Core as a novel viral target for hepatitis C drugs

Hepatitis C virus (HCV) infects over 130 million people worldwide and is a major cause of liver disease. No vaccine is available. Novel specific drugs for HCV are urgently required, since the standard-of-care treatment of pegylated interferon combined with ribavirin is poorly tolerated and cures less than half of the treated patients. Promising, effective direct-acting drugs currently in the clinic have been described for three of the ten potential HCV target proteins: NS3/NS4A protease, NS5B polymerase and NS5A, a regulatory phosphoprotein. We here present core, the viral capsid protein, as another attractive, non-enzymatic target, against which a new class of anti-HCV drugs can be raised. Core plays a major role in the virion's formation, and interacts with several cellular proteins, some of which are involved in host defense mechanisms against the virus. This most conserved of all HCV proteins requires oligomerization to function as the organizer of viral particle assembly. Using core dimerization as the basis of transfer-of-energy screening assays, peptides and small molecules were identified which not only inhibit core-core interaction, but also block viral production in cell culture. Initial chemical optimization resulted in compounds active in single digit micromolar concentrations. Core inhibitors could be used in combination with other HCV drugs in order to provide novel treatments of Hepatitis C.     

Chaperones in hepatitis C virus infection

The hepatitis C virus(HCV) infects approximately 3% of the world population or more than 185 million people worldwide. Each year, an estimated 350000-500000 deaths occur worldwide due to HCV-associated diseases including cirrhosis and hepatocellular carcinoma. HCV is the most common indication for liver transplantation in patients with cirrhosis worldwide. HCV is an enveloped RNA virus classified in the genus Hepacivirus in the Flaviviridae family. The HCV viral life cycle in a cell can be divided into six phases:(1) binding and internalization;(2) cytoplasmic release and uncoating;(3) viral polyprotein translation and processing;(4) RNA genome replication;(5) encapsidation(packaging) and assembly; and(6) virus morphogenesis(maturation) and secretion. Many host factors are involved in the HCV life cycle. Chaperones are an important group of host cytoprotective molecules that coordinate numerous cellular processes including protein folding, multimeric protein assembly, protein trafficking, and protein degradation. All phases of the viral life cycle require chaperone activity and the interaction of viral proteins with chaperones. This review will present our current knowledge and understanding of the role of chaperones in the HCV life cycle. Analysis of chaperones in HCV infection will provide further insights into viral/host interactions and potential therapeutic targets for both HCV and other viruses.     

Nonstructural protein 3-4A: the Swiss army knife of hepatitis C virus

Hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) is a complex composed of NS3 and its cofactor NS4A. It harbours serine protease as well as NTPase/RNA helicase activities and is essential for viral polyprotein processing, RNA replication and virion formation. Specific inhibitors of the NS3-4A protease significantly improve sustained virological response rates in patients with chronic hepatitis C when combined with pegylated interferon-α and ribavirin. The NS3-4A protease can also target selected cellular proteins, thereby blocking innate immune pathways and modulating growth factor signalling. Hence, NS3-4A is not only an essential component of the viral replication complex and prime target for antiviral intervention but also a key player in the persistence and pathogenesis of HCV. This review provides a concise update on the biochemical and structural aspects of NS3-4A, its role in the pathogenesis of chronic hepatitis C and the clinical development of NS3-4A protease inhibitors.     

Hepatitis C virus (HCV) NS5A protein: role in HCV replication and resistance to interferon-alpha

The hepatitis C virus (HCV) non-structural (NS) 5A protein appears to play an important regulatory role on viral replication and could also be involved in viral pathogenesis. HCV resistance to interferon is a complex mechanism involving multiple causes, among which certain NS5A functions could play a role.     

Hepatitis C virus (HCV) NS5A protein: role in HCV replication and resistance to interferon-α

The hepatitis C virus (HCV) non-structural (NS) 5A protein appears to play an important regulatory role on viral replication and could also be involved in viral pathogenesis. HCV resistance to interferon is a complex mechanism involving multiple causes, among which certain NS5A functions could play a role.     

丙型肝炎病毒NS5B的体外表达、纯化和RNA多聚酶活性鉴定

目的 研究丙型肝炎病毒NS5B蛋白的表达及其生物学功能。方法 构建长度为1676bp(7261-9036bp,编码558aa)的HCV NS5B编码区cDNA,定向克隆到表达质粒pET16b中,并在大肠杆菌BL21中表达。应用合成的多聚A或寡聚U为模板进行~3H-UTP掺入实验分析纯化蛋白的活性。结果 表达的NS5B蛋白分子量为65KD,以包涵体形式存在细胞内,改变部分表达条件未能减少包涵体的形成及可溶性蛋白的增加,该包涵体蛋白在6M尿素浓度、pH:10及500mM NaCl的溶液中完全溶解。~3H-UTP掺入实验表明纯化的蛋白具有RNA多聚酶活性并依赖多聚A的存在。结论 对NS5B蛋白生物学活性的研究,有助于了解HCV的复制及抗病毒药物的开发。     

