抗丙型肝炎药物研究进展

近些年来,丙型肝炎病毒(HCV)生物学的快速进展使人们对HCV的感染和复制有了突破性认识,对抗HCV药物的研究与开发产生了重要影响.HCV NS3/4蛋白抑制剂telaprevir和boceprevir的开发进入临床Ⅲ期试验,一些NS5B抑制剂也进入临床研究,在很好降低感染病人病毒载量的同时显示出较好的耐受性.另外,通过干预与HCV复制有关的宿主细胞蛋白进行抗HCV的研究也受到普遍关注,并取得众多突破.本文将着重介绍一些近年来以病毒蛋白为靶点及以细胞因子为靶点的抗HCV约物的研究进展.     

丙型肝炎病毒入侵宿主细胞的机制

丙型肝炎病毒(hepatitis C virus,HCV)入侵宿主细胞是一个复杂的多步过程.病毒对宿主细胞的附着与糖胺聚糖及低密度脂蛋白受体有关,附着后病毒连续与3种入侵因子:B类Ⅰ型清除受体(scavenger receptor)、CD81以及密蛋白1相互作用.新近又发现了CD81配偶体EWI-2wint,它可以阻断HCV颗粒与CD81的相互作用,表达该蛋白的细胞可免受HCV感染.HCV与宿主细胞结合后,通过网格蛋白介导的胞吞作用进入胞内,并可能在早期内体中与其融合.本文旨在介绍目前对HCV入侵细胞机制的最新认识.     

基于丙型肝炎病毒非结构蛋白NS5B晶体结构的抑制剂筛选

丙型肝炎病毒（hepatitis C virus,HCV）是引起慢性肝炎的主要病原体之一,主要通过血源传播,严重危害人类的健康,寻找有效的抗病毒药物具有重要意义。随着HCV复制过程中一些重要蛋白以及这些蛋白与相关配体或抑制剂的精确三维结构的解析,对这些蛋白的三维结构进行设计和筛选,成为目前开发治疗HCV感染药物的重要手段。NS5BRNA聚合酶是HCV复制过程中的关键酶,是研究抗HCV病毒药物的一个重要靶点。本文以NS5B的晶体结构为基础,用晶体浸泡的方法进行NS5B蛋白的抑制剂筛选,得到了小分子抑制剂与NS5B蛋白的精确三维结构,从原子水平上阐释了抑制剂对HCVNS5B蛋白的抑制机理。     

丙型肝炎治疗新药替拉瑞韦

丙型肝炎病毒（hepatitis C virus,HCV）是一种高度变异的正链RNA黄病毒,具有慢性转化率高、病变率高等特征。有研究表明,NS3/NS4A多功能蛋白酶是HCV复制所必须的,因而NS3/NS4A蛋白酶抑制剂在HCV治疗中的意义备受关注。替拉瑞韦（Telaprevir）是一种可逆的HCVNS3/NS4A蛋白酶抑制剂,动物试验和临床试验都表明它可以有效地直接攻击HCV并阻断其复制,对HCV的抑制作用持久,将成为丙型肝炎病毒的新型治疗药物。     

DNA疫苗

慢性乙型和丙型肝炎病毒 (HBV,HCV)感染常常导致慢性肝病及肝细胞癌。实验小鼠接种编码 HCV和 HBV结构蛋白或非结构蛋白的 DNA疫苗后 ,产生了宿主清除病毒所需的广泛的 CD4 及 CD8 细胞免疫应答。 DNA免疫是开发抗 HBV和 HCV治疗和预防性疫苗的有希望的抗病毒方法。     

直接抗HCV药物特拉泼维和博赛泼维的研究进展

直接抗病毒药物(DAA)的研究是慢性丙型病毒性肝炎(CHC)治疗药物的重要研究方向,丙型肝炎病毒(HCV)NS3/4A丝氨酸蛋白酶(NS3/4A SP)对病毒蛋白前体加工成熟和病毒复制过程十分重要,是抗HCV治疗的理想靶点。本文重点综述NS3/4A丝氨酸蛋白酶抑制剂特拉泼维(telaprevir)和博赛泼维(boceprevir)治疗HCV感染的最新进展。     

丙型肝炎病毒全长cDNA克隆在Huh7细胞中的表达与复制

目的 了解丙型肝炎病毒(HCV)在体外的复制和表达情况。方法 利用HCV全长cDNA克隆构建表达质粒p3．1HCV并转染Huh7细胞。应用RT—PCR，Western blotting分析病毒正、负链RNA，蛋白表达和剪切。结果 在转染p3．1HCV的Huh7细胞中可检出相应病毒的正、负链RNA、Western blotting在转染p3．1HCV的Huh7细胞检测中可检到针对HCVNS3蛋白、分子量约70kD的特异性条带。结论 HCV可以在体外培养的Huh7细胞中复制且其前体蛋白能在Huh7细胞中完成剪切。     

宿主因素与丙型肝炎慢性化

丙型肝炎病毒(HCV)感染呈世界性分布,约5 0 %～80 %可发展为慢性肝炎,部分病例可发展为肝纤维化和肝细胞癌[1 ,2 ] 。丙型肝炎慢性化的影响因素主要包括病毒和宿主两方面。病毒因素主要包括HCV的高度变异性、泛嗜性、基因型的差异、与抗体结合表面蛋白的缺失、HCV与脂蛋白的结合等;宿主因素包括宿主的遗传性差异、免疫耐受、CD4 和CD8 T细胞应答低下、Th1 /Th2失调、树突状细胞功能低下、中和抗体的免疫压力选择等。本文主要综述宿主相关的一些因素对丙型肝炎慢性化的影响。1　CD4 和CD8 T细胞与丙型肝炎慢性化在慢性丙型肝炎,HCV…     

抗丙型肝炎药 Boceprevir

全球丙型肝炎病毒（HCV）感染者约有1．7亿之多，若不及时治疗，可发展为慢性肝炎，并可导致肝硬化、肝衰竭或肝细胞癌。HCV为一种包膜正链RNA病毒，一旦进人适宜的宿主细胞，其基因组便作为模板，翻译一个含3000个氨基酸的多聚蛋白，此蛋白经宿主和被命名为NS3的病毒编码的蛋白酶水解成熟。NS3与可增强酶活性的一种必需辅助因子——NS4A蛋白形成异二聚体复合物，作用于多聚蛋白的非结构部分。此外，NS3还可直接破坏宿主细胞，抑制其对于扰素的应答。     

全反式维甲酸通过脂代谢途径抗丙型肝炎相关肝癌的研究进展

丙型肝炎病毒(HCV)作为肝癌的主要危险因素之一,其复制、组装和感染均可以引起宿主肝细胞内过量脂滴累积。脂滴内三酰甘油等中性脂的代谢产物可破坏细胞稳态,导致细胞变性坏死,推动肝硬化进程。直接抗病毒药物(DAA)尽管已在临床广泛应用,但在国内仍无统一的一线治疗方案。且有数据表明,接受DAA治疗的患者,其肝癌复发率较未接受者更高。近期研究发现全反式维甲酸(ATRA)具有抗HCV及其相关肝癌的作用,可能的机制为ATRA抑制宿主感染肝细胞的脂肪合成、促进脂肪干细胞分化,缓解感染肝细胞的脂变性,进而终止肝硬化的进程,从而起到抗癌的作用。本文主要对HCV感染与肝癌发生发展的脂代谢机制以及ATRA抗癌及缓解脂代谢紊乱的主要信号分子和通路进行综述。     

Modulation of host metabolism as a target of new antivirals

The therapy for chronic hepatitis C (CH-C) started with interferon (IFN) monotherapy in the early 1990s and this therapy was considered effective in about 10% of cases. The present standard therapy of pegylated IFN with ribavirin achieves a sustained virologic response in about 50% of patients. However, about half of the CH-C patients are still at risk of fatal liver cirrhosis and hepatocellular carcinoma. The other significant event in hepatitis C virus (HCV) research has been the development of a cell culture system. The subgenomic replicon system enables robust HCV RNA replication in hepatoma cells. And recently, the complete life cycle of HCV has been achieved using a genotype 2a strain, JFH1. These hallmarks have provided much information about the mechanisms of HCV replication, including information on the host molecules required for the replication. Anti-HCV reagents targeting HCV proteins have been developed, and some of them are now in clinical trials. However, the RNA-dependent RNA polymerase frequently causes mutations in the HCV genome, which lead to the emergence of drug-resistant HCV mutants. Some of the cellular proteins essential for HCV RNA replication have already been discovered using the HCV cell culture system. These host molecules are also candidate targets for antivirals. Here, we describe the recent progress regarding the anti-HCV reagents targeting host metabolism.     

Evaluation systems for anti-HCV drugs

Development of therapeutics for chronic hepatitis C has been hampered by the lack of an efficient cell culture system and a small animal model for the hepatitis C virus (HCV). An RNA replicon system, in which the HCV genome replicates autonomously in cells, and replication competent viruses derived from an HCV genotype 2a JFH1 strain efficiently propagating in Huh7 cells have been developed, and these systems have contributed to the evaluation of anti-HCV drugs targeted to viral and host proteins involved in the replication of HCV. Several compounds counteracting the viral enzymes, such as RNA polymerase and proteases, and host proteins involved in the lipid synthesis and protein folding are reported to have anti-HCV activities based on assessments using these in vitro systems. Furthermore, a mouse model transplanted with human liver fragments was shown to be capable of replicating HCV and has been used to evaluate the efficacy of antiviral drugs in vivo. In this review, we summarize information regarding systems for studying the HCV life cycle and potential new targets for therapeutic intervention for chronic hepatitis C.     

