Potential Anti-Mpox Virus Activity of Atovaquone, Mefloquine, and Molnupiravir, and Their Potential Use as Treatments.

BACKGROUND: Mpox virus (MPXV) is a zoonotic orthopoxvirus and caused an outbreak in 2022. Although tecovirimat and brincidofovir are approved as anti-smallpox drugs, their effects in mpox patients have not been well documented. In this study, by a drug repurposing approach, we identified potential drug candidates for treating mpox and predicted their clinical impacts by mathematical modeling. METHODS: We screened 132 approved drugs using an MPXV infection cell system. We quantified antiviral activities of potential drug candidates by measuring intracellular viral DNA and analyzed the modes of action by time-of-addition assay and electron microscopic analysis. We further predicted the efficacy of drugs under clinical concentrations by mathematical simulation and examined combination treatment. RESULTS: Atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 µM, which was more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted postentry processes. Atovaquone was suggested to exert its activity through inhibiting dihydroorotate dehydrogenase. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by 7 days at clinically relevant drug concentrations. CONCLUSIONS: These data suggest that atovaquone would be a potential candidate for treating mpox.

X-ray absorption spectra of aqueous cellobiose: Experiment and theory.

The hydration structure of cellulose is very important for understanding the hydrolysis of cellulose at the molecular level. In this paper, we report a joint experimental and theoretical study on x-ray absorption spectroscopy (XAS) of aqueous cellobiose, a disaccharide unit of cellulose. In the experimental part, high resolution measurements of the carbon K-edge XAS spectra were taken. In the theoretical part, ab initio molecular dynamics simulations and ensemble calculations of electronic excited states were performed to obtain the continuous XAS spectra. The XAS spectra were found to have three characteristic peaks at 289.3, 290.7, and 293.6 eV, each representing the absorption by carbon atoms of the alcohol group, the hemiacetal group, and both of these functional groups. It was found that the peak heights in the spectrum change considerably over the temperature range of 25-60 °C, which is a reflection of the number of hydrogen bonds between cellobiose and water. We suggest that this spectral change could be useful information for identifying the hydration of cellulose in various environments.

A novel MitoNEET ligand, TT01001, improves diabetes and ameliorates mitochondrial function in db/db mice.

The mitochondrial outer membrane protein mitoNEET is a binding protein of the insulin sensitizer pioglitazone (5-[[4-[2-(5-ethylpyridin-2-yl)ethoxy]phenyl]methyl]-1,3-thiazolidine-2,4-dione) and is considered a novel target for the treatment of type II diabetes. Several small-molecule compounds have been identified as mitoNEET ligands using structure-based design or virtual docking studies. However, there are no reports about their therapeutic potential in animal models. Recently, we synthesized a novel small molecule, TT01001 [ethyl-4-(3-(3,5-dichlorophenyl)thioureido)piperidine-1-carboxylate], designed on the basis of pioglitazone structure. In this study, we assessed the pharmacological properties of TT01001 in both in vitro and in vivo studies. We found that TT01001 bound to mitoNEET without peroxisome proliferator-activated receptor-γ activation effect. In type II diabetes model db/db mice, TT01001 improved hyperglycemia, hyperlipidemia, and glucose intolerance, and its efficacy was equivalent to that of pioglitazone, without the pioglitazone-associated weight gain. Mitochondrial complex II + III activity of the skeletal muscle was significantly increased in db/db mice. We found that TT01001 significantly suppressed the elevated activity of the complex II + III. These results suggest that TT01001 improved type II diabetes without causing weight gain and ameliorated mitochondrial function of db/db mice. This is the first study that demonstrates the effects of a mitoNEET ligand on glucose metabolism and mitochondrial function in an animal disease model. These findings support targeting mitoNEET as a potential therapeutic approach for the treatment of type II diabetes.

