MicroRNA-185-5p mediates regulation of SREBP2 expression by hepatitis C virus core protein

AIM: To investigate the molecular mechanism for regulation of cholesterol metabolism by hepatitis C virus(HCV) core protein in Hep G2 cells.METHODS: HCV genotype 1b core protein was cloned and expressed in Hep G2 cells. The cholesterol content was determined after transfection. The expression of sterol regulatory element binding protein 2(SREBP2) and the rate-limiting enzyme in cholesterol synthesis(HMGCR) was measured by quantitative real-time PCR and immunoblotting after transfection. The effects of core protein on the SREBP2 promoter and 3'-untranslated region were analyzed by luciferase assay. We used different target predictive algorithms, micro RNA(mi RNA) mimics/inhibitors, and site-directed mutation to identify a putative target of a particular mi RNA.RESULTS: HCV core protein expression in Hep G2 cells increased the total intracellular cholesterol level(4.05 ± 0.17 vs 6.47 ± 0.68, P = 0.001), and this increase corresponded to an increase in SREBP2 and HMGCR m RNA levels(P = 0.009 and 0.037, respectively) and protein expression. The molecular mechanism studyrevealed that the HCV core protein increased the expression of SREBP2 by enhancing its promoter activity(P = 0.004). In addition, mi R-185-5p expression was tightly regulated by the HCV core protein(P = 0.041). Moreover, overexpression of mi R-185-5p repressed the SREBP2 m RNA level(P = 0.022) and protein expression. In contrast, inhibition of mi R-185-5p caused upregulation of SREBP2 protein expression. mi R-185-5p was involved in the regulation of SREBP2 expression by HCV core protein. CONCLUSION: HCV core protein disturbs the cholesterol homeostasis in Hep G2 cells via the SREBP2 pathway; mi R-185-5p is involved in the regulation of SREBP2 by the core protein.

Hepatitis C virus core protein modulates several signaling pathways involved in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is the fifth most common cancer, and hepatitis C virus(HCV) infection plays a major role in HCC development. The molecular mechanisms by which HCV infection leads to HCC are varied. HCV core protein is an important risk factor in HCV-associated liver pathogenesis and can modulate several signaling pathways involved in cell cycle regulation, cell growth promotion, cell proliferation, apoptosis, oxidative stress and lipid metabolism. The dysregulation of signaling pathways such as transforming growth factor β(TGF-β), vascular endothelial growth factor(VEGF), Wnt/β-catenin(WNT), cyclooxygenase-2(COX-2) and peroxisome proliferator-activated receptor α(PPARα) by HCV core protein is implicated in the development of HCC. Therefore, it has been suggested that this protein be considered a favorable target for further studies in the development of HCC. In addition, considering the axial role of these signaling pathways in HCC, they are considered druggable targets for cancer therapy. Therefore, using strategies to limit the dysregulation effects of core protein on these signaling pathways seems necessary to prevent HCV-related HCC.

Effect of hepatitis C virus core protein on modulation of cellular proliferation and apoptosis in hilar cholangiocarcinoma

BACKGROUND: Hepatitis C virus (HCV) is believed to be an important human pathogen causing carcinoma. But the effect of HCV infection on the alteration of cellular pro- liferation and apoptosis and the relationship between the effect and the development of hilar cholangiocarcinoma are largely unknown. The aim of this study was to assess the effect of HCV core protein on proliferation and apoptosis of hilar cholangiocarcinoma. METHODS: HCV core protein (HCV C protein) was de- tected by peroxidase-antiperoxidase assay in surgical speci- mens from 48 patients with hilar cholangiocarcinoma. The apoptosis index ( AI) and PCNA index ( PI) in hilar cholangiocarcinoma were detected by in situ end labeling assay and streptavidin-biotin assay respectively. RESULTS: The expression of HCV C protein was observed in 32 (67.7%) of the 48 specimens of hilar cholangiocarci- noma. The mean ± standard deviation for AI and PI was 3.52%±0.64% and 46.24%±11.46% respectively. The AI of hilar cholangiocarcinoma specimens with HCV C protein expression was significantly lower than that of HCV C pro- tein negative specimens (P<0.01), whereas the PI of HCV C protein positive specimens was significantly higher than that of HCV C protein negative specimens (P<0.01). CONCLUSION: HCV C protein may promote the cellular proliferation of hilar cholangiocarcinoma and inhibit its cel- lular apoptosis.

