Deep learning-based segmentation of subcellular organelles in high-resolution phase-contrast images.

Although quantitative analysis of biological images demands precise extraction of specific organelles or cells, it remains challenging in broad-field grayscale images, where traditional thresholding methods have been hampered due to complex image features. Nevertheless, rapidly growing artificial intelligence technology is overcoming obstacles. We previously reported the fine-tuned apodized phase-contrast microscopy system to capture high-resolution, label-free images of organelle dynamics in unstained living cells (Shimasaki, K. et al. (2024). Cell Struct. Funct., 49:21-29). We here showed machine learning-based segmentation models for subcellular targeted objects in phase-contrast images using fluorescent markers as origins of ground truth masks. This method enables accurate segmentation of organelles in high-resolution phase-contrast images, providing a practical framework for studying cellular dynamics in unstained living cells.Key words: Label-free imaging, Organelle dynamics, Apodized phase contrast, Deep learning-based segmentation.

A high-resolution phase-contrast microscopy system for label-free imaging in living cells.

Cell biologists have long sought the ability to observe intracellular structures in living cells without labels. This study presents procedures to adjust a commercially available apodized phase-contrast (APC) microscopy system for better visualizing the dynamic behaviors of various subcellular organelles in living cells. By harnessing the versatility of this technique to capture sequential images, we could observe morphological changes in cellular geometry after virus infection in real time without probes or invasive staining. The tune-up APC microscopy system is a highly efficient platform for simultaneously observing the dynamic behaviors of diverse subcellular structures with exceptional resolution.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne.

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3ß,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.

The combination of levodopa with levodopa-metabolizing enzyme inhibitors prevents severe fever with thrombocytopenia syndrome virus infection in vitro more effectively than single levodopa.

Severe fever with thrombocytopenia syndrome is a hemorrhagic fever caused by a tick-borne infection. The causative agent, Dabie bandavirus, is also called the severe fever with thrombocytopenia syndrome virus (SFTSV). Ogawa et al. (2022) reported that levodopa, an antiparkinsonian drug with an o-dihydroxybenzene backbone, which is important for anti-SFTSV activity, inhibited SFTSV infection. Levodopa is metabolized by dopa decarboxylase (DDC) and catechol-O-methyltransferase (COMT) in vivo. We evaluated the anti-SFTSV efficacy of two DDC inhibitors, benserazide hydrochloride and carbidopa, and two COMT inhibitors, entacapone and nitecapone, which also have an o-dihydroxybenzene backbone. Only DDC inhibitors inhibited SFTSV infection with pretreatment of the virus (half-maximal inhibitory concentration [IC50]: 9.0-23.6 µM), whereas all the drugs inhibited SFTSV infection when infected cells were treated (IC50: 21.3-94.2 µM). Levodopa combined with carbidopa and/or entacapone inhibited SFTSV infection in both conditions: pretreatment of the virus (IC50: 2.9-5.8 µM) and treatment of infected cells (IC50: 10.7-15.4 µM). The IC50 of levodopa in the above-mentioned study for pretreatment of the virus and treatment of infected cells were 4.5 and 21.4 µM, respectively. This suggests that a synergistic effect was observed, especially for treatment of infected cells, although the effect is unclear for pretreatment of the virus. This study demonstrates the anti-SFTSV efficacy of levodopa-metabolizing enzyme inhibitors in vitro. These drugs may increase the time for which the levodopa concentration is maintained in vivo. The combination of levodopa and levodopa-metabolizing enzyme inhibitors might be a candidate for drug repurposing.

Global Proteomics for Identifying the Alteration Pathway of Niemann-Pick Disease Type C Using Hepatic Cell Models.