Up-regulation of glycolipid transfer protein by bicyclol causes spontaneous restriction of hepatitis C virus replication

Bicyclol is a synthetic drug for hepatoprotection in clinic since 2004. Preliminary clinical observations suggest that bicyclol might be active against hepatitis C virus (HCV) with unknown mechanism. Here, we showed that bicyclol significantly inhibited HCV replication in vitro and in hepatitis C patients. Using bicyclol as a probe, we identified glycolipid transfer protein (GLTP) to be a novel restrictive factor for HCV replication. The GLTP preferentially bound host vesicle-associated membrane protein-associated protein-A (VAP-A) in competition with the HCV NS5A, causing an interruption of the complex formation between VAP-A and HCV NS5A. As the formation of VAP-A/NS5A complex is essential for viral RNA replication, up-regulation of GLTP by bicyclol reduced the level of VAP-A/NS5A complex and thus inhibited HCV replication. Bicyclol also exhibited an inhibition on HCV variants resistant to direct-acting antiviral agents (DAAs) with an efficacy identical to that on wild type HCV. In combination with bicyclol, DAAs inhibited HCV replication in a synergistic fashion. GLTP appears to be a newly discovered host restrictive factor for HCV replication, Up-regulation of GLTP causes spontaneous restriction of HCV replication.     

Roles of phosphoinositides and phosphoinositides kinases in hepatitis C virus RNA replication

Phosphoinositides (PIs) play an essential role in mediating key signaling pathways on biological membranes. Hepatitis C virus (HCV) replicates its RNA genome by establishing a viral replication complex (RC) on host cell membranes. Recently, an increasing body of literature reported that not only PIs themselves but also several PIs-specific kinases are required for efficient replication of HCV RNA genome. Especially, PI 4-kinases type III alpha, beta as well as their enzymatic products including phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) are consistently identified to be host factors essential for HCV replication. In this article, the current state of our knowledge of PIs and PIs-specific kinases together with their roles in modulating HCV replication is reviewed. The effects of various PIsspecific kinases inhibitors on HCV replication are also highlighted, proposing them as promising candidate targets to which a new class of anti-HCV therapeutics can be envisaged.     

Hepatitis C virus clearance: the enigma of failure despite an impeccable survival strategy

Infection with human hepatitis C virus (HCV) as a result of a bilateral process of host-virus interactions. There are factors on both sides that contribute to clearance and to chronicity. Virus strategy to survive is built on several basic features. The first, recently recognized, is a wide cell tropism. HCV can infect not only hepatocytes, but also cells of immune system (B-cells, monocytes, macrophages, dendritic cells), epithelium, and immunologically privileged sites such as the central nervous system. Dendritic cells and platelets can also be passive virus carriers. Possibilities of virus clearance or abortive inapparent HCV infection at the stage of adsorption are considered. The second feature is rapid error-prone replication that leads to accumulation within one host of multiple virus variants (quasispecies). Viral heterogeneity could be multiplied by recombination of HCV genomic/subgenomic RNA molecules. Quasispecies nature gives virus an advantage in adaptation to varying host environment including availability of permissive cells, the presence of innate and adaptive immune response, and antiviral treatment. Analysis of HCV polymorphisms and their evolution rates may pinpoint the molecular (sequence) correlates of HCV clearance. The third feature is the capacity to modify or adapt host milieu. HCV core, envelope E2 and nonstructural NS2, NS3, NS5A proteins seem to hold a grip over the host cellular functions by down-regulating processes unfavorable and up-regulating processes favorable for virus replication and persistence. The relevance of the latter interactions to HCV infection outcome remains to be demonstrated. This review discusses recent developments in this area of HCV research.     

A target on the move: innate and adaptive immune escape strategies of hepatitis C virus

Obligate intracellular parasites such as the hepatitis C virus (HCV) have to cope intensively with immune responses in order to establish persistent infection. Powerful antiviral mechanisms of the host act on several levels. The innate immune response is able to slow down viral replication and activate cytokines which trigger the synthesis of antiviral proteins. The adaptive immune system neutralizes virus particles and destroys infected cells. Viruses have therefore developed a number of countermeasures to stay moving targets for the immune system. Here, we attempt to summarize the current state of research regarding innate and adaptive immune responses against HCV and the different escape strategies evolved by this virus.     

Membrane association of hepatitis C virus nonstructural proteins and identification of the membrane alteration that harbors the viral replication complex

Hepatitis C virus (HCV) replicates its genome in a membrane-associated complex composed of viral proteins, replicating RNA, and altered cellular membranes. Determinants for membrane association of the HCV nonstructural proteins involved in genome replication have been defined. In addition, a specific membrane alteration, designated membranous web, was recently identified as the site of viral RNA synthesis and, therefore, represents the HCV replication complex. These findings add to our current understanding of the HCV life cycle and may ultimately allow to design novel antiviral strategies against hepatitis C.     

NS5A--from obscurity to new target for HCV therapy

The hepatitis C virus (HCV) non-structural 5A (NS5A) protein is essential for viral RNA replication and may play a role in subverting host intracellular signaling pathways. Although no intrinsic enzymatic activity has been ascribed to NS5A, this proline-rich hydrophilic phosphoprotein is likely to exert its functions by interacting with viral and cellular factors. Recent studies using the HCV replicon cell culture system as a model for HCV RNA replication as well as for high-throughput screening of pharmacological inhibitors have revealed blockade of NS5A as a promising therapeutic strategy for the treatment of HCV. This review will summarize our progress in understanding the role of NS5A in HCV RNA replication and will introduce the most recent patents on inhibitors of NS5A.