scholarly journals A cis-Acting Replication Element in the Sequence Encoding the NS5B RNA-Dependent RNA Polymerase Is Required for Hepatitis C Virus RNA Replication

2004 ◽  
Vol 78 (3) ◽  
pp. 1352-1366 ◽  
Author(s):  
Shihyun You ◽  
Decherd D. Stump ◽  
Andrea D. Branch ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Structure ◽  
Mutational Analysis ◽  
Rna Replication ◽  
Site Directed Mutagenesis ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Structure Probing

ABSTRACT RNA structures play key roles in the replication of RNA viruses. Sequence alignment software, thermodynamic RNA folding programs, and classical comparative phylogenetic analysis were used to build models of six RNA elements in the coding region of the hepatitis C virus (HCV) RNA-dependent RNA polymerase, NS5B. The importance of five of these elements was evaluated by site-directed mutagenesis of a subgenomic HCV replicon. Mutations disrupting one of the predicted stem-loop structures, designated 5BSL3.2, blocked RNA replication, implicating it as an essential cis-acting replication element (CRE). 5BSL3.2 is about 50 bases in length and is part of a larger predicted cruciform structure (5BSL3). As confirmed by RNA structure probing, 5BSL3.2 consists of an 8-bp lower helix, a 6-bp upper helix, a 12-base terminal loop, and an 8-base internal loop. Mutational analysis and structure probing were used to explore the importance of these features. Primary sequences in the loops were shown to be important for HCV RNA replication, and the upper helix appears to serve as an essential scaffold that helps maintain the overall RNA structure. Unlike certain picornavirus CREs, whose function is position independent, 5BSL3.2 function appears to be context dependent. Understanding the role of 5BSL3.2 and determining how this new CRE functions in the context of previously identified elements at the 5′ and 3′ ends of the RNA genome should provide new insights into HCV RNA replication.

scholarly journals Membrane Association of the RNA-Dependent RNA Polymerase Is Essential for Hepatitis C Virus RNA Replication

2004 ◽  
Vol 78 (23) ◽  
pp. 13278-13284 ◽  
Author(s):  
Darius Moradpour ◽  
Volker Brass ◽  
Elke Bieck ◽  
Peter Friebe ◽  
Rainer Gosert ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Insertion Sequence ◽  
Rna Replication ◽  
Hcv Rna ◽  
Membrane Association ◽  
Rna Dependent Rna Polymerase ◽  
Terminal Domain

ABSTRACT 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.

scholarly journals A Cell-Based Model of HCV-Negative-Strand RNA Replication Utilizing a Chimeric Hepatitis C Virus/Reporter RNA Template

2002 ◽  
Vol 13 (6) ◽  
pp. 353-362 ◽  
Author(s):  
Robert W King ◽  
Marianne Zecher ◽  
Matthew W Jeffries ◽  
Denise R Carroll ◽  
Joseph M Parisi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Cell Line ◽  
Rna Replication ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Negative Strand ◽  
A Cell ◽  
Chimeric Rna

The inability of hepatitis C virus (HCV) to replicate in cell culture has hindered the discovery of antiviral agents against this virus. One of the biggest challenges has been to find a model that allows one to easily and accurately quantify the level of HCV RNA replication that is occurring inside the cell. In an attempt to solve this problem, we have created a plasmid pMJ050 that encodes a chimeric ‘HCV-like’ RNA that can act as a reporter for HCV RNA replication. This RNA consists of an antisense copy of the firefly luciferase sequence flanked by the 5′ and 3′ untranslated regions of the negative strand of the HCV RNA. If, in cells that contain functional HCV proteins, the chimeric RNA is recognized as a substrate for the viral RNA-dependent RNA polymerase, the chimeric RNA will be transcribed into the complementary strand. This RNA has a 5′ HCV internal ribosome entry site and the luciferase sequence in the coding orientation, allowing translation of the RNA into biologically active luciferase. When pMJ050 was transfected into a cell line that is stably transfected with a cDNA copy of the HCV 1b genome, luciferase was produced in a manner that was dependent upon the presence of at least a functional HCV RNA-dependent RNA polymerase. In addition, we constructed a cell line, 293B4α that constitutively produced luciferase in response to the presence of functional HCV proteins. This system permits the accurate determination of the level of HCV RNA replication by the quantification of luciferase.

