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Previously, we have reported that the active vitamin D metabolite, calcitriol and vitamin D 3 (cholecalciferol), both remarkably inhibit hepatitis C virus production. The mechanism by which vitamin D 3 exerts its effect is puzzling due to the low levels of calcitriol produced in vitamin D 3 -treated Huh7.5 cells. In this study, we aimed to explore the mechanism of vitamin D 3 anti-hepatitis C virus effect. We show that vitamin D 3 activity is not mediated by its metabolic conversion to calcitriol, but may be due to its primary metabolic product 25(OH)D 3 . This is inferred from the findings that 25(OH)D 3 could inhibit hepatitis C virus production in our system, and that adequate concentrations needed to exert this effect are produced in Huh7.5 cells treated with vitamin D 3 . Using the CRISPR-Cas9 editing technology to knockout the vitamin D receptor, we found that the antiviral activity of vitamin D 3 and 25(OH)D 3 was not impaired in the vitamin D receptor knockout cells. This result indicates that 25(OH)D 3 anti-hepatitis C virus effect is exerted by a vitamin D receptor-independent mode of action. The possibility that vitamin D 3 and 25(OH)D 3 , being 3β-hydroxysteroids, affect hepatitis C virus production by direct inhibition of the Hedgehog pathway in a vitamin D receptor-independent manner was ruled out. Taken together, this study proposes a novel mode of action for the anti-hepatitis C virus activity of vitamin D 3 that is mediated by 25(OH)D 3 in a vitamin D receptor-independent mechanism.

Serodiagnosis of infectious disease is often based on the detection of pathogen-specific antibodies in a patient's blood. For this, mixtures of pathogen-related antigens are used as bait to capture corresponding antibodies in solid phase immunoassays such as enzyme immunoassay (EIA). Western blots provide improved diagnostic power as compared with EIA due to the fact that the mixture of markers in the EIA well is resolved and tested as individual antigens on the Western blot. Hence, confirmation of EIA results is accomplished using the antigen arrays of Western blots. Here we took this approach one step further and tested the attributes of using epitope arrays in a diagnostic platform coined "combinatorial diagnostics." As a case in point, we tested a panel of phage-displayed epitope-based markers in the serodiagnosis of hepatitis C virus (HCV). The repertoire of HCV antigens was deconvoluted into panels of distinct linear and conformational epitopes and tested individually by quantitative EIA. Combinatorial diagnostics proved to be effective for the discrimination between positive and negative sera as well as serotyping of HCV., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication., (© 2011 Blackwell Publishing Ltd.)

Unlabelled: Vitamin D supplementation was reported to improve the probability of achieving a sustained virological response when combined with antiviral treatment against hepatitis C virus (HCV). Our aim was to determine the in vitro potential of vitamin D to inhibit HCV infectious virus production and explore the mechanism(s) of inhibition. Here we show that vitamin D(3) remarkably inhibits HCV production in Huh7.5 hepatoma cells. These cells express CYP27B1, the gene encoding for the enzyme responsible for the synthesis of the vitamin D hormonally active metabolite, calcitriol. Treatment with vitamin D(3) resulted in calcitriol production and induction of calcitriol target gene CYP24A1, indicating that these cells contain the full machinery for vitamin D metabolism and activity. Notably, treatment with calcitriol resulted in HCV inhibition. Collectively, these findings suggest that vitamin D(3) has an antiviral activity which is mediated by its active metabolite. This antiviral activity involves the induction of the interferon signaling pathway, resulting in expression of interferon-β and the interferon-stimulated gene, MxA. Intriguingly, HCV infection increased calcitriol production by inhibiting CYP24A1 induction, the enzyme responsible for the first step in calcitriol catabolism. Importantly, the combination of vitamin D(3) or calcitriol and interferon-α synergistically inhibited viral production., Conclusion: This study demonstrates for the first time a direct antiviral effect of vitamin D in an in vitro infectious virus production system. It proposes an interplay between the hepatic vitamin D endocrine system and HCV, suggesting that vitamin D has a role as a natural antiviral mediator. Importantly, our study implies that vitamin D might have an interferon-sparing effect, thus improving antiviral treatment of HCV-infected patients., (Copyright © 2011 American Association for the Study of Liver Diseases.)

HBV and HCV have major roles in hepatocarcinogenesis. More than 500 million people are infected with hepatitis viruses and, therefore, HCC is highly prevalent, especially in those countries endemic for HBV and HCV. Viral and host factors contribute to the development of HCC. The main viral factors include the circulating load of HBV DNA or HCV RNA and specific genotypes. Various mechanisms are involved in the host-viral interactions that lead to HCC development, among which are genetic instability, self-sufficiency in growth signals, insensitivity to antigrowth signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and tissue invasiveness. Prevention of HBV by vaccination, as well as antiviral therapy against HBV and for HCV seem able to inhibit the development of HCC., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins"). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small-molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Background/aims: Hepatitis C virus infection is a major worldwide health problem, causing chronic hepatitis, cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be important. With the aim of inhibiting NS3 protease activity as a means to inhibit HCV replication we used a novel bacterial genetic screen to isolate NS3-inhibiting peptide aptamers., Methods: We have isolated and characterized seven NS3-inhibiting peptide aptamers. We investigated the phenotypic changes that SEAP-secreting subgenomic RNA replicons undergo upon intracellular expression of these peptide aptamers, assayed by real-time RT-PCR and inhibition of SEAP secretion by transfected replicon cells., Results and Conclusions: The peptide aptamers inhibited NS3 protease activity in vitro with an IC50 in the low micromolar range. Upon transfection, aptamers inhibited the replication of SEAP-secreting genotype 1b subgenomic RNA replicons. Aptamer-based intracellular immunization may emerge as a promising antiviral approach to interfere with the life cycle and pathogenicity of HCV.

