The Experts below are selected from a list of 159752115 Experts worldwide ranked by ideXlab platform
Charles M Rice - One of the best experts on this subject based on the ideXlab platform.
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fast hepatitis c virus rna elimination and NS5A redistribution by NS5A inhibitors studied by a multiplex assay approach
Antimicrobial Agents and Chemotherapy, 2015Co-Authors: Juan Ji, Tanya P Ndongwe, Eleftherios Michailidis, Robert Ralston, Charles M Rice, Stefan G SarafianosAbstract:ABSTRACT While earlier therapeutic strategies for the treatment of hepatitis C virus (HCV) infection relied exclusively on interferon (IFN) and ribavirin (RBV), four direct-acting antiviral agents (DAAs) have now been approved, aiming for an interferon-free strategy with a short treatment duration and fewer side effects. To facilitate studies on the mechanism of action (MOA) and efficacy of DAAs, we established a multiplex assay approach, which employs flow cytometry, a Gaussia luciferase reporter system, Western blot analysis, reverse transcription-quantitative PCR (RT-qPCR), a limited dilution assay (50% tissue culture infectious dose [TCID 50 ]), and an image profiling assay that follows the NS5A redistribution in response to drug treatment. We used this approach to compare the relative potency of various DAAs and the kinetics of their antiviral effects as a potential preclinical measure of their potential clinical utility. We evaluated the NS5A inhibitors ledipasvir (LDV) and daclatasvir (DCV), the NS3/4A inhibitor danoprevir (DNV), and the NS5B inhibitor sofosbuvir (SOF). In terms of kinetics, our data demonstrate that the NS5A inhibitor LDV, followed closely by DCV, has the fastest effect on suppression of viral proteins and RNA and on redistribution of NS5A. In terms of MOA, LDV has a more pronounced effect than DCV on the viral replication, assembly, and infectivity of released virus. Our approach can be used to facilitate the study of the biological processes involved in HCV replication and help identify optimal drug combinations.
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The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication
Journal of Virology, 2007Co-Authors: Brett D Lindenbach, Roland Montserret, Anna Marie Pyle, Béla M. Prágai, Rudolf K. F. Beran, Francois Penin, Charles M RiceAbstract:Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.
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mutations in the yellow fever virus nonstructural protein ns2a selectively block production of infectious particles
Journal of Virology, 2002Co-Authors: Beate M Kummerer, Charles M RiceAbstract:The Yellow fever virus (YF) belongs to the genus Flavivirus within the family Flaviviridae. Members of the Flavivirus genus are typically transmitted to vertebrates by mosquitoes or ticks and frequently cause significant human morbidity and mortality (reviewed in reference 25). Human pathogens include dengue virus, Japanese encephalitis virus, tick-borne encephalitis virus, West Nile virus, and YF. The estimated 100 million cases of dengue virus infection per year worldwide (5) and the emergence and spread of West Nile virus in the Eastern United States underscore the need for continued efforts to develop effective and inexpensive flavivirus vaccines. For YF, a live attenuated vaccine strain (17D) has been used effectively for almost 65 years. However, YF remains an enduring global public health problem due to the endemic persistence of mosquito-borne disease in sub-Saharan Africa and South America and recent reports of six fatalities temporally associated with live-virus vaccination (22, 33). The YF genome is a positive-sense RNA approximately 11 kb in length that is capped at the 5′ end but lacks a 3′ poly(A) tract. The RNA contains a single large open reading frame that is cleaved co- and posttranslationally by host cell and viral proteases (reviewed in reference 16). The polyprotein is arranged with the structural proteins at the amino terminus (C-prM-E), followed by the nonstructural (NS) proteins (NS1 through NS5). The arrangement of the proteins is NH2-C-(pr)M-E-NS1-NS2A-NS2B-NS3-NS4A-2K-NS4B-NS5-COOH. Most of the cleavages releasing the structural proteins are mediated by host cell signal peptidase. Exceptions include prM cleavage into pr and M by the host cell enzyme furin shortly before virus release and the cleavage generating the C terminus of the virion C protein by the virus-encoded serine protease (NS2B-3 protease). This serine protease, consisting of NS2B and the N-terminal part of NS3, produces the N termini of NS2B, NS3, NS4A, 2K, and NS5. The enzyme responsible for cleavage at the NS1/2A site is unknown. Although all flavivirus proteins stem from a single polyprotein, the structural and NS proteins have been viewed as functionally distinct modules responsible for virion formation and RNA replication, respectively. For example, coexpression of prM and E is sufficient for secretion of subviral particles that mimic the subunit structure and fusogenic capabilities of the mature virion envelope (2, 29). Subgenomic replicons lacking the structural proteins replicate efficiently and can be packaged by trans expression of the structural proteins (11). Although initially thought to be involved in virion morphogenesis or release, secreted glycoprotein NS1 plays an essential role in RNA replication (18). These observations have reinforced a modular view of the flavivirus polyprotein with the dividing line between structural proteins and replicase drawn at the E/NS1 junction. The only known exception is the NS2B-3 serine protease-mediated cleavage at the C terminus of mature C, a cleavage that is a necessary prerequisite for signalase generation of the prM N terminus and virus production (4). Specific functions have been attributed to many flavivirus proteins (reviewed in reference 16), but little is known about the function of NS2A. NS2A is a small hydrophobic protein of about 22 kDa (8). Consistent with a role in RNA replication, studies with Kunjin virus (KUN) have shown that NS2A colocalizes with double-stranded RNA in discrete cytoplasmic foci and interacts with the 3′ untranslated region of KUN RNA, as well as NS3 and NS5 (20). NS2A has also been implicated in the Japanese encephalitis virus-induced cytopathic effect (CPE) (10). In YF-infected cells, 22- and 20-kDa forms of NS2A have been identified and both of these forms possess the same N-terminal sequence (8). The 20-kDa form (called NS2Aα) is believed to result from an additional internal cleavage by the NS2B-3 serine protease. Cleavage by the YF serine protease occurs at a consensus sequence consisting of two basic amino acids followed by an amino acid with a short side chain (RR↓S/G) (6). However, in the case of the NS4A/2K junction, the cleavage site is QR↓S (14). Based on the known cleavage sites, as well as substitutions at these sites that are tolerated (9, 15, 26), NS2A residues 189 to 191 (QK↓T) were identified as a possible target for the viral serine protease. Consistent with this hypothesis, replacement of Lys-190 with Ser resulted in loss of the 20-kDa protein (26). Inhibition of cleavage at the NS2A/2B site did not abrogate production of NS2Aα, indicating that processing at the NS2A/2B site was not a prerequisite for cleavage at the NS2Aα site (26). It was further shown that the NS2A Lys-190-Ser mutation blocked production of infectious virus (26). Although the roles of NS2A and this additional cleavage in YF replication were unknown, a block at the level of YF RNA replication seemed most likely. In this report, we show that NS2Aα cleavage site mutants have unimpaired RNA replication but are unable to produce infectious virus. This defect in virus production can be complemented by NS2A or NS2Aα supplied in trans and compensated for by mutations at the NS2Aα cleavage site or by second-site changes in the helicase domain of NS3. These results reveal a more complex interplay between the NS proteins and virus production than previously suspected.
