Oligomerization and cooperative RNA synthesis activity of hepatitis C virus RNA-dependent RNA polymerase.
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, K(d), of approximately 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 A. 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.
Wang
Q May
QM
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305.
Hockman
Michelle A
MA
Staschke
Kirk
K
Johnson
Robert B
RB
Case
Katharine A
KA
Lu
Jirong
J
Parsons
Steve
S
Zhang
Faming
F
Rathnachalam
Radhakrishnan
R
Kirkegaard
Karla
K
Colacino
Joseph M
JM
D003432:Cross-Linking Reagents; D003460:Crystallization; D004795:Enzyme Stability; D016174:Hepacivirus / Q000201:enzymology*; D006358:Hot Temperature; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase