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.     

丙型肝炎病毒5＇端非编码区和NS3蛋白酶共调控外分泌性碱性磷酸酶表达细胞模型的建立及意义

目的 构建丙型肝炎病毒5'端非编码区(HCV 5'NCR)和NS3丝氨酸蛋白酶共调控外分泌性碱性磷酸酶(SEAP)表达细胞模型,并分析其用于抗HCV药物筛选和评价的可行性。 方法 用聚合酶链反应技术扩增HCV 5'NCR和NS3/4A片段,定向克隆至表达质粒pSEAP2-Control的SEAP基因上游,构建含HCV 5'NCR-NS3/4A-SEAP嵌合基因的重组表达质粒pNCR-NS3/4A-SEAP。将重组质粒转染至肝细胞株QSG7701,用化学发光法检测SEAP的表达,并观察HCV 5'NCR区对应的反义寡聚核苷酸(ASODN)和丝氨酸蛋白酶抑制剂TPCK对SEAP表达的影响。 结果 重组质粒pNCR-NS3/4A-SEAP有高强度SEAP表达,5μmol/L、10μmol/L ASODN和100μmol/L TPCK对SEAP表达有显著抑制作用(t值分别为4.315、6.985、6.949,P值均<0.01)。 结论 重组质粒pNCR-NS3/4A-SEAP的SEAP表达受HCV 5'NCR和NS 3蛋白酶共调控,建立的细胞模型可用于以HCV 5'NCR和NS3蛋白酶为靶位点的药物筛选和评价。     

Structural determinants for membrane association and dynamic organization of the hepatitis C virus NS3-4A complex

Hepatitis C virus (HCV) NS3-4A is a membrane-associated multifunctional protein harboring serine protease and RNA helicase activities. It is an essential component of the HCV replication complex and a prime target for antiviral intervention. Here, we show that membrane association and structural organization of HCV NS3-4A are ensured in a cooperative manner by two membrane-binding determinants. We demonstrate that the N-terminal 21 amino acids of NS4A form a transmembrane α-helix that may be involved in intramembrane protein–protein interactions important for the assembly of a functional replication complex. In addition, we demonstrate that amphipathic helix α₀, formed by NS3 residues 12–23, serves as a second essential determinant for membrane association of NS3-4A, allowing proper positioning of the serine protease active site on the membrane. These results allowed us to propose a dynamic model for the membrane association, processing, and structural organization of NS3-4A on the membrane. This model has implications for the functional architecture of the HCV replication complex, proteolytic targeting of host factors, and drug design.     

慢性丙型肝炎直接抗病毒药物的研究进展和应用前景

针对HCV感染的标准治疗方案是聚乙二醇干扰素α联合利巴韦林。按此治疗方案部分HCV感染者是可治愈的。但仍有一小部分患者不能治愈。随着分子生物学的进展,针对HCV生活周期中病毒蛋白靶向特异性治疗的许多小分子化合物的研究得到了迅速发展,提高了抗病毒的疗效。这些药物统一命名为抗HCV的直接抗病毒药物(direct-acting antiviralagents,DAAs),包括非结构蛋白(non-structural protein,NS)3/4A蛋白酶抑制剂、NS5B聚合酶抑制剂和NS5A蛋白抑制剂等。本文对慢性丙型肝炎DAAs的研究进展、临床应用、应用中的问题及应用前景进行概述。     

Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.     

Cell transformation induced by hepatitis C virus NS3 serine protease

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.     

Inflammatory markers neopterin and alanine aminotransferase in HCV patients treated with HCV NS3•4A protease inhibitor telaprevir (VX-950) and/or peginterferon alfa-2a