Ethanol inhibition: the humoral and cellular immune response to hepatitis C virus NS5 protein after genetic immunization

BACKGROUND: The combination of chronic hepatitis C virus (HCV) and ethanol may increase viral replication, impair cellular immunity, and result in severe and progressive liver disease. Because HCV nonstructural proteins play a major role in viral elimination, we examined the cellular and humoral immune responses after genetic immunization against NS5 in a chronic ethanol mouse model. METHODS: Mice were fed an ethanol or isocaloric pair-fed control liquid diet and were immunized with HCV NS5-expression plasmid. RESULTS: The humoral and cellular arms of the immune system were significantly impaired in ethanol-fed mice. Abstinence partially reversed the inhibitory effects on antibody levels and the CD4+ proliferative immune response but did not restore the CD8+ cytotoxic T-cell response to this HCV nonstructural protein. Furthermore, we determined whether murine interleukin-2 coadministration with the NS5 expression plasmid would reverse the inhibitory effects of chronic ethanol consumption; again, partial restoration was observed for B-cell and CD4+ T-cell activity, but not for cytotoxic T cells. CONCLUSIONS: These results suggest that the high rate of chronic HCV infection in alcoholics may be due to ethanol's effects on antiviral immune responses.     

Hepatitis C core and nonstructural 3 proteins trigger toll-like receptor 2-mediated pathways and inflammatory activation

BACKGROUND AND AIMS: Recent evidence suggests that toll-like receptors (TLRs) recognize certain viruses. We reported that hepatitis C virus (HCV) core and nonstructural 3 (NS3) proteins activate inflammatory pathways in monocytes. The aim of this study was to investigate the role of TLRs in innate immune cell activation by core and NS3 proteins. METHODS: Human monocytes, human embryonic kidney cells transfected with TLR2, and peritoneal macrophages from TLR2, MyD88 knockout, and wild-type mice were studied to determine intracellular signaling and proinflammatory cytokine induction by HCV proteins. RESULTS: HCV core and NS3 proteins triggered inflammatory cell activation via the pattern recognition receptor TLR2 and failed to activate macrophages from TLR2 or MyD88-deficient mice. HCV core and NS3 induced interleukin (IL)-1 receptor-associated kinase (IRAK) activity, phosphorylation of p38, extracellular regulated (ERK), and c-jun N-terminal (JNK) kinases and induced AP-1 activation. Activation of nuclear factor-kappaB by core and NS3 was associated with increased IkappaBalpha phosphorylation. TLR2-mediated cell activation was dependent on the conformation of core and NS3 proteins and required sequences in the regions of aa 2-122 in core and aa 1450-1643 in NS3. Although cellular uptake of core and NS3 proteins was independent of TLR2 expression, cell activation required TLR2. HCV core protein and TLR2 showed intracellular colocalization. The hyper-elevated TNF-alpha induction by TLR2 ligands in monocytes of HCV-infected patients was not due to increased TLR2 expression. CONCLUSIONS: HCV core and NS3 proteins trigger inflammatory pathways via TLR2 that may affect viral recognition and contribute to activation of the innate immune system.     

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

ＰｒｅｐａｒａｔｉｏｎａｎｄａｐｐｌｉｃａｔｉｏｎｏｆｍｏｎｏｃｌｏｎａｌａｎｔｉｂｏｄｉｅｓａｇａｉｎｓｔｈｅｐａｔｉｔｉｓＣｖｉｒｕｓｎｏｎｓｔｒｕｃｔｕｒａｌｐｒｏｔｅｉｎｓＧＡＯＪｉａｎＥｎ，ＴＡＯＱｉＭｉｎ，ＧＵＯＪｉａｎＰｉｎ...     

丙型肝炎病毒非结构蛋白NS4B反式激活基因的克隆化研究

目的应用抑制性消减杂交(suppression subtractive hybridization,SSH)技术构建丙型肝炎病毒(HCV)非结构蛋白4B(NS4B)转染细胞差异表达cDNA消减文库,克隆HCV NS4B蛋白反式激活相关基因.方法以HCV NS4B表达质粒pcDNA3.1(-)-NS4B转染HepG2细胞,以空载体pcDNA3.1(-)为对照,制备转染后的细胞裂解液,提取mRNA并逆转录为cDNA,进行抑制性消减杂交分析.将富集的二次PCR产物与T/A载体连接,并转染大肠杆菌进行文库扩增,随机挑取克隆聚合酶链反应(PCR)扩增后进行测序及同源性分析.结果文库扩增后得到33个阳性克隆,经菌落PCR分析显示其中28个克隆含有大小不等的200～1000 bp插入片段.测序及同源性分析显示,12种已知基因编码蛋白,包括一些与细胞周期、信号传导及肿瘤发生等细胞生长调节密切相关的蛋白编码基因,可能是NS4B反式激活靶基因.结论成功构建了HCV NS4B反式激活基因差异表达的cDNA消减文库,为进一步阐明HCV NS4B反式调节的靶基因在肝炎、肝纤维化和肝细胞癌发生的分子生物学机制提供理论依据.     

丙型肝炎病毒非结构蛋白5A反式激活基因4的克隆化研究

目的 丙型肝炎病毒(HCV)非结构蛋白5A(NS5A)病毒蛋白反式激活作用的新的靶基因NS5ATP4的基因序列的确立及基因克隆化研究。方法 依据我室构建的NS5A反式激活基因差异表达的cDNA消减文库，利用生物信息学技术获得新基因NS5ATP4的编码序列，对其可能的氨基酸序列进行分析比较，并对其基因利用多聚酶链反应技术(PCR)进行克隆化研究。结果 发现了HCVNS5A反式激活作用的新的靶基因NS5ATP4。结论 这一发现，为阐明HCVV NS5A蛋白的反式激活作用及其机制，开辟了新的研究方向。     

人胰腺细胞cDNA文库中丙型肝炎病毒核心蛋白结合蛋白基因的筛选

目的 为进一步研究HCV影响糖、脂代谢机制奠定研究基础.方法 扩增人胰腺cDNA文库并经纯化鉴定后,将文库质粒转化酵母菌株Y 187.诱饵质粒pGBKT7-core转化酵母菌株AH109,在色氨酸缺陷型培养基(SD/-Trp)上筛选阳性菌落.应用酵母双杂交系统3将阳性重组AH109菌株与重组酵母菌株Y187进行配合,在四缺培养基(SD/-Trp/-Leu/-His/-Ade)和铺有X-α-gal的四缺培养基上进行筛选,提取蓝色酵母菌落质粒,电转化大肠埃希菌DH5α后再提取质粒测序,测序结果进行序列比对.结果 筛选出11种与HCV核心蛋白相结合的蛋白基因,包括胰凝乳蛋白酶原B1前体、羧肽酶A1,胰蛋白酶原2、胰凝乳蛋白C、胰蛋白酶1,羧肽酶B1、驱动蛋白家族3B、胰蛋白酶2,线粒体蛋白基因、弹性蛋白酶3A,辅脂肪酶.结论 在筛选出的HCV核心蛋白结合的人胰腺细胞蛋白基因中,部分与糖,脂代谢密切相关.     

Contributions of transgenic mouse studies on the research of hepatitis B virus and hepatitis C virus-induced hepatocarcinogenesis

Transgenic mouse technology has enabled the investigation of the pathogenic effects, including those on development, immunological reactions and carcinogenesis, of viral genes directly in living organism in a real-time manner. Although viral hepatocarcinogenesis comprises multiple sequences of pathological events, that is, chronic necroinflammation and the subsequent regeneration of hepatocytes that induces the accumulation of genetic alterations and hepatocellular carcinoma(HCC), the direct action of viral proteins also play significant roles. The pathogenesis of hepatitis B virus X and hepatitis C virus(HCV) core genes has been extensively studied by virtue of their functions as a transactivator and a steatosis inducer, respectively. In particular, the mechanism of steatosis in HCV infection and its possible association with HCC has been well studied using HCV core gene transgenic mouse models. Although transgenic mouse models have remarkable advantages, they are intrinsically accompanied by some drawbacks when used to study human diseases. Therefore, the results obtained from transgenic mouse studies should be carefully interpreted in the context of whether or not they are well associated with human pathogenesis.     

Hepatitis C virus comes for dinner: How the hepatitis C virus interferes with autophagy

Autophagy is a highly-regulated, conserved cellular process for the degradation of intracellular components in lysosomes to maintain the energetic balance of the cell. It is a pro-survival mechanism that plays an important role during development, differentiation, apoptosis, ageing and innate and adaptive immune response. Besides, autophagy has been described to be involved in the development of various human diseases, e.g., chronic liver diseases and the development of hepatocellular carcinoma. The hepatitis C virus (HCV) is a major cause of chronic liver diseases. It has recently been described that HCV, like other RNA viruses, hijacks the autophagic machinery to improve its replication. However, the mechanisms underlying its activation are conflicting. HCV replication and assembly occurs at the so-called membranous web that consists of lipid droplets and rearranged endoplasmic reticulum-derived membranes including single-, double- and multi-membrane vesicles. The double-membrane vesicles have been identified to contain NS3, NS5A, viral RNA and the autophagosomal marker microtubule-associated protein 1 light chain 3, corroborating the involvement of the autophagic pathway in the HCV life-cycle. In this review, we will highlight the crosstalk of the autophagosomal compartment with different steps of the HCV life-cycle and address its implications on favoring the survival of infected hepatocytes.     