Niemann-Pick disease type C (NPC) is an autosomal recessive disorder with progressive neurodegeneration. Although the causative genes were previously identified, NPC has unclear pathophysiological aspects, and patients with NPC present various symptoms and onset ages. However, various novel biomarkers and metabolic alterations have been investigated; at present, few comprehensive proteomic alterations have been reported in relation to NPC. In this study, we aimed to elucidate proteomic alterations in NPC and perform a global proteomics analysis for NPC model cells. First, we developed two NPC cell models by knocking out NPC1 using CRISPR/Cas9 (KO1 and KO2). Second, we performed a label-free (LF) global proteomics analysis. Using the LF approach, more than 300 proteins, defined as differentially expressed proteins (DEPs), changed in the KO1 and/or KO2 cells, while the two models shared 35 DEPs. As a bioinformatics analysis, the construction of a protein-protein interaction (PPI) network and an enrichment analysis showed that common characteristic pathways such as ferroptosis and mitophagy were identified in the two model cells. There are few reports of the involvement of NPC in ferroptosis, and this study presents ferroptosis as an altered pathway in NPC. On the other hand, many other pathways and DEPs were previously suggested to be associated with NPC, supporting the link between the proteome analyzed here and NPC. Therapeutic research based on these results is expected in the future.

The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione.

Viral spike proteins play important roles in the viral entry process, facilitating attachment to cellular receptors and fusion of the viral envelope with the cell membrane. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor angiotensin converting enzyme-2 (ACE2) via its receptor-binding domain (RBD). The cysteine residue at position 488, consisting of a disulfide bridge with cysteine 480 is located in an important structural loop at ACE2-binding surface of RBD, and is highly conserved among SARS-related coronaviruses. We showed that the substitution of Cys-488 with alanine impaired pseudotyped SARS-CoV-2 infection, syncytium formation, and cell-cell fusion triggered by SARS-CoV-2 spike expression. Consistently, in vitro binding of RBD and ACE2, spike-mediated cell-cell fusion, and pseudotyped viral infection of VeroE6/TMPRSS2 cells were inhibited by the thiol-reactive compounds N-acetylcysteine (NAC) and a reduced form of glutathione (GSH). Furthermore, we demonstrated that the activity of variant spikes from the SARS-CoV-2 alpha and delta strains were also suppressed by NAC and GSH. Taken together, these data indicate that Cys-488 in spike RBD is required for SARS-CoV-2 spike functions and infectivity, and could be a target of anti-SARS-CoV-2 therapeutics.

L-DOPA, a treatment for Parkinson's disease, and its enantiomer D-DOPA inhibit severe fever with thrombocytopenia syndrome virus infection in vitro.

Severe fever with thrombocytopenia syndrome (SFTS) is a hemorrhagic fever. Patients mainly develop fever, thrombocytopenia, and leukopenia. A high case fatality rate of 16.2-47% has been reported. Vaccines and antivirals that are effective against SFTS virus (SFTSV) are not yet available in clinical practice. We previously showed that o-dihydroxybenzene is the important chemical core structure for anti-SFTSV activity. In this study, we evaluated the anti-SFTSV efficacy of 3-Hydroxy-L-tyrosine (L-DOPA), a treatment for Parkinson's disease and its enantiomer, 3-hydroxy-D-tyrosine (D-DOPA), both of which have an o-dihydroxybenzene backbone. SFTSV was preincubated with L- or D-DOPA and then inhibition of viral infection as well as viral attachment to host cells were evaluated by viral quantification. Both L- and D-DOPA inhibited SFTSV infection in a dose-dependent manner, mainly by blocking viral attachment to host cells. The half-maximal inhibitory concentration (IC50) of L-DOPA was 4.46-5.09 µM. IC50 of D-DOPA was 4.23-6.72 µM. IC50 of L-DOPA is very close to its maximum blood concentration after oral administration as a therapy for Parkinson's disease. D-DOPA, which IC50 was almost the same as that of L-DOPA, might not cause side effect. Thus, our present study demonstrated that L- and D-DOPA are potentially useful candidates for anti-SFTSV drugs.

SARS-CoV-2 Spike Protein Mutation at Cysteine-488 Impairs Its Golgi Localization and Intracellular S1/S2 Processing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds to the cellular receptor-angiotensin-converting enzyme-2 (ACE2) as the first step in viral cell entry. SARS-CoV-2 spike protein expression in the ACE2-expressing cell surface induces cell-cell membrane fusion, thus forming syncytia. To exert its fusogenic activity, the spike protein is typically processed at a specific site (the S1/S2 site) by cellular proteases such as furin. The C488 residue, located at the spike-ACE2 interacting surface, is critical for the fusogenic and infectious roles of the SARS-CoV-2 spike protein. We have demonstrated that the C488 residue of the spike protein is involved in subcellular targeting and S1/S2 processing. C488 mutant spike localization to the Golgi apparatus and cell surface were impaired. Consequently, the S1/S2 processing of the spike protein, probed by anti-Ser-686-cleaved spike antibody, markedly decreased in C488 mutant spike proteins. Moreover, brefeldin-A-mediated endoplasmic-reticulum-to-Golgi traffic suppression also suppressed spike protein S1/S2 processing. As brefeldin A treatment and C488 mutation inhibited S1/S2 processing and syncytia formation, the C488 residue of spike protein is required for functional spike protein processing.

