scholarly journals Characterization of Soluble Hepatitis C Virus RNA-Dependent RNA Polymerase Expressed in Escherichia coli

1999 ◽  
Vol 73 (2) ◽  
pp. 1649-1654 ◽  
Author(s):  
Eric Ferrari ◽  
Jacquelyn Wright-Minogue ◽  
Jane W. S. Fang ◽  
Bahige M. Baroudy ◽  
Johnson Y. N. Lau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Nonstructural Protein ◽  
Full Length ◽  
Dependent Manner ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Domain ◽  
Hcv Ns5b

ABSTRACT Production of soluble full-length nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) has been shown to be problematic and requires the addition of salts, glycerol, and detergents. In an effort to improve the solubility of NS5B, the hydrophobic C terminus containing 21 amino acids was removed, yielding a truncated NS5B (NS5BΔCT) which is highly soluble and monodispersed in the absence of detergents. Fine deletional analysis of this region revealed that a four-leucine motif (LLLL) in the hydrophobic domain is responsible for the solubility profile of the full-length NS5B. Enzymatic characterization revealed that the RNA-dependent RNA polymerase (RdRp) activity of this truncated NS5B was comparable to those reported previously by others. For optimal enzyme activity, divalent manganese ions (Mn2+) are preferred rather than magnesium ions (Mg2+), whereas zinc ions (Zn2+) inhibit the RdRp activity. Gliotoxin, a known poliovirus 3D RdRp inhibitor, inhibited HCV NS5B RdRp in a dose-dependent manner. Kinetic analysis revealed that HCV NS5B has a rather low processivity compared to those of other known polymerases.

scholarly journals De Novo Initiation of RNA Synthesis by the RNA-Dependent RNA Polymerase (NS5B) of Hepatitis C Virus

2000 ◽  
Vol 74 (2) ◽  
pp. 851-863 ◽  
Author(s):  
Guangxiang Luo ◽  
Robert K. Hamatake ◽  
Danielle M. Mathis ◽  
Jason Racela ◽  
Karen L. Rigat ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Oligonucleotide Primer ◽  
Transferase Activity ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn2+ than in the presence of Mg2+. When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a “copy-back” mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3′ end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (≥50 μM) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.

scholarly journals Stimulation of Hepatitis C Virus (HCV) Nonstructural Protein 3 (NS3) Helicase Activity by the NS3 Protease Domain and by HCV RNA-Dependent RNA Polymerase

2005 ◽  
Vol 79 (14) ◽  
pp. 8687-8697 ◽  
Author(s):  
Chen Zhang ◽  
Zhaohui Cai ◽  
Young-Chan Kim ◽  
Ranjith Kumar ◽  
Fenghua Yuan ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Enzyme Activities ◽  
Nonstructural Protein ◽  
Helicase Activity ◽  
Protease Domain ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Ns3 Protease

ABSTRACT Hepatitis C virus (HCV) nonstructural protein 3 (NS3) possesses multiple enzyme activities. The N-terminal one-third of NS3 primarily functions as a serine protease, while the remaining two-thirds of NS3 serve as a helicase and nucleoside triphosphatase. Whether the multiple enzyme activities of NS3 are functionally interdependent and/or modulated by other viral NS proteins remains unclear. We performed biochemical studies to examine the functional interdependence of the NS3 protease and helicase domains and the modulation of NS3 helicase by NS5B, an RNA-dependent RNA polymerase (RdRp). We found that the NS3 protease domain of the full-length NS3 (NS3FL) enhances the NS3 helicase activity. Additionally, HCV RdRp stimulates the NS3FL helicase activity by more than sevenfold. However, the helicase activity of the NS3 helicase domain was unaffected by HCV RdRp. Glutathione S-transferase pull-down as well as fluorescence anisotropy results revealed that the NS3 protease domain is required for specific NS3 and NS5B interaction. These findings suggest that HCV RdRp regulates the functions of NS3 during HCV replication. In contrast, NS3FL does not increase NS5B RdRp activity in vitro, which is contrary to a previously published report that the HCV NS3 enhances NS5B RdRp activity.

scholarly journals Hepatitis C Virus RNA-Dependent RNA Polymerase Is Regulated by Cysteine S-Glutathionylation

2019 ◽  
Vol 2019 ◽  
pp. 1-11
Author(s):  
Marina K. Kukhanova ◽  
Vera L. Tunitskaya ◽  
Olga A. Smirnova ◽  
Olga A. Khomich ◽  
Natalia F. Zakirova ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Recombinant Protein ◽  
Rna Polymerase ◽  
Reducing Agents ◽  
Cysteine Residues ◽  
Rdrp Activity ◽  
Rna Dependent Rna Polymerase ◽  
Redox Switches ◽  
Ns5b Protein