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bovine viral diarrhea virus ns3 serine proteinase polyprotein cleavage sites cofactor requirements and molecular model of an enzyme essential for pestivirus replication
Journal of Virology, 1997Co-Authors: Ernesto Mendez, Paul R Caron, Chao Lin, Mark A Murcko, Marc S Collett, Charles M RiceAbstract:Members of the Flaviviridae encode a serine proteinase termed NS3 that is responsible for processing at several sites in the viral polyproteins. In this report, we show that the NS3 proteinase of the pestivirus bovine viral diarrhea virus (BVDV) (NADL strain) is required for processing at nonstructural (NS) protein sites 3/4A, 4A/4B, 4B/5A, and 5A/5B but not for cleavage at the junction between NS2 and NS3. Cleavage sites of the proteinase were determined by amino-terminal sequence analysis of the NS4A, NS4B, NS5A, and NS5B proteins. A conserved leucine residue is found at the P1 position of all four cleavage sites, followed by either serine (3/4A, 4B/5A, and 5A/5B sites) or alanine (4A/4B site) at the P1' position. Consistent with this cleavage site preference, a structural model of the pestivirus NS3 proteinase predicts a highly hydrophobic P1 specificity pocket. trans-Processing experiments implicate the 64-residue NS4A protein as an NS3 proteinase cofactor required for cleavage at the 4B/5A and 5A/5B sites. Finally, using a full-length functional BVDV cDNA clone, we demonstrate that a catalytically active NS3 serine proteinase is essential for pestivirus replication.
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characterization of the hepatitis c virus encoded serine proteinase determination of proteinase dependent polyprotein cleavage sites
Journal of Virology, 1993Co-Authors: Arash Grakoui, Czeslaw Wychowski, Stephen M Feinstone, David W Mccourt, Charles M RiceAbstract:Processing of the hepatitis C virus (HCV) H strain polyprotein yields at least nine distinct cleavage products: NH2-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-CO OH. As described in this report, site-directed mutagenesis and transient expression analyses were used to study the role of a putative serine proteinase domain, located in the N-terminal one-third of the NS3 protein, in proteolytic processing of HCV polyproteins. All four cleavages which occur C terminal to the proteinase domain (3/4A, 4A/4B, 4B/5A, and 5A/5B) were abolished by substitution of alanine for either of two predicted residues (His-1083 and Ser-1165) in the proteinase catalytic triad. However, such substitutions have no observable effect on cleavages in the structural region or at the 2/3 site. Deletion analyses suggest that the structural and NS2 regions of the polyprotein are not required for the HCV NS3 proteinase activity. NS3 proteinase-dependent cleavage sites were localized by N-terminal sequence analysis of NS4A, NS4B, NS5A, and NS5B. Sequence comparison of the residues flanking these cleavage sites for all sequenced HCV strains reveals conserved residues which may play a role in determining HCV NS3 proteinase substrate specificity. These features include an acidic residue (Asp or Glu) at the P6 position, a Cys or Thr residue at the P1 position, and a Ser or Ala residue at the P1' position.
Mark Harris - One of the best experts on this subject based on the ideXlab platform.
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hepatitis c virus NS5A enigmatic but still promiscuous 10 years on
Journal of General Virology, 2015Co-Authors: Douglas Rossthriepland, Mark HarrisAbstract:Since one of us co-authored a review on NS5A a decade ago, the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone, which allowed the study of all aspects of the virus life cycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We shall review the recent structural insights – NS5A is predicted to comprise three domains; X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains, which are predicted to be largely unstructured. Recent insights into the phosphorylation of NS5A will be discussed, and we shall highlight a few pertinent examples from the ever-expanding list of NS5A-binding partners identified over the past decade. Lastly, we shall review the literature showing that NS5A is a potential target for a new class of highly potent small molecules that function to inhibit virus replication. These direct-acting antivirals (DAAs) are now either licensed, or in the late stages of approval for clinical use both in the USA and in the UK/Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionizing treatment for HCV infection.
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Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!