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus.

In microbiological research, it is important to understand the time course of each step in a pathogen's lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3-4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5' UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.

Direct Inhibition of SARS-CoV-2 Spike Protein by Peracetic Acid.

Peracetic acid (PAA) disinfectants are effective against a wide range of pathogenic microorganisms, including bacteria, fungi, and viruses. Several studies have shown the efficacy of PAA against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); however, its efficacy in SARS-CoV-2 variants and the molecular mechanism of action of PAA against SARS-CoV-2 have not been investigated. SARS-CoV-2 infection depends on the recognition and binding of the cell receptor angiotensin-converting enzyme 2 (ACE2) via the receptor-binding domain (RBD) of the spike protein. Here, we demonstrated that PAA effectively suppressed pseudotyped virus infection in the Wuhan type and variants, including Delta and Omicron. Similarly, PAA reduced the authentic viral load of SARS-CoV-2. Computational analysis suggested that the hydroxyl radicals produced by PAA cleave the disulfide bridges in the RBD. Additionally, the PAA treatment decreased the abundance of the Wuhan- and variant-type spike proteins. Enzyme-linked immunosorbent assay showed direct inhibition of RBD-ACE2 interactions by PAA. In conclusion, the PAA treatment suppressed SARS-CoV-2 infection, which was dependent on the inhibition of the interaction between the spike RBD and ACE2 by inducing spike protein destabilization. Our findings provide evidence of a potent disinfection strategy against SARS-CoV-2.

Role of the cytosolic domain of occludin in trafficking and hepatitis C virus infection.

The role of the tight-junction (TJ) protein occludin (OCLN) in hepatitis C virus (HCV) entry remains elusive. Here, we investigated the OCLN C-terminal cytosolic domain in HCV infection. We expressed a series of C-terminal deletion mutants in Huh-7 cells KO for OCLN and characterized their functionality in HCV infection and trafficking. Deleting the OCLN cytosolic domain led to protein instability and intracellular retention. The first 15 residues (OCLN-C15 mutant) of the cytosolic domain were sufficient for OCLN stability, but led to its accumulation in the trans-Golgi network (TGN) due to a deficient cell surface export after synthesis. In contrast, the OCLN-C18 mutant, containing the first 18 residues of the cytosolic domain, was expressed at the cell surface and could mediate HCV infection. Point mutations in the context of C18 showed that I279 and W281 are crucial residues for cell surface expression of OCLN-C18. However, in the context of full-length OCLN, mutation of these residues only partially affected infection and cell surface localization. Importantly, the characterization of OCLN-C18 in human-polarized hepatocytes revealed a defect in its TJ localization without affecting HCV infection. These data suggest that TJ localization of OCLN is not a prerequisite for HCV infection in polarized hepatocytes.

Sphingomyelin Is Essential for the Structure and Function of the Double-Membrane Vesicles in Hepatitis C Virus RNA Replication Factories.

