with a selectivity index (SI=CC50 /EC50 ) of 9-37. Structure-activity relationship analysis demonstrated that substitution at the C-2, N-1, and C-5 positions of the BMZ ring were important for anti-ZIKV activity. The hybrid structure of BMZ and naphthalene rings was a structural feature responsible for the high anti-ZIKV activity. Importantly, BMZs inhibited ZIKV in human neural stem cells, a physiologically relevant system considering the severe congenital anomalies, like microcephaly, caused by ZIKV infection. Compound 39 displayed the highest antiviral efficacy against the African ZIKV strain in Huh-7 (SI>37) and neural stem cells (SI=12). Compound 35 possessed the highest activity in Vero cells (SI=115). Together, our data indicate that BMZs derivatives have to be considered for the development of ZIKV therapeutic interventions.

The Beginning of Ending Hepatitis C Virus: A Summary of the 26th International Symposium on Hepatitis C Virus and Related Viruses.

: Hepatitis C virus (HCV) infects ~71 million people worldwide, and 399,000 people die annually due to HCV-related liver cirrhosis and hepatocellular carcinoma. The use of direct-acting antivirals results in a sustained virologic response in >95% of patients with chronic HCV infection. However, several issues remain to be solved to eradicate HCV. At the 26th International Symposium on Hepatitis C Virus and Related Viruses (HCV2019) held in Seoul, South Korea, October 5-8, 2019, virologists, immunologists, and clinical scientists discussed these remaining issues and how we can achieve the elimination of HCV.

An Engineered Microvirin Variant with Identical Structural Domains Potently Inhibits Human Immunodeficiency Virus and Hepatitis C Virus Cellular Entry.

Microvirin (MVN) is one of the human immunodeficiency virus (HIV-1) entry inhibitor lectins, which consists of two structural domains sharing 35% sequence identity and contrary to many other antiviral lectins, it exists as a monomer. In this study, we engineered an MVN variant, LUMS1, consisting of two domains with 100% sequence identity, thereby reducing the chemical heterogeneity, which is a major factor in eliciting immunogenicity. We determined carbohydrate binding of LUMS1 through NMR chemical shift perturbation and tested its anti-HIV activity in single-round infectivity assay and its anti-hepatitis C virus (HCV) activity in three different assays including HCVcc, HCVpp, and replicon assays. We further investigated the effect of LUMS1 on the activation of T helper (Th) and B cells through flow cytometry. LUMS1 showed binding to (1-2)mannobiose, the minimum glycan epitope of MVN, potently inhibited HIV-1 and HCV with EC50 of 37.2 and 45.3 nM, respectively, and showed negligible cytotoxicity with CC50 > 10 µM against PBMCs, Huh-7.5 and HepG2 cells, and 4.9 µM against TZM-bl cells. LUMS1 did not activate Th cells, and its stimulatory effect on B cells was markedly less as compared to MVN. Together, with these effects, LUMS1 represents a potential candidate for the development of antiviral therapies.

Identification and characterization of a novel hepatitis B virus pregenomic RNA encapsidation inhibitor.

Currently, therapies to treat chronic hepatitis B (CHB) infection are based on the use of interferon-α or nucleos(t)ide analogs (NAs) to prevent viral DNA synthesis by inhibiting the reverse transcriptase activity of the hepatitis B virus (HBV) polymerase (Pol). However, these therapies are not curative; thus, the development of novel anti-HBV agents is needed. In accordance with this unmet medical need, we devised a new target- and cell-based, high-throughput screening assay to identify novel small molecules that block the initial interaction of the HBV Pol with its replication template the viral pregenomic RNA (pgRNA). We screened approximately 110,000 small molecules for the ability to prevent HBV Pol recognition of the pgRNA 5' epsilon (ε) stem-loop structure, identifying (Z)-2-(allylamino)-4-amino-N'-cyanothiazole-5-carboximidamide (AACC). Viral nucleocapsid-captured quantitative RT-PCR and Western blot results revealed that AACC significantly decreased encapsidated pgRNA levels and blocked capsid assembly without affecting core protein expression in stable HBV-replicating cells. As a result, both intra- and extracellular accumulation of viral DNA was strongly reduced. AACC treatment of HepG2-sodium taurocholate transporting polypeptide (NTCP) cells and primary human hepatocytes infected with cell culture- or patient-derived HBV isolates showed both time- and dose-dependent inhibition of infectious viral progeny and rcDNA production. Furthermore, AACC showed cross-genotypic activity against genotypes B, C, and D. Of note, AACC inhibited the viral replication of lamivudine and a capsid inhibitor-resistant HBV, and showed synergistic effects with NAs and a capsid inhibitor. In conclusion, we identified a novel class of compounds specifically targeting the ε-Pol interaction and thereby preventing the encapsidation of pgRNAs into viral capsids. This promising new HBV inhibitor class potently inhibits HBV amplification with distinct characteristics from existing NAs and other drugs currently under development, promising to add value to existing therapies for CHB.