Genes transactivated by hepatitis C virus core protein, a microarray assay

AIM: To explore the new target genes transactivated by hepatitis C virus (HCV) core protein and to elucidate the pathogenesis of HCV infection.METHODS: Reverse transcribed cDNA was subjected tomicroarray assay. The coding gene transactivated by HCV core protein was cloned and analyzed with bioinformatics methods.RESULTS: The expressive vector of pcDNA3.1(-)-core was constructed and confirmed by restriction enzyme digestion and DNA sequencing and approved correct. mRNA was purified from HepG2 and HepG2 cells transfected with pcDNA3.1(-)-core, respectively. The cDNA derived was subjected to microarray assay. A new gene namedHCTP4 was cloned with molecular biological method in combination with bioinformatics method.CONCLUSION: HCV core is a potential transactivator.Microarray is an efficient and convenient method for analysis of differentially expressed genes.

Proapoptotic and pronecrosis effect of different truncated hepatitis C virus core proteins

Objective: To study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells. Methods: Seven truncated CORE-GFP (green fluorescent protein) fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1?172 aa, 1?126 aa, 1?58 aa, 59?126 aa, 127?172 aa; BNT: 1?172 aa and C191: 1?172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry. Results: Different truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1?172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1?172 aa>1?126 aa>1?58 aa>127?172 aa>59?126 aa). Conclusion: These results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of dif- ferent HCV quasi-species might have some difference in their pathogenesis.

Lethality in mice infected with recombinant vaccinia virus expressing hepatitis C virus core protein

OBJECTIVE: To establish a mouse model of HCV core expression and investigate the toxicity of HCV core protein or the possible pathogenic effects. METHODS: A series of vaccinia viral expression vectors were engineered to express 5' portion of HCV genes including 5' non-translated region (NTR), core protein, and portion of the E1 gene. These HCV sequences were fused to a luciferase reporter gene and inserted into a vaccinia virus expression vector (pSC11) adjacent to the vaccinia virus promoter, p7.5. The recombinant DNA constructs were packed into infectious recombinant chimeric viruses. The expression of HCV core protein was examined in cultured cells after infection with these viruses. Death of the infected mice was investigated by specific correlation to the expression of HCV core protein and its expression levels. RESULTS: The recombinant virus (VNCE-LUA) expressed HCV core protein and an envelope-luciferase fusion protein in cultured cells. When Balb/c mice were inoculated intraperitoneally with more than 10~7 pfu per mouse of VNCE-LUA, death occurred immediately. The mortality was dependent on the amount of VNCE-LUA virus inoculated. All mice inoculated with 3×10~8 pfu of VNCE-LUA died within 4 days of infection and 50% of mice inoculated with 3×10~7 pfu of VNCE-LUA died within 7 days of infection. No death occurred in mice inoculated with 3×10~8 pfu of a control recombinant vaccinia virus, which expressed luciferase but not the HCV core and envelope proteins. Deletion of core sequences from VNCE-LUA rapidly reduced the mortality of infected mice whereas deletion of envelope sequence did not. SCID mice infected with VNCE-LUA died 2-3 days after infection, suggesting that the HCV-core induced mortality is not dependent on host T-or B-cell responses to core protein. CONCLUSIONS: HCV core protein can be lethal to mice when expressed in vivo and this specific lethality is independent of T-cells or B-cells. The findings and model itself provide a useful tool for further investigation on potential pathological effects as well as the potential toxicity of the HCV core protein.

Screening and identification of interacting proteins with hepatitis B virus core protein in leukocytes and cloning of new gene C1

瞄准：在外部血在 HBV 复制的致病调查 HBcAg 的生物功能单音的原子房间(PBMC ) 。方法：HBcAg 区域被聚合酶链反应(PCR ) 放大， HBV HBcAg 诱饵原生质标志 pGBKT7-HBcAg 被平淡的分子的生物方法构造。然后， recombinant 原生质标志 DNA 被转变成酵母 AH109。在 HBV 核心蛋白质在 AH109 酵母紧张(西方的污点分析) 被表示以后，酵母 -- 屏蔽的二个混血儿被与包含白血球 cDNA 图书馆的 Y187 交配 AH109 执行原生质标志。双酵母房间是合成退学学生营养培养基(SD/-Trp-Leu-His-Ade )(QDO ) 和合成退学学生营养培养基(SD/-Trp-Leu-His-Ade )(TDO ) 上的 plated。第二屏蔽与 LacZ 报告基因被执行(酵母房间在 QDO 被种中等包含 X-alpha-gal ) 。在从积极殖民地获得的 HBV 核心蛋白质和蛋白质之间的相互作用被重复酵母进一步证实 -- 二个混血儿。在原生质标志 DNA 从蓝殖民地被提取并且定序以后，结果被生物信息的方法分析。结果：十八个殖民地被获得并且定序，包括在癌症 2 的亢奋的甲基化( 3 关口)，真核细胞的翻译延伸因素 2 ( 2 关口)，乙酰辅酶 A 合成酶 3 ( 1 个关口一个)， DNA 聚合酶鲸鱼群妈( 1 个关口一个)，通常认为的翻译开始因素( 1 个关口一个)， chemokine ( C-C 主题)受体 5 ( 1 个关口一个)， mitochondrial ribosomal 蛋白质 L41 ( 1 个关口一个)， kyot 绑定蛋白质基因( 1 个关口一个)， RanBPM ( 1 个关口一个)，HBeAg有约束力的蛋白质 3 ( 1 个关口一个)，规划了细胞死亡 2 ( 1 个关口一个)。有未知功能的四新基因被识别。结论：在白血球的核心蛋白质交往蛋白质可以提供的 HBV 的基因的成功的克隆为学习 HBV 的生物功能的一些新线索核心蛋白质。

