3I5K

Crystal structure of the NS5B polymerase from Hepatitis C Virus (HCV) strain JFH1

Summary for 3I5K

• Entry DOI: 10.2210/pdb3i5k/pdb
• Descriptor: RNA-directed RNA polymerase, PHOSPHATE ION (3 entities in total)
• Functional Keywords: rdrp structure (fingers, palm, thumb domains), apoptosis, atp-binding, capsid protein, cell membrane, disulfide bond, endoplasmic reticulum, envelope protein, fusion protein, glycoprotein, helicase, host-virus interaction, hydrolase, interferon antiviral system evasion, lipid droplet, lipoprotein, membrane, metal-binding, mitochondrion, multifunctional enzyme, nucleotide-binding, nucleotidyltransferase, nucleus, oncogene, palmitate, phosphoprotein, protease, ribonucleoprotein, rna replication, rna-binding, rna-directed rna polymerase, secreted, serine protease, sh3-binding, thiol protease, transcription, transcription regulation, transferase, transmembrane, viral nucleoprotein, virion
• Biological source: Hepatitis C virus JFH-1 (HCV)
• Cellular location: Core protein p21: Host endoplasmic reticulum membrane; Single-pass membrane protein (By similarity). Core protein p19: Virion (By similarity). Envelope glycoprotein E1: Virion membrane; Single-pass type I membrane protein (Potential). Envelope glycoprotein E2: Virion membrane; Single-pass type I membrane protein (Potential). p7: Host endoplasmic reticulum membrane; Multi-pass membrane protein. Protease NS2-3: Host endoplasmic reticulum membrane; Multi-pass membrane protein (Potential). Serine protease NS3: Host endoplasmic reticulum membrane; Peripheral membrane protein (Probable). Non-structural protein 4A: Host endoplasmic reticulum membrane; Single-pass type I membrane protein (Potential). Non-structural protein 4B: Host endoplasmic reticulum membrane; Multi-pass membrane protein (By similarity). Non-structural protein 5A: Host endoplasmic reticulum membrane; Peripheral membrane protein (By similarity). RNA-directed RNA polymerase: Host endoplasmic reticulum membrane; Single-pass type I membrane protein (Potential): Q99IB8
• Total number of polymer chains: 4
• Total formula weight: 253503.54

Authors

Simister, P.C.,Schmitt, M.,Lohmann, V.,Bressanelli, S. (deposition date: 2009-07-05, release date: 2009-09-22, Last modification date: 2024-03-20)

Primary citation

Simister, P.,Schmitt, M.,Geitmann, M.,Wicht, O.,Danielson, U.H.,Klein, R.,Bressanelli, S.,Lohmann, V.
Structural and functional analysis of hepatitis C virus strain JFH1 polymerase
J.Virol., 83:11926-11939, 2009

PubMed Abstract: The hepatitis C virus (HCV) isolate JFH1 represents the only cloned wild-type sequence capable of efficient replication in cell culture, as well as in chimpanzees. Previous reports have pointed to the viral polymerase NS5B as a major determinant for efficient replication of this isolate. To understand the underlying mechanisms, we expressed and purified NS5B of JFH1 and of the closely related isolate J6, which replicates below the limit of detection in cell culture. The JFH1 enzyme exhibited a 5- to 10-fold-higher specific activity in vitro, consistent with the polymerase activity itself contributing to efficient replication of JFH1. The higher in vitro activity of the JFH1 enzyme was not due to increased RNA binding, elongation rate, or processivity of the polymerase but to higher initiation efficiency. By using homopolymeric and heteropolymeric templates, we found that purified JFH1 NS5B was significantly more efficient in de novo initiation of RNA synthesis than the J6 counterpart, particularly at low GTP concentrations, probably representing an important prerequisite for the rapid replication kinetics of JFH1. Furthermore, we solved the crystal structure of JFH1 NS5B, which displays a very closed conformation that is expected to facilitate de novo initiation. Structural analysis shows that this closed conformation is stabilized by a sprinkle of substitutions that together promote extra hydrophobic interactions between the subdomains "thumb" and "fingers." These analyses provide deeper insights into the initiation of HCV RNA synthesis and might help to establish more efficient cell culture models for HCV using alternative isolates.

PubMed: 19740982
DOI: 10.1128/JVI.01008-09

Experimental method

X-RAY DIFFRACTION (2.2 Å)