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Zhi Hong - One of the best experts on this subject based on the ideXlab platform.
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Effects of genotypic variations on hepatitis C virus Nonstructural Protein 5B structure and activity
Frontiers in Viral Hepatitis, 2003Co-Authors: Zhi Hong, Eric Ferrari, Angela Skelton, Jacquelyn Wright-minogue, Weidong Zhong, Charles A. LesburgAbstract:Publisher Summary Hepatitis C virus (HCV) is the causative agent for most cases of non-A and non-B hepatitis. HCV, a member of the Flaviviridae family, is a positive-stranded RNA virus. Its life cycle consists of several interrelated processes that occur primarily in the cytoplasm of the host cells. Nonstructural Protein 5B (NS5B) of HCV possesses an RNA-dependent RNA polymerase (RdRp) activity responsible for viral genome replication. It presents an excellent target for antiviral development. Recent studies revealed that removal of the C-terminal hydrophobic domain improved the solubility of NS5B to a level suitable for enzymatic characterization and structural determination. This hydrophobic C-terminal tail is highly conserved among all six genotypes of HCV, indicating an important functional and structural role, presumably as a membrane anchor for the assembly of a replication complex. Hydrophobic domains were also identified in related viruses, such as pestiviruses and GB viruses. Structure-based surface variability analysis identified highly conserved regions in the active site and predicted asymmetric distribution of important functionality and critical structural elements essential for replication.
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Selection of 3'-template bases and initiating nucleotides by hepatitis C virus NS5B RNA-dependent RNA polymerase.
Journal of virology, 2002Co-Authors: Jae Hoon Shim, Gary Larson, Zhi HongAbstract:De novo RNA synthesis by hepatitis C virus (HCV) Nonstructural Protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3′-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3′-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3′ end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3′ end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.
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Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture.
Virology, 2002Co-Authors: I. Wayne Cheney, Zhi Hong, Vicky C. H. Lai, Michelle P. Walker, Suhaila Naim, Shannon Dempsey, Daniel Bellows, Jae Hoon Shim, Nigel Horscroft, Weidong ZhongAbstract:Abstract Hepatitis C virus (HCV) Nonstructural Protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique β-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and β-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the β-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMT R 345), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416–8428).
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functional properties of a monoclonal antibody inhibiting the hepatitis c virus rna dependent rna polymerase
Journal of Biological Chemistry, 2002Co-Authors: Darius Moradpour, Zhi Hong, E. Bieck, T. Hugle, He Blum, Winfried Wels, Jim Zhen Wu, R. BartenschlagerAbstract:Abstract The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), has recently emerged as a promising target for antiviral intervention. Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp. This mAb, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyProtein or subgenomic replicons. Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected. mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level. The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells. The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV. More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents.
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A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase.
Virology, 2001Co-Authors: Zhi Hong, Craig E. Cameron, Johnson Y.n. Lau, Michelle P. Walker, Christian Castro, Nanhua Yao, Weidong ZhongAbstract:Hepatitis C virus (HCV) Nonstructural Protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique beta-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this beta-hairpin plays an important role in positioning the 3' terminus of the viral RNA genome for correct initiation of replication. The presence of this beta-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site. We propose that this beta-hairpin may serve as a "gate" which prevents the 3' terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a beta-hairpin deletion mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses.
Weidong Zhong - One of the best experts on this subject based on the ideXlab platform.
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Effects of genotypic variations on hepatitis C virus Nonstructural Protein 5B structure and activity
Frontiers in Viral Hepatitis, 2003Co-Authors: Zhi Hong, Eric Ferrari, Angela Skelton, Jacquelyn Wright-minogue, Weidong Zhong, Charles A. LesburgAbstract:Publisher Summary Hepatitis C virus (HCV) is the causative agent for most cases of non-A and non-B hepatitis. HCV, a member of the Flaviviridae family, is a positive-stranded RNA virus. Its life cycle consists of several interrelated processes that occur primarily in the cytoplasm of the host cells. Nonstructural Protein 5B (NS5B) of HCV possesses an RNA-dependent RNA polymerase (RdRp) activity responsible for viral genome replication. It presents an excellent target for antiviral development. Recent studies revealed that removal of the C-terminal hydrophobic domain improved the solubility of NS5B to a level suitable for enzymatic characterization and structural determination. This hydrophobic C-terminal tail is highly conserved among all six genotypes of HCV, indicating an important functional and structural role, presumably as a membrane anchor for the assembly of a replication complex. Hydrophobic domains were also identified in related viruses, such as pestiviruses and GB viruses. Structure-based surface variability analysis identified highly conserved regions in the active site and predicted asymmetric distribution of important functionality and critical structural elements essential for replication.
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Mutations in NS5B polymerase of hepatitis C virus: impacts on in vitro enzymatic activity and viral RNA replication in the subgenomic replicon cell culture.
Virology, 2002Co-Authors: I. Wayne Cheney, Zhi Hong, Vicky C. H. Lai, Michelle P. Walker, Suhaila Naim, Shannon Dempsey, Daniel Bellows, Jae Hoon Shim, Nigel Horscroft, Weidong ZhongAbstract:Abstract Hepatitis C virus (HCV) Nonstructural Protein 5B (NS5B) is an RNA-dependent RNA polymerase (RdRp) essential for virus replication. Several consensus sequence motifs have been identified in NS5B, some of which have been shown to be critical for its enzymatic activity. A unique β-hairpin structure located between amino acids 443 and 454 in the thumb subdomain has also been shown to play an important role in ensuring terminal initiation of RNA synthesis in vitro. However, the importance of these sequence and structural elements in viral RNA replication in infected cells has not been established, mainly due to the lack of a reliable cell culture system for HCV. In this study, we investigated the effect of several single amino acid substitutions and β-hairpin truncations in NS5B on viral RNA replication by using the subgenomic replicon cell culture system. A strong correlation between in vitro polymerase activity and viral RNA replication was observed with most of the substitutions. Interestingly, truncations of the β-hairpin (by four and eight amino acid residues, respectively), which did not reduce the in vitro enzymatic activity, completely abolished the ability of the replicon RNA to replicate in Huh-7 cells, demonstrating its essential role in viral RNA replication. Furthermore, a conservative substitution in motif D, from an arginine residue (AMT R 345), which is conserved among all HCV isolates, to a lysine residue, resulted in significant improvements in both transient RNA replication and colony formation efficiencies. This result also correlates with a previous observation that the enzymatic activity of NS5B increased by about 50% when the same NS5B substitution was introduced (V. Lohmann, F. Korner, U. Herian, and R. Bartenschlager, J. Virol. 1997, 71, 8416–8428).
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A novel mechanism to ensure terminal initiation by hepatitis C virus NS5B polymerase.
Virology, 2001Co-Authors: Zhi Hong, Craig E. Cameron, Johnson Y.n. Lau, Michelle P. Walker, Christian Castro, Nanhua Yao, Weidong ZhongAbstract:Hepatitis C virus (HCV) Nonstructural Protein 5B (NS5B) RNA-dependent RNA polymerase (RdRp) has acquired a unique beta-hairpin in the thumb subdomain which protrudes toward the active site. We report here that this beta-hairpin plays an important role in positioning the 3' terminus of the viral RNA genome for correct initiation of replication. The presence of this beta-hairpin interferes with polymerase binding to preannealed double-stranded RNA (dsRNA) molecules and allows only the single-stranded 3' terminus of an RNA template to bind productively to the active site. We propose that this beta-hairpin may serve as a "gate" which prevents the 3' terminus of the template RNA from slipping through the active site and ensures initiation of replication from the terminus of the genome. This hypothesis is supported by the ability of a beta-hairpin deletion mutant that utilizes dsRNA substrates and initiates RNA synthesis internally. The proposed terminal initiation mechanism may represent a novel replication strategy adopted by HCV and related viruses.
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Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus Nonstructural Protein 5B polymerase.
Virus research, 2001Co-Authors: Paul Ingravallo, Zhi Hong, Frederick C. Lahser, Ellen Xia, Bruce I. Sodowich, Vicky C. H. Lai, Weidong ZhongAbstract:The Nonstructural Protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B Protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the Nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B Proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B Proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.
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Template/primer requirements and single nucleotide incorporation by hepatitis C virus Nonstructural Protein 5B polymerase
Journal of virology, 2000Co-Authors: Weidong Zhong, Eric Ferrari, Charles A. Lesburg, David Maag, Saikat Kumar B. Ghosh, Craig E. Cameron, Johnson Y.n. Lau, Zhi HongAbstract:Nonstructural Protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.
Darius Moradpour - One of the best experts on this subject based on the ideXlab platform.