Some plus-stranded RNA viruses generate double-membrane vesicles (DMVs), one type of the membrane replication factories, as replication sites. Little is known about the lipid components involved in the biogenesis of these vesicles. Sphingomyelin (SM) is required for hepatitis C virus (HCV) replication, but the mechanism of SM involvement remains poorly understood. SM biosynthesis starts in the endoplasmic reticulum (ER) and gives rise to ceramide, which is transported from the ER to the Golgi by the action of ceramide transfer protein (CERT), where it can be converted to SM. In this study, inhibition of SM biosynthesis, either by using small-molecule inhibitors or by knockout (KO) of CERT, suppressed HCV replication in a genotype-independent manner. This reduction in HCV replication was rescued by exogenous SM or ectopic expression of the CERT protein, but not by ectopic expression of nonfunctional CERT mutants. Observing low numbers of DMVs in stable replicon cells treated with a SM biosynthesis inhibitor or in CERT-KO cells transfected with either HCV replicon or with constructs that drive HCV protein production in a replication-independent system indicated the significant importance of SM to DMVs. The degradation of SM of the in vitro-isolated DMVs affected their morphology and increased the vulnerability of HCV RNA and proteins to RNase and protease treatment, respectively. Poliovirus, known to induce DMVs, showed decreased replication in CERT-KO cells, while dengue virus, known to induce invaginated vesicles, did not. In conclusion, these findings indicated that SM is an essential constituent of DMVs generated by some plus-stranded RNA viruses.IMPORTANCE Previous reports assumed that sphingomyelin (SM) is essential for HCV replication, but the mechanism was unclear. In this study, we showed for the first time that SM and ceramide transfer protein (CERT), which is in the SM biosynthesis pathway, are essential for the biosynthesis of double-membrane vesicles (DMVs), the sites of viral replication. Low numbers of DMVs were observed in CERT-KO cells transfected with replicon RNA or with constructs that drive HCV protein production in a replication-independent system. HCV replication was rescued by ectopic expression of the CERT protein, but not by CERT mutants, that abolishes the binding of CERT to vesicle-associated membrane protein-associated protein (VAP) or phosphatidylinositol 4-phosphate (PI4P), indicating new roles for VAP and PI4P in HCV replication. The biosynthesis of DMVs has great importance to replication by a variety of plus-stranded RNA viruses. Understanding of this process is expected to facilitate the development of diagnosis and antivirus.

Several catechins and flavonols from green tea inhibit severe fever with thrombocytopenia syndrome virus infection in vitro.

INTRODUCTION: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne hemorrhagic fever caused by SFTS virus (SFTSV). The mortality rate of SFTS is pretty high, but no vaccines and antiviral drugs are currently available. METHODS: The antiviral effects of six green tea-related polyphenols, including four catechins and two flavonols, on SFTSV were evaluated to identify natural antiviral compounds. RESULTS: Pretreatment with all polyphenols inhibited SFTSV infection in a concentration-dependent manner. The half-maximal inhibitory concentrations of (-)-epigallocatechin gallate (EGCg) and (-)-epigallocatechin (EGC) were 1.7-1.9 and 11-39 µM, respectively. The selectivity indices of EGCg and EGC were larger than those of the other polyphenols. Furthermore, pretreatment with EGCg and EGC dose-dependently decreased viral attachment to the host cells. Additionally, the treatment of infected cells with EGCg and EGC inhibited infection more significantly at a lower multiplicity of infection (MOI) than at a higher MOI, and this effect was less effective than that of pretreatment. Pyrogallol, a trihydroxybenzene that is the structural backbone of both EGCg and EGC, also inhibited SFTSV infection, as did gallic acid. CONCLUSIONS: Our study revealed that green tea-related polyphenols, especially EGCg and EGC, are useful as candidate anti-SFTSV drugs. Furthermore, the structural basis of their antiviral activity was identified, which should enable investigations of more active drugs in the future.

Structural basis of antiviral activity of caffeic acid against severe fever with thrombocytopenia syndrome virus.

Caffeic acid (CA), a coffee-related natural compound, has various beneficial biological effects, including antiviral effects. Our former studies demonstrated that the CA dose-dependently inhibited the in vitro infection with Dabie bandavirus, which was previously named as severe fever with thrombocytopenia syndrome virus (SFTSV), mainly at the step of virus attachment. Therefore, we studied the structural basis of CA for conferring anti-SFTSV activity to clarify the mechanism of action of CA against SFTSV. In this study, the anti-SFTSV activity of nine CA analogs were examined. The treatment of SFTSV with the 3,4-dihydroxyhydrocinnamic acid (DHCA) as well as CA inhibited the SFTSV infection in a dose-dependent manner, whereas other CA analogs did not. Both CA and DHCA only possessed the o-dihydroxybenzene backbone. When SFTSV was treated with catechol (o-dihydroxybenzene), SFTSV infection was also dose-dependently inhibited. Additionally, four compounds having the o-dihydroxybenzene backbone; CA phenethyl ester, methyl CA, 3,4-dihydroxyphenylacetic acid, and 3,4-dihydroxybenzoic acid, dose-dependently inhibited the viral infection, although these compounds were more toxic or less effective than CA. In conclusion, the o-dihydroxybenzene backbone in CA and its analogs was a critical structure for the anti-SFTSV activity. Based on these findings, modifications of the o-dihydroxybenzene backbone with various other residues might improve the antiviral effect and cytotoxicity for SFTSV.

