Diverse effects of cyclosporine on hepatitis C virus strain replication

Recently, a production system for infectious particles of hepatitis C virus (HCV) utilizing the genotype 2a JFH1 strain has been developed. This strain has a high capacity for replication in the cells. Cyclosporine (CsA) has a suppressive effect on HCV replication. In this report, we characterize the anti-HCV effect of CsA. We observe that the presence of viral structural proteins does not influence the anti-HCV activity of CsA. Among HCV strains, the replication of genotype 1b replicons was strongly suppressed by treatment with CsA. In contrast, JFH1 replication was less sensitive to CsA and its analog, NIM811. Replication of JFH1 did not require the cellular replication cofactor, cyclophilin B (CyPB). CyPB stimulated the RNA binding activity of NS5B in the genotype 1b replicon but not the genotype 2a JFH1 strain. These findings provide an insight into the mechanisms of diversity governing virus-cell interactions and in the sensitivity of these strains to antiviral agents.     

Design and evaluation of a potential mutagen for Hepatitis C virus

Pyrrolopyrimidine nucleoside 1 was designed and synthesized as a potential mutagen for HCV. An in vitro HCV NS5B enzymatic assay indicated that pyrrolopyrimidine triphosphate acts as a CTP analog rather than a UTP analog. The SATE-prodrug of pyrrolopyrimidine monophosphate showed a weak inhibitory activity in an HCV replicon system (EC(50)=60 microM) and did not exhibit cytotoxicity (CC(50)>100 microM). Investigation of phosphorylation events using nucleoside kinases and LC-MS analysis revealed that the second phosphorylation step, from monophosphate ester to diphosphate ester, is unfavorable.     

Structure-function studies on the amphibian peptide brevinin 1E: translocating the cationic segment from the C-terminal end to a central position favors selective antibacterial activity.

Brevinin 1E, which has the sequence FLPLLAGLAANFLPKIFCKITRKC, is an antimicrobial peptide isolated from the skin secretions of the European frog Rana esculenta. Both the linear and the disulfide-bridged forms have relatively broad-spectrum antibacterial as well as hemolytic activities. The antibacterial and hemolytic activities and biophysical properties of synthetic peptides corresponding to brevinin 1E and its analog in which the segment CKITRKC has been transposed to a central location resulting in the sequence FLPLLAGLCKITRKCAANFLPKIF have been investigated. Our studies indicate that the analog peptide has antibacterial activity comparable with brevinin 1E, but with considerably reduced hemolytic activity. The linear variant of the analog has no hemolytic activity, unlike the linear form of brevinin 1E. The biological activities can be explained on the basis of relative affinities for anionic and zwitterionic lipids. A cluster of cationic amino acids flanked on one side by a hydrophobic stretch of amino acids and another side composed of apolar amino acids appears to favor preferential antibacterial activity.     

Electrochemical nucleic acid hybridization biosensor based on poly(L-Aspartic acid)-modified electrode for the detection of short oligonucleotide sequences related to hepatitis C virus 1a

Abstract

This work describes, for the first time, the fabrication of poly(L-aspartic acid) (PAA) film modified pencil graphite electrode (PGE) for the detection of hepatitis C Virus 1a (HCV1a). The presence of PAA on the electrode surface can provide free carboxyl groups for covalent binding of biomolecules. The PGE surface was first coated with PAA via electropolymerization of the L-aspartic acid, and avidin was subsequently attached to the PAA modified electrode by covalent attachment. Biotinylated HCV1a probes were immobilized on avidin/PAA/PGE via avidin-biotin interaction. The morphology of PAA/PGE was examined using a scanning electron microscope. The hybridization events were monitored with square wave voltammetry using Meldola’s blue (MDB). Compared to non-complementary oligonucleotide sequences, when hybridization was carried out between the probe and its synthetic targets or the synthetic polymerase chain reaction analog of HCV1a, the highest MDB signal was observed. The linear range of the biosensor was 12.5 to 100?nM and limit of detection was calculated as 8.7?nM. The biosensor exhibited favorable stability over relatively long-term storage. All these results suggest that PAA-modified electrode can be used to nucleic acid biosensor application and electropolymerization of L-aspartic acid can be considered as a good candidate for the immobilization of biomolecules.     

