Notch signaling facilitates hepatitis B virus covalently closed circular DNA transcription via cAMP response element-binding protein with E3 ubiquitin ligase-modulation.

Notch1 is regulated by E3 ubiquitin ligases, with proteasomal degradation of the Notch intracellular domain affecting the transcription of target genes. cAMP response element-binding protein (CREB) mediates the transcription of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). We assessed the relationship between HBV cccDNA and Notch signaling activities. HBV cccDNA levels and relative gene expression were evaluated in HBV-replicating cells treated with Jagged1 shRNA and a γ-secretase inhibitor. The effects of these factors in surgically resected clinical samples were also assessed. Notch inhibition suppressed HBV cccDNA and CREB-related expression but increased ITCH and NUMB levels. Proteasome inhibitor augmented HBV cccDNA, restored Notch and CREB expression, and inhibited ITCH and NUMB function. Increased HBV cccDNA was observed after ITCH and NUMB blockage, even after treatment with the adenylate cyclase activator forskolin; protein kinase A (PKA) inhibitor had the opposite effect. Notch activation and E3 ligase inactivation were observed in HBV-positive cells in clinical liver tissue. Collectively, these findings reveal that Notch signaling activity facilitates HBV cccDNA transcription via CREB to trigger the downstream PKA-phospho-CREB cascade and is regulated by E3 ubiquitin ligase-modulation of the Notch intracellular domain.

Hepatitis B virus X protein induces hepatic stem cell-like features in hepatocellular carcinoma by activating KDM5B.

AIM: To determine the role of hepatitis B virus X protein (HBx), HBx in regulating hepatic progenitor cell (HPC)-like features in hepatocellular carcinoma (HCC) and the underlying molecular mechanisms. METHODS: We used a retrovirus vector to introduce wild type HBx or empty vector into HepG2 cells. We then used these cells to analyze cell proliferation, senescence, transformation, and stem-like features. Gene expression profiling was carried out on Affymetrix GeneChip Human U133A2.0 ver.2 arrays according to the manufacturer's protocol. Unsupervised hierarchical clustering analysis and Class Comparison analysis were performed by BRB-Array Tools software Version 4.2.2. A total of 238 hepatitis B virus (HBV)-related HCC patients' array data were used for analyzing clinical features. RESULTS: The histone demethylase KDM5B was significantly highly expressed in HBV-related HCC cases (P < 0.01). In HBV proteins, only HBx up-regulated KDM5B by activating c-myc. Hepatic stem cell (HpSC) markers (EpCAM, AFP, PROM1, and NANOG) were significantly highly expressed in KDM5B-high HCC cases (P < 0.01). KDM5B played an important role in maintaining HpSC-like features and was associated with a poor prognosis. Moreover, inhibition of KDM5B suppressed spheroid formation and cell invasion in vitro. CONCLUSION: HBx activates the histone demethylase KDM5B and induces HPC-like features in HCC. Histone demethylases KDM5B may be an important therapeutic target against HBV-related HCC cases.

Efficient Suppression of Hepatitis C Virus Replication by Combination Treatment with miR-122 Antagonism and Direct-acting Antivirals in Cell Culture Systems.

Direct-acting antivirals (DAAs) against Hepatitis C virus (HCV) show effective antiviral activity with few side effects. However, the selection of DAA-resistance mutants is a growing problem that needs to be resolved. In contrast, miR-122 antagonism shows extensive antiviral effects among all HCV genotypes and a high barrier to drug resistance. In the present study, we evaluated three DAAs (simeprevir, daclatasvir, and sofosbuvir) in combination with anti-miR-122 treatment against HCV genotype 1a in cell cultures. We found that combination treatments with anti-miR-122 and a DAA had additive or synergistic antiviral effects. The EC50 values of simeprevir in simeprevir-resistant mutants were significantly decreased by combining simeprevir with anti-miR-122. A similar reduction in EC50 in daclatasvir-resistant mutants was achieved by combining daclatasvir with anti-miR-122. Combination treatment in HCV-replicating cells with DAA and anti-miR-122 sharply reduced HCV RNA amounts. Conversely, DAA single treatment with simeprevir or daclatasvir reduced HCV RNA levels initially, but the levels later rebounded. DAA-resistant mutants were less frequently observed in combination treatments than in DAA single treatments. In summary, the addition of miR-122 antagonism to DAA single treatments had additive or synergistic antiviral effects and helped to efficiently suppress HCV replication and the emergence of DAA-resistant mutants.