The Journal of general virology, 2014Co-Authors: Douglas Ross-thriepland, Mark HarrisAbstract:Since one of us co-authored a review on NS5A a decade ago, the hepatitis C virus (HCV) field has changed dramatically, primarily due to the advent of the JFH-1 cell culture infectious clone, which allowed the study of all aspects of the virus life cycle from entry to exit. This review will describe advances in our understanding of NS5A biology over the past decade, highlighting how the JFH-1 system has allowed us to determine that NS5A is essential not only in genome replication but also in the assembly of infectious virions. We shall review the recent structural insights - NS5A is predicted to comprise three domains; X-ray crystallography has revealed the structure of domain I but there is a lack of detailed structural information about the other two domains, which are predicted to be largely unstructured. Recent insights into the phosphorylation of NS5A will be discussed, and we shall highlight a few pertinent examples from the ever-expanding list of NS5A-binding partners identified over the past decade. Lastly, we shall review the literature showing that NS5A is a potential target for a new class of highly potent small molecules that function to inhibit virus replication. These direct-acting antivirals (DAAs) are now either licensed, or in the late stages of approval for clinical use both in the USA and in the UK/Europe. In combination with other DAAs targeting the viral protease (NS3) and polymerase (NS5B), they are revolutionizing treatment for HCV infection.
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all three domains of the hepatitis c virus nonstructural NS5A protein contribute to rna binding
Journal of Virology, 2010Co-Authors: Toshana L Foster, Tamara Belyaeva, Nicola J Stonehouse, Arwen R Pearson, Mark HarrisAbstract:The hepatitis C virus (HCV) nonstructural protein NS5A is critical for viral genome replication and is thought to interact directly with both the RNA-dependent RNA polymerase, NS5B, and viral RNA. NS5A consists of three domains which have, as yet, undefined roles in viral replication and assembly. In order to define the regions that mediate the interaction with RNA, specifically the HCV 3' untranslated region (UTR) positive-strand RNA, constructs of different domain combinations were cloned, bacterially expressed, and purified to homogeneity. Each of these purified proteins was probed for its ability to interact with the 3' UTR RNA using filter binding and gel electrophoretic mobility shift assays, revealing differences in their RNA binding efficiencies and affinities. A specific interaction between domains I and II of NS5A and the 3' UTR RNA was identified, suggesting that these are the RNA binding domains of NS5A. Domain III showed low in vitro RNA binding capacity. Filter binding and competition analyses identified differences between NS5A and NS5B in their specificities for defined regions of the 3' UTR. The preference of NS5A, in contrast to NS5B, for the polypyrimidine tract highlights an aspect of 3' UTR RNA recognition by NS5A which may play a role in the control or enhancement of HCV genome replication.
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a link between translation of the hepatitis c virus polyprotein and polymerase function possible consequences for hyperphosphorylation of NS5A
Journal of General Virology, 2006Co-Authors: Christopher Mccormick, David Brown, Stephen Griffin, Lisa J Challinor, D J Rowlands, Mark HarrisAbstract:Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.
Francois Penin - One of the best experts on this subject based on the ideXlab platform.
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The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection
Nature Reviews Microbiology, 2013Co-Authors: Ralf Bartenschlager, Volker Lohmann, Francois PeninAbstract:Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Cell culture systems for HCV, especially the replicon and cell culture-derived HCV (HCVcc) systems, have been essential for researchers to gain insights into the viral replication cycle and for the development of selective drugs. Prime targets for direct-acting antiviral agents (DAAs) against HCV are the protease NS3-4A, the replicase factor NS5A and the RNA-dependent RNA polymerase NS5B. Knowledge of the biochemical and structural properties of NS3-4A, NS5A and NS5B has been a key factor for the development of highly efficient drugs targeting these proteins. Additional viral proteins, such as the ion channel formed by p7 or the membrane-active protein NS4B, represent alternative targets for antiviral therapy. Drugs directed against certain host cell factors on which HCV is dependent, such as cyclophilin A or microRNA miR-122, are highly efficient in vitro and in vivo . New drug regimens based on the combination of DAAs and independent of interferon and, eventually, ribavirin (both of which drugs account for serious side effects) appear to be within reach in the near future. Hepatitis C virus infection is a major cause of liver cirrhosis and cancer, and current therapies are often ineffective or have severe side effects. Here, Bartenschlager and colleagues review how structural and functional insights into the viral life cycle have allowed the development of novel direct-acting antiviral agents. The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.
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Hepatitis C virus proteins: from structure to function.
Current Topics in Microbiology and Immunology, 2013Co-Authors: Darius Moradpour, Francois PeninAbstract:Great progress has been made over the past years in elucidating the structure and function of the hepatitis C virus (HCV) proteins, most of which are now actively being pursued as antiviral targets. The structural proteins, which form the viral particle, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins include the p7 viroporin, the NS2 protease, the NS3-4A complex harboring protease and NTPase/RNA helicase activities, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. NS4B is a master organizer of replication complex formation while NS5A is a zinc-containing phosphoprotein involved in the regulation of HCV RNA replication versus particle production. Core to NS2 make up the assembly module while NS3 to NS5B represent the replication module (replicase). However, HCV proteins exert multiple functions during the viral life cycle, and these may be governed by different structural conformations and/or interactions with viral and/or cellular partners. Remarkably, each viral protein is anchored to intracellular membranes via specific determinants that are essential to protein function in the cell. This review summarizes current knowledge of the structure and function of the HCV proteins and highlights recent advances in the field.
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The C Terminus of Hepatitis C Virus NS4A Encodes an Electrostatic Switch That Regulates NS5A Hyperphosphorylation and Viral Replication
Journal of Virology, 2007Co-Authors: Brett D Lindenbach, Roland Montserret, Anna Marie Pyle, Béla M. Prágai, Rudolf K. F. Beran, Francois Penin, Charles M RiceAbstract:Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation. By selecting for replication-competent mutants, several second-site changes in NS3 were identified and shown to suppress these defects in replication and NS5A hyperphosphorylation. Circular-dichroism spectroscopy and nuclear magnetic resonance spectroscopy on a peptide corresponding to the C-terminal 19 aa of NS4A revealed that this region can adopt an alpha-helical conformation, but that this folding requires neutralization of a cluster of acidic residues. Taken together, these data suggest that the C terminus of NS4A acts as a dynamic regulator of NS3-4A interaction, NS5A hyperphosphorylation, and HCV replicase activity.
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Conserved determinants for membrane association of nonstructural protein 5A from hepatitis C virus and related viruses.
Journal of Virology, 2007Co-Authors: V. Brass, Francois Penin, Z. Pal, N. Sapay, G. Deleage, He Blum, D. MoradpourAbstract:Nonstructural protein 5A (NS5A) is a membrane-associated essential component of the hepatitis C virus (HCV) replication complex. An N-terminal amphipathic alpha helix mediates in-plane membrane association of HCV NS5A and at the same time is likely involved in specific protein-protein interactions required for the assembly of a functional replication complex. The aim of this study was to identify the determinants for membrane association of NS5A from the related GB viruses and pestiviruses. Although primary amino acid sequences differed considerably, putative membrane anchor domains with amphipathic features were predicted in the N-terminal domains of NS5A proteins from these viruses. Confocal laser scanning microscopy, as well as membrane flotation analyses, demonstrated that NS5As from GB virus B (GBV-B), GBV-C, and bovine viral diarrhea virus, the prototype pestivirus, display membrane association characteristics very similar to those of HCV NS5A. The N-terminal 27 to 33 amino acid residues of these NS5A proteins were sufficient for membrane association. Circular dichroism analyses confirmed the capacity of these segments to fold into alpha helices upon association with lipid-like molecules. Despite structural conservation, only very limited exchanges with sequences from related viruses were tolerated in the context of functional HCV RNA replication, suggesting virus-specific interactions of these segments. In conclusion, membrane association of NS5A by an N-terminal amphipathic alpha helix is a feature shared by HCV and related members of the family Flaviviridae. This observation points to conserved roles of the N-terminal amphipathic alpha helices of NS5A in replication complex formation.
Stanley M Lemon - One of the best experts on this subject based on the ideXlab platform.
