Hepatitis C Virus Polyprotein Processing Research Articles

Epoxide based inhibitors of the hepatitis C virus non-structural 2 autoprotease

Hepatitis C virus (HCV) non-structural 2 (NS2) encodes an essential protease activity responsible for processing at the NS2–NS3 junction which represents an attractive antiviral target. Attempts to inhibit the NS2 autoprotease with mechanism-based protease inhibitors and substrate peptides have had limited success. We report a series of epoxide-containing small molecules capable of blocking NS2–NS3 proteolysis in vitro and demonstrate the potential for selectivity towards the NS2 autoprotease. A compound within this series was able to perturb HCV genome replication in a subgenomic replicon system only when polyprotein processing was dependent on NS2 autoprotease activity, in addition it inhibited replication of full length HCV. These findings suggest blocking HCV polyprotein processing through inhibition of the NS2 autoprotease represents a viable route to exert an antiviral effect.

NS2 proteins of GB virus B and hepatitis C virus share common protease activities and membrane topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

ACH-806, an NS4A Antagonist, Inhibits Hepatitis C Virus Replication by Altering the Composition of Viral Replication Complexes

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

Scotomas in molecular virology and epidemiology of hepatitis C virus

In the 1970s, scientists learned of a new pathogen causing non-A, non-B hepatitis. Classical approaches were used to isolate and characterize this new pathogen, but it could be transmitted experimentally only to chimpanzees and progress was slow until the pathogen was identified as hepatitis C virus (HCV) in 1989. Since then, research and treatment of HCV have expanded with the development of modern biological medicine: HCV genome organization and polyprotein processing were delineated in 1993; the first three-dimensional structure of HCV nonstructural protein (NS3 serine protease) was revealed in 1996; an infectious clone of HCV complementary DNA was first constructed in 1997; interferon and ribavirin combination therapy was established in 1998 and the therapeutic strategy gradually optimized; the HCV replicon system was produced in 1999; functional HCV pseudotyped viral particles were described in 2003; and recombinant infectious HCV in tissue culture was produced successfully in 2005. Recently, tremendous advances in HCV receptor discovery, understanding the HCV lifecycle, decryption of the HCV genome and proteins, as well as new anti-HCV compounds have been reported. Because HCV is difficult to isolate and culture, researchers have had to avail themselves to the best of modern biomedical technology; some of the major achievements in HCV research have not only advanced the understanding of HCV but also promoted knowledge of virology and cellular physiology. In this review, we summarize the advancements and remaining scotomas in the molecular virology and epidemiology of HCV.

Hepatitis B and C

Hepatitis B and C

A Data Mining Approach for the Prediction of Hepatitis C Virus protease Cleavage Sites

Summary: Several papers have been published about the prediction of hepatitis C virus (HCV) polyprotein cleavage sites, using symbolic and non-symbolic machine learning techniques. The published papers achieved different Levels of prediction accuracy. the achieved results depends on the used technique and the availability of adequate and accurate HCV polyprotein sequences with known cleavage sites. We tried here to achieve more accurate prediction results, and more Informative knowledge about the HCV protein cleavage sites using Decision tree algorithm. There are several factors that can affect the overall prediction accuracy. One of the most important factors is the availably of acceptable and accurate HCV polyproteins sequences with known cleavage sites. We collected latest accurate data sets to build the prediction model. Also we collected another dataset for the model testing. Motivation: Hepatitis C virus is a global health problem affecting a significant portion of the world’s population. The World Health Organization estimated that in1999; 170 million hepatitis C virus (HCV) carriers were present worldwide, with 3 to 4 million new cases per year. Several approaches have been performed to analyze HCV life cycle to find out the important factors of the viral replication process. HCV polyprotein processing by the viral protease has a vital role in the virus replication. The prediction of HCV protease cleavage sites can help the biologists in the design of suitable viral inhibitors. Results: The ease to use and to understand of the decision tree enabled us to create simple prediction model. We used here the latest accurate viral datasets. Decision tree achieved here acceptable prediction accuracy results. Also it generated informative knowledge about the cleavage process itself. These results can help the researchers in the development of effective viral inhibitors. Using decision tree to predict HCV protein cleavage sites achieved high prediction accuracy.