Molecular virology of the hepatitis C virus

Infection with the hepatitis C virus (HCV) is the major cause of nonA-nonB hepatitis worldwide. Although this virus cannot be cultivated in vitro, several of its key features have been elucidated in the past few years. The viral genome is a positive-sense, single-stranded, 9.6 kb long RNA molecule. The viral genome is translated into a single polyprotein of about 3000 amino acids. The viral polyprotein is proteolytically processed by the combination of cellular and viral proteinases in order to yield all the mature viral gene products. The genomic order of HCV has been shown to be C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B. C, E1 and E2 are the virion.structural proteins. The function of p7 is currently unknown. These proteins have been shown to arise from the viral polyprotein via proteolytic processing by the host signal peptidases. Generation of the mature nonstructural proteins, NS2 to NS5B, relies on the activity of viral proteinases. Cleavage at the NS2/NS3 junction is accomplished by a metal-dependent autocatalytic proteinase encoded within NS2 and the N-terminus of NS3. The remaining cleavages downstream from this site are effected by a serine proteinase also contained within the N-terminal region of NS3. NS3 also contains an RNA helicase domain at its C-terminus. NS3 forms a heterodimeric complex with NS4A. The latter is a membrane protein that has been shown to act as a cofactor of the proteinase. While no function has yet been attributed to NS4B, it has recently been suggested that NS5A is involved in mediating the resistance of the hepatitis C virus to the action of interferon. Finally, the NS5B protein has been shown to be the viral RNA-dependent RNA polymerase.     

HCV NS3 Blocking Effect on IFN Induced ISGs Like Viperin and IL28 With and Without NS4A

Background:

Hepatitis C virus (HCV) is able to down-regulate innate immune response. It is important to know the immune pathways that this virus interacts with. HCV non-structural protein 3 (NS3) plays an important role in this viral feature. HCV NS3 protein could affect the expression of antiviral protein such as viperin, and interleukin 28whichare important proteins in antiviral response.

Objectives:

HCV has developed different mechanisms to maintain a persistent infection, especially by disrupting type I interferon response and subsequent suppression of expression of Interferon stimulatory genes (ISGs). Viperin, a member of ISGs, is considered as a host antiviral protein, which interferes with viral replication. Since it is a good target for some viruses to evade host responses, it is interesting to study if HCV has evolved a mechanism to interfere with this member of ISGs.

Materials and Methods:

We evaluated the impact of NS3, NS3/4A and a mutated nonfunctional NS3 on ISGs expression such as viperin and IL-28 after the induction of IFN signaling Jak-STAT pathway using IFN-.

Results:

NS3 protein disrupted the expressions of viperin gene and IL-28, an inducer for the expression of ISGs and viperin itself. By comparing the roles of NS3 and NS3/4A protease activities in suppressing the innate immune responses, we also showed that NS3 (without NS4A) has the ability to down-regulate ISGs expression, similar to that of NS3/4A.

Conclusions:

ISGs expression is impeded by NS3 protease activity and its interaction with Jak-STAT pathway proteins. In addition, the NS3/4A substrates spectrum seems to be similar to those of NS3.     

HCVcore/NS3/NS5A对内源性IFN-β表达影响及其调控机制初步研究

目的探讨丙型肝炎病毒(HCV)core、NS3、NS5A对内源性IFN-β表达的影响及其调控机制。方法将HCVcore、NS3、NS5A表达载体pcDNA3.1/myc-His-core/NS3/NS5A转染至HepG2细胞,验证蛋白表达之后,采用实时荧光定量PCR和Westernblot及ELISA方法观察3种蛋白对IFN-βmRNA及蛋白水平表达的影响。构建IFN-β全长启动子报告基因表达载体,借助双萤虫素酶活性检测,探讨HCVcore、NS3、NS5A对IFN-β转录水平的调控机制。结果 pcDNA3.1/myc-His-core/NS3/NS5A在HepG2细胞中成功表达,与转染pcDNA3.1/myc-His空载体相比,pcDNA3.1/myc-His-NS3/NS5A过表达时,在mRNA及蛋白水平均能抑制HepG2细胞内IFN-β的表达,与转染空载体的对照组相比,差异有统计学意义(P0.05)。双萤虫素酶活性检测显示,转染IFN-β全长启动子报告基因表达质粒后,与对照组相比,双萤虫素酶活性降低,差异有统计学意义(P0.05)。pcDNA3.1/myc-His-core过表达时对IFN-β的表达无明显影响。结论 HCVNS3/NS5A在mRNA及蛋白水平能抑制IFN-β表达,并通过其转录水平影响IFN-β表达,core对IFN-β的表达无明显影响。其具体调控机制有待进一步研究。