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NS5A inhibitors unmask differences in functional replicase complex half life between different hepatitis c virus strains
PLOS Pathogens, 2017Co-Authors: Tiffany Benzine, Petra Neddermann, Raffaele De Francesco, Ryan Brandt, William Lovell, Ruian Ke, Stanley M Lemon, Alan S. Perelson, Daisuke Yamane, David R McgivernAbstract:Hepatitis C virus (HCV) RNA is synthesized by the replicase complex (RC), a macromolecular assembly composed of viral non-structural proteins and cellular co-factors. Inhibitors of the HCV NS5A protein block formation of new RCs but do not affect RNA synthesis by pre-formed RCs. Without new RC formation, existing RCs turn over and are eventually lost from the cell. We aimed to use NS5A inhibitors to estimate the half-life of the functional RC of HCV. We compared different cell culture-infectious strains of HCV that may be grouped based on their sensitivity to lipid peroxidation: robustly replicating, lipid peroxidation resistant (LPOR) viruses (e.g. JFH-1 or H77D) and more slowly replicating, lipid peroxidation sensitive (LPOS) viruses (e.g. H77S.3 and N.2). In luciferase assays, LPOS HCV strains declined under NS5A inhibitor therapy with much slower kinetics compared to LPOR HCV strains. This difference in rate of decline was not observed for inhibitors of the NS5B RNA-dependent RNA polymerase suggesting that the difference was not simply a consequence of differences in RNA stability. In further analyses, we compared two isoclonal HCV variants: the LPOS H77S.3 and the LPOR H77D that differ only by 12 amino acids. Differences in rate of decline between H77S.3 and H77D following NS5A inhibitor addition were not due to amino acid sequences in NS5A but rather due to a combination of amino acid differences in the non-structural proteins that make up the HCV RC. Mathematical modeling of intracellular HCV RNA dynamics suggested that differences in RC stability (half-lives of 3.5 and 9.9 hours, for H77D and H77S.3, respectively) are responsible for the different kinetics of antiviral suppression between LPOS and LPOR viruses. In nascent RNA capture assays, the rate of RNA synthesis decline following NS5A inhibitor addition was significantly faster for H77D compared to H77S.3 indicating different half-lives of functional RCs.
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selectable subgenomic and genome length dicistronic rnas derived from an infectious molecular clone of the hcv n strain of hepatitis c virus replicate efficiently in cultured huh7 cells
Journal of Virology, 2002Co-Authors: Masanori Ikeda, Stanley M LemonAbstract:Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.
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virus specific cofactor requirement and chimeric hepatitis c virus gb virus b nonstructural protein 3
Journal of Virology, 2000Co-Authors: Nancy Butkiewicz, Jacquelyn Wrightminogue, James Durkin, David Standring, Bahige M Baroudy, D V Sangar, Rumin Zhang, Paul Ingravallo, Weidong Zhong, Stanley M LemonAbstract:GB virus B (GBV-B) is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species), making it an attractive surrogate virus for in vivo testing of anti-HCV inhibitors in a small monkey model. It has been reported that the nonstructural protein 3 (NS3) serine protease of GBV-B shares similar substrate specificity with its counterpart in HCV. Authentic proteolytic processing of the HCV polyprotein junctions (NS4A/4B, NS4B/5A, and NS5A/5B) can be accomplished by the GBV-B NS3 protease in an HCV NS4A cofactor-independent fashion. We further characterized the protease activity of a full-length GBV-B NS3 protein and its cofactor requirement using in vitro-translated GBV-B substrates. Cleavages at the NS4A/4B and NS5A/5B junctions were readily detectable only in the presence of a cofactor peptide derived from the central region of GBV-B NS4A. Interestingly, the GBV-B substrates could also be cleaved by the HCV NS3 protease in an HCV NS4A cofactor-dependent manner, supporting the notion that HCV and GBV-B share similar NS3 protease specificity while retaining a virus-specific cofactor requirement. This finding of a strict virus-specific cofactor requirement is consistent with the lack of sequence homology in the NS4A cofactor regions of HCV and GBV-B. The minimum cofactor region that supported GBV-B protease activity was mapped to a central region of GBV-B NS4A (between amino acids Phe22 and Val36) which overlapped with the cofactor region of HCV. Alanine substitution analysis demonstrated that two amino acids, Val27 and Trp31, were essential for the cofactor activity, a finding reminiscent of the two critical residues in the HCV NS4A cofactor, Ile25 and Ile29. A model for the GBV-B NS3 protease domain and NS4A cofactor complex revealed that GBV-B might have developed a similar structural strategy in the activation and regulation of its NS3 protease activity. Finally, a chimeric HCV/GBV-B bifunctional NS3, consisting of an N-terminal HCV protease domain and a C-terminal GBV-B RNA helicase domain, was engineered. Both enzymatic activities were retained by the chimeric protein, which could lead to the development of a chimeric GBV-B virus that depends on HCV protease function.