The Novel Cyclophilin Inhibitor CPI-431-32 Concurrently Blocks HCV and HIV-1 Infections via a Similar Mechanism of Action

HCV-related liver disease is the main cause of morbidity and mortality of HCV/HIV-1 co-infected patients. Despite the recent advent of anti-HCV direct acting antivirals (DAAs), the treatment of HCV/HIV-1 co-infected patients remains a challenge, as these patients are refractory to most therapies and develop liver fibrosis, cirrhosis and liver cancer more often than HCV mono-infected patients. Until the present study, there was no suitable in vitro assay to test the inhibitory activity of drugs on HCV/HIV-1 co-infection. Here we developed a novel in vitro “co-infection” model where HCV and HIV-1 concurrently replicate in their respective main host target cells—human hepatocytes and CD4+ T-lymphocytes. Using this co-culture model, we demonstrate that cyclophilin inhibitors (CypI), including a novel cyclosporin A (CsA) analog, CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre- and post-infection. In contrast, the HIV-1 protease inhibitor nelfinavir or the HCV NS5A inhibitor daclatasvir only blocks the replication of a single virus in the “co-infection” system. CPI-431-32 efficiently inhibits HCV and HIV-1 variants, which are normally resistant to DAAs. CPI-431-32 is slightly, but consistently more efficacious than the most advanced clinically tested CypI—alisporivir (ALV)—at interrupting an established HCV/HIV-1 co-infection. The superior antiviral efficacy of CPI-431-32 over ALV correlates with its higher potency inhibition of cyclophilin A (CypA) isomerase activity and at preventing HCV NS5A-CypA and HIV-1 capsid-CypA interactions known to be vital for replication of the respective viruses. Moreover, we obtained evidence that CPI-431-32 prevents the cloaking of both the HIV-1 and HCV genomes from cellular sensors. Based on these results, CPI-431-32 has the potential, as a single agent or in combination with DAAs, to inhibit both HCV and HIV-1 infections.     

ATP binding modulates the nucleic acid affinity of hepatitis C virus helicase

The helicase of hepatitis C virus (HCV) unwinds nucleic acid using the energy of ATP hydrolysis. The ATPase cycle is believed to induce protein conformational changes to drive helicase translocation along the length of the nucleic acid. We have investigated the energetics of nucleic acid binding by HCV helicase to understand how the nucleotide ligation state of the helicase dictates the conformation of its nucleic acid binding site. Because most of the nucleotide ligation states of the helicase are transient due to rapid ATP hydrolysis, several compounds were analyzed to find an efficient unhydrolyzable ATP analog. We found that the beta-gamma methylene/amine analogs of ATP, ATPgammaS, or [AlF4]ADP were not effective in inhibiting the ATPase activity of HCV helicase. On the other hand, [BeF3]ADP was found to be a potent inhibitor of the ATPase activity, and it binds tightly to HCV helicase with a 1:1 stoichiometry. Equilibrium binding studies showed that HCV helicase binds single-stranded nucleic acid with a high affinity in the absence of ATP or in the presence of ADP. Upon binding to the ATP analog, a 100-fold reduction in affinity for ssDNA was observed. The reduction in affinity was also observed in duplex DNA with 3' single-stranded tail and in RNA but not in duplex DNA. The results of this study indicate that the nucleic acid binding site of HCV helicase is allosterically modulated by the ATPase reaction. The binding energy of ATP is used to bring HCV helicase out of a tightly bound state to facilitate translocation, whereas ATP hydrolysis and product release steps promote tight rebinding of the helicase to the nucleic acid. On the basis of these results we propose a Brownian motor model for unidirectional translocation of HCV helicase along the nucleic acid length.     