Hepatitis C virus core proteins derived from different quasispecies of genotype 1b inhibit the growth of Chang liver cells

AIM: To investigate the influence of different quasispecies of hepatitis C virus (HCV) genotype 1b core protein on growth of Chang liver cells. METHODS: Three eukaryotic expression plasmids (pEGFP-N1/core) that contained different quasispecies truncated core proteins of HCV genotype 1b were constructed. These were derived from tumor (T) and non- tumor (NT) tissues of a patient infected with HCV and C191 (HCV-J6). The core protein expression plasmids were transiently transfected into Chang liver cells. At different times, the cell cycle and apoptosis was assayed by flow cytometry, and cell proliferation was assayed by methyl thiazolyl tetrazolium (MTT) assay. RESULTS: The proportion of S-phase Chang liver cells transfected with pEGFP-N1/core was significantly lower than that of cells transfected with blank plasmid at three different times after transfection (all P < 0.05). The proliferation ratio of cells transfected with pEGFP-N1/corewas significantly lower than that of cells transfected with blank plasmid. Among three different quasispecies, T, NT and C191 core expression cells, there was no significant difference in the proportion of S- and G0/G1-phase cells. The percentage of apoptotic cells was highest for T (T > NT > C191), and apoptosis was increased in cells transfected with pEGFP-N1/core as the transfection time increased (72 h > 48 h > 24 h). CONCLUSION: These results suggest that HCV genotype 1b core protein induces apoptosis, and inhibits cell- cycle progression and proliferation of Chang liver cells. Different quasispecies core proteins of HCV genotype 1b might have some differences in the pathogenesis of HCV persistent infection and hepatocellular carcinoma.

The Identification of Three Sizes of Core Proteins during the Establishment of Persistent Hepatitis C Virus Infection in vitro

Similar to Hepatitis C virus (HCV) infection in humans, HCVcc infection can also result in persistent and chronic infection. The core protein is a variable protein and exists in several sizes. Some sizes of core proteins have been reported to be related to chronic HCV infection. To study the possible role of the core protein in persistent HCV infection, a persistent HCVcc infection was established, and the expression of the core protein was analysed over the course of the infection. The results show that there are three sizes of core proteins (p24, p21 and p19) expressed during the establishment of persistent HCVcc infection. Of these, the p21 core protein is the mature form of the HCV core protein. The p24 core protein is the phosphorylated form of p21. The p19 core protein appears to be a functional by-product generated during the course of infection. These three core proteins are all localized in the cytoplasm and can be encapsidated into the HCV virion. The appearance of the p19 and p24 core proteins might be related to acute HCVcc infection and chronic infection respectively and may play an important role in the pathology of a HCV infection.

C-terminal domain of hepatitis C virus core protein is essential for secretion

AIM: We have previously demonstrated that hepatitis C virus (HCV) core protein is efficiently released into the culture medium in insect cells. The objective of this study is to characterize the HCV core secretion in insect cells.METHODS: We constructed recombinant baculoviruses expressing various-length of mutant core proteins, expressed these proteins in insect cells, and examined core protein secretion in insect cells.RESULTS: Only wild type core was efficiently released into the culture medium, although the protein expression level of wild type core was lower than those of other mutant core proteins. We found that the shorter form of the core construct expressed the higher level of protein. However, if more than 18 amino acids of the core were truncated at the C-terminus,core proteins were no longer seareted into the culture medium.Membrane flotation data show that the secreted core proteins are associated with the cellular membrane protein, indicating that HCV core is secreted as a membrane complex.CONCLUSION: The C-terminal 18 amino acids of HCV core were crucial for core secretion into the culture media.Since HCV replication occurs on lipid raft membrane structure,these results suggest that HCV may utilize a unique core release mechanism to escape immune surveillance, thereby potentially representing the feature of HCV morphogenesis.

Specific activation of 2'-5'oligoadenylate synthetase gene promoter by hepatitis C virus-core protein:A potential for developing hepatitis C virus targeting gene therapy

AIM:To examine whether 2'-5'oligoadenylate synthetase (OAS) gene promoter can be specifically activated by hepatitis C virus (HCV)-core protein.METHODS: Human embryo hepatic cell line L02 wa transfected with pcDNA3.1-core plasmid and selected by G418. Expression of HCV-core was detected by reverse transcription polymerase chain reaction and Western blotting. The OAS promoter sequence wa amplified from the genomic DNA and inserted into pGL3-basic vector. The resultant pGL3-OAS-Luc plasmid was transiently transfected into L02/core cell and luciferase activity was assayed. RESULTS: L02/core cell line stably expressing HCV core protein was established. The pGL3-OAS-Luc construct exhibited significant transcriptional activity in the L02/core cells but not in the L02 cells.CONCLUSION: HCV-core protein activates the OAS gene promoter specifically and effectively. Utilization of OAS gene promoter would be an ideal strategy for developing HCV-specific gene therapy.

Effect of Hepatitis C Virus Core Protein on Interferon-Induced Antiviral Genes Expression and Its Mechanisms

Emerging data indicated that HCV subverts the antiviral activity of interferon(IFN);however,whether HCV core protein contributes to the process remains controversial.In the present study,we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway.Our results showed that,following treatment with IFN-α,the transcription of PKR,MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein.In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased.Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1,whereas the level of STAT1 expression was unchanged.Accordingly,SOCS3,the negative regulator of the Jak/STAT pathway,was induced by HCV core protein.These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.

Preparation and application of monoclonal antibodies against hepatitis C virus nonstructural proteins

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Production and pathogenicity of hepatitis C virus core gene products

Hepatitis C virus(HCV)is a major cause of chronic liver diseases,including steatosis,cirrhosis and hepatocellular carcinoma,and its infection is also associated with insulin resistance and type 2 diabetes mellitus.HCV,belonging to the Flaviviridae family,is a small enveloped virus whose positive-stranded RNA genome encoding a polyprotein.The HCV core protein is cleaved first at residue 191 by the host signal peptidase and further cleaved by the host signal peptide peptidase at about residue 177 to generate the mature core protein(a.a.1-177)and the cleaved peptide(a.a.178-191).Core protein could induce insulin resistance,steatosis and even hepatocellular carcinoma through various mechanisms.The peptide(a.a.178-191)may play a role in the immune response.The polymorphism of this peptide is associated with the cellular lipid drop accumulation,contributing to steatosis development.In addition to the conventional open reading frame(ORF),in the+1 frame,an ORF overlaps with the core proteincoding sequence and encodes the alternative reading frame proteins(ARFP or core+1).ARFP/core+1/F protein could enhance hepatocyte growth and may regulate iron metabolism.In this review,we briefly summarized the current knowledge regarding the production of different core gene products and their roles in viral pathogenesis.

Detection of hepatitis C virus core antigen for early diagnosis of hepatitis C virus infection in plasma donor in China

AIM: To evaluate the effi cacy of a new hepatitis C virus (HCV) core antigen assay developed in China. METHODS: After the determination of HCV infection, 49 serial samples were selected from 11 regular plasma donors in 5 different plasma stations. To compare the performance of HCV core antigen detection and HCV PCR, these samples were genotyped, and each specimen was analyzed by ELISA for the detection of HCV core antigen and by qualitative HCV PCR. RESULTS: Among all of the sequential samples, the original 13 specimens were HCV RNA-negative, and 36 samples were HCV RNA-positive. Twenty-seven samples (75%) were HCV core antigen-positive from these HCV RNA-positive specimens. Conversely, 27 samples (93.1%) were found HCV RNA-positive in HCV core antigen-positive samples. Intervals between HCV RNA and HCV core antigen-positive, as well as between HCV core antigen-positive and HCV antibody-positive were 36.0 and 32.8 d, respectively. CONCLUSION: This HCV core antigen assay, developed in China, is able to detect much of anti-HCV-negative, HCV RNA-positive preseroconversion window period (PWP) plasma donations.