In trans interaction of hepatitis C virus helicase domains mediates protease activity critical for internal NS3 cleavage and cell transformation

Hepatitis C virus (HCV) internal non-structural protein 3 (NS3) cleavage can occur in trans in the presence of NS4A. In this study, we have further demonstrated a critical role of the helicase domain in the internal NS3 cleavage, different from HCV polyprotein processing which requires only the serine protease domain. The NTPase domain of NS3 helicase interacts with the RNA binding domain to facilitate internal NS3 cleavage. In addition, NS3 protease activity contributes to the transforming ability of the major internal cleavage product NS3(1–402). These findings imply important roles of the internal cleavage and protease activity of the NS3 protein in the pathogenesis of HCV. Structured summaryMINT-7306465: NS3 (uniprotkb:P29846) physically interacts (MI:0915) with NS3 (uniprotkb:P29846) by anti tag coimmunoprecipitation (MI:0007).

Hepatitis C virus NS2/3 protease regulates HCV IRES-dependent translation and NS5B RdRp activity

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. NS2/3 protease is the first of two virally encoded proteases required for HCV polyprotein processing. In this report, we investigated the function of NS2/3 protease on HCV replication and translation. Cells transfected with plasmids encoding wild-type or mutant NS2/3 and a dual-luciferase reporter construct containing an HCV internal ribosome entry site (IRES) were used to examine the effect of NS2/3 protease on translation of HCV RNA. Cells transfected with plasmids encoding wild-type or mutant NS2/3, pcDNA-NS5B and a reporter plasmid were used to examine the effect of NS2/3 protease on HCV replication. The results showed that both autocleavage processing and the uncleaved form of NS2/3 protease specifically decrease HCV IRES-directed translation, while the uncleaved form of NS2/3 protease decreases HCV NS5B RdRp activity (replication), indicating that autoregulation by NS2/3 protease of HCV replication and translation may play an important role in persistent HCV infection.

Essential Role of Cyclophilin A for Hepatitis C Virus Replication and Virus Production and Possible Link to Polyprotein Cleavage Kinetics

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication.

Virological characterization of the hepatitis C virus JFH-1 strain in lymphocytic cell lines

While hepatocytes are the major site of hepatitis C virus (HCV) infection, a number of studies have suggested that HCV can replicate in lymphocytes. However, in vitro culture systems to investigate replication of HCV in lymphocytic cells are severely limited. Robust HCV culture systems have been established using the HCV JFH-1 strain and Huh-7 cells. To gain more insights into the tissue tropism of HCV, we investigated the infection, replication, internal ribosome entry site (IRES)-dependent translation and polyprotein processing of the HCV JFH-1 strain in nine lymphocytic cell lines. HCV JFH-1 failed to infect lymphocytes and replicate, but exhibited efficient polyprotein processing and IRES-dependent translation in lymphocytes as well as in Huh-7 cells. Our results suggest that lymphocytic cells can support HCV JFH-1 translation and polyprotein processing, but may lack some host factors essential for HCV JFH-1 infection and replication.

Structural biology of hepatitis C virus.

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Studies of this virus have been hampered by the lack of a productive cell culture system; most information thus has been obtained from analysis of the HCV genome, heterologous expression systems, in vitro and in vivo models, and structural analyses. Structural analyses of HCV components provide an essential framework for understanding of the molecular mechanisms of HCV polyprotein processing, RNA replication, and virion assembly and may contribute to a better understanding of the pathogenesis of hepatitis C. Moreover, these analyses should allow the identification of novel targets for antiviral intervention and development of new strategies to prevent and combat viral hepatitis. This article reviews the current knowledge of HCV structural biology.

Peptide inhibitors of hepatitis C virus NS3 protease.

Hepatitis C virus (HCV) is a highly prevalent virus and one of the major agents of chronic hepatitis. Since HCV NS3 protease is essential for the processing of HCV polyprotein, this protease is a target of choice to control HCV replication. Peptide inhibitors of NS3 were developed by selective amino acid replacement of six leader sequences, corresponding to regions of HCV polyprotein that are cleaved by NS3. The large numbers of potential 14-mer and 16-mer peptide inhibitors thus obtained were tested against NS3 using the fluorescent probe RETS1 and peptide cofactor SVVIVGRIILSGRA from NS4A protein. This afforded several peptide inhibitors with an IC50 of around 2 microM. These peptides may be good leading compounds for the development of peptidomimetics to control HCV replication in the treatment of chronic hepatitis C.

Full-length core sequence dependent complex-type glycosylation of hepatitis C virus E2 glycoprotein.