The aryl hydrocarbon receptor-cytochrome P450 1A1 pathway controls lipid accumulation and enhances the permissiveness for hepatitis C virus assembly.

Viruses hijack and modify host cell functions to maximize viral proliferation. Hepatitis C virus (HCV) reorganizes host cell metabolism to produce specialized membrane structures and to modify organelles such as double-membrane vesicles and enlarged lipid droplets (LDs), thereby enabling virus replication and assembly. However, the molecular bases of these host-HCV interactions are largely unknown. Here, using a chemical screen, we demonstrate that the benzamide derivative flutamide reduces the host capacity to produce infectious HCV. Flutamide disrupted the formation of enlarged LDs in HCV-infected cells, thereby abolishing HCV assembly. We also report that aryl hydrocarbon receptor (AhR), a known flutamide target, plays a key role in mediating LD accumulation and HCV production. This AhR function in lipid production was also observed in HCV-uninfected Huh-7 cells and primary human hepatocytes, suggesting that AhR signaling regulates lipid accumulation independently of HCV infection. We further observed that a downstream activity, that of cytochrome P450 1A1 (CYP1A1), was the primary regulator of AhR-mediated lipid production. Specifically, blockade of AhR-induced CYP1A1 up-regulation counteracted LD overproduction, and overproduction of CYP1A1, but not of CYP1B1, in AhR-inactivated cells restored lipid accumulation. Of note, HCV infection up-regulated the AhR-CYP1A1 pathway, resulting in the accumulation of enlarged LDs. In conclusion, we demonstrate that the AhR-CYP1A1 pathway has a significant role in lipid accumulation, a hallmark of HCV infection that maximizes progeny virus production. Our chemical-genetic analysis reveals a new strategy and lead compounds to control hepatic lipid accumulation as well as HCV infection.

Human-rat chimeric anti-occludin monoclonal antibodies inhibit hepatitis C virus infection.

Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention. We previously generated rat anti-OCLN monoclonal antibodies (mAbs) that strongly prevented HCV infection in vitro and in vivo. In the present study, we attempted to improve the druggability of the extracellular loop domain-recognizing anti-OCLN mAbs, namely clones 1-3 and 37-5, using genetic engineering. To avoid adverse reactions induced by antibody-dependent cellular cytotoxicity and enhance the antibody stability, we developed human-rat chimeric immunoglobulin G4 S228P mutant (IgG4m) forms of clones 1-3 and 37-5 (named Xi 1-3 and Xi 37-5, respectively) by grafting the variable regions of the light and heavy chains of each rat anti-OCLN mAb into those of human IgG4m. The constructed Xi 1-3 and Xi 37-5 chimeras demonstrated levels of affinity and specificity similar to each parental rat anti-OCLN mAb, and the Fcγ receptor Ⅲa was not activated by the antigen-bound chimeric mAbs, as expected. Both chimeric mAbs inhibited in vitro infection with various HCV genotypes. These results indicate that the IgG4m forms of human-rat chimeric anti-OCLN mAbs may be potential candidate molecules of host-targeting antivirals with pan-genotypic anti-HCV activity.

Monoclonal Antibodies against Occludin Completely Prevented Hepatitis C Virus Infection in a Mouse Model.

Hepatitis C virus (HCV) entry into host cells is a multistep process requiring various host factors, including the tight junction protein occludin (OCLN), which has been shown to be essential for HCV infection in in vitro cell culture systems. However, it remains unclear whether OCLN is an effective and safe target for HCV therapy, owing to the lack of binders that can recognize the intact extracellular loop domains of OCLN and prevent HCV infection. In this study, we successfully generated four rat anti-OCLN monoclonal antibodies (MAbs) by the genetic immunization method and unique cell differential screening. These four MAbs bound to human OCLN with a very high affinity (antibody dissociation constant of <1 nM). One MAb recognized the second loop of human and mouse OCLN, whereas the three other MAbs recognized the first loop of human OCLN. All MAbs inhibited HCV infection in Huh7.5.1-8 cells in a dose-dependent manner without apparent cytotoxicity. Additionally, the anti-OCLN MAbs prevented both cell-free HCV infection and cell-to-cell HCV transmission. Kinetic studies with anti-OCLN and anti-claudin-1 (CLDN1) MAbs demonstrated that OCLN interacts with HCV after CLDN1 in the internalization step. Two selected MAbs completely inhibited HCV infection in human liver chimeric mice without apparent adverse effects. Therefore, OCLN would be an appropriate host target for anti-HCV entry inhibitors, and anti-OCLN MAbs may be promising candidates for novel anti-HCV agents, particularly in combination with direct-acting HCV antiviral agents.IMPORTANCE HCV entry into host cells is thought to be a very complex process involving various host entry factors, such as the tight junction proteins claudin-1 and OCLN. In this study, we developed novel functional MAbs that recognize intact extracellular domains of OCLN, which is essential for HCV entry into host cells. The established MAbs against OCLN, which had very high affinity and selectivity for intact OCLN, strongly inhibited HCV infection both in vitro and in vivo Using these anti-OCLN MAbs, we found that OCLN is necessary for the later stages of HCV entry. These anti-OCLN MAbs are likely to be very useful for understanding the OCLN-mediated HCV entry mechanism and might be promising candidates for novel HCV entry inhibitors.

Inhibition Mechanisms of Hepatitis C Virus Infection by Caffeic Acid and Tannic Acid.

Previously, we reported that coffee extract and its constituents, caffeic acid (CA) and p-coumaric acid, inhibit infection by the hepatitis C virus (HCV). In the present report, we identified another coffee-related compound, tannic acid (TA), which also inhibits HCV infection. We systematically evaluated which steps of the viral lifecycle were affected by CA and TA. TA substantially inhibits HCV RNA replication and egression, while CA does not. The infectivity of the HCV pretreated with CA or TA was almost lost. Cellular attachment of HCV particles and their interaction with apolipoprotein E, which is essential for HCV infectivity, were significantly reduced by CA. These results indicate that CA inhibits HCV entry via its direct effect on viral particles and TA inhibits HCV RNA replication and particle egression as well as entry into host cells. Taken together, our findings may provide insights into CA and TA as potential anti-HCV strategies.

Thermostable hepatitis C virus JFH1-derived variant isolated by adaptation to Huh7.5.1 cells.

Hepatitis C virus (HCV) infection and propagation in cultured cells have mainly been investigated using the infectious clinical clone JFH1. However, its infectivity is not high enough for infection to be detected easily. In this study, we attempted to isolate HCV-JFH1 variants adapted to human hepatoma Huh7.5.1 cells. By performing serial passages of the wild-type HCV-JFH1 in Huh7.5.1 cells, we obtained a variant that was capable of inducing severe cytopathic effects and showed approximately 700-fold higher infectivity than the wild-type HCV-JFH1. Further, when highly permissive Huh7.5.1-8 cells were infected with this variant, viral particles were produced at >1011 copies ml-1, making this variant one of the most efficient HCV production systems. Two adaptive mutations were noted in the variant genome: a1994c (K74T) in the core protein region and t3014c (I414T) in the E2 protein region. Both mutations contributed to enhanced infectivity and their combination showed synergistic effects in this regard. An examination of recombinant viruses carrying K74T, I414T and K74T/I414T mutations revealed that none of the mutations had an effect on the steps after viral entry (genome replication, particle assembly and egress), but led to the viral infection becoming less dependent on scavenger receptor class B type I, changes of the infectious particles to a broader and lower range of densities, and enhanced thermal stability of the infectious viruses. Thus, this Huh7.5.1-adapted HCV-JFH1 variant with higher and stable infectivity should be a valuable tool for studying the molecular mechanisms behind the life cycle of HCV and for antiviral screening.