Impaired interferon signaling in chronic hepatitis C patients with advanced fibrosis via the transforming growth factor beta signaling pathway.

UNLABELLED: Malnutrition in the advanced fibrosis stage of chronic hepatitis C (CH-C) impairs interferon (IFN) signaling by inhibiting mammalian target of rapamycin complex 1 (mTORC1) signaling. However, the effect of profibrotic signaling on IFN signaling is not known. Here, the effect of transforming growth factor (TGF)-ß signaling on IFN signaling and hepatitis C virus (HCV) replication was examined in Huh-7.5 cells by evaluating the expression of forkhead box O3A (Foxo3a), suppressor of cytokine signaling 3 (Socs3), c-Jun, activating transcription factor 2, ras homolog enriched in brain, and mTORC1. The findings were confirmed in liver tissue samples obtained from 91 patients who received pegylated-IFN and ribavirin combination therapy. TGF-ß signaling was significantly up-regulated in the advanced fibrosis stage of CH-C. A significant positive correlation was observed between the expression of TGF-ß2 and mothers against decapentaplegic homolog 2 (Smad2), Smad2 and Foxo3a, and Foxo3a and Socs3 in the liver of CH-C patients. In Huh-7.5 cells, TGF-ß1 activated the Foxo3a promoter through an AP1 binding site; the transcription factor c-Jun was involved in this activation. Foxo3a activated the Socs3 promoter and increased HCV replication. TGF-ß1 also inhibited mTORC1 and IFN signaling. Interestingly, c-Jun and TGF-ß signaling was up-regulated in treatment-resistant IL28B minor genotype patients (TG/GG at rs8099917), especially in the early fibrosis stage. Branched chain amino acids or a TGF-ß receptor inhibitor canceled these effects and showed an additive effect on the anti-HCV activity of direct-acting antiviral drugs (DAAs). CONCLUSION: Blocking TGF-ß signaling could potentiate the antiviral efficacy of IFN- and/ or DAA-based treatment regimens and would be useful for the treatment of difficult-to-cure CH-C patients.

The acyclic retinoid Peretinoin inhibits hepatitis C virus replication and infectious virus release in vitro.

Clinical studies suggest that the oral acyclic retinoid Peretinoin may reduce the recurrence of hepatocellular carcinoma (HCC) following surgical ablation of primary tumours. Since hepatitis C virus (HCV) infection is a major cause of HCC, we assessed whether Peretinoin and other retinoids have any effect on HCV infection. For this purpose, we measured the effects of several retinoids on the replication of genotype 1a, 1b, and 2a HCV in vitro. Peretinoin inhibited RNA replication for all genotypes and showed the strongest antiviral effect among the retinoids tested. Furthermore, it reduced infectious virus release by 80-90% without affecting virus assembly. These effects could be due to reduced signalling from lipid droplets, triglyceride abundance, and the expression of mature sterol regulatory element-binding protein 1c and fatty acid synthase. These negative effects of Peretinoin on HCV infection may be beneficial in addition to its potential for HCC chemoprevention in HCV-infected patients.

MicroRNA-27a regulates lipid metabolism and inhibits hepatitis C virus replication in human hepatoma cells.