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Hepatitis C Virus RNA Replication Requires a Conserved Structural Motif within the Transmembrane Domain of the NS5B RNA-Dependent RNA Polymerase
Journal of virology, 2010Co-Authors: Volker Brass, Jérôme Gouttenoire, Anja Wahl, Zsuzsanna Pal, Hubert E. Blum, François Penin, Darius MoradpourAbstract:Hepatitis C virus (HCV) Nonstructural Protein 5B (NS5B), the viral RNA-dependent RNA polymerase (RdRp), is a tail-anchored Protein with a highly conserved C-terminal transmembrane domain (TMD) that is required for the assembly of a functional replication complex. Here, we report that the TMD of the HCV RdRp can be functionally replaced by a newly identified analogous membrane anchor of the GB virus B (GBV-B) NS5B RdRp. Replicons with a chimeric RdRp consisting of the HCV catalytic domain and the GBV-B membrane anchor replicated with reduced efficiency. Compensatory amino acid changes at defined positions within the TMD improved the replication efficiency of these chimeras. These observations highlight a conserved structural motif within the TMD of the HCV NS5B RdRp that is required for RNA replication.
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Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication
Journal of virology, 2004Co-Authors: Darius Moradpour, Volker Brass, Hubert E. Blum, François Penin, E. Bieck, P. Friebe, R. Gosert, R. Bartenschlager, V. LohmannAbstract:The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), belongs to a class of integral membrane Proteins termed tail-anchored Proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus Protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane Protein-Protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored Proteins.
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Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication.
Journal of Virology, 2004Co-Authors: Darius Moradpour, Volker Brass, François Penin, E. Bieck, He Blum, P. Friebe, R. Gosert, R. Bartenschlager, V. LohmannAbstract:The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), belongs to a class of integral membrane Proteins termed tail-anchored Proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus Protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane Protein-Protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored Proteins.The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), belongs to a class of integral membrane Proteins termed tail-anchored Proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus Protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane Protein-Protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored Proteins.
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functional properties of a monoclonal antibody inhibiting the hepatitis c virus rna dependent rna polymerase
Journal of Biological Chemistry, 2002Co-Authors: Darius Moradpour, Zhi Hong, E. Bieck, T. Hugle, He Blum, Winfried Wels, Jim Zhen Wu, R. BartenschlagerAbstract:Abstract The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), has recently emerged as a promising target for antiviral intervention. Here, we describe the isolation, functional characterization, and molecular cloning of a monoclonal antibody (mAb) inhibiting the HCV RdRp. This mAb, designated 5B-12B7, binds with high affinity to a conformational epitope in the palm subdomain of the HCV RdRp and recognizes native NS5B expressed in the context of the entire HCV polyProtein or subgenomic replicons. Complete inhibition of RdRp activity in vitro was observed at equimolar concentrations of NS5B and mAb 5B-12B7, whereas RdRp activities of classical swine fever virus NS5B and poliovirus 3D polymerase were not affected. mAb 5B-12B7 selectively inhibited NTP binding to HCV NS5B, whereas binding of template RNA was unaffected, thus explaining the mechanism of action at the molecular level. The mAb 5B-12B7 heavy and light chain variable domains were cloned by reverse transcription-PCR, and a single chain Fv fragment was assembled for expression in Escherichia coli and in eukaryotic cells. The mAb 5B-12B7 single chain Fv fragment bound to NS5B both in vitro and in transfected human cell lines and therefore may be potentially useful for intracellular immunization against HCV. More important, detailed knowledge of the mAb 5B-12B7 contact sites on the enzyme may facilitate the development of small molecule RdRp inhibitors as novel antiviral agents.
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Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase.
The Journal of biological chemistry, 2001Co-Authors: J. Schmidt-mende, Hubert E. Blum, François Penin, E. Bieck, T. Hugle, Charles M. Rice, Darius MoradpourAbstract:Abstract The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), is believed to form a membrane-associated RNA replication complex together with other Nonstructural Proteins and as yet unidentified host components. However, the determinants for membrane association of this essential viral enzyme have not been defined. By double label immunofluorescence analyses, NS5B was found in the endoplasmic reticulum (ER) or an ER-like modified compartment both when expressed alone or in the context of the entire HCV polyProtein. The carboxyl-terminal 21 amino acid residues were necessary and sufficient to target NS5B or a heterologous Protein to the cytosolic side of the ER membrane. This hydrophobic domain is highly conserved among 269 HCV isolates analyzed and predicted to form a transmembrane α-helix. Association of NS5B with the ER membrane occurred by a posttranslational mechanism that was ATP-independent. These features define the HCV RdRp as a new member of the tail-anchored Protein family, a class of integral membrane Proteins that are membrane-targeted posttranslationally via a carboxyl-terminal insertion sequence. Formation of the HCV replication complex, therefore, involves specific determinants for membrane association that represent potential targets for antiviral intervention.