To study HCV polyprotein processing is important for the understanding of the natural history of HCV and the design of vaccines against HCV. The purpose of this study is to investigate the affection of context sequences on hepatitis C virus (HCV) E2 processing. HCV genes of different lengths were expressed and compared in vaccinia virus/T7 system with homologous patient serum S94 and mouse anti-serum M( E2116) raised against E.coli -derived E2 peptide, respectively. Deglycosylation analysis and GNA ( Galanthus nivalus ) lectin binding assay were performed to study the post-translational processing of the expressed products. E2 glycoproteins with different molecular weights (-75 kDa and -60 kDa) were detected using S94 and M( E2116), respectively. Deglycosylation analysis showed that this difference was mainly due to different glycosylation. Endo H resistance and its failure to bind to GNA lectin demonstrated that the higher molecular weight form (75 kDa) of E2 was complex-type glycosylated, which was readily recognized by homologous patient serum S94. Expression of complex-type glycosylated E2 could not be detected in all of the core-truncated constructs tested, but readily detected in constructs encoding full-length core sequences. The upstream conserved full-length core coding sequence was required for the production of E2 glycoproteins carrying complex-type N-glycans which reacted strongly with homologous patient serum and therefore possibly represented more mature forms of E2. As complex-type N-glycans indicated modification by Golgi enzymes, the results suggest that the presence of full-length core might be critical for E1/E2 complex to leave ER. Our data may contribute to a better understanding of the processing of HCV structural proteins as well as HCV morphogenesis.

Internally located signal peptides direct hepatitis C virus polyprotein processing in the ER membrane.

An endoplasmic reticulum (ER) signal peptide is an amino acid sequence motif that directs the translocation of nascent polypeptides to the lumen of ER membrane. Most of known ER signal peptides are either N-terminal cleavable or internally uncleavable. In the structural protein region of hepatitis C virus (HCV) polyprotein, however, four internally located cleavable signal peptides are arranged in a tandem array. The published experimental results indicated that the nascent HCV polyprotein is processed in the ER membrane by host signal peptidase(s) to the respective viral proteins. Here we propose that the four ER signal peptides lead the nascent HCV polyprotein to ER membrane, and the four internally located cleavable signal peptides are the sole determinant for the compartment localization of the matured viral proteins. After cleavage at the C-terminus, the signal peptides retain at the C-terminus of mature proteins, and serve as ER membrane anchors. The signal peptide directed polyprotein processing in the ER membrane preludes the virion assembly and budding from the ER membrane. This unique processing may be a general mechanism adopted by many types of virus for virion assembly and replication. The revelation of signal peptidase involved in HCV polyprotein processing presents a novel drug target to suppress HCV viral replication for the much needed HCV therapy.

Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein.

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.

Ultrastructural and immunocytochemical evidences of core-particle formation in the methylotrophicPichia pastorisyeast when expressing HCV structural proteins (core-E1)

Particulate antigens of the Hepatitis C virus (HCV) are reported for the first time by transmission electron microscopy inPichia pastoris. The yeast was cloned to express the first 339 NH2-terminal amino acids of the HCV polyprotein (C-E1.339 polypeptide). The C-E1.339 polypeptide covers the putative 191 aa of the core protein (aa 1–191) and 148 aa of the E1 envelope antigen (aa 192–339). Virus-like particles (VLP) with diameters ranging from 20 nm to 30 nm were specifically observed in those cells expressing the HCV polyprotein. The VLP appeared along the membrane of the endoplasmic reticulum, but were fundamentally localized in vacuoles, either free or inside autophagic bodies. Clustered particles, chains of particles, high-density reticular structures, and crystalloid bodies were also detected, the last one being an orderly arrangement of particles with 20 nm diameters. The crystal-associated particles are well differentiated from the intracellular VLP because of their uniform size and shape. We argue that membrane components are retained in the architecture of the VLP, conferring to this particle certain heterogeneity. Both kinds of particles, the VLP formed after treatment with NP-40 and the crystal-associated particles, were core protein-positives. Whether they reflect mature HCV nucleocapsid or intermediary states in the viral nucleocapsid morphogenesis remains unknown. We conclude that, like mammalian cell lines, theP. pastorisyeast could be an appropriate host for the analysis of HCV polyprotein processing and, eventually, virus assembly.

The NS3 proteinase domain of hepatitis C virus is a zinc-containing enzyme.