The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence with unknown biological function. In this study we sought to characterize the prevalence of specific anti-F antibodies in patients with chronic HCV infection and to analyze the anti-F antibody profile before, during, and after antiviral treatment in order to gain a better understanding of the role of F protein in HCV pathogenesis. Serum samples were collected from 44 patients with chronic HCV infection and from 19 healthy controls. Consecutive samples from 27 patients taken before, during, and after treatment with antiviral therapy. The F and the core proteins were cloned from the HCV genome. The recombinant proteins were expressed in Escherichia coli and affinity purified. A sensitive and specific enzyme-linked immunosorbent assay was developed to assess the prevalence of anti-F antibodies. Eighty-nine percent of chronic HCV patients had evidence of anti-F antibodies, and 95% of them had anti-core antibodies. No correlation of anti-F antibodies was found with response to treatment, genotype, or seroconversion. We conclude that the F protein elicits specific antibodies in most individuals chronically infected with HCV with no correlation with response to treatment. Our results confirm the expression of F protein during natural HCV infection.

Background: Transmission of hepatitis C virus (HCV) from infected health care workers to patients rarely occurs. In 2003, a cluster of patients with HCV infection was identified at a medical center in Israel. All patients had a common history of various surgical procedures performed during the period 2001-2003. All patients had been anesthetized by an anesthesiologist who was an injection drug user and was infected with genotype 2a HCV. Screening was initiated by the hospital to identify newly infected patients with HCV infection and to determine the source of the iatrogenic HCV infection outbreak using comparative molecular analysis of the HCV E1 and HCV E2 hypervariable regions (HVR1 and HVR2)., Methods: A total of 1200 patients who were anesthetized by the anesthesiologist (the related group) and 873 hospital personnel and patients anesthetized by other anesthetists (the unrelated group) were examined. Serum samples were screened for anti-HCV antibodies, HCV RNA, and genotype. Sequence analysis of HVR1 and HVR2 was performed after reverse-transcriptase polymerase chain reaction., Results: HCV type 2a was found in 33 patients in the related group but in only 1 patient in the unrelated group. The differences between the sequences isolated from the related group serum samples and the sequences isolated from genotype 2a control group serum samples (obtained from 15 patients) were highly statistically significant. The genetic distances from the anesthesiologist sequence were 1.4%-4.4% in the HVR1 and 0%-3% in the HVR2 in the related group serum samples, whereas in the HCV genotype 2a control group serum samples, the genetic distances were 22%-45% and 10%-35%, respectively., Conclusions: Molecular analysis revealed sequence similarity of HVR1 and HVR2 in the related group, suggesting that the anesthesiologist with chronic HCV infection may have transmitted HCV to 33 patients.

Hepatitis C virus (HCV) infection is a major world-wide health problem causing chronic hepatitis, liver cirrhosis and primary liver cancer. The high frequency of treatment failure points to the need for more specific, less toxic and more active antiviral therapies for HCV. The HCV NS3 is currently regarded as a prime target for anti-viral drugs, thus specific inhibitors of its activity are of utmost importance. Here, we report the development of a novel bacterial genetic screen for inhibitors of NS3 catalysis and its application for the isolation of single-chain antibody-inhibitors. Our screen is based on the concerted co-expression of a reporter gene, of recombinant NS3 protease and of fusion-stabilized single-chain antibodies (scFvs) in Escherichia coli. The reporter system had been constructed by inserting a short peptide corresponding to the NS5A/B cleavage site of NS3 into a permissive site of the enzyme beta-galactosidase. The resulting engineered lacZ gene, coding for an NS3-cleavable beta-galactosidase, is carried on a low copy plasmid that also carried the NS3 protease-coding sequence. The resultant beta-galactosidase enzyme is active, conferring a Lac+ phenotype (blue colonies on indicator 5-bromo-4-chloro-3-indolyl beta-D-galactoside (X-gal) plates), while induction of NS3 expression results in loss of beta-galactosidase activity (transparent colonies on X-gal plates). The identification of inhibitors, as shown here by isolating NS3-inhibiting single-chain antibodies, expressed from a compatible high copy number plasmid, is based on the appearance of blue colonies (NS3 inhibited) on the background of colorless colonies (NS3 active). Our source of inhibitory scFvs was an scFv library that we prepared from spleens of NS3-immunized mice and subjected to limited affinity selection. Once isolated, the inhibitors were validated as genuine and specific NS3 binders by an enzyme-linked immunosorbent assay and as bone fide NS3 serine protease inhibitors by an in vitro catalysis assay. We further show that upon expression as cytoplasmic intracellular antibodies (intrabodies) in NS3-expressing mammalian cells, three of the scFvs inhibit NS3-mediated cell proliferation. Although applied here for the isolation of antibody-based inhibitors, our genetic screen should be applicable for the identification of candidate inhibitors from other sources.

