4RY4

C-terminal mutant (Y448F) of HCV/J4 RNA polymerase

Summary for 4RY4

• Entry DOI: 10.2210/pdb4ry4/pdb
• Related: 1NB4 4RY5 4RY6 4RY7
• Descriptor: HCV J4 RNA polymerase (NS5B) (2 entities in total)
• Functional Keywords: hepatitis c virus, replication, function analysis, viral rna polymerase, rna rntp mg, nucleotide transfer, transferase
• Biological source: Hepatitis C virus isolate HC-J4 (HCV)
• Cellular location: Core protein p21: Host endoplasmic reticulum membrane ; Single-pass membrane protein . Core protein p19: Virion . Envelope glycoprotein E1: Virion membrane ; Single-pass type I membrane protein . Envelope glycoprotein E2: Virion membrane ; Single-pass type I membrane protein . p7: Host endoplasmic reticulum membrane ; Multi-pass membrane protein . Protease NS2-3: Host endoplasmic reticulum membrane ; Multi-pass membrane protein . Serine protease NS3: Host endoplasmic reticulum membrane ; Peripheral membrane protein . Non-structural protein 4A: Host endoplasmic reticulum membrane ; Single-pass type I membrane protein . Non-structural protein 4B: Host endoplasmic reticulum membrane ; Multi-pass membrane protein . Non-structural protein 5A: Host endoplasmic reticulum membrane ; Peripheral membrane protein . RNA-directed RNA polymerase: Host endoplasmic reticulum membrane ; Single-pass type I membrane protein : O92972
• Total number of polymer chains: 2
• Total formula weight: 126881.48

Authors

Jaeger, J.,Cherry, A.,Dennis, C. (deposition date: 2014-12-13, release date: 2014-12-31, Last modification date: 2023-09-20)

Primary citation

Cherry, A.L.,Dennis, C.A.,Baron, A.,Eisele, L.E.,Thommes, P.A.,Jaeger, J.
Hydrophobic and Charged Residues in the C-Terminal Arm of Hepatitis C Virus RNA-Dependent RNA Polymerase Regulate Initiation and Elongation.
J.Virol., 89:2052-2063, 2015

PubMed Abstract: The 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 for in vitro analysis of RdRp de novo initiation 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.

PubMed: 25428878
DOI: 10.1128/JVI.01106-14

Experimental method

X-RAY DIFFRACTION (2.59 Å)