Objective. Neopterin is a marker of monocyte/macrophage activity. Alanine aminotransferase (ALAT) is a marker of hepatocyte injury. The aim of this study was to determine changes in neopterin and ALAT levels, as markers of inflammation, in two ancillary studies during two-phase 1b trials of hepatitis C virus (HCV) NS3?4A protease inhibitor telaprevir (VX-950), with or without peginterferon alfa-2a (Peg-IFN). Material and methods. Fifty-four chronic hepatitis C patients (genotype 1) received placebo or telaprevir, with or without Peg-IFN, for 14 days in two multiple-dose studies. Results. During administration of telaprevir, every patient demonstrated a >2-log decrease in HCV RNA. Mean neopterin and ALAT levels decreased in all four groups receiving telaprevir alone. In contrast, mean neopterin levels increased and ALAT levels decreased in the Peg-IFN plus telaprevir and Peg-IFN plus placebo groups. Conclusions. These data suggest that treatment of chronic hepatitis C patients with an HCV NS3?4A protease inhibitor ameliorates inflammation. The increase in neopterin levels and the decrease in ALAT levels during administration of Peg-IFN with or without telaprevir are in accordance with earlier observations showing that IFN reduces hepatocyte injury but increases monocyte/macrophage activity. The IFN-mediated immunomodulatory effects appear to remain intact when IFN is combined with telaprevir.     

Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors

BACKGROUND/AIMS: Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV METHODS: Active NS3 protease domains of genotype 1-3 (representing five subtypes: la, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B. RESULTS: Our data revealed that NS3 proteases cross-interacted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner. CONCLUSION: Our study suggests that the NS4A cofactor function is well conserved among HCV It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.     

The role of resistance in HCV treatment

The recent development of small molecule compounds that directly inhibit the viral life cycle represents a major milestone for the treatment of chronic hepatitis C virus (HCV) infection. These new drugs that are collectively termed direct-acting antivirals (DAA) include a range of inhibitors of the non-structural (NS) 3/4A protease, NS5B polymerase and NS5A protein. Two NS3/4A protease inhibitors (boceprevir and telaprevir) in combination with pegylated interferon and ribavirin have now been approved for the treatment of chronic HCV genotype 1 infection and cure rates could be increased by 20–30%. However, the majority of DAAs is still in early clinical development. The rapid replication rate of HCV, along with the error-prone polymerase activity leads to a high genetic diversity among HCV virions that includes mutants with reduced susceptibility to DAA-therapy. These resistance-associated variants often occur at very low frequencies. However, during DAA-based treatment, rapid selection of resistance mutations may occur, eventually leading to viral break-through. A number of variants with different levels of resistance have been described in vitro and in vivo for virtually all DAAs. We review the parameters that determine DAA resistance as well as the clinical implications of resistance testing. In addition, the most recent literature and conference data on resistance profiles of DAAs in clinical development and future strategies to avoid the emergence of viral resistance are also discussed.     

BILN2061、telaprevir和boceprevir治疗慢性丙型肝炎临床疗效比较

HCV感染易导致慢性肝炎、肝硬化及肝癌等相关性疾病,病死率较高.HCV在体内的翻译是经宿主信号肽和非结构蛋白(non-structural protein,NS)3病毒蛋白酶水解成熟.此外,NS3还可直接破坏宿主细胞,抑制其对干扰素的应答,因此研究HCV NS3具有重要的临床意义.本文通过对3种NS3/4A蛋白酶抑制剂(BILN 2061、telaprevir和boceprevir)的药理作用、药动学与代谢、安全性和临床研究进行分析,总结其作用特性和临床疗效.同时,对boceprevir和telaprevir(VX-950)分别联合聚乙二醇干扰素和利巴韦林的疗效进行评价,为临床医生治疗慢性丙型肝炎提供指导.     

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.     

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed protease-helicase domain assembly in solution.     

Non-structural protein NS4B: HCV replication web inducer

Hepatitis C virus (HCV) genome consists of a positive strand RNA encoding a polyprotein, having 3 structural and 6 non-structural components including non structural protein 4B (NS4B). NS4B is a 27 KDa protein, of 261 amino acids, released after polyprotein cleavage by NS3 serine protease and localized on endoplasmic reticulum (ER). NS4B has 2 alpha helices each in its N and C terminal domains and 4 transmembrane domains in its central region. N-terminal domain resides in the ER-lumen while C-terminal domain resides in the cytoplasm. Around its middle it has a nucleotide binding motif (NBM) which plays a role in ATP and GTP hydrolysis. It is involved in hyperphosphorylation of the NS5A protein and is also thought to be involved in production of various cytokines by the activation of NF-kB pathway. NS4B plays a major role in HCV replication by inducing membranous web and facilitating other HCV proteins necessary for replication. Here we discuss various functional aspects of this protein and their potential for targeted antiviral approaches.     