Hepatitis C virus (HCV) triggers massive production of reactive oxygen species (ROS) and affects expression of genes encoding ROS-scavenging enzymes. Multiple lines of evidence show that levels of ROS production contribute to the development of various virus-associated pathologies. However, investigation of HCV redox biology so far remained in the paradigm of oxidative stress, whereas no attention was given to the identification of redox switches among viral proteins. Here, we report that one of such redox switches is the NS5B protein that exhibits RNA-dependent RNA polymerase (RdRp) activity. Treatment of the recombinant protein with reducing agents significantly increases its enzymatic activity. Moreover, we show that the NS5B protein is subjected to S-glutathionylation that affects cysteine residues 89, 140, 170, 223, 274, 521, and either 279 or 295. Substitution of these cysteines except C89 and C223 with serine residues led to the reduction of the RdRp activity of the recombinant protein in a primer-dependent assay. The recombinant protein with a C279S mutation was almost inactive in vitro and could not be activated with reducing agents. In contrast, cysteine substitutions in the NS5B region in the context of a subgenomic replicon displayed opposite effects: most of the mutations enhanced HCV replication. This difference may be explained by the deleterious effect of oxidation of NS5B cysteine residues in liver cells and by the protective role of S-glutathionylation. Based on these data, redox-sensitive posttranslational modifications of HCV NS5B and other proteins merit a more detailed investigation and analysis of their role(s) in the virus life cycle and associated pathogenesis.

scholarly journals Identification of a C-Terminal Regulatory Motif in Hepatitis C Virus RNA-Dependent RNA Polymerase: Structural and Biochemical Analysis

2003 ◽  
Vol 77 (16) ◽  
pp. 9020-9028 ◽  
Author(s):  
Vincent J.-P. Lévêque ◽  
Robert B. Johnson ◽  
Stephen Parsons ◽  
Jianxin Ren ◽  
Congping Xie ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Dimensional Structure ◽  
Synthesis Rate ◽  
Rna Dependent Rna Polymerase ◽  
Hcv Ns5b ◽  
Ns5b Polymerase

ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-Å resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to ∼50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

scholarly journals Selection of 3′-Template Bases and Initiating Nucleotides by Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase

2002 ◽  
Vol 76 (14) ◽  
pp. 7030-7039 ◽  
Author(s):  
Jae Hoon Shim ◽  
Gary Larson ◽  
Jim Zhen Wu ◽  
Zhi Hong
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Nonstructural Protein ◽  
Initiation Site ◽  
De Novo Synthesis ◽  
Rna Dependent Rna Polymerase ◽  
Selection Of

ABSTRACT 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.

scholarly journals Oligomerization and Cooperative RNA Synthesis Activity of Hepatitis C Virus RNA-Dependent RNA Polymerase

2002 ◽  
Vol 76 (8) ◽  
pp. 3865-3872 ◽  
Author(s):  
Q. May Wang ◽  
Michelle A. Hockman ◽  
Kirk Staschke ◽  
Robert B. Johnson ◽  
Katharine A. Case ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Order Structure ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Region ◽  
Synthesis Activity ◽  
Hcv Ns5b ◽  
Ns5b Polymerase

ABSTRACT The NS5B RNA-dependent RNA polymerase encoded by hepatitis C virus (HCV) plays a key role in viral replication. Reported here is evidence that HCV NS5B polymerase acts as a functional oligomer. Oligomerization of HCV NS5B protein was demonstrated by gel filtration, chemical cross-linking, temperature sensitivity, and yeast cell two-hybrid analysis. Mutagenesis studies showed that the C-terminal hydrophobic region of the protein was not essential for its oligomerization. Importantly, HCV NS5B polymerase exhibited cooperative RNA synthesis activity with a dissociation constant, Kd , of ≈22 nM, suggesting a role for the polymerase-polymerase interaction in the regulation of HCV replicase activity. Further functional evidence includes the inhibition of the wild-type NS5B polymerase activity by a catalytically inactive form of NS5B. Finally, the X-ray crystal structure of HCV NS5B polymerase was solved at 2.9 Å. Two extensive interfaces have been identified from the packing of the NS5B molecules in the crystal lattice, suggesting a higher-order structure that is consistent with the biochemical data.

scholarly journals Sphingomyelin Activates Hepatitis C Virus RNA Polymerase in a Genotype-Specific Manner

2010 ◽  
Vol 84 (22) ◽  
pp. 11761-11770 ◽  
Author(s):  
Leiyun Weng ◽  
Yuichi Hirata ◽  
Masaaki Arai ◽  
Michinori Kohara ◽  
Takaji Wakita ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Lipid Raft ◽  
Binding Activity ◽  
Rdrp Activity ◽  
Biochemical Effect ◽  
Virus Rna ◽  
Membrane Localization Domain ◽  
Hcv Ns5b

ABSTRACT Hepatitis C virus (HCV) replication and infection depend on the lipid components of the cell, and replication is inhibited by inhibitors of sphingomyelin biosynthesis. We found that sphingomyelin bound to and activated genotype 1b RNA-dependent RNA polymerase (RdRp) by enhancing its template binding activity. Sphingomyelin also bound to 1a and JFH1 (genotype 2a) RdRps but did not activate them. Sphingomyelin did not bind to or activate J6CF (2a) RdRp. The sphingomyelin binding domain (SBD) of HCV RdRp was mapped to the helix-turn-helix structure (residues 231 to 260), which was essential for sphingomyelin binding and activation. Helix structures (residues 231 to 241 and 247 to 260) are important for RdRp activation, and 238S and 248E are important for maintaining the helix structures for template binding and RdRp activation by sphingomyelin. 241Q in helix 1 and the negatively charged 244D at the apex of the turn are important for sphingomyelin binding. Both amino acids are on the surface of the RdRp molecule. The polarity of the phosphocholine of sphingomyelin is important for HCV RdRp activation. However, phosphocholine did not activate RdRp. Twenty sphingomyelin molecules activated one RdRp molecule. The biochemical effect of sphingomyelin on HCV RdRp activity was virologically confirmed by the HCV replicon system. We also found that the SBD was the lipid raft membrane localization domain of HCV NS5B because JFH1 (2a) replicon cells harboring NS5B with the mutation A242C/S244D moved to the lipid raft while the wild type did not localize there. This agreed with the myriocin sensitivity of the mutant replicon. This sphingomyelin interaction is a target for HCV infection because most HCV RdRps have 241Q.

scholarly journals Hepatitis C Virus RNA Replication Requires a Conserved Structural Motif within the Transmembrane Domain of the NS5B RNA-Dependent RNA Polymerase

2010 ◽  
Vol 84 (21) ◽  
pp. 11580-11584 ◽  
Author(s):  
Volker Brass ◽  
Jérôme Gouttenoire ◽  
Anja Wahl ◽  
Zsuzsanna Pal ◽  
Hubert E. Blum ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Catalytic Domain ◽  
Transmembrane Domain ◽  
Nonstructural Protein ◽  
Rna Replication ◽  
Structural Motif ◽  
Rna Dependent Rna Polymerase ◽  
Membrane Anchor

ABSTRACT 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.

scholarly journals Template Requirements for RNA Synthesis by a Recombinant Hepatitis C Virus RNA-Dependent RNA Polymerase

2000 ◽  
Vol 74 (23) ◽  
pp. 11121-11128 ◽  
Author(s):  
C. Cheng Kao ◽  
Xueyong Yang ◽  
Allen Kline ◽  
Q. May Wang ◽  
Donna Barket ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Nonstructural Protein ◽  
Resonance Spectroscopy ◽  
Rna Dependent Rna Polymerase ◽  
Systematic Analysis ◽  
Stem Loop

ABSTRACT The RNA-dependent RNA polymerase (RdRp) from hepatitis C virus (HCV), nonstructural protein 5B (NS5B), has recently been shown to direct de novo initiation using a number of complex RNA templates. In this study, we analyzed the features in simple RNA templates that are required to direct de novo initiation of RNA synthesis by HCV NS5B. NS5B was found to protect RNA fragments of 8 to 10 nucleotides (nt) from RNase digestion. However, NS5B could not direct RNA synthesis unless the template contained a stable secondary structure and a single-stranded sequence that contained at least one 3′ cytidylate. The structure of a 25-nt template, named SLD3, was determined by nuclear magnetic resonance spectroscopy to contain an 8-bp stem and a 6-nt single-stranded sequence. Systematic analysis of changes in SLD3 revealed which features in the stem, loop, and 3′ single-stranded sequence were required for efficient RNA synthesis. Also, chimeric molecules composed of DNA and RNA demonstrated that a DNA molecule containing a 3′-terminal ribocytidylate was able to direct RNA synthesis as efficiently as a sequence composed entirely of RNA. These results define the template sequence and structure sufficient to direct the de novo initiation of RNA synthesis by HCV RdRp.

Sign in / Sign up

Export Citation Format

Share Document