THE QSAR RESEARCH ON AURONE INHIBITORS OF HEPATITIS C VIRUS RNA-DEPENDENT RNA POLYMERASE BY APPLYING MULTI-DIMENSIONAL SCALING METHOD

2013 ◽  
Vol 06 (01) ◽  
pp. 1250062
Author(s):  
YONG-HONG HU ◽  
BAO-HUA ZHANG
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Training Set ◽  
Rna Dependent Rna Polymerase ◽  
Dimensional Scaling ◽  
Naturally Occurring ◽  
Virus Rna ◽  
Multi Dimensional Scaling

In this paper, we take naturally occurring 2-benzylidenebenzofuran-3-ones (aurones) inhibitors of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) as an example to study the Multi-dimensional scaling (MDS) method for structure-activity relationship. By analyzing training set molecules, our MDS method combined with a PROXSCAL algorithm can predict inhibitory activity of most compounds correctly. Thus, a new sample's activity can be estimated and judged conveniently, and whether it should be synthesized can be known. The MDS method is applicable to optimize the structure for a compound and to provide suggestions for drug design.

scholarly journals Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation

2014 ◽  
Vol 89 (4) ◽  
pp. 2052-2063 ◽  
Author(s):  
Amy L. Cherry ◽  
Caitriona A. Dennis ◽  
Andrew Baron ◽  
Leslie E. Eisele ◽  
Pia A. Thommes ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
De Novo ◽  
Structural Features ◽  
Primer Extension ◽  
Hcv Rna ◽  
Genome Replication ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Replicon

ABSTRACTThe RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV) is essential for viral genome replication. Crystal structures of the HCV RdRp reveal two C-terminal features, a β-loop and a C-terminal arm, suitably located for involvement in positioning components of the initiation complex. Here we show that these two elements intimately regulate template and nucleotide binding, initiation, and elongation. We constructed a series of β-loop and C-terminal arm mutants, which were used forin vitroanalysis of RdRpde novoinitiation and primer extension activities. All mutants showed a substantial decrease in initiation activities but a marked increase in primer extension activities, indicating an ability to form more stable elongation complexes with long primer-template RNAs. Structural studies of the mutants indicated that these enzyme properties might be attributed to an increased flexibility in the C-terminal features resulting in a more open polymerase cleft, which likely favors the elongation process but hampers the initiation steps. A UTP cocrystal structure of one mutant shows, in contrast to the wild-type protein, several alternate conformations of the substrate, confirming that even subtle changes in the C-terminal arm result in a more loosely organized active site and flexible binding modes of the nucleotide. We used a subgenomic replicon system to assess the effects of the same mutations on viral replication in cells. Even the subtlest mutations either severely impaired or completely abolished the ability of the replicon to replicate, further supporting the concept that the correct positioning of both the β-loop and C-terminal arm plays an essential role during initiation and in HCV replication in general.IMPORTANCEHCV RNA polymerase is a key target for the development of directly acting agents to cure HCV infections, which necessitates a thorough understanding of the functional roles of the various structural features of the RdRp. Here we show that even highly conservative changes, e.g., Tyr→Phe or Asp→Glu, in these seemingly peripheral structural features have profound effects on the initiation and elongation properties of the HCV polymerase.

scholarly journals The Hepatitis C Virus RNA-Dependent RNA Polymerase Membrane Insertion Sequence Is a Transmembrane Segment

2002 ◽  
Vol 76 (24) ◽  
pp. 13088-13093 ◽  
Author(s):  
Natalia Ivashkina ◽  
Benno Wölk ◽  
Volker Lohmann ◽  
Ralf Bartenschlager ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Insertion Sequence ◽  
Transmembrane Protein ◽  
Phospholipid Bilayer ◽  
Hcv Rna ◽  
Membrane Insertion ◽  
Transmembrane Segment ◽  
Rna Dependent Rna Polymerase

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) belongs to a class of membrane proteins termed tail-anchored proteins. Here, we show that the HCV RdRp C-terminal membrane insertion sequence traverses the phospholipid bilayer as a transmembrane segment. Moreover, the HCV RdRp was found to be retained in the endoplasmic reticulum (ER) or an ER-derived modified compartment both following transient transfection and in the context of a subgenomic replicon. An absolutely conserved GVG motif was not essential for membrane insertion but possibly provides a docking site for transmembrane protein-protein interactions. These findings have important implications for the functional architecture of the HCV replication complex.

scholarly journals Evidence for Internal Initiation of RNA Synthesis by the Hepatitis C Virus RNA-Dependent RNA Polymerase NS5B In Cellulo

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Philipp Schult ◽  
Maren Nattermann ◽  
Chris Lauber ◽  
Stefan Seitz ◽  
Volker Lohmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Viruses ◽  
Rna Replication ◽  
Rna Dependent Rna Polymerase ◽  
Internal Initiation ◽  
Positive Strand Rna

ABSTRACT Initiation of RNA synthesis by the hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) NS5B has been extensively studied in vitro and in cellulo. Intracellular replication is thought to rely exclusively on terminal de novo initiation, as it conserves all genetic information of the genome. In vitro, however, additional modes of initiation have been observed. In this study, we aimed to clarify whether the intracellular environment allows for internal initiation of RNA replication by the HCV replicase. We used a dual luciferase replicon harboring a terminal and an internal copy of the viral genomic 5′ untranslated region, which was anticipated to support noncanonical initiation. Indeed, a shorter RNA species was detected by Northern blotting with low frequency, depending on the length and sequence composition upstream of the internal initiation site. By introducing mutations at either site, we furthermore established that internal and terminal initiation shared identical sequence requirements. Importantly, lethal point mutations at the terminal site resulted exclusively in truncated replicons. In contrast, the same mutations at the internal site abrogated internal initiation, suggesting a competitive selection of initiation sites, rather than recombination or template-switching events. In conclusion, our data indicate that the HCV replicase is capable of internal initiation in its natural environment, although functional replication likely requires only terminal initiation. Since many other positive-strand RNA viruses generate subgenomic messenger RNAs during their replication cycle, we surmise that their capability for internal initiation is a common and conserved feature of viral RdRps. IMPORTANCE Many aspects of viral RNA replication of hepatitis C virus (HCV) are still poorly understood. The process of RNA synthesis is driven by the RNA-dependent RNA polymerase (RdRp) NS5B. Most mechanistic studies on NS5B so far were performed with in vitro systems using isolated recombinant polymerase. In this study, we present a replicon model, which allows the intracellular assessment of noncanonical modes of initiation by the full HCV replicase. Our results add to the understanding of the biochemical processes underlying initiation of RNA synthesis by NS5B by the discovery of internal initiation in cellulo. Moreover, they validate observations made in vitro, showing that the viral polymerase acts very similarly in isolation and in complex with other viral and host proteins. Finally, these observations provide clues about the evolution of RdRps of positive-strand RNA viruses, which might contain the intrinsic ability to initiate internally.

scholarly journals De Novo Initiation Pocket Mutations Have Multiple Effects on Hepatitis C Virus RNA-Dependent RNA Polymerase Activities

2004 ◽  
Vol 78 (22) ◽  
pp. 12207-12217 ◽  
Author(s):  
C. T. Ranjith-Kumar ◽  
R. T. Sarisky ◽  
L. Gutshall ◽  
M. Thomson ◽  
C. C. Kao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
De Novo ◽  
Specific Interaction ◽  
Binding Pocket ◽  
Hcv Rna ◽  
Elongation Complex ◽  
Rna Dependent Rna Polymerase ◽  
Template Switch

ABSTRACT The hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp) has several distinct biochemical activities, including initiation of RNA synthesis by a de novo mechanism, extension from a primed template, nontemplated nucleotide addition, and synthesis of a recombinant RNA product from two or more noncovalently linked templates (template switch). All of these activities require specific interaction with nucleoside triphosphates (NTPs). Based on the structure of the HCV RdRp bound to NTP (S. Bressanelli, L. Tomei, F. A. Rey, and R. DeFrancesco, J. Virol. 76:3482-3492, 2002), we mutated the amino acid residues that contact the putative initiation GTP and examined the effects on the various activities. Although all mutations retained the ability for primer extension, alanine substitution at R48, R158, R386, R394, or D225 decreased de novo initiation, and two or more mutations abolished de novo initiation. While the prototype enzyme had a Km for GTP of 3.5 μM, all of the mutations except one had Km s that were three- to sevenfold higher. These results demonstrate that the affected residues are functionally required to interact with the initiation nucleotide. Unexpectedly, many of the mutations also affected the addition of nontemplated nucleotide, indicating that residues in the initiating NTP (NTPi)-binding pocket are required for nontemplated nucleotide additions. Interestingly, mutations in D225 are dramatically affected in template switch, indicating that this residue of the NTPi pocket also interacts with components in the elongation complex. We also examined the interaction of ribavirin triphosphate with the NTPi-binding site.