The Discovery and Development of Boceprevir: A Novel,First-generation Inhibitor of the Hepatitis C Virus NS3/4A Serine Protease

An estimated 2–3% of the world''s population is infected with hepatitis C virus (HCV), making it a major global health problem. Consequently, over the past 15 years, there has been a concerted effort to understand the pathophysiology of HCV infection and the molecular virology of replication, and to utilize this knowledge for the development of more effective treatments. The virally encoded non-structural serine protease (NS3) is required to process the HCV polyprotein and release the individual proteins that form the viral RNA replication machinery. Given its critical role in the replication of HCV, the NS3 protease has been recognized as a potential drug target for the development of selective HCV therapies. In this review, we describe the key scientific discoveries that led to the approval of boceprevir, a first-generation, selective, small molecule inhibitor of the NS3 protease. We highlight the early studies that reported the crystal structure of the NS3 protease, its role in the processing of the HCV polyprotein, and the structural requirements critical for substrate cleavage. We also consider the novel attributes of the NS3 protease-binding pocket that challenged development of small molecule inhibitors, and the studies that ultimately yielded milligram quantities of this enzyme in a soluble, tractable form suitable for inhibitor screening programs. Finally, we describe the discovery of boceprevir, from the early chemistry studies, through the development of high-throughput assays, to the phase III clinical development program that ultimately provided the basis for approval of this drug. This latest phase in the development of boceprevir represents the culmination of a major global effort to understand the pathophysiology of HCV and develop small molecule inhibitors for the NS3 protease.     

The NS3/4A proteinase of the hepatitis C virus: unravelling structure and function of an unusual enzyme and a prime target for antiviral therapy

The hepatitis C virus (HCV) is a major causative agent of transfusion-acquired and sporadic non-A, non-B hepatitis worldwide. Infections most often persist and lead, in 50% of all patients, to chronic liver disease. As is characteristic for a member of the family Flaviviridae , HCV has a plus-strand RNA genome encoding a polyprotein, which is cleaved co- and post-translationally into at least 10 different products. These cleavages are mediated, among others, by a virally encoded chymotrypsin-like serine proteinase located in the N-terminal domain of non-structural protein 3 (NS3). Activity of this enzyme requires NS4A, a 54-residue polyprotein cleavage product, to form a stable complex with the NS3 domain. This review will describe the biochemical properties of the NS3/4A proteinase, its X-ray crystal structure and current attempts towards development of efficient inhibitors.     

Hepatitis C virus resistance to protease inhibitors

Recent advances in molecular biology have led to the development of novel small molecules that target specific viral proteins of the hepatitis C virus (HCV) life cycle. These drugs, collectively termed directly acting antivirals (DAA) against HCV, include a range of non-structural (NS) 3/NS4A protease, NS5B polymerase, and NS5A inhibitors at various stages of clinical development. The rapid replication rate of HCV, along with the low fidelity of its polymerase, gives rise to generations of mutations throughout the viral genome resulting in remarkable sequence variation in the HCV population, known as a quasispecies. The efficacy of DAAs is limited by the presence of those mutations that give rise to amino-acid substitutions within the targeted protein, and that affect the viral sensitivity to these compounds. Thus, due to the high genetic variability of HCV, variants with reduced susceptibility to DAA can occur naturally even before treatment begins, but usually at low levels. Not surprisingly then, these changes are selected in patients either breaking through or not responding to potent DAA treatment. In vitro or in vivo, six major position mutations in the NS3 HCV protease (36, 54, 155, 156, 168, and 170) have now been reported associated with different levels of resistance. The amino acid composition at several of the drug resistance sites can vary between the HCV genotypes/subtypes, resulting in different consensus amino acids leading to a reduction in replicative fitness as well as reduced DAA sensitivity. Different amino acid diversity profiles for HCV genotypes/subtypes suggest differences in the position/type of immune escape and drug resistance mutations. Also, different pathways of resistance profiles based on the chemical scaffold (linear or macrocyclic) of the protease inhibitors have been described. This review first describes how resistance to a protease inhibitor can develop and then provides an overview of the mechanism of how particular mutations confer varying levels of resistance to protease inhibitor, which have been identified and characterized using both genotypic and phenotypic tools. Future potential therapeutic strategies to assist patients who do develop resistance to protease inhibitors are also outlined. The challenge developing new HCV protease inhibitors should take into consideration not only the antiviral potency of the drugs, the occurrence and importance of side effects, the frequency of oral administration, but also the resistance profiles of these agents.     