The replication and infectivity of the lipotropic hepatitis C virus (HCV) are regulated by cellular lipid status. Among differentially expressed microRNAs (miRNAs), we found that miR-27a was preferentially expressed in HCV-infected liver over hepatitis B virus (HBV)-infected liver. Gene expression profiling of Huh-7.5 cells showed that miR-27a regulates lipid metabolism by targeting the lipid synthetic transcription factor RXRα and the lipid transporter ATP-binding cassette subfamily A member 1. In addition, miR-27a repressed the expression of many lipid metabolism-related genes, including FASN, SREBP1, SREBP2, PPARα, and PPARγ, as well as ApoA1, ApoB100, and ApoE3, which are essential for the production of infectious viral particles. miR-27a repression increased the cellular lipid content, decreased the buoyant density of HCV particles from 1.13 to 1.08 g/cm(3), and increased viral replication and infectivity. miR-27a overexpression substantially decreased viral infectivity. Furthermore, miR-27a enhanced in vitro interferon (IFN) signaling, and patients who expressed high levels of miR-27a in the liver showed a more favorable response to pegylated IFN and ribavirin combination therapy. Interestingly, the expression of miR-27a was upregulated by HCV infection and lipid overload through the adipocyte differentiation transcription factor C/EBPα. In turn, upregulated miR-27a repressed HCV infection and lipid storage in cells. Thus, this negative feedback mechanism might contribute to the maintenance of a low viral load and would be beneficial to the virus by allowing it to escape host immune surveillance and establish a persistent chronic HCV infection.

La protein required for internal ribosome entry site-directed translation is a potential therapeutic target for hepatitis C virus replication.

BACKGROUND: Translation of the hepatitis C virus (HCV) is mediated by an internal ribosome entry site (IRES). Here, we analyzed the functional relevance of La protein for replication of HCV using an infectious HCV clone, JFH-1. METHODS: A single-nucleotide mutation from A to U was introduced at the 338th nucleotide in the stem-loop domain IV structure of HCV IRES, which stabilized stem-loop IV and abolished translation and replication of JFH-1 almost completely. RESULTS: During JFH-1 replication, translation initiation factors required for HCV IRES activity, including La protein, polypyrimidine tract binding protein (PTB), PSMA7, and PCBP2, were significantly induced in Huh-7.5 cells. Interestingly, JFH-1 infection increased telomerase activity and induced the expression of human telomerase RNA (hTR) in Huh-7.5 cells. In 37 tissue specimens from patients with chronic hepatitis C, La protein significantly correlated with the representative essential telomerase components hTR, p23, and HSP90 (P<.001). Recombinant adenovirus that expressed short-hairpin RNA against La protein successfully suppressed the levels of La protein and core protein of JFH-1 to 30% of that in the control cells. CONCLUSIONS: HCV infection might be strongly related to telomerase activity in the liver through La protein induction. Inhibition of La protein substantially repressed JFH-1 replication; therefore, La protein is a potential therapeutic target for HCV.

Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein.

The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on ("Tet-on") gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.

The scaffold protein c-Jun NH2-terminal kinase-associated leucine zipper protein regulates cell migration through interaction with the G protein G(alpha 13).

Scaffold proteins for MAP kinase (MAPK) signalling modules play an important role in the specific and efficient signal transduction of the relevant MAPK cascades. Here, we investigated the function of the scaffolding protein c-Jun NH(2)-terminal kinase (JNK)-associated leucine zipper protein (JLP) by depleting it in cultured cells using a short hairpin RNA (shRNA) against human JLP. HeLa and DLD-1 cells stably expressing the shRNA showed a defect in cell migration. The re-expression of full-length shRNA-resistant mouse JLP rescued the impaired cell migration of the JLP-depleted HeLa cells; whereas, a C-terminal deletion mutant of mouse JLP, which failed to bind the G protein G(alpha13), showed little or no effect on the cell migration defect. Furthermore, although a constitutively active G(alpha13) enhanced the migration of control HeLa cells, the G(alpha13)-induced cell migration was significantly suppressed in the JLP-depleted HeLa cells. Taken together, these results suggest that JLP regulates cell migration through an interaction with G(alpha13).

Mutations in Ran system affected telomere silencing in Saccharomyces cerevisiae.

The Ran GTPase system regulates the direction and timing of several cellular events, such as nuclear-cytosolic transport, centrosome formation, and nuclear envelope assembly in telophase. To gain insight into the Ran system's involvement in chromatin formation, we investigated gene silencing at the telomere in several mutants of the budding yeast Saccharomyces cerevisiae, which had defects in genes involved in the Ran system. A mutation of the RanGAP gene, rna1-1, caused reduced silencing at the telomere, and partial disruption of the nuclear Ran binding factor, yrb2-delta2, increased this silencing. The reduced telomere silencing in rna1-1 cells was suppressed by a high dosage of the SIR3 gene or the SIT4 gene. Furthermore, hyperphosphorylated Sir3 protein accumulated in the rna1-1 mutant. These results suggest that RanGAP is required for the heterochromatin structure at the telomere in budding yeast.