Ralf Bartenschlager - One of the best experts on this subject based on the ideXlab platform.
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The molecular and structural basis of advanced antiviral therapy for hepatitis C virus infection
Nature Reviews Microbiology, 2013Co-Authors: Ralf Bartenschlager, Volker Lohmann, Francois PeninAbstract:Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma worldwide. Cell culture systems for HCV, especially the replicon and cell culture-derived HCV (HCVcc) systems, have been essential for researchers to gain insights into the viral replication cycle and for the development of selective drugs. Prime targets for direct-acting antiviral agents (DAAs) against HCV are the protease NS3-4A, the replicase factor NS5A and the RNA-dependent RNA polymerase NS5B. Knowledge of the biochemical and structural properties of NS3-4A, NS5A and NS5B has been a key factor for the development of highly efficient drugs targeting these proteins. Additional viral proteins, such as the ion channel formed by p7 or the membrane-active protein NS4B, represent alternative targets for antiviral therapy. Drugs directed against certain host cell factors on which HCV is dependent, such as cyclophilin A or microRNA miR-122, are highly efficient in vitro and in vivo . New drug regimens based on the combination of DAAs and independent of interferon and, eventually, ribavirin (both of which drugs account for serious side effects) appear to be within reach in the near future. Hepatitis C virus infection is a major cause of liver cirrhosis and cancer, and current therapies are often ineffective or have severe side effects. Here, Bartenschlager and colleagues review how structural and functional insights into the viral life cycle have allowed the development of novel direct-acting antiviral agents. The availability of the first molecular clone of the hepatitis C virus (HCV) genome allowed the identification and biochemical characterization of two viral enzymes that are targets for antiviral therapy: the protease NS3-4A and the RNA-dependent RNA polymerase NS5B. With the advent of cell culture systems that can recapitulate either the intracellular steps of the viral replication cycle or the complete cycle, additional drug targets have been identified, most notably the phosphoprotein NS5A, but also host cell factors that promote viral replication, such as cyclophilin A. Here, we review insights into the structures of these proteins and the mechanisms by which they contribute to the HCV replication cycle, and discuss how these insights have facilitated the development of new, directly acting antiviral compounds that have started to enter the clinic.