RT套式PCR检测血浆HCV RNA及与抗HCV检测的比较

应用微量血清热变性法提取核酸,逆转录套式聚合酶链反应(RT-nest PCR)检测血浆HCV RNA,并与抗HCV ELISA检测结果比较,对HCV RNA阳性标本进行HGV RNA的筛查.结果在32例抗HCV阳性和20例抗HCV阴性血浆中,HCV RNA分别检出18例和2例,总符合率为70%,20例HCV RNA阳性者中有2例合并感染HBV,1例合并感染HGV.证明血浆样本中抗HCV与HCV RNA间存在很大的相关性.     

Synthesis and inhibitory activity on hepatitis C virus RNA replication of 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analogs

Using our recently developed assay system for full-genome-length hepatitis C virus (HCV) RNA replication in human hepatoma-derived Li23 cells (ORL8), we identified 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analog 1a as a novel HCV inhibitor. Structural modifications of 1a provided a series of sulfonamides 7 with much more potent HCV RNA replication-inhibitory activity than ribavirin. Compound 7a showed an additive anti-HCV effect in combination with standard anti-HCV therapy (IFN-α plus ribavirin). Since 7a generated reactive oxygen species (ROS) in the ORL8 system and its anti-HCV activity was blocked by vitamin E, its anti-HCV activity may be mediated at least in part by ROS.     

Genome of human hepatitis C virus (HCV): gene organization, sequence diversity, and variation

Hepatitis C virus (HCV) is the major etiologic agent of non-A, non-B hepatitis. HCV infection frequently causes chronic hepatitis, which progresses to liver cirrhosis and hepatocellular carcinoma. Since the discovery of HCV in 1989, a large number of genetic analyses of HCV have been reported, and the viral genome structure has been elucidated. An enveloped virus, HCV belongs to the family Flaviviridae, whose genome consists of a positive-stranded RNA molecule of about 9.6 kilobases and encodes a large polyprotein precursor (about 3000 amino acids). This precursor protein is cleaved by the host and viral proteinase to generate at least 10 proteins: the core, envelope 1 (E1), E2, p7, nonstructural (NS) 2, NS3, NS4A, NS4B, NS5A, and NS5B. These HCV proteins not only function in viral replication but also affect a variety of cellular functions. HCV has been found to have remarkable genetic heterogeneity. To date, more than 30 HCV genotypes have been identified worldwide. Furthermore, HCV may show quasispecies distribution in an infected individual. These findings may have important implications in diagnosis, pathogenesis, treatment, and vaccine development. The hypervariable region 1 found within the envelope E2 protein was shown to be a major site for the genetic evolution of HCV after the onset of hepatitis, and might be involved in escape from the host immunesurveillance system.     

Synthesis, antiviral activity, and conformational studies of a P3 aza-peptide analog of a potent macrocyclic tripeptide HCV protease inhibitor

BILN 2061 is a macrocyclic tripeptide inhibitor of hepatitis C virus NS3-4A protease that has shown efficacy in the clinic for treating patients infected with HCV. We have synthesized a P3 aza-peptide analog of a potent macrocyclic tripeptide inhibitor closely related to BILN 2061. This aza-derivative was found to be >2 orders of magnitude less active than the parent macrocycle in both isolated enzyme (HCV NS3-4A) and HCV subgenomic replicon assays. NMR studies of P3 aza-peptides revealed these compounds adopt a beta-turn conformation stabilized by an intramolecular H-bonding interaction. Molecular models of these structures indicate a D-like configuration of the P3 aza-residue. Thus, the configurationally undefined nature at P3 in the aza-peptide allows the compound to adopt an H-bond stabilized conformation that is substantially different from that necessary for tight binding to the active site of HCV NS3 protease.     