Neutralizing antibodies induced by cell culture-derived hepatitis C virus protect against infection in mice.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is a major cause of liver cancer, so strategies to prevent infection are needed. A system for cell culture of infectious HCV particles (HCVcc) has recently been established; the inactivated HCVcc particles might be used as antigens in vaccine development. We aimed to confirm the potential of HCVcc as an HCV particle vaccine. METHODS: HCVcc derived from the J6/JFH-1 chimeric genome was purified from cultured cells by ultrafiltration and ultracentrifugation purification steps. Purified HCV particles were inactivated and injected into female BALB/c mice with adjuvant. Sera from immunized mice were collected and their ability to neutralize HCV was examined in naive Huh7.5.1 cells and urokinase-type plasminogen activator-severe combined immunodeficiency mice (uPA(+/+)-SCID mice) given transplants of human hepatocytes (humanized livers). RESULTS: Antibodies against HCV envelope proteins were detected in the sera of immunized mice; these sera inhibited infection of cultured cells with HCV genotypes 1a, 1b, and 2a. Immunoglobulin G purified from the sera of HCV-particle-immunized mice (iHCV-IgG) inhibited HCV infection of cultured cells. Injection of IgG from the immunized mice into uPA(+/+)-SCID mice with humanized livers prevented infection with the minimum infectious dose of HCV. CONCLUSIONS: Inactivated HCV particles derived from cultured cells protect chimeric liver uPA(+/+)-SCID mice against HCV infection, and might be used in the development of a prophylactic vaccine.

Mpox Neutralizing Antibody Response to LC16m8 Vaccine in Healthy Adults.

BACKGROUND: Vaccination against mpox (formerly known as monkeypox), an infectious disease caused by the monkeypox virus (MPXV), is needed to prevent outbreaks and consequent public health concerns. The LC16m8 vaccine, a dried cell-cultured proliferative live attenuated vaccinia virus­based vaccine, was approved in Japan against smallpox and mpox. However, its immunogenicity and efficacy against MPXV have not been fully assessed. We assessed the safety and immunogenicity of LC16m8 against MPXV in healthy adults. METHODS: We conducted a single-arm study that included 50 participants who were followed up for 168 days postvaccination. The primary end point was the neutralizing antibody seroconversion rate against MPXVs, including the Zr599 and Liberia strains, on day 28. The secondary end points included the vaccine "take" (major cutaneous reaction) rate, neutralizing titer kinetics against MPXV and vaccinia virus (LC16m8) strains, and safety outcomes. RESULTS: Seroconversion rates on day 28 were 72% (36 of 50), 70% (35 of 50), and 88% (44 of 50) against the Zr599 strain, the Liberia strain, and LC16m8, respectively. On day 168, seroconversion rates decreased to 30% (15 of 50) against the Zr599 and Liberia strains and to 76% (38 of 50) against LC16m8. The vaccine "take" (broad definition) rate on day 14 was 94% (46 of 49). Adverse events (AEs), including common solicited cutaneous reactions, occurred in 98% (45 of 48) of participants; grade 3 severity AEs occurred in 16% (8 of 50). No deaths, serious AEs, or mpox onset incidences were observed up to day 168. CONCLUSIONS: The LC16m8 vaccine generated neutralizing antibody responses against MPXV in healthy adults. No serious safety concerns occurred with LC16m8 use. (Funded by the Ministry of Health, Labour and Welfare of Japan; Japan Registry of Clinical Trials number, jRCTs031220171.)

Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone.

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.

Hepatitis C virus reveals a novel early control in acute immune response.