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modulation of hepatitis c virus NS5A hyperphosphorylation by nonstructural proteins ns3 ns4a and ns4b
Journal of Virology, 1999Co-Authors: Jan Oliver Koch, Ralf BartenschlagerAbstract:NS5A of the hepatitis C virus (HCV) is a highly phosphorylated protein involved in resistance against interferon and required most likely for replication of the viral genome. Phosphorylation of this protein is mediated by a cellular kinase(s) generating multiple proteins with different electrophoretic mobilities. In the case of the genotype 1b isolate HCV-J, in addition to the basal phosphorylated NS5A (designated pp56), a hyperphosphorylated form (pp58) was found on coexpression of NS4A (T. Kaneko, Y. Tanji, S. Satoh, M. Hijikata, S. Asabe, K. Kimura, and K. Shimotohno, Biochem. Biophys. Res. Commun. 205:320‐326, 1994). Using a comparative analysis of two full-length genomes of genotype 1b, competent or defective for NS5A hyperphosphorylation, we investigated the requirements for this NS5A modification. We found that hyperphosphorylation occurs when NS5A is expressed as part of a continuous NS3-5A polyprotein but not when it is expressed on its own or trans complemented with one or several other viral proteins. Results obtained with chimeras of both genomes show that single amino acid substitutions within NS3 that do not affect polyprotein cleavage can enhance or reduce NS5A hyperphosphorylation. Furthermore, mutations in the central or carboxy-terminal NS4A domain as well as small deletions in NS4B can also reduce or block hyperphosphorylation without affecting polyprotein processing. These requirements most likely reflect the formation of a highly ordered NS3-5A multisubunit complex responsible for the differential phosphorylation of NS5A and probably also for modulation of its biological activities. Hepatitis C virus (HCV) is the major causative agent of sporadic and transfusion-associated non-A, non-B hepatitis (10, 24). Although most infections are inapparent or initially associated with only mild symptoms, due to the high persistence the long-term effects are dramatic. About 50% of all infections lead to chronic liver disease, which can range from an apparently healthy carrier state to chronic active hepatitis, liver cirrhosis, or hepatocellular carcinoma. It is estimated that 100 million to 200 million people worldwide are infected with this insidious agent. HCV was classified as the distinct genus Hepacivirus together with the genera Flavivirus and Pestivirus in the family Flaviviridae (41). These viruses are characterized by an enveloped virion harboring a plus-strand RNA genome. In case of HCV, this genome has a length of ca. 9.6 kb and carries a single long open reading frame (ORF) flanked at the 59 and 39 ends by nontranslated regions required for RNA translation and replication (for reviews, see references 4, 9, and 45). The viral genes are expressed as a polyprotein, just 3,000 amino acids in length, which is cleaved co- and posttranslationally by host cell signal peptidases and two viral proteinases. At least 10 different cleavage products have been identified, which are ordered within the polyprotein (from the amino to the carboxy terminus): NH2-core-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5ANS5B-COOH. The structural proteins core, envelope protein 1 (E1), and E2 are the major constituents of the virion. The function of p7, a small highly hydrophobic peptide is not known. NS2 and the amino-terminal domain of NS3 constitute the NS2-3 proteinase responsible for cleavage at the NS2/3 junction (18, 20). NS3 is a bifunctional molecule. The aminoterminal domain carries a serine-type proteinase required for
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complex formation between the ns3 serine type proteinase of the hepatitis c virus and ns4a and its importance for polyprotein maturation
Journal of Virology, 1995Co-Authors: Ralf Bartenschlager, Volker Lohmann, T Wilkinson, Jan Oliver KochAbstract:Processing of the hepatitis C virus polyprotein is mediated by host cell signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third of NS3 is responsible for cleavage at the four sites carboxy terminal of NS3. The activity of this proteinase is modulated by NS4A, a 54-amino-acid polyprotein cleavage product essential for processing at the NS3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to express hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase and NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the minimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Similarly, deletion of amino-terminal NS3 sequences impairs complex formation as well as cleavage at the NS4B/5A site but not at the NS4A-independent NS5A/5B site.TheseresultssuggestthatastableNS3-NS4AinteractionisimportantforcleavageattheNS4A-dependent sites and that amino-terminal NS3 sequences and the central NS4A domain are directly involved in complex formation.
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Kinetic and structural analyses of hepatitis C virus polyprotein processing.
Journal of Virology, 1994Co-Authors: Ralf Bartenschlager, L Ahlborn-laake, Jan Mous, Helmut JacobsenAbstract: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.