Inhibition of hepatitis C virus RNA replication by 2'-modified nucleoside analogs

The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is essential for the replication of viral RNA and thus constitutes a valid target for the chemotherapeutic intervention of HCV infection. In this report, we describe the identification of 2'-substituted nucleosides as inhibitors of HCV replication. The 5'-triphosphates of 2'-C-methyladenosine and 2'-O-methylcytidine are found to inhibit NS5B-catalyzed RNA synthesis in vitro, in a manner that is competitive with substrate nucleoside triphosphate. NS5B is able to incorporate either nucleotide analog into RNA as determined with gel-based incorporation assays but is impaired in its ability to extend the incorporated analog by addition of the next nucleotide. In a subgenomic replicon cell line, 2-C-methyladenosine and 2'-O-methylcytidine inhibit HCV RNA replication. The 5'-triphosphates of both nucleosides are detected intracellularly following addition of the nucleosides to the media. However, significantly higher concentrations of 2'-C-methyladenosine triphosphate than 2'-O-methylcytidine triphosphate are detected, consistent with the greater potency of 2'-C-methyladenosine in the replicon assay, despite similar inhibition of NS5B by the triphosphates in the in vitro enzyme assays. Thus, the 2'-modifications of natural substrate nucleosides transform these molecules into potent inhibitors of HCV replication.     

A cellular RNA-binding protein enhances internal ribosomal entry site-dependent translation through an interaction downstream of the hepatitis C virus polyprotein initiation codon

Translational initiation of hepatitis C virus (HCV) mRNA occurs by internal entry of ribosomes into an internal ribosomal entry site (IRES) at the 5' nontranslated region. A region encoding the N-terminal part of the HCV polyprotein has been shown to augment the translation of HCV mRNA. Here we show that a cellular protein, NS1-associated protein 1 (NSAP1), augments HCV mRNA translation through a specific interaction with an adenosine-rich protein-coding region within the HCV mRNA. The overexpression of NSAP1 specifically enhanced HCV IRES-dependent translation, and knockdown of NSAP1 by use of a small interfering RNA specifically inhibited the translation of HCV mRNA. An HCV replicon RNA capable of mimicking the HCV proliferation process in host cells was further used to confirm that NSAP1 enhances the translation of HCV mRNA. These results suggest the existence of a novel mechanism of translational enhancement that acts through the interaction of an RNA-binding protein with a protein coding sequence.     

The Akt/GSK-3β pathway mediates flurbiprofen-induced neuroprotection against focal cerebral ischemia/reperfusion injury in rats

The advent of infectious molecular clones of Hepatitis C virus (HCV) has unlocked the understanding of HCV life cycle. However, packaging of the genomic RNA, which is crucial to generate infectious viral particles, remains poorly understood. Molecular interactions of the domain 1 (D1) of HCV Core protein and HCV RNA have been described in vitro. Since compaction of genetic information within HCV genome has hampered conventional mutational approach to study packaging in vivo, we developed a novel heterologous system to evaluate the interactions between HCV RNA and Core D1. For this, we took advantage of the recruitment of Vpr fusion-proteins into HIV-1 particles. By fusing HCV Core D1 to Vpr we were able to package and transfer a HCV subgenomic replicon into a HIV-1 based lentiviral vector. We next examined how deletion mutants of basic sub-domains of Core D1 influenced HCV RNA recruitment. The results emphasized the crucial role of the first and third basic regions of D1 in packaging. Interestingly, the system described here allowed us to mobilise full-length JFH1 genome in CD81 defective cells, which are normally refractory to HCV infection. This finding paves the way to an evaluation of the replication capability of HCV in various cell types.     

Tupaia CD81, SR-BI, claudin-1, and occludin support hepatitis C virus infection

Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.     

Baculovirus Mediated Production of Infectious Hepatitis C Virus in Human Hepatoma Cells Stably Expressing T7 RNA Polymerase