scholarly journals Preclinical Characterization of JTK-853, a Novel Nonnucleoside Inhibitor of the Hepatitis C Virus RNA-Dependent RNA Polymerase

2012 ◽  
Vol 56 (8) ◽  
pp. 4250-4256 ◽  
Author(s):  
Izuru Ando ◽  
Tsuyoshi Adachi ◽  
Naoki Ogura ◽  
Yukiyo Toyonaga ◽  
Kazuyuki Sugimoto ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Rna Polymerase ◽  
Antiviral Agents ◽  
Hcv Rna ◽  
Resistance Mutations ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna ◽  
Polymerase Inhibitor

ABSTRACTJTK-853 is a novel piperazine derivative nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase. JTK-853 showed potent inhibitory activity against genotype 1 HCV polymerase, with a 50% inhibitory concentration in the nanomolar range, and showed potent antiviral activity against the genotype 1b replicon, with a 50% effective concentration of 0.035 μM. The presence of human serum at up to 40% had little effect on the antiviral activity of JTK-853. Structure analysis of HCV polymerase with JTK-853 revealed that JTK-853 associates with the palm site and β-hairpin region of HCV polymerase, and JTK-853 showed decreased antiviral activity against HCV replicons bearing the resistance mutations C316Y, M414T, Y452H, and L466V in the palm site region of HCV polymerase. JTK-853 showed an additive combination effect with other DAAs (direct antiviral agents), such as nucleoside polymerase inhibitor, thumb pocket-binding nonnucleoside polymerase inhibitor, NS5A inhibitor, and protease inhibitor. Collectively, these data demonstrate that JTK-853 is a potent and novel nonnucleoside palm site-binding HCV polymerase inhibitor, suggesting JTK-853 as a potentially useful agent in combination with other DAAs for treatment of HCV infections.

scholarly journals Mutations Conferring Resistance to a Hepatitis C Virus (HCV) RNA-Dependent RNA Polymerase Inhibitor Alone or in Combination with an HCV Serine Protease Inhibitor In Vitro

2005 ◽  
Vol 49 (10) ◽  
pp. 4305-4314 ◽  
Author(s):  
Hongmei Mo ◽  
Liangjun Lu ◽  
Tami Pilot-Matias ◽  
Ron Pithawalla ◽  
Rubina Mondal ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Serine Protease ◽  
Hcv Rna ◽  
Protease Gene ◽  
Polymerase Gene ◽  
Rna Dependent Rna Polymerase ◽  
Resistant Mutants ◽  
Ns5b Polymerase

ABSTRACT Compounds A-782759 (an N-1-aza-4-hydroxyquinolone benzothiadiazine) and BILN-2061 are specific anti-hepatitis C virus (HCV) agents that inhibit the RNA-dependent RNA polymerase and the NS3 serine protease, respectively. Both compounds display potent activity against HCV replicons in tissue culture. In order to characterize the development of resistance to these anti-HCV agents, HCV subgenomic 1b-N replicon cells were cultured with A-782759 alone or in combination with BILN-2061 at concentrations 10 times above their corresponding 50% inhibitory concentrations in the presence of neomycin. Single substitutions in the NS5B polymerase gene (H95Q, N411S, M414L, M414T, or Y448H) resulted in substantial decreases in susceptibility to A-782759. Similarly, replicons containing mutations in the NS5B polymerase gene (M414L or M414T), together with single mutations in the NS3 protease gene (A156V or D168V), conferred high levels of resistance to both A-782759 and BILN-2061. However, the A-782759-resistant mutants remained susceptible to nucleoside and two other classes of nonnucleoside NS5B polymerase inhibitors, as well as interferon. In addition, we found that the frequency of replicons resistant to both compounds was significantly lower than the frequency of resistance to the single compound. Furthermore, the dually resistant mutants displayed significantly reduced replication capacities compared to the wild-type replicon. These findings provide strategic guidance for the future treatment of HCV infection.

Sign in / Sign up

Export Citation Format

Share Document