Recognition of viral RNA structures by the intracytosolic RNA helicase RIG-I triggers induction of innate immunity. Efficient induction requires RIG-I ubiquitination by the E3 ligase TRIM25, its interaction with the mitochondria-bound MAVS protein, recruitment of TRAF3, IRF3- and NF-κB-kinases and transcription of Interferon (IFN). In addition, IRF3 alone induces some of the Interferon-Stimulated Genes (ISGs), referred to as early ISGs. Infection of hepatocytes with Hepatitis C virus (HCV) results in poor production of IFN despite recognition of the viral RNA by RIG-I but can lead to induction of early ISGs. HCV was shown to inhibit IFN production by cleaving MAVS through its NS3/4A protease and by controlling cellular translation through activation of PKR, an eIF2α-kinase containing dsRNA-binding domains (DRBD). Here, we have identified a third mode of control of IFN induction by HCV. Using HCVcc and the Huh7.25.CD81 cells, we found that HCV controls RIG-I ubiquitination through the di-ubiquitine-like protein ISG15, one of the early ISGs. A transcriptome analysis performed on Huh7.25.CD81 cells silenced or not for PKR and infected with JFH1 revealed that HCV infection leads to induction of 49 PKR-dependent genes, including ISG15 and several early ISGs. Silencing experiments revealed that this novel PKR-dependent pathway involves MAVS, TRAF3 and IRF3 but not RIG-I, and that it does not induce IFN. Use of PKR inhibitors showed that this pathway requires the DRBD but not the kinase activity of PKR. We then demonstrated that PKR interacts with HCV RNA and MAVS prior to RIG-I. In conclusion, HCV recruits PKR early in infection as a sensor to trigger induction of several IRF3-dependent genes. Among those, ISG15 acts to negatively control the RIG-I/MAVS pathway, at the level of RIG-I ubiquitination.These data give novel insights in the machinery involved in the early events of innate immune response.

Antiviral Activity of Micafungin and Its Derivatives against SARS-CoV-2 RNA Replication.

Echinocandin antifungal drugs, including micafungin, anidulafungin, and caspofungin, have been recently reported to exhibit antiviral effects against various viruses such as flavivirus, alphavirus, and coronavirus. In this study, we focused on micafungin and its derivatives and analyzed their antiviral activities against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The micafungin derivatives Mi-2 and Mi-5 showed higher antiviral activity than micafungin, with 50% maximal inhibitory concentration (IC50) of 5.25 and 6.51 µM, respectively (3.8 to 4.7-fold stronger than micafungin) and 50% cytotoxic concentration (CC50) of >64 µM in VeroE6/TMPRSS2 cells. This high anti-SARS-CoV-2 activity was also conserved in human lung epithelial cell-derived Calu-3 cells. Micafungin, Mi-2, and Mi-5 were suggested to inhibit the intracellular virus replication process; additionally, these compounds were active against SARS-CoV-2 variants, including Delta (AY.122, hCoV-19/Japan/TY11-927/2021), Omicron (BA.1.18, hCoV-19/Japan/TY38-873/2021), a variant resistant to remdesivir (R10/E796G C799F), and a variant resistant to casirivimab/imdevimab antibody cocktail (E406W); thus, our results provide basic evidence for the potential use of micafungin derivatives for developing antiviral agents.

Identification of Membrane-expressed CAPRIN-1 as a Novel and Universal Cancer Target, and Generation of a Therapeutic Anti-CAPRIN-1 Antibody TRK-950.

Specific targets for cancer treatment are highly desirable, but still remain to be discovered. While previous reports suggested that CAPRIN-1 localizes in the cytoplasm, here we now show that part of this molecule is strongly expressed on the cell membrane surface in most solid cancers, but not normal tissues. Notably, the membrane expression of CAPRIN-1 extended to the subset of highly tumorigenic cancer stem cells and epithelial-mesenchymal transition (EMT)-induced metastatic cancer cells. In addition, we revealed that cancer cells with particularly high CAPRIN-1 surface expression exhibited enhanced tumorigenicity. We generated a therapeutic humanized anti-CAPRIN-1 antibody (TRK-950), which strongly and specifically binds to various cancer cells and shows antitumor effects via engagement of immune cells. TRK-950 was further developed as a new cancer drug and a series of preclinical studies demonstrates its therapeutic potency in tumor-bearing mouse models and safety in a relevant cynomolgus monkey model. Together, our data demonstrate that CAPRIN-1 is a novel and universal target for cancer therapies. A phase I clinical study of TRK-950 has been completed (NCT02990481) and a phase Ib study (combination with approved drugs) is currently underway (NCT03872947) in the United States and France. In parallel, a phase I study in Japan is in progress as well (NCT05423262). Significance: Antibody-based cancer therapies have been demonstrated to be effective, but are only approved for a limited number of targets, because the majority of these markers is shared with healthy tissue, which may result in adverse effects. Here, we have successfully identified CAPRIN-1 as a novel truly cancer-specific target, universally expressed on membranes of various cancer cells including cancer stem cells. Clinical studies are underway for the anti-CAPRIN-1 therapeutic antibody TRK-950.