R. Bartenschlager - One of the best experts on this subject based on the ideXlab platform.
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Conserved RNA secondary structures and long-range interactions in hepatitis C viruses.
RNA (New York N.Y.), 2015Co-Authors: Markus Fricke, R. Bartenschlager, Nadia Dünnes, Margarita Zayas, Michael Niepmann, Manja MarzAbstract:Hepatitis C virus (HCV) is a hepatotropic virus with a plus-strand RNA genome of ∼9.600 nt. Due to error-prone replication by its RNA-dependent RNA polymerase (RdRp) residing in Nonstructural Protein 5B (NS5B), HCV isolates are grouped into seven genotypes with several subtypes. By using whole-genome sequences of 106 HCV isolates and secondary structure alignments of the plus-strand genome and its minus-strand replication intermediate, we established refined secondary structures of the 5' untranslated region (UTR), the cis-acting replication element (CRE) in NS5B, and the 3' UTR. We propose an alternative structure in the 5' UTR, conserved secondary structures of 5B stem-loop (SL)1 and 5BSL2, and four possible structures of the X-tail at the very 3' end of the HCV genome. We predict several previously unknown long-range interactions, most importantly a possible circularization interaction between distinct elements in the 5' and 3' UTR, reminiscent of the cyclization elements of the related flaviviruses. Based on analogy to these viruses, we propose that the 5'-3' UTR base-pairing in the HCV genome might play an important role in viral RNA replication. These results may have important implications for our understanding of the nature of the cis-acting RNA elements in the HCV genome and their possible role in regulating the mutually exclusive processes of viral RNA translation and replication.
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essential role of cyclophilin a for hepatitis c virus replication and virus production and possible link to polyProtein cleavage kinetics
PLOS Pathogens, 2009Co-Authors: Artur Kaul, V. Lohmann, Margarita Zayas, Sarah Stauffer, Carola Berger, Thomas Pertel, Jennifer Schmitt, Stephanie Kallis, Jeremy Luban, R. BartenschlagerAbstract: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.
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Identification of Determinants Involved in Initiation of Hepatitis C Virus RNA Synthesis by Using Intergenotypic Replicase Chimeras
Journal of virology, 2007Co-Authors: Marco Binder, R. Bartenschlager, Doris Quinkert, Olga Bochkarova, Rahel Klein, Nikolina Kezmic, V. LohmannAbstract:The 5′ nontranslated region (NTR) and the X tail in the 3′ NTR are the least variable parts of the hepatitis C virus (HCV) genome and play an important role in the initiation of RNA synthesis. By using subgenomic replicons of the HCV isolates Con1 (genotype 1) and JFH1 (genotype 2), we characterized the genotype specificities of the replication signals contained in the NTRs. The replacement of the JFH1 5′ NTR and X tail with the corresponding Con1 sequence resulted in a significant decrease in replication efficiency. Exchange of the X tail specifically reduced negative-strand synthesis, whereas substitution of the 5′ NTR impaired the generation of progeny positive strands. In search for the Proteins involved in the recognition of genotype-specific initiation signals, we analyzed recombinant Nonstructural Protein 5B (NS5B) RNA polymerases of both isolates and found some genotype-specific template preference for the 3′ end of positive-strand RNA in vitro. To further address genotype specificity, we constructed a series of intergenotypic replicon chimeras. When combining NS3 to NS5A of Con1 with NS5B of JFH1, we observed more-efficient replication with the genotype 2a X tail, indicating that NS5B recognizes genotype-specific signals in this region. In contrast, a combination of the NS3 helicase with NS5A and NS5B was required to confer genotype specificity to the 5′ NTR. These results present the first genetic evidence for an interaction between helicase, NS5A, and NS5B required for the initiation of RNA synthesis and provide a system for the specific analysis of HCV positive- and negative-strand syntheses.
