PDBsum entry 2giq

Transferase

PDB id: 2giq

Contents

Protein chains

557 a.a. *

Ligands

NN2 ×2

Waters ×518

* Residue conservation analysis

PDB id:

2giq

Name:

Transferase

Title:

Hepatitis c virus RNA-dependent RNA polymerase ns5b with nni-2 inhibitor

Structure:

RNA-directed RNA polymerase. Chain: a, b. Fragment: hcv ns5b. Synonym: ns5b, p68. Engineered: yes

Source:

Hepatitis c virus (isolate bk). Organism_taxid: 11105. Strain: genotype 1b. Strain bk. Gene: ns5b. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.65Å R-factor: 0.204 R-free: 0.228

Authors:

S.F.Harris

Key ref:

S.Le Pogam et al. (2006). Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus. J Virol, 80, 6146-6154. PubMed id: 16731953

Date:

29-Mar-06 Release date: 03-Apr-07

Protein chains

P26663  (POLG_HCVBK) -  Genome polyprotein from Hepatitis C virus genotype 1b (isolate BK)

Seq: 3010 a.a.

Struc: 557 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 1: E.C.2.7.7.48 - RNA-directed Rna polymerase.
• Reaction: RNA(n) + a ribonucleoside 5'-triphosphate = RNA(n+1) + diphosphate
• Enzyme class 2: E.C.3.4.21.98 - hepacivirin.
• Reaction: Hydrolysis of four peptide bonds in the viral precursor polyprotein, commonly with Asp or Glu in the P6 position, Cys or Thr in P1 and Ser or Ala in P1'.
• Enzyme class 3: E.C.3.4.22.- - ?????
• Enzyme class 4: E.C.3.6.1.15 - nucleoside-triphosphate phosphatase.
• Reaction: a ribonucleoside 5'-triphosphate + H2O = a ribonucleoside 5'-diphosphate + phosphate + H⁺
• Enzyme class 5: E.C.3.6.4.13 - Rna helicase.
• Reaction: ATP + H2O = ADP + phosphate + H⁺

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

J Virol 80:6146-6154 (2006)

PubMed id: 16731953

Selection and characterization of replicon variants dually resistant to thumb- and palm-binding nonnucleoside polymerase inhibitors of the hepatitis C virus.

S.Le Pogam, H.Kang, S.F.Harris, V.Leveque, A.M.Giannetti, S.Ali, W.R.Jiang, S.Rajyaguru, G.Tavares, C.Oshiro, T.Hendricks, K.Klumpp, J.Symons, M.F.Browner, N.Cammack, I.Nájera.

ABSTRACT

Multiple nonnucleoside inhibitor binding sites have been identified within the hepatitis C virus (HCV) polymerase, including in the palm and thumb domains. After a single treatment with a thumb site inhibitor (thiophene-2-carboxylic acid NNI-1), resistant HCV replicon variants emerged that contained mutations at residues Leu419, Met423, and Ile482 in the polymerase thumb domain. Binding studies using wild-type (WT) and mutant enzymes and structure-based modeling showed that the mechanism of resistance is through the reduced binding of the inhibitor to the mutant enzymes. Combined treatment with a thumb- and a palm-binding polymerase inhibitor had a dramatic impact on the number of replicon colonies able to replicate in the presence of both inhibitors. A more exact characterization through molecular cloning showed that 97.7% of replicons contained amino acid substitutions that conferred resistance to either of the inhibitors. Of those, 65% contained simultaneously multiple amino acid substitutions that conferred resistance to both inhibitors. Double-mutant replicons Met414Leu and Met423Thr were predominantly selected, which showed reduced replication capacity compared to the WT replicon. These findings demonstrate the selection of replicon variants dually resistant to two NS5B polymerase inhibitors binding to different sites of the enzyme. Additionally, these findings provide initial insights into the in vitro mutational threshold of the HCV NS5B polymerase and the potential impact of viral fitness on the selection of multiple-resistant mutants.