Identification of IMP Dehydrogenase as a Potential Target for Anti-Mpox Virus Agents.

Mpox virus (formerly monkeypox virus [MPXV]) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.026 to 0.89 µM) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the postentry step to reduce the intracellular production of virions. Knockdown of IMP dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. This evidence shows that IMPDH is a potential target for the development of anti-MPXV agents. IMPORTANCE Mpox is a zoonotic disease caused by infection with the mpox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against mpox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against mpox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-mpox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited mpox virus propagation and exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested IMP dehydrogenase as a potential target for the development of anti-mpox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-mpox virus activity than mycophenolic acid.

RNA polymerase activity and specific RNA structure are required for efficient HCV replication in cultured cells.

We have previously reported that the NS3 helicase (N3H) and NS5B-to-3'X (N5BX) regions are important for the efficient replication of hepatitis C virus (HCV) strain JFH-1 and viral production in HuH-7 cells. In the current study, we investigated the relationships between HCV genome replication, virus production, and the structure of N5BX. We found that the Q377R, A450S, S455N, R517K, and Y561F mutations in the NS5B region resulted in up-regulation of J6CF NS5B polymerase activity in vitro. However, the activation effects of these mutations on viral RNA replication and virus production with JFH-1 N3H appeared to differ. In the presence of the N3H region and 3' untranslated region (UTR) of JFH-1, A450S, R517K, and Y561F together were sufficient to confer HCV genome replication activity and virus production ability to J6CF in cultured cells. Y561F was also involved in the kissing-loop interaction between SL3.2 in the NS5B region and SL2 in the 3'X region. We next analyzed the 3' structure of HCV genome RNA. The shorter polyU/UC tracts of JFH-1 resulted in more efficient RNA replication than J6CF. Furthermore, 9458G in the JFH-1 variable region (VR) was responsible for RNA replication activity because of its RNA structures. In conclusion, N3H, high polymerase activity, enhanced kissing-loop interactions, and optimal viral RNA structure in the 3'UTR were required for J6CF replication in cultured cells.

In vivo adaptation of hepatitis C virus in chimpanzees for efficient virus production and evasion of apoptosis.

UNLABELLED: Hepatitis C virus (HCV) employs various strategies to establish persistent infection that can cause chronic liver disease. Our previous study showed that both the original patient serum from which the HCV JFH-1 strain was isolated and the cell culture-generated JFH-1 virus (JFH-1cc) established infection in chimpanzees, and that infected JFH-1 strains accumulated mutations after passage through chimpanzees. The aim of this study was to compare the in vitro characteristics of JFH-1 strains emerged in each chimpanzee at early and late stages of infection, as it could provide an insight into the phenomenon of viral persistence. We generated full-genome JFH-1 constructs with the mutations detected in patient serum-infected (JFH-1/S1 and S2) and JFH-1cc-infected (JFH-1/C) chimpanzees, and assessed their effect on replication, infectious virus production, and regulation of apoptosis in cell culture. The extracellular HCV core antigen secreted from JFH-1/S1-, S2-, and C-transfected HuH-7 cells was 2.5, 8.9, and 2.1 times higher than that from JFH-1 wild-type (JFH-1/wt) transfected cells, respectively. Single cycle virus production assay with a CD81-negative cell line revealed that the strain JFH-1/S2, isolated from the patient serum-infected chimpanzee at a later time point of infection, showed lower replication and higher capacity to assemble infectious virus particles. This strain also showed productive infection in human hepatocyte-transplanted mice. Furthermore, the cells harboring this strain displayed lower susceptibility to the apoptosis induced by tumor necrosis factor α or Fas ligand compared with the cells replicating JFH-1/wt. CONCLUSION: The ability of lower replication, higher virus production, and less susceptibility to cytokine-induced apoptosis may be important for prolonged infection in vivo. Such control of viral functions by specific mutations may be a key strategy for establishing persistent infection.