Hepatitis B virus X protein overcomes oncogenic RAS-induced senescence in human immortalized cells.

Chronic infection with hepatitis B virus (HBV) is a major risk factor for hepatocellular carcinoma. The HBV X protein (HBx) is thought to have oncogenic potential, although the molecular mechanism remains obscure. Pathological roles of HBx in the carcinogenic process have been examined using rodent systems and no report is available on the oncogenic roles of HBx in human cells in vitro. We therefore examined the effect of HBx on immortalization and transformation in human primary cells. We found that HBx could overcome active RAS-induced senescence in human immortalized cells and that these cells could form colonies in soft agar and tumors in nude mice. HBx alone, however, could contribute to neither immortalization nor transformation of these cells. In a population doubling analysis, an N-terminal truncated mutant of HBx, HBx-D1 (amino acids 51-154), which harbors the coactivation domain, could overcome active RAS-induced cellular senescence, but these cells failed to exhibit colonigenic and tumorigenic abilities, probably due to the low expression level of the protein. By scanning a HBx expression library of the clustered-alanine substitution mutants, the N-terminal domain was found to be critical for overcoming active RAS-induced senescence by stabilizing full-length HBx. These results strongly suggest that HBx can contribute to carcinogenesis by overcoming active oncogene-induced senescence.

Functional interaction of hepatitis C Virus NS5B with Nucleolin GAR domain.

Hepatitis C Virus (HCV) non-structural proteins are major components of replication complex that is modulated by several host factors. We previously reported that nucleolin, a representative nucleolar marker, interacts with the NS5B through two separated sequences, amino acids (aa) 208-214 and 500-506, and that W208 in the former stretch is essential for both nucleolin-binding and HCV replication. Here we evaluated the role of the latter stretch aa 500-506 of WRHRARS in nucleolin-binding and HCV replication scanned by alanine-substituted clustered mutant (cm) or point mutant (pm). One tryptophan and three arginine residues in the sequence were found to be essential both for nucleolin-binding in vivo and HCV replication detected with a HCV subgenomic replicon transfected into Huh7 cells. NS5B-binding of nucleolin was further delineated by truncation and clustered mutants of nucleolin. Arginine-glycine-glycine (RGG) repeat in the Glycine arginine rich (GAR) domain were defined to be indispensable for NS5B-binding immunologically detected in in vivo and in vitro although short internal-truncations of RGG repeat are tolerable for NS5B-binding. These results indicate that nucleolin is a critical host factor for HCV replication through the direct interaction between W208 and several residues at the sequence, aa 500-505, of NS5B, and the long-turn motif including RGG repeat at nucleolin C-terminal.

Human telomerase exists in two distinct active complexes in vivo.

Telomerase, a stable complex of telomerase reverse transcriptase (TERT) and template RNA (TERC), is responsible for telomere maintenance. During purification trials of recombinant human telomerase of the two components reconstituted in insect cells, we identified two complexes of human telomerase of molecular masses 680 and 380 kDa, both of which retain telomerase activity in vitro. We show here that the former complex does not include Hsp90 (heat shock protein 90) and its telomerase activity is resistant to Hsp90 inhibitors, whereas the latter contains Hsp90 and its telomerase activity is sensitive to Hsp90 inhibitors. N-terminal of FLAG-hTERT in the former is exposed, as this complex was efficiently purified with anti-FLAG M2 affinity resin. We also identified two different telomerase complexes in HeLa cells, in addition to ectopically expressed hTERT. Most of endogenous hTERT and FLAG-hTERT was detected around 680 kDa. These two complexes in HeLa cells have the same properties as their respective reconstituted telomerases. The unstable property of the telomerase complex with Hsp90, especially in the presence of Hsp90 inhibitors, was due to proteasome-mediated degradation of hTERT, since proteasome inhibitors prevented hTERT degradation in vivo. To our knowledge, this is the first demonstration of two distinct active complexes of human telomerase ectopically expressed in insect and mammalian cells.

Temperature-sensitive defects of the GSP1gene, yeast Ran homologue, activate the Tel1-dependent pathway.