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Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication
Journal of virology, 2004Co-Authors: Darius Moradpour, Volker Brass, Hubert E. Blum, François Penin, E. Bieck, P. Friebe, R. Gosert, R. Bartenschlager, V. LohmannAbstract:The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), belongs to a class of integral membrane Proteins termed tail-anchored Proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus Protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane Protein-Protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored Proteins.
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Membrane association of the RNA-dependent RNA polymerase is essential for hepatitis C virus RNA replication.
Journal of Virology, 2004Co-Authors: Darius Moradpour, Volker Brass, François Penin, E. Bieck, He Blum, P. Friebe, R. Gosert, R. Bartenschlager, V. LohmannAbstract:The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), belongs to a class of integral membrane Proteins termed tail-anchored Proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus Protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane Protein-Protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored Proteins.The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), represented by Nonstructural Protein 5B (NS5B), belongs to a class of integral membrane Proteins termed tail-anchored Proteins. Its membrane association is mediated by the C-terminal 21 amino acid residues, which are dispensable for RdRp activity in vitro. For this study, we investigated the role of this domain, termed the insertion sequence, in HCV RNA replication in cells. Based on a structural model and the amino acid conservation among different HCV isolates, we designed a panel of insertion sequence mutants and analyzed their membrane association and RNA replication. Subgenomic replicons with a duplication of an essential cis-acting replication element overlapping the sequence that encodes the C-terminal domain of NS5B were used to unequivocally distinguish RNA versus Protein effects of these mutations. Our results demonstrate that the membrane association of the RdRp is essential for HCV RNA replication. Interestingly, certain amino acid substitutions within the insertion sequence abolished RNA replication without affecting membrane association, indicating that the C-terminal domain of NS5B has functions beyond serving as a membrane anchor and that it may be involved in critical intramembrane Protein-Protein interactions. These results have implications for the functional architecture of the HCV replication complex and provide new insights into the expanding spectrum of tail-anchored Proteins.
Kenneth A. Johnson - One of the best experts on this subject based on the ideXlab platform.
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Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation
The Journal of biological chemistry, 2016Co-Authors: Kenneth A. JohnsonAbstract:Replication of the hepatitis C viral genome is catalyzed by the NS5B (Nonstructural Protein 5B) RNA-dependent RNA polymerase, which is a major target of antiviral drugs currently in the clinic. Prior studies established that initiation of RNA replication could be facilitated by starting with a dinucleotide (pGG). Here we establish conditions for efficient initiation from GTP to form the dinucleotide and subsequent intermediates leading to highly processive elongation, and we examined the effects of four classes of nonnucleoside inhibitors on each step of the reaction. We show that palm site inhibitors block initiation starting from GTP but not when starting from pGG. In addition we show that nonnucleoside inhibitors binding to thumb site-2 (NNI2) lead to the accumulation of abortive intermediates three-five nucleotides in length. Our kinetic analysis shows that NNI2 do not significantly block initiation or elongation of RNA synthesis; rather, they block the transition from initiation to elongation, which is thought to proceed with significant structural rearrangement of the enzyme-RNA complex including displacement of the β-loop from the active site. Direct measurement in single turnover kinetic studies show that pyrophosphate release is faster than the chemistry step, which appears to be rate-limiting during processive synthesis. These results reveal important new details to define the steps involved in initiation and elongation during viral RNA replication, establish the allosteric mechanisms by which NNI2 inhibitors act, and point the way to the design of more effective allosteric inhibitors that exploit this new information.
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Thumb Site 2 Inhibitors of Hepatitis C Viral RNA-Dependent RNA Polymerase Allosterically Block the Transition from Initiation to Elongation
Biophysical Journal, 2016Co-Authors: Daniel Deredge, Patrick L. Wintrode, Kenneth A. JohnsonAbstract:Hepatitis C virus (HCV) is a small, single stranded, positive-sense (+) RNA virus that replicates its viral genome by an RNA-dependent RNA polymerase (RdRp), encoded as Nonstructural Protein 5B (NS5B), which is the primary target for anti-HCV treatment. Non-nucleoside inhibitors binding to the thumb site-2 (NNI2) of NS5B have shown success in clinical trials and although numerous crystal structures have been solved of NS5B with small molecule nonnucleoside inhibitors, little is known about the mechanism of inhibition.