Different efficacies of neutralizing antibodies and antiviral drugs on SARS-CoV-2 Omicron subvariants, BA.1 and BA.2.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron subvariant BA.2 has spread in many countries, replacing the earlier Omicron subvariant BA.1 and other variants. Here, using a cell culture infection assay, we quantified the intrinsic sensitivity of BA.2 and BA.1 compared with other variants of concern, Alpha, Gamma, and Delta, to five approved-neutralizing antibodies and antiviral drugs. Our assay revealed the diverse sensitivities of these variants to antibodies, including the loss of response of both BA.1 and BA.2 to casirivimab and of BA.1 to imdevimab. In contrast, EIDD-1931 and nirmatrelvir showed a more conserved activities to these variants. The viral response profile combined with mathematical analysis estimated differences in antiviral effects among variants in the clinical concentrations. These analyses provide essential evidence that gives insight into variant emergence's impact on choosing optimal drug treatment.

Biological properties of purified recombinant HCV particles with an epitope-tagged envelope.

To establish a simple system for purification of recombinant infectious hepatitis C virus (HCV) particles, we designed a chimeric J6/JFH-1 virus with a FLAG (FL)-epitope-tagged sequence at the N-terminal region of the E2 hypervariable region-1 (HVR1) gene (J6/JFH-1/1FL). We found that introduction of an adaptive mutation at the potential N-glycosylation site (E2N151K) leads to efficient production of the chimeric virus. This finding suggests the involvement of glycosylation at Asn within the envelope protein(s) in HCV morphogenesis. To further analyze the biological properties of the purified recombinant HCV particles, we developed a strategy for large-scale production and purification of recombinant J6/JFH-1/1FL/E2N151K. Infectious particles were purified from the culture medium of J6/JFH-1/1FL/E2N151K-infected Huh-7 cells using anti-FLAG affinity chromatography in combination with ultrafiltration. Electron microscopy of the purified particles using negative staining showed spherical particle structures with a diameter of 40-60 nm and spike-like projections. Purified HCV particle-immunization induced both an anti-E2 and an anti-FLAG antibody response in immunized mice. This strategy may contribute to future detailed analysis of HCV particle structure and to HCV vaccine development.

Characterization of infectious hepatitis C virus from liver-derived cell lines.

The efficient production of infectious HCV from the JFH-1 strain is restricted to the Huh7 cell line and its derivatives. However, the factors involved in this restriction are unknown. In this study, we examined the production of infectious HCV from other liver-derived cell lines, and characterized the produced viruses. Clones of the Huh7, HepG2, and IMY-N9, harboring the JFH-1 full-genomic replicon, were obtained. The supernatant of each cell clone exhibited infectivity for naïve Huh7. Each infectious supernatant was then characterized by sucrose density gradient. For all of the cell lines, the main peak of the HCV-core protein and RNA exhibited at approximately 1.15g/mL of buoyant density. However, the supernatant from the IMY-N9 differed from that of Huh7 in the ratio of core:RNA at 1.15g/mL and significant peaks were also observed at lower density. The virus particles produced from the different cell lines may have different characteristics.

An infectious and selectable full-length replicon system with hepatitis C virus JFH-1 strain.

AIM: The hepatitis C virus (HCV) strain JFH-1 was cloned from a patient with fulminant hepatitis. A JFH-1 subgenomic replicon and full-length JFH-1 RNA efficiently replicate in cultured cells. In this study, an infectious, selectable HCV replicon containing full-length JFH-1 cDNA was constructed. METHODS: The full-genome replicon was constructed using the neomycin-resistant gene, EMCV IRES and wild-type JFH-1 cDNA. Huh7 cells were transfected with RNA synthesized in vitro, and then cultured with G418. Independent colonies were cloned to establish cell lines that replicate the full-length HCV replicon. RESULTS: HCV RNA replication was detected in each isolated cell line. HCV proteins and HCV RNA were secreted into culture medium, and exhibited identical density profiles. Interestingly, culture supernatants of the replicon cells were infectious for naïve Huh7 cells. Long-term culture did not affect replication of replicon RNA in the replicon cells, but it reduced core protein secretion and infectivity of culture supernatant. Culture supernatant obtained after serial passage of replicon virus was infectious for Huh7 cells. CONCLUSIONS: Selectable infection was established using HCV replicon containing full-length genotype 2a JFH-1 cDNA. This system might be useful for HCV research.

Trans-complemented hepatitis C virus particles as a versatile tool for study of virus assembly and infection.

In this study, we compared the entry processes of trans-complemented hepatitis C virus particles (HCVtcp), cell culture-produced HCV (HCVcc) and HCV pseudoparticles (HCVpp). Anti-CD81 antibody reduced the entry of HCVtcp and HCVcc to almost background levels, and that of HCVpp by approximately 50%. Apolipoprotein E-dependent infection was observed with HCVtcp and HCVcc, but not with HCVpp, suggesting that the HCVtcp system is more relevant as a model of HCV infection than HCVpp. We improved the productivity of HCVtcp by introducing adapted mutations and by deleting sequences not required for replication from the subgenomic replicon construct. Furthermore, blind passage of the HCVtcp in packaging cells resulted in a novel mutation in the NS3 region, N1586D, which contributed to assembly of infectious virus. These results demonstrate that our plasmid-based system for efficient production of HCVtcp is beneficial for studying HCV life cycles, particularly in viral assembly and infection.

Production and characterization of HCV particles from serum-free culture.

Hepatitis C virus (HCV) is a major cause of liver cancer, and it is therefore important to develop a prophylactic strategy for HCV infection. In recent years, a system for cell culture of the infectious HCV particle has been established, and the inactivated particle has potential as an antigen for vaccine development. In this study, we aimed to establish highly efficient HCV particle purification procedures using the following serum-free culture of HCV particles. First, naïve human hepatoma Huh7 cells were grown in serum-free medium that was supplemented with human-derived insulin, transferrin and sodium selenite. Then, in vitro transcribed JFH-1 or J6/JFH-1 chimeric HCV-RNA was transfected into the serum-free conditioned Huh7 cells. Infectious HCV was secreted into the culture supernatant with the same efficiency as that from cells cultured in FBS-containing medium. The HCV-core protein and RNA continued to be detected in the culture supernatant when the infected cells were subcultured in serum-free medium. Sucrose gradient centrifugation analyses indicated that the profiles of HCV-core, HCV-RNA and the infectivity of HCV particles were almost identical between HCV from FBS-supplemented and serum-free cultures. We further determined that anti-CD81, anti-SR-BI and anti-E2 antibodies inhibited infection by serum-free cultured HCV to a greater extent than infection by HCV from FBS-supplemented cultures. These HCV particles also differed in the level of associated apoplipoproteins: the ApoE level was lower in serum-free cultured HCV. ApoB and ApoE antibody-depletion assays suggested that infection of serum-free cultured HCV was independent of ApoB and ApoE proteins. These data suggest that lipids conjugated with HCV affect infection and neutralization.

Structural basis for platelet antiaggregation by angiotensin II type 1 receptor antagonist losartan (DuP-753) via glycoprotein VI.

GPVI is a key receptor for collagen-induced platelet activation. Loss or inhibition of GPVI causes only mildly prolonged bleeding times but prevents arterial thrombus formation in animal models. Therefore, GPVI is considered to be a potent target molecule for therapy of thrombotic diseases. Recently, it was reported that the AT(1)-receptor antagonist losartan (DuP-753) and EXP3179 inhibit platelet adhesion and aggregation via GPVI. However, it is still not clear how losartan is associated with inhibition of binding between GPVI and collagen at the molecular level. Here, we show by NMR that losartan directly interacts with the hydrophobic region consisting of strands C' and E in the N-terminal Ig-like domain of GPVI. A reliable GPVI-losartan complex model is presented by using a combination of NMR data and in silico tools. These data indicated that the phenyl group with the tetrazole ring in losartan plays a crucial role in the interaction with GPVI.