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.

Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies.

A current challenge is the emergence of SARS-CoV-2 variants, such as BQ.1.1 and XBB.1.5, that can evade immune defenses, thereby limiting antibody drug effectiveness. Emergency-use antibody drugs, including the widely effective bebtelovimab, are losing their benefits. One potential approach to address this issue are bispecific antibodies which combine the targeting abilities of two antibodies with distinct epitopes. We engineered neutralizing bispecific antibodies in the IgG-scFv format from two initially non-neutralizing antibodies, CvMab-6 (which binds to the receptor-binding domain [RBD]) and CvMab-62 (targeting a spike protein S2 subunit epitope adjacent to the known anti-S2 antibody epitope). Furthermore, we created a bispecific antibody by incorporating the scFv of bebtelovimab with our anti-S2 antibody, demonstrating significant restoration of effectiveness against bebtelovimab-resistant BQ.1.1 variants. This study highlights the potential of neutralizing bispecific antibodies, which combine existing less effective anti-RBD antibodies with anti-S2 antibodies, to revive the effectiveness of antibody therapeutics compromised by immune-evading variants.

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.

Hazara orthonairovirus nucleoprotein facilitates viral cell-to-cell spread by modulating tight junction protein, claudin-1.

Background: Tight junctions act as a barrier that prevents invasion of pathogens through epithelial cells. This study aims to elucidate the correlation between tight junctions and nairoviruses using Hazara orthonairovirus (HAZV) as a surrogate model for Crimean-Congo hemorrhagic fever virus. Methods: mRNA, total protein, and cell surface protein levels of tight junction proteins were examined by quantitative real-time reverse transcription polymerase chain reaction, immunoblot and flow cytometry, respectively. HAZV growth was measured by plaque assay. Immunofluorescence assay was used to examine viral cell-to-cell spread. The interaction between HAZV nucleoprotein and claudin-1 was analyzed by immunoprecipitation. Results: HAZV infection induced mRNA of several tight junction proteins, especially claudin-1. HAZV infection also induced cell surface expression of claudin-1 protein. Claudin-1 overexpression inhibited the growth of HAZV by blocking its cell-to-cell spread. In contrast, HAZV nucleoprotein completely inhibited HAZV-induced cell surface expression of claudin-1, and this inhibition required interaction between HAZV nucleoprotein and claudin-1. Conclusion: HAZV nucleoprotein was shown to bind to claudin-1 to negatively regulate its cell surface expression, and so can promote cell-to-cell spread of HAZV. This is the first presentation of a possible mechanism behind how nairoviruses counteract tight junction barrier function.

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.

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.

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.

A single mutation in the E2 glycoprotein of hepatitis C virus broadens the claudin specificity for its infection.

Entry of the hepatitis C virus (HCV) into host cells is a multistep process mediated by several host factors, including a tight junction protein claudin-1 (CLDN1). We repeatedly passaged HCV-JFH1-tau, an HCV substrain with higher infectivity, on Huh7.5.1-8 cells. A multi-passaged HCV-JFH1-tau lot was infectious to CLDN1-defective S7-A cells, non-permissive to original HCV-JFH1-tau infection. We identified a single mutation, M706L, in the E2 glycoprotein of the HCV-JFH1-tau lot as an essential mutation for infectivity to S7-A cells. The pseudovirus JFH1/M706L mutant could not infect human embryonic kidney 293 T (HEK293T) cells lacking CLDN family but infected HEK293T cells expressing CLDN1, CLDN6, or CLDN9. Thus, this mutant virus could utilize CLDN1, and other CLDN6 and CLDN9, making HCV possible to infect cells other than hepatocytes. iPS cells, one of the stem cells, do not express CLDN1 but express CLDN6 and other host factors required for HCV infection. We confirmed that the HCV-JFH1-tau-derived mutant with an M706L mutation infected iPS cells in a CLDN6-dependent manner. These results demonstrated that a missense mutation in E2 could broaden the CLDN member specificity for HCV infection. HCV may change its receptor requirement through a single amino acid mutation and infect non-hepatic cells.

Establishment of Vero cell lines persistently harboring a yellow fever virus 17D subgenomic replicon.