Background/aims: Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer. In addition to its role in the viral polyprotein-processing, the viral NS3 serine protease has been implicated in interactions with various cell constituents resulting in phenotypic changes including malignant transformation. NS3 is currently regarded a prime target for anti-viral drugs thus specific inhibitors of its activities should be of importance. With the aim of inhibiting NS3-mediated cell transformation, we used antibody-phage display to isolate NS3-specific single-chain antibodies (scFv)., Methods: We have isolated and characterized eight anti-NS3 scFvs. We investigated the phenotypic changes that NS3-expressing cells undergo upon intracellular expression of these antibodies in different subcellular compartments (intracellular immunization), assayed by their proliferation rate and their ability to grow anchorage independently. The intracellular location of NS3 and the scFvs were analyzed by immunofluorescent staining using confocal microscopy., Results: Nuclear targeted anti-NS3 intrabodies shuttle NS3 from the cytosol to the nucleus with concomitant inhibition of cell proliferation and loss of the transformed phenotype., Conclusions: Intracellular immunization-based gene therapy strategies may emerge as a promising antiviral approach to interfere with the life cycle and tumorigenicity of HCV.

Hepatitis C virus (HCV) infection is a major worldwide health problem, causing chronic hepatitis, liver cirrhosis and primary liver cancer (Hepatocellular carcinoma). HCV encodes a precursor polyprotein that is enzymatically cleaved to release the individual viral proteins. The viral non-structural proteins are cleaved by the HCV NS3 serine protease. NS3 is regarded currently as a potential target for anti-viral drugs thus specific inhibitors of its enzymatic activity should be of importance. A prime requisite for detailed biochemical studies of the protease and its potential inhibitors is the availability of a rapid reliable in vitro assay of enzyme activity. A novel assay for measurement of HCV NS3 serine protease activity was developed for screening of HCV NS3 serine protease potential inhibitors. Recombinant NS3 serine protease was isolated and purified, and a fluorometric assay for NS3 proteolytic activity was developed. As an NS3 substrate we engineered a recombinant fusion protein where a green fluorescent protein is linked to a cellulose-binding domain via the NS5A/B site that is cleavable by NS3. Cleavage of this substrate by NS3 results in emission of fluorescent light that is easily detected and quantitated by fluorometry. Using our system we identified NS3 serine protease inhibitors from extracts obtained from natural Indian Siddha medicinal plants. Our unique fluorometric assay is very sensitive and has a high throughput capacity making it suitable for screening of potential NS3 serine protease inhibitors.

Persistent infection with hepatitis C virus (HCV) may lead to hepatocellular carcinoma (HCC). It has been suggested that HCV-encoded proteins are directly involved in the tumorigenic process. The HCV nonstructural protein NS3 has been identified as a virus-encoded serine protease. To study whether HCV NS3 has oncogenic activity, nontumorigenic rat fibroblast (RF) cells were stably transfected with an expression vector containing cDNA for the NS3 serine protease (nucleotides 3356-4080). The NS3 serine protease activity was determined in the transfected cells. The transfected cells grew rapidly and proliferated serum independently, lost contact inhibition, grew anchorage independently in soft agar and induced significant tumour formation in nude mice. Cells transfected with an expression vector containing a mutated NS3 serine protease (serine 139 to alanine at the catalytic site) showed no transforming abilities; their growth was dependent on serum and they did not grow anchorage independently in soft agar. Moreover, cells transfected with the NS3 serine protease and treated with the chymotrypsin inhibitors TPCK and PMSF (a serine protease inhibitor) lost their transforming feature. These results suggest that the NS3 serine protease of HCV is involved in cell transformation and that the ability to transform requires an active enzyme.

The mechanism of hepatitis C virus (HCV) -induced hepatotocellular carcinoma (HCC) is still unknown, but in vitro studies clearly suggest that HCV proteins exert a direct effect on liver carcinogenesis. HCV NS3 serine protease is known to play a key role in the life cycle of the virus and may interact with the host cellular regulatory proteins. The aim of the present study was to conduct a genetic analysis of the HCV NS3 gene coding for the serine protease isolated from serum, tumor, and nontumor tissue of HCC patients. RNA was extracted and HCV cDNA was amplified by nested reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence comparison yielded unique changes at the vicinity of the catalytic sites of the NS3 clones isolated only from HCC tissue. These changes included the insertion of a "large" and charged amino acid, substitution of a polar with a hydrophobic amino acid, and substitution of a charged with a polar amino acid. Those changes affect the electrostatic charge around the active site, and thus the activity and substrate specificity of the serine protease. This is the first study to define significant amino acid changes at the catalytic domain of the NS3 serine protease gene isolated from HCC tissue.