Although much has been learned about Hepatitis C virus (HCV), research progress has been hindered by the lack of a suitable cell culture system supporting its replication. Recently, a unique HCV strain JFH1 has been found to replicate efficiently in cell culture with production of infectious HCV (HCVcc). Baculovirus vectors were found to be efficient delivery vehicles and a HBV recombinant baculovirus/HepG2 system efficiently delivered the HBV genome into HepG2 resulting in HBV replication. In this study, we developed a recombinant baculovirus expression system to generate infectious HCV particles in hepatoma cell line Huh7-lunetT7 by using cDNA from the HCV JFH1 genotype. Results show that HCV positive, negative RNA strands and proteins were produced in this system. Furthermore, HCV particles were produced and secreted into the culture medium. Sucrose density gradient centrifugation of the culture medium revealed co-localization of HCV RNA and structural proteins in the fraction with a density of 1.08–1.13 g/ml. Electron microscopy (EM) showed viral particles approximately 55 nm in diameter, which could be recognized by anti-HCV E2 antibodies. Real-time RT-PCR detected that the level of HCV vRNA in the supernatant was 10⁷ copies/ml at 72 h post-transduction (hpt). In addition, the JFH1 virus produced by the recombinant baculovirus was confirmed to be infectious in vitro. In summary, this system provides a novel tool not only for the analysis of the replication and pathogenesis of HCV but also to screen for potential therapeutic targets.     

Hepatitis C Virus Genotype 1b Core Protein Does Not Exert Immunomodulatory Effects on Virus-Induced Cellular Immunity

The hepatitis C virus (HCV) core protein is among the most conserved proteins in HCV and is known to induce sensitization of cytotoxic T lymphocytes (CTL). Therefore, it is a prime candidate for a component of a potential HCV vaccine. The HCV core protein has, however, been reported to exert multiple effects on cell functions, raising questions as to its suitability for this purpose. This question was investigated here with mice into which replication-deficient adenoviruses expressing core protein of an HCV genotype 1b isolate were injected. We show that induction of cytokines in response to the infection, infiltration of lymphocytes into the infected liver, priming of virus-specific CTL, and liver injury are not modulated by expression of the core protein in the liver. Moreover, no changes in the sensitivity to tumor necrosis factor alpha- or Fas-mediated liver injury are demonstrable. A similar lack of demonstrable effects of the core protein on immune functions has also been obtained using transgenic mice expressing another HCV genotype 1b core protein. It is concluded that the HCV core protein of genotype 1b has no modulatory effects on induction of virus-specific immune responses and may therefore be a suitable component of an HCV vaccine.     

丙型肝炎病毒C+E1区基因在原核细胞中的克隆与表达

将丙型肝炎病毒Ｃ＋Ｅ１区基因插入到原核高效表达载体ｐＢＶ２２１质粒中,构建了质粒ｐＢＶ２２１ＨＣＶ／Ｃ＋Ｅ１作为表达载体,然后,将含有该质粒的宿主大肠杆菌进行升温诱导表达ＨＣＶ／Ｃ＋Ｅ１区基因,并对表达产物进行了生物活性的检测。结果表明,插入到表达载体ｐＢＶ２２１中的ＨＣＶ／Ｃ＋Ｅ１基因片段能够得到有效的表达,表达产物主要为非融合蛋白形式存在于细胞中,同时这种Ｃ区和Ｅ１区连接共表达的产物保持了良好的抗原活性     

Assembly of a functional HCV glycoprotein heterodimer

The two HCV envelope glycoproteins E1 and E2 are released from HCV polyprotein by signal peptidase cleavages. These glycoproteins are type I transmembrane proteins with a highly glycosylated N-terminal ectodomain and a C-terminal hydrophobic anchor. After their synthesis, HCV glycoproteins E1 and E2 associate as a noncovalent heterodimer. The transmembrane domains of HCV envelope glycoproteins play a major role in E1E2 heterodimer assembly and subcellular localization. The envelope glycoprotein complex E1E2 has been proposed to be essential for HCV entry. However, for a long time, HCV entry studies have remained limited because of the lack of a robust cell culture system to amplify this virus. A few years ago, a model mimicking the entry process of HCV lifecycle has been developed by pseudotyping retroviral particles with native HCV envelope glycoproteins. This model allowed the characterization of functional E1E2 envelope glycoproteins. The data obtained can now be confirmed with the help of a newly developed cell-culture system that allows efficient amplification of HCV (HCVcc). Here, we present the recent data that have been accumulated on the assembly of the functional HCV glycoprotein heterodimer.