Yellow fever virus (YFV), a member of the genus Flavivirus, family Flaviviridae, is the etiological agent for an acute viral hemorrhagic disease, yellow fever. Although effective live attenuated vaccines based on the strain YFV 17D are currently available, no specific antiviral drug is available, and the disease remains a major public health concern. Hence, the discovery and development of antiviral drugs should lead to great benefits in controlling the disease. To provide a screening platform for antiviral agents targeting YFV RNA translation/replication, we have established and characterized two Vero cell lines that persistently harbor a subgenomic replicon derived from YFV 17D-204 (referred to as replicon cells). The replicon carries YFV nucleotides (1 - 176 and 2382-10,862) and a green fluorescent protein (GFP)-Zeocin resistance fusion gene as a selection marker and indicator of persistent replication. Immunofluorescence analysis revealed that both replicon cells and YFV 17D-infected cells showed similar distribution patterns of viral NS4B protein and replication intermediate, double-stranded RNA. Sequencing analysis of persistent replicons from the two replicon cell lines suggested that their nucleotide sequences did not vary greatly following multiple passages. We examined the effect of five agents, the antiviral cytokines interferon-ß and -γ, the nucleoside analog ribavirin, the squalene synthase inhibitor zaragozic acid A, and the antibiotic rifapentine, a recently reported entry and replication inhibitor against YFV, on the persistent replication in the two replicon cell lines. These agents were selected because they inhibited both production of YFV 17D and transient replication of a luciferase-expressing replicon in Vero cells, without greatly affecting cell viability. We found that each of the agents decreased GFP fluorescence in the replicon cells, albeit to varying degrees. The agents other than rifapentine also showed a decrease in viral RNA levels in the replicon cells comparable to that seen for GFP fluorescence. These results indicate that persistent replication is susceptible to each of these five agents, although their mechanisms of action may differ. Taken together, these results provide evidence that translation/replication of the replicon in the replicon cells mimics that of the viral genome upon YFV 17D infection, indicating that the replicon cell lines can serve as a useful tool for high-throughput antiviral drug screening.

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.

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.

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.

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.

Seroprevalence of Flavivirus Neutralizing Antibodies in Thailand by High-Throughput Neutralization Assay: Endemic Circulation of Zika Virus before 2012.

Thailand is a hyperendemic country for flavivirus infections in Southeast Asia. Although the reporting system for flavivirus surveillance in Thailand is well established, syndromic surveillance tends to underestimate the true epidemiological status of flaviviruses due to the majority of infections being asymptomatic. To accurately understand the prevalence of flaviviruses in endemic regions, we performed neutralization tests against multiple flaviviruses using 147 serum samples from healthy donors collected from four distinct regions in Thailand. Single-round infectious particles (SRIP) for six flaviviruses, dengue virus types 1 to 4 (DENV-1 to -4), Japanese encephalitis virus (JEV), and Zika virus (ZIKV), were used as antigens for developing a safe, high-throughput neutralization assay. Titers of neutralizing antibodies (NAbs) against the six flaviviruses revealed that DENV-1 and DENV-2, followed by ZIKV were the predominant circulating flaviviruses in a total of four regions, whereas the prevalence of NAbs against JEV varied among regions. Although the seroprevalence of ZIKV was low relative to that of DENV-1 and DENV-2, the findings strongly suggested that ZIKV has been circulating at a sustained level in Thailand since before 2012. These findings not only demonstrated the application of an SRIP-neutralization test in a serological study, but also elucidated the circulation and distribution trends of different flaviviruses in Thailand. IMPORTANCE Neutralization tests are the most reliable assay for flavivirus antibody detection; however, these assays are not suitable for high-throughput processing due to their time-consuming and labor-intensive nature. In this study, we developed single-round infectious particles (SRIPs) with a luciferase gene for dengue virus types 1 to 4, Japanese encephalitis virus, and Zika virus for use in a safe, high-throughput neutralization assay. We performed neutralization tests against multiple flaviviruses using 147 serum samples that were collected from healthy donors residing in four distinct regions of Thailand in 2011 to 2012. The assay was useful for surveys of flavivirus seroprevalence. The data revealed that dengue virus type 1 (DENV-1) and DENV-2 were the predominant circulating flaviviruses in Thailand and that Zika virus has been circulating at a sustained level in Thailand since before 2012.