NS3 proteinase of hepatitis C virus (HCV), contained within the N-terminal domain of the NS3 protein, is a chymotrypsin-like serine proteinase responsible for processing of the nonstructural region of the HCV polyprotein. In this study, we examined the sensitivity of the NS3 proteinase to divalent metal ions, which is unusual behavior for this proteinase class. By using a cell-free coupled transcription-translation system, we found that HCV polyprotein processing can be activated by Zn2+ (and, to a lesser degree, by Cd2+, Pb2+, and Co2+) and inhibited by Cu2+ and Hg2+ ions. Elemental analysis of the purified NS3 proteinase domain revealed the presence of zinc in an equimolar ratio. The zinc content was unchanged in a mutated NS3 proteinase in which active-site residues His-57 and Ser-139 were replaced with Ala, suggesting that the zinc atom is not directly involved in catalysis but rather may have a structural role. Based on data from site-directed mutagenesis combined with zinc content determination, we propose that Cys-97, Cys-99, Cys-145, and His-149 coordinate the structural zinc in the HCV NS3 proteinase. A similar metal binding motif is found in 2A proteinases of enteroviruses and rhinoviruses, suggesting that these 2A proteinases and HCV NS3 proteinase are structurally related.

Virology of hepatitis C virus

Hepatitis C virus (HCV) has been identified as the main causative agent of post-transfusion non-A, non-B hepatitis. Through recently developed diagnostic assays, routine serologic screening of blood donors has prevented most cases of post-transfusion hepatitis. The purpose of this paper is to comprehensively review current information regarding the virology of HCV. Recent findings on the genome organization, its relationship to other viruses, the replication of HCV ribonucleic acid, HCV translation, and HCV polyprotein expression and processing are discussed. Also reviewed are virus assembly and release, the variability of HCV and its classification into genotypes, the geographic distribution of HCV genotypes, and the biologic differences between HCV genotypes. The assays used in HCV genotyping are discussed in terms of reliability and consistency of results, and the molecular epidemiology of HCV infection is reviewed. These approaches to HCV epidemiology will prove valuable in documenting the spread of HCV in different risk groups, evaluating alternative (nonparenteral) routes of transmission, and in understanding more about the origins and evolution of HCV.

Kinetic and structural analyses of hepatitis C virus polyprotein processing.

Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites. Processing of the NS3'-5B polyprotein was complex and occurred rapidly. Discrete polypeptide species corresponding to various processing intermediates were detected. With the exception of NS4AB-5A/NS5A, no clear precursor-product relationships were detected. Using double infection of cells with vaccinia virus recombinants expressing either a proteolytically inactive NS3'-5B polyprotein or an active NS3 proteinase, we found that cleavage at the NS4A/4B, NS4B/5A, and NS5A/5B sites could be mediated in trans. Absence of trans cleavage at the NS3/4A junction together with the finding that processing at this site was insensitive to dilution of the enzyme suggested that cleavage at this site is an intramolecular reaction. The trans-cleavage assay was also used to show that (i) the first 211 amino acids of NS3 were sufficient for processing at all trans sites and (ii) small deletions from the amino terminus of NS3 selectively affected cleavage at the NS4B/5A site, whereas more extensive deletions also decreased processing efficiencies at the other sites. Using a series of amino-terminally truncated substrate polyproteins in the trans-cleavage assay, we found that NS4A is essential for cleavage at the NS4B/5A site and that processing at this site could be restored by NS4A provided in cis (i.e., together with the substrate) or in trans (i.e., together with the proteinase). These results suggest that in addition to the NS3 proteinase, NS4A sequences play an important role in HCV polyprotein processing.

Identification of the domain required for trans-cleavage activity of hepatitis C viral serine proteinase

A serine proteinase, Cpro-2, encoded in the hepatitis C virus (HCV) genome, is considered to be located in the N-terminal part of HCV p70, one of the putative nonstructural (NS) proteins of HCV. Cpro-2 is suggested to be responsible for producing several kinds of NS proteins by processing of the HCV precursor polyprotein. We identified the active domain of Cpro-2 and clarified the mechanism of HCV polyprotein processing; various HCV mutants deleted around this serine proteinase structure were cosynthesized with unprocessed HCV polypeptides containing Cpro-2-dependent cleavage sites in COS-1 cells. We showed that Cpro-2 cleaved the HCV precursor polyprotein intermolecularly ( trans) and that Cpro-2 domain which is necessary and sufficient for that cleavage mapped to within 167 aa, from Gly 1049 to Ser 1215 of the HCV precursor polyprotein.