RanGTPase is involved in many cellular processes. It functions in nuclear-cytosolic transport and centrosome formation. Ran also localizes to chromatin as RCC1 does, its guanine nucleotide exchange factor, but Ran's function on chromatin is not known. We found that gsp1, a temperature-sensitive mutant of GSP1, a Saccharomyces cerevisiae Ran homologue, suppressed the hydroxyurea (HU) and ultra violet (UV) sensitivities of the mec1 mutant. In UV-irradiated mec1 gsp1 cells, Rad53 was phosphorylated despite the lack of Mec1. This suppression depended on the TEL1 gene, given that the triple mutant, mec1 gsp1 tel1, was unable to grow. The gsp1 mutations also suppressed the HU sensitivity of the rad9 mutant in a Tel1-dependent manner, but not the HU sensitivity of the rad53 mutant. These results indicated that Rad53 was activated by the Tel1 pathway in mec1 gsp1 cells, suggesting that Gsp1 helps regulate the role switching the ATM family kinases Mec1 and Tel1.

Molecular functions and biological roles of hepatitis B virus x protein.

Chronic infection of hepatitis B virus (HBV) is one of the major causes of hepatocellular carcinoma (HCC) in the world. Hepatitis B virus X protein (HBx) has been long suspected to be involved in hepatocarcinogenesis, although its oncogenic role remains controversial. HBx is a multifunctional regulator that modulates transcription, signal transduction, cell cycle progress, protein degradation pathways, apoptosis, and genetic stability by directly or indirectly interacting with host factors. This review focuses on the biological roles of HBx in HBV replication and cellular transformation in terms of the molecular functions of HBx. Using the transient HBV replication assay, ectopically expressed HBx could stimulate HBV transcription and replication with the X-defective replicon to the level of those with the wild one. The transcription coactivation is mainly contributing to the stimulatory role of HBx on HBV replication although the other functions may affect HBV replication. Effect of HBx on cellular transformation remains controversial and was never addressed with human primary or immortal cells. Using the human immortalized primary cells, HBx was found to retain the ability to overcome active oncogene RAS-induced senescence that requires full-length HBx. At least two functions of HBx, the coactivation function and the ability to overcome oncogene-induced senescence, may be cooperatively involved in HBV-related hepatocarcinogenesis.

Effect of hepatitis C virus (HCV) NS5B-nucleolin interaction on HCV replication with HCV subgenomic replicon.

We previously reported that nucleolin, a representative nucleolar marker, interacts with nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) through two independent regions of NS5B, amino acids 208 to 214 and 500 to 506. We also showed that truncated nucleolin that harbors the NS5B-binding region inhibited the RNA-dependent RNA polymerase activity of NS5B in vitro, suggesting that nucleolin may be involved in HCV replication. To address this question, we focused on NS5B amino acids 208 to 214. We constructed one alanine-substituted clustered mutant (CM) replicon, in which all the amino acids in this region were changed to alanine, as well as seven different point mutant (PM) replicons, each of which harbored an alanine substitution at one of the amino acids in the region. After transfection into Huh7 cells, the CM replicon and the PM replicon containing NS5B W208A could not replicate, whereas the remaining PM replicons were able to replicate. In vivo immunoprecipitation also showed that the W208 residue of NS5B was essential for its interaction with nucleolin, strongly suggesting that this interaction is essential for HCV replication. To gain further insight into the role of nucleolin in HCV replication, we utilized the small interfering RNA (siRNA) technique to investigate the knockdown effect of nucleolin on HCV replication. Cotransfection of replicon RNA and nucleolin siRNA into Huh7 cells moderately inhibited HCV replication, although suppression of nucleolin did not affect cell proliferation. Taken together, our findings strongly suggest that nucleolin is a host component that interacts with HCV NS5B and is indispensable for HCV replication.

Mutational analysis of human RNA polymerase II subunit 5 (RPB5): the residues critical for interactions with TFIIF subunit RAP30 and hepatitis B virus X protein.

RNA polymerase II (RNAPII) subunit 5 (RPB5) is positioned close to DNA downstream of the initiation site and is the site of interaction with several regulators. Hepatitis B virus X protein (HBx) binds the central part of RPB5 to modulate activated transcription, and TFIIF subunit RAP30 interacts with the same part of RPB5 that is critical for the association between TFIIF and RNAPII. However the residues necessary for these interactions remain unknown. Here we report systematic mutagenesis of the central part of RPB5 using two-step alanine scanning libraries to pinpoint critical residues for its binding to RAP30 in the TFIIF complex and/or to HBx, and identified these residues in both mammalian cells and in an in vitro binding assay. Four residues, F76, I104, T111 and S113, are critical for both TFIIF- and HBx-binding, indicating the overlapping nature of the sites of interaction. In addition, V74 and N98 are required for HBx-binding, and T56 and L58 are needed for RAP30-binding. Interestingly the residues exposed to solvent, T111 and S113, are very close to the DNA, implying that two factors may modulate the interaction between DNA and RPB5.

The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication.

The role and functional domain of hepatitis B virus (HBV) X protein (HBx) in regulating HBV transcription and replication were investigated with a transient transfection system in the human hepatoma cell line HepG2 using wild-type or HBx-minus HBV genome constructs and a series of deletion or mutation HBx expression plasmids. We show here that HBx has augmentation effects on HBV transcription and replication as a HBV mutant genome with defective X gene led to decreased levels of 3.5-kb HBV RNA and HBV replication intermediates and that these decreases can be restored by either transient ectopic expression of HBx or a stable HBx expression cell line. The C-terminal two-thirds (amino acids [aa] 51 to 154), which contain the transactivation domain, is required for this function of HBx; the N-terminal one-third (aa 1 to 50) is not required. Using the alanine scanning mutagenesis strategy, we demonstrated that the regions between aa 52 to 65 and 88 to 154 are important for the augmentation function of HBx in HBV replication. By the luciferase reporter gene analysis, we found that the transactivation and coactivation activities of HBx coincide well with its augmentation function in HBV transcription and replication. These results suggest that HBx has an important role in stimulating HBV transcription and replication and that the transcriptional transactivation function of HBx may be critical for its augmentation effect on HBV replication.

The SIT4 gene, which encodes protein phosphatase 2A, is required for telomere function in Saccharomyces cerevisiae.

Life span and number of cell divisions in eukaryotes are limited. The accumulation of stress-associated damage due to ageing may cause irreversible cell cycle arrest, so-called "cellular senescence". Although many genes have been implicated in determining life span, regulatory systems that counteract age-related stress have not yet been clarified. We examined senescence during a stress of Saccharomyces cerevisiae strains carrying disruptions in protein phosphatase (PPase)-encoding genes in order to identify the system counteracting senescence. Among these strains, short telomeres were found in the sit4 disruptant that lacks one form of protein phosphatase 2A (PP2A). Silencing ability in the subtelomeric region was impaired and hyperphosphorylation of Sir3 was also observed in this mutant. The sit4 mutant was found to have altered nucleoli and a life span as short as an sgs1 mutant. These observations suggest that the PP2A pathway regulates life span in yeast.

[Purification and partial characterization of hepatitis C virus (HCV) non-structural protein 5A (NS5A) expressed in Escherichia coli].

It has been suggested that non-structural protein 5A (NS5A) of hepatitis C virus (HCV) may have a regulatory role similar to other RNA viruses in RNA replication. In order to investigate the replication function of NS5A, we tried to purify recombinant His(6) tagged NS5A expressed in Escherichia coli by a denature-renaturing method. The native lysis buffer was used to remove most of the soluble non-specific proteins. His-NS5A protein was solublized with the denaturing lysis buffer containing 8 mol/L Urea, and then bound to Ni2+ -NTA resin. The protein bound resin was successively washed with buffer containing reducing concentrations of Urea in the presence of NaCl and DTT to renature the protein. The renatured His-NS5A protein was eluted from the resin and it was capable of interacting with glutathione S-transferase fused form NS5Bt (GST-NS5Bt). The purified His-NS5A exhibited an inhibitory effect on RNA-dependent RNA Polymerase (RdRP) activity of GST-NS5Bt in vitro. Conclusively, in this study, we have established a purification method of bacterial recombinant HCV NS5A, and the results support the notion that NS5A may involve in the regulation of HCV replication through direct interaction with NS5B.