Hepatitis C virus core antigen testing: Role in diagnosis, disease monitoring and treatment

While hepatitis B virus(HBV)screening relies on hepatitis B surface antigen to confirm HBV infection since the early days of hepatitis B disease management,hepatitis C virus(HCV)infection screening is based on anti-HCV testing which does not discriminate active from past infection.Thus to confirm infection HCV RNA testing has been required;recently a HCV core antigen assay became widely commercially available which could serve to confirm infection.That assay is less sensitive than current HCV RNA assays,but as more than 50%of anti-HCV positive persons will be HCV core antigen positive,HCV core antigen testing can be a cost effective and reflex test to confirm HCV infection in anti-HCV positive individuals and will be easier as it can be applied on the same platform.For treatment monitoring,more data need to be generated,but the early data available at present suggest that HCV core antigen may be an alternative to HCV RNA monitoring.With direct antivirals,HCV core antigen could even be superior to HCV RNA testing,as direct antivirals might already prevent virus formation when HCV core antigen is still produced and thereby correlates better with eventual viral clearance.

Biological impact of hepatitis B virus X-hepatitis C virus core fusion gene on human hepatocytes

AIM: To investigate the biological impact of hepatitis B virus X- hepatitis C virus core (HBV X-HCV C) fusion gene on hepatoma cells. METHODS: The recombinant adenoviruses Ad- XC, Ad-X and Ad-C expressing HBV X-HCV C fusion gene, HBV X gene and HCV C gene were constructed, respectively. Hepatoma cells were infected with different recombinant adenoviruses. MTT, colonyforming experiment, FCM, TUNEL assay were performed to observe the biological impact of the HBV X-HCV C fusion gene on liver cells. RESULTS: MTT showed that the Ad-XC group cells grew faster than the other group cells. Colony-forming experiment showed that the colony-forming rate for the Ad-XC group cells was significantly higher than that for the other group cells. FCM analysis showed that Ad-XC/Ad-X/Ad-C infection enhanced the progression of G1→S phase in the HepG2 cell cycle. The apoptosis index of the Ad-XC, Ad-X, Ad-C group cells was significantly lower than that of the Ad0 and control group cells. Semi-quantitative RT-PCR showed that the expression level of c-myc was the highest in Ad- XC infected cells. Tumor formation was found at the injected site of mice inoculated with Ad-XC-infected LO2 cells, but not in control mice. CONCLUSION: Ad-XC, Ad-X and Ad-C facilitate the proliferation activity of HepG2 cells and inhibit their apoptosis in vitro. The effect of Ad-XC is significantly stronger than that of Ad-X and Ad-C. Up-regulation of c-myc may be one of the mechanisms underlying the synergism of HBV X and HCV C genes on hepatocarcinogenesis in athymic nude mice.

Interaction of hepatitis C virus envelope glycoprotein E2 with the large extracellular loop of tupaia CD81

AIM: To further analyze the interaction of tupaia CD81 with hepatitis C virus (HCV) envelope protein E2. METHODS: A tupaia CD81 large extracellular loop (CD81 LEL), which binds to HCV E2 protein, was cloned and expressed as a GST-fusion protein, and interaction of HCV E2 protein with a tupaia CD81 LEL was evaluated by enzyme-linked immunosorbent assay (EIA). RESULTS: Although tupaia and human CD81 LEL differed in 6 amino acid changes, tupaia CD81 LEL was strongly recognized by anti-CD81 antibodies against human CD81 LEL conformation-dependent epitopes. Investigating LEL CD81-E2 interactions by EIA, we demonstrated that binding of tupaia CD81 LEL GST fusion protein to recombinant HCV E2 protein was markedly reduced compared to binding of human CD81 LEL GST fusion protein to recombinant HCV E2 protein. CONCLUSION: These data suggest that the structural differences in-between the tupaia and human CD81 may alter the interaction of the large extracellular loop with HCV envelope glycoprotein E2. These findings may be important for the understanding of the mechanisms of binding and entry of HCV to PTHs.

Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencing of plasmids from blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.

Screening of hepatocyte proteins binding to F protein of hepatitis C virus by yeast two-hybrid system

AIM: To investigate the biological function of F protein by yeast two-hybrid system.METHODS: We constructed F protein bait plasmid by cloning the gene of F protein into pGBKT7, then recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-HisAde) containing X-α-gal for selection and screening.After extracting and sequencing plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics.RESULTS: Thirty-six colonies were selected and sequenced.Among them, 11 colonies were zymogen granule protein,5 colonies were zinc finger protein, 4 colonies were zinc-α-2-glycoprotein, 1 colony was sialyltransferase, 1 colony was complement control protein factor Ⅰ, 1 colony was vitronectin, and 2 colonies were new genes with unknown function.CONCLUSION: The yeast two-hybrid system is an effective method for identifying hepatocyte proteins interacting with F protein of hepatitis C virus. F protein may bind to different proteins.