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.

Occludin-binding single-chain variable fragment and antigen-binding fragment antibodies prevent hepatitis C virus infection.

Occludin (OCLN) is a tetraspan membrane component of epithelial tight junctions and a known receptor for hepatitis C virus (HCV). Previously, we established functional monoclonal antibodies (mAbs) that bind to each extracellular loop of OCLN and showed their ability to prevent in vitro and in vivo HCV infection. In this study, we converted these mAbs to corresponding monovalent antigen-binding fragments (Fabs) and single-chain variable fragment (scFv) antibodies. These Fab fragments and scFv antibodies demonstrate similar binding specificity and affinity to parental anti-OCLN mAbs. Moreover, Fab fragments and scFv antibodies inhibit in vitro HCV infection. The small functional monovalent OCLN-binding probes reported in our study have high potential as drug candidates and tools for biological and pharmaceutical studies of OCLN.

Identification of Characteristic Genomic Markers in Human Hepatoma HuH-7 and Huh7.5.1-8 Cell Lines.

The human hepatoma-derived HuH-7 cell line and its derivatives (Huh7.5 and Huh7.5.1) have been widely used as a convenient experimental substitute for primary hepatocytes. In particular, these cell lines represent host cells suitable for propagating the hepatitis C virus (HCV) in vitro. The Huh7.5.1-8 cell line, a subline of Huh7.5.1, can propagate HCV more efficiently than its parental cells. To provide genomic information for cells' quality control, we performed whole-genome sequencing of HuH-7 and Huh7.5.1-8 and identified their characteristic genomic deletions, some of which are applicable to an in-house test for cell authentication. Among the genes related to HCV infection and replication, 53 genes were found to carry missense or loss-of-function mutations likely specific to the HuH-7 and/or Huh7.5.1-8. Eight genes, including DDX58 (RIG-I), BAX, EP300, and SPP1 (osteopontin), contained mutations observed only in Huh7.5.1-8 or mutations with higher frequency in Huh7.5.1-8. These mutations might be relevant to phenotypic differences between the two cell lines and may also serve as genetic markers to distinguish Huh7.5.1-8 cells from the ancestral HuH-7 cells.

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.

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero.

The Flaviviridae is a family of enveloped viruses with a positive-sense single-stranded RNA genome. It contains many viruses that threaten human health, such as Japanese encephalitis virus (JEV) and yellow fever virus (YFV) of the genus Flavivirus as well as hepatitis C virus of the genus Hepacivirus. Cell culture systems highly permissive for the Flaviviridae viruses are very useful for their isolation, propagation, and diagnosis, an understanding of their biology, and the development of vaccines and antiviral agents. Previously, we isolated a human hepatoma HuH-7-derived cell clone, Huh7.5.1-8, which is highly permissive to hepatitis C virus infection. Here, we have characterized flavivirus infection in the Huh7.5.1-8 cell line by comparing with that in the African green monkey kidney-derived Vero cell line, which is permissive for a wide spectrum of viruses. Upon infection with JEV, Huh7.5.1-8 cells produced a higher amount of virus particles early in infection and were more susceptible to virus-induced cell death than Vero cells. Similar outcomes were obtained when the cells were infected with another flavivirus, YFV (17D-204 strain). Quantification of cellular and extracellular viral RNA revealed that high JEV production in Huh7.5.1-8 cells can be attributed to rapid viral replication kinetics and efficient virus release early in infection. In a plaque assay, Huh7.5.1-8 cells developed JEV plaques more rapidly than Vero cells. Although this was not the case with YFV plaques, Huh7.5.1-8 cells developed higher numbers of YFV plaques than Vero cells. Sequence analysis of cDNA encoding an antiviral RNA helicase, RIG-I, showed that Huh7.5.1-8 cells expressed not only a full-length RIG-I mRNA with a known dominant-negative missense mutation but also variants without the mutation. However, the latter mRNAs lacked exon 5/6-12, indicating functional loss of RIG-I in the cells. These characteristics of the Huh7.5.1-8 cell line are helpful for flavivirus detection, titration, and propagation.