scholarly journals The C-Terminal Hydrophobic Domain of Hepatitis C Virus RNA Polymerase NS5B Can Be Replaced with a Heterologous Domain of Poliovirus Protein 3A

2006 ◽  
Vol 80 (22) ◽  
pp. 11343-11354 ◽  
Author(s):  
Haekyung Lee ◽  
Ying Liu ◽  
Edison Mejia ◽  
Aniko V. Paul ◽  
Eckard Wimmer
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Decisive Role ◽  
Amino Acid Residues ◽  
Subgenomic Replicon ◽  
Hydrophobic Domain ◽  
Virus Rna ◽  
Membrane Bound

ABSTRACT Replication of the plus-stranded RNA genome of hepatitis C virus (HCV) occurs in a membrane-bound replication complex consisting of viral and cellular proteins and viral RNA. NS5B, the RNA polymerase of HCV, is anchored to the membranes via a C-terminal 20-amino-acid-long hydrophobic domain, which is flanked on each side by a highly conserved positively charged arginine. Using a genotype 1b subgenomic replicon (V. Lohmann, F. Korner, J. O. Koch, U. Herian, L. Theilmann, and R. Bartensclager, Science 285:110-113, 1999), we determined the effect of mutations of some highly conserved residues in this domain. The replacement of arginine 570 with alanine completely abolished the colony-forming ability by the replicon, while a R591A change was found to be highly detrimental to replication, viability, and membrane binding by the mutant NS5B protein. Mutations of two other highly conserved amino acids (L588A and P589A) reduced but did not eliminate colony formation. It was of interest, if specific amino acid residues play a role in membrane anchoring of NS5B and replication, to determine whether a complete exchange of the NS5B hydrophobic domain with a domain totally unrelated to NS5B would ablate replication. We selected the 22-amino-acid-long hydrophobic domain of poliovirus polypeptide 3A that is known to adopt a transmembrane configuration, thereby anchoring 3A to membranes. Surprisingly, either partial or full replacement of the NS5B hydrophobic domain with the anchor sequences of poliovirus polypeptide 3A resulted in the replication of replicons whose colony-forming abilities were reduced compared to that of the wild-type replicon. Upon continued passage of the replicon in Huh-7 cells in the presence of neomycin, the replication efficiency of the replicon increased. However, the sequence of the poliovirus polypeptide 3A hydrophobic domain, in the context of the subgenomic HCV replicon, was stably maintained throughout 40 passages. Our results suggest that anchoring NS5B to membranes is necessary but that the amino acid sequence of the anchor per se does not require HCV origin. This suggests that specific interactions between the NS5B hydrophobic domain and other membrane-bound factors may not play a decisive role in HCV replication.

scholarly journals An interferon lambda 4-associated variant in the hepatitis C virus RNA polymerase affects viral replication in infected cells

2020 ◽  
Author(s):  
Connor G.G. Bamford ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Nucleotide Polymorphisms ◽  
Virus Rna ◽  
Infected Cells ◽  
Presence And Absence ◽  
Amino Acid Variants

Host IFNL4 haplotype status contributes to the development of chronic hepatitis C virus (HCV) infection in individuals who are acutely infected with the virus. In silico studies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single-nucleotide polymorphisms (SNPs) in the IFNL4 locus. Thus, SNPs at the IFNL4 locus may select variants that influence virus replication and thereby the outcome of infection. Here, we examine the most significantly IFNL4-associated amino acid variants that lie in the ‘lambda (L) 2 loop’ of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication was examined in the presence and absence of exogenous IFNλ4. Our data demonstrate that while mutation of the NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication in both the presence and absence of IFNλ4. Given the strong genetic association between these variants and IFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFNλ4.

scholarly journals An interferon lambda 4-associated variant in the hepatitis C virus RNA polymerase affects viral replication in infected cells

2020 ◽  
Author(s):  
Connor G G Bamford ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Rna Polymerase ◽  
Viral Replication ◽  
Nucleotide Polymorphisms ◽  
Virus Rna ◽  
Infected Cells ◽  
Presence And Absence ◽  
Amino Acid Variants

AbstractHost IFNL4 haplotype status contributes to the development of chronic hepatitis C virus infection in individuals who are acutely infected with the virus. In silico studies revealed that specific amino acid variants at multiple sites on the HCV polyprotein correlate with functional single nucleotide polymorphisms (SNPs) in the IFNL4 locus. Thus, SNPs at the IFNL4 locus may select variants that influence virus replication and thereby outcome of infection. Here, we examine the most significantly IFNL4-associated amino acid variants that lie in the ‘Lambda (L) 2 loop’ of the HCV NS5B RNA polymerase. L2 loop variants were introduced into both sub-genomic replicon and full-length infectious clones of HCV and viral replication examined in the presence and absence of exogenous IFNλ4. Our data demonstrate that while mutation of NS5B L2 loop affects replication, individual IFNL4-associated variants have modest but consistent effects on replication both in the presence and absence of IFNλ4. Given the strong genetic association between these variants and IFNL4, these data suggest a nuanced effect of each individual position on viral replication, the combined effect of which might mediate resistance to the effects of IFNλ4.

Binding Mode Determination of Benzimidazole Inhibitors of the Hepatitis C Virus RNA Polymerase by a Structure and Dynamics Strategy

2004 ◽  
Vol 116 (33) ◽  
pp. 4406-4411 ◽  
Author(s):  
Steven R. LaPlante ◽  
Araz Jakalian ◽  
Norman Aubry ◽  
Yves Bousquet ◽  
Jean-Marie Ferland ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Binding Mode ◽  
Hepatitis C Virus Rna ◽  
Structure And Dynamics ◽  
Virus Rna

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 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.

B-ring modified aurones as promising allosteric inhibitors of hepatitis C virus RNA-dependent RNA polymerase

2014 ◽  
Vol 80 ◽  
pp. 579-592 ◽  
Author(s):  
Amel Meguellati ◽  
Abdelhakim Ahmed-Belkacem ◽  
Wei Yi ◽  
Romain Haudecoeur ◽  
Marie Crouillère ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hepatitis C Virus Rna ◽  
Allosteric Inhibitors ◽  
Rna Dependent Rna Polymerase ◽  
Virus Rna
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