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

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

The practical management of chronic hepatitis C infection in Japan - dual therapy of daclatasvir + asunaprevir

Introduction: Without treatment, many of the 200 million people worldwide with chronic hepatitis C virus (HCV) infection will develop cirrhosis or liver cancer. Japan was the first nation to approve an interferon-free therapy for HCV, and sustained viral response (SVR) rates >90% have been achieved with asunaprevir, a protease inhibitor, plus daclatasvir, an inhibitor of the non-structural 5A (NS5A) protein.

Areas covered: This review provides an overview of the results from both clinical trials and real world experience with asunaprevir and daclatasvir therapy focused primarily on Japan. A literature search using the keywords ‘asunaprevir,’ ‘daclatasvir,’ ‘interferon-free therapy,’ and ‘direct-acting antiviral drugs’ was initially used to select relevant literature for inclusion in the review.

Expert commentary: While not approved in the United States, dual therapy with asunaprevir plus daclatasvir has already been successfully used in Japan and throughout East Asia to treat many thousands of patients. Pre-existing or treatment-emergent NS5A-Y93 or -L31 resistance-associated variants (RAVs) may lead to viral breakthrough, and alternative therapies should be considered for these patients, but patients who harbor NS5A RAVs only at low frequency are likely to achieve SVR. The therapy has also been shown to be safe and effective with renal dysfunction or liver cirrhosis.     

Natural prevalence of HCV minority variants that are highly resistant to NS3/4A protease inhibitors

Summary. Minority drug‐resistant hepatitis C virus (HCV) variants may go undetected yet be clinically important. NS3/4A protease resistance substitutions V36A and A156S/T/V were selected in patients treated with protease inhibitors. The aim of this study was to investigate whether these substitutions pre‐existed in HCV infected patients. An allele‐specific PCR protocol that detected the NS3/4A protease resistance substitutions V36A and A156S/T/V was used to determine the prevalence of naturally occurring variants in 45 patients. All patient samples were infected with HCV of genotype 1b and were naïve for pegIFNα/ribavirin treatment. Thirty samples (67%) had at least one HCV PI‐resistant variant. A156T (23, 51%) was detected more frequently than A156V (13, 29%) or A156S (1, 2%). V36A was detected in 12 samples (27%). These results demonstrate the high prevalence of minority drug‐resistant NS3/4 protease resistance substitutions. Our results also demonstrate that allele‐specific PCR can be used to detect minor HCV NS3 protease resistant variants in pretreatment samples and to study in detail the evolution of mutant viruses during targeted antiviral therapy.     

Virologic Tools for HCV Drug Resistance Testing

Recent advances in molecular biology have led to the development of new antiviral drugs that target specific steps of the Hepatitis C Virus (HCV) lifecycle. These drugs, collectively termed direct-acting antivirals (DAAs), include non-structural (NS) HCV protein inhibitors, NS3/4A protease inhibitors, NS5B RNA-dependent RNA polymerase inhibitors (nucleotide analogues and non-nucleoside inhibitors), and NS5A inhibitors. Due to the high genetic variability of HCV, the outcome of DAA-based therapies may be altered by the selection of amino-acid substitutions located within the targeted proteins, which affect viral susceptibility to the administered compounds. At the drug developmental stage, preclinical and clinical characterization of HCV resistance to new drugs in development is mandatory. In the clinical setting, accurate diagnostic tools have become available to monitor drug resistance in patients who receive treatment with DAAs. In this review, we describe tools available to investigate drug resistance in preclinical studies, clinical trials and clinical practice.