Hepatitis C virus (HCV) entry into host cells is a complex process requiring multiple host factors, including claudin-1 (CLDN1).Safe and effective therapeutic entry inhibitors need to be developed. We isolated a human hepatic Huh7.5.1-derived cell mutant that is nonpermissive to HCV, and comparative microarray analysis showed that the mutant was CLDN1 defective. Four hybridomas were obtained, which produced monoclonal antibodies (MAbs) that interacted with the parental Huh7.5.1 cell but not with the CLDN1-defective mutant. All MAbs produced by these hybridomas specifically bound to human CLDN1 with a very high affinity and prevented HCV infection of Huh7.5.1 cells in a dose-dependent manner, without apparent cytotoxicity. Two selected MAbs also inhibited HCV infection of human liver-chimeric mice without significant adverse effects. CLDN1 may be a potential target to prevent HCV infection in vivo. Anti-CLDN1 MAbs may hence be promising candidates as novel anti-HCV agents. IMPORTANCESafe and effective therapeutic entry inhibitors against hepatitis C virus (HCV) are very useful for combination therapies with other anti-HCV drugs, such as direct-acting antivirals. In this study, we first showed an effective strategy for developing functional monoclonal antibodies (MAbs) against extracellular domains of a multimembrane-spanning target protein, claudin-1 (CLDN1), by using parental cells expressing the intact target membrane protein and target-defective cells. The established MAbs against CLDN1, which had a very high affinity for intact CLDN1, efficiently inhibited in vitro and in vivo HCV infections. These anti-CLDN1 MAbs are promising leads for novel entry inhibitors against HCV. W orldwide, 170 million people are infected with hepatitis C virus (HCV), which is a major cause of liver cirrhosis and hepatocellular carcinoma. Thus, overcoming HCV infection is an important global health care issue (1). HCV is an enveloped, positive-sense, single-stranded RNA virus in the Flaviviridae family (2). Recent clinical research using direct-acting antivirals that target HCV enzymes, such as sofosbuvir and simeprevir, has provided new insights into combination therapy with inhibitors of multiple targets (3-5).Preventing viral entry into hepatocytes is an attractive target for anti-HCV agents, but strategies for preventing HCV entry into host cells are clinically unavailable (6). Host factors involved in initiating infection include heparan sulfate (7), low-density lipoprotein receptor (8), CD81 (9), scavenger receptor class B type I (SRBI) (10), claudin-1 (CLDN1) (11), occludin (12, 13), epidermal growth factor receptor (EGFR) (14), and Niemann-Pick C1-like 1 (15). Among these, CLDN1 is considered a potent target because it is essential for HCV entry into cells via interaction with CD81 and for cell-to-cell HCV transmission (16,17). Anti-CLDN1 antibodies (Abs) that inhibit HCV infection in vitro were reported by Baumert et al. (18,19) and Hötzel et al. (20), but a CLDN1 binder that prevents HCV infection in vivo has not...

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.

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We established a novel cell-free protein synthesis system derived from Trichoplusia ni (HighFive) insect cells by a simple extraction method. Luciferase and beta-galactosidase were synthesized in this system with active forms. We analyzed and optimized (1) the preparation method of the insect cell extract, (2) the concentration of the reaction components, and (3) the 5'-untranslated region (5'-UTR) of mRNA. The extract was prepared by freeze-thawing insect cells suspended in the extraction buffer. This preparation method was a simple and superior method compared with the conventional method using a Dounce homogenizer. Furthermore, protein synthesis efficiency was improved by the addition of 20% (v/v) glycerol to the extraction buffer. Concentrations of the reaction components were optimized to increase protein synthesis efficiency. Moreover, mRNAs containing 5'-UTRs derived from baculovirus polyhedrin genes showed high protein synthesis activity. Especially, the leader composition of the Ectropis obliqua nucleopolyhedrovirus polyhedrin gene showed the highest enhancement activity among the six 5'-UTRs tested. As a result, in a batch reaction approximately 71 microg of luciferase was synthesized per milliliter of reaction volume at 25 degrees C for 6 h. Moreover, this method for the establishment of a cell-free system was applied also to Spodoptera frugiperda 21 (Sf21) insect cells. After optimizing the concentrations of the reaction components and the 5'-UTR of mRNA, approximately 45 microg/mL of luciferase was synthesized in an Sf21 cell-free system at 25 degrees C for 3 h. These productivities were sufficient to perform gene expression analyses. Thus, these cell-free systems may be a useful tool for simple synthesis in post-genomic studies as a novel protein production method.

Exogenous addition of copper stimulates cellular differentiation inStreptomyces spp. Several lines of evidence suggested a parallel correlation between the stimulatory effect of copper and phenol-oxidizing enzyme activities in Streptomyces griseus. Here a novel extracytoplasmic phenol oxidase (EpoA) associated with cellular development of this organism was identified and characterized. EpoA activity, examined by an in-gel stain procedure with N,N'-dimethyl-p-phenylenediamine sulfate as a substrate, was repressed by glucose and induced by copper supplied in the medium. The enzyme activity was abolished and markedly reduced in the mutants for A-factor biosynthesis and amfR, respectively, which suggested that the activity of the enzyme depends on those essential regulators for morphogenesis in S. griseus. EpoA protein was purified to homogeneity and the N-terminal amino acid sequence was determined. A homologous sequence identified in the genomic database of Streptomyces coelicolorA3(2) was used as a probe to clone the complete epoA gene of S. griseus. The deduced amino acid sequence of EpoA revealed that the mature protein with a molecular mass of 34 kDa was preceded by a signal peptide consisting of 34 aa, consistent with EpoA being a secreted enzyme. EpoA was predicted to be a laccase-type oxidase by not only the sequence similarity, but its substrate selectivity, oxidizing not tyrosine but dihydroxyphenylalanine (DOPA) to generate melanin pigment. Introduction of epoA on a plasmid partially restored both the EpoA activity and aerial mycelium productivity in an A-factor-deficient mutant. Exogenous supplementation of a substance synthesized by purified EpoA from DOPA stimulated cellular differentiation in S. griseus and several other species. Ultrafiltration indicated that the molecular mass of the putative stimulant synthesized by EpoA is between 500 and 1000 Da.