RNA unwinding activity of the hepatitis C virus NS3 helicase is modulated by the NS5B polymerase.
Hepatitis C virus (HCV) infects over 170 million persons worldwide. It is the leading cause of liver disease in the U.S. and is responsible for most liver transplants. Current treatments for this infectious disease are inadequate; therefore, new therapies must be developed. Several labs have obtained evidence for a protein complex that involves many of the nonstructural (NS) proteins encoded by the virus. NS3, NS4A, NS4B, NS5A, and NS5B appear to interact structurally and functionally. In this study, we investigated the interaction between the helicase, NS3, and the RNA polymerase, NS5B. Pull-down experiments and surface plasmon resonance data indicate a direct interaction between NS3 and NS5B that is primarily mediated through the protease domain of NS3. This interaction reduces the basal ATPase activity of NS3. However, NS5B stimulates product formation in RNA unwinding experiments under conditions of excess nucleic acid substrate. When the concentrations of NS3 and NS5B are in excess of nucleic acid substrate, NS5B reduces the rate of NS3-catalyzed unwinding. Under pre-steady-state conditions, in which NS3 and substrate concentrations are similar, product formation increased in the presence of NS5B. The increase was consistent with 1:1 complex formed between the two proteins. A fluorescently labeled form of NS3 was used to investigate this interaction through fluorescence polarization binding assays. Results from this assay support interactions that include a 1:1 complex formed between NS3 and NS5B. The modulation of NS3 by NS5B suggests that these proteins may function together during replication of the HCV genome.
Jennings
Thomas A
TA
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Chen
Yingfeng
Y
Sikora
Deniz
D
Harrison
Melody K
MK
Sikora
Bartek
B
Huang
Luyun
L
Jankowsky
Eckhard
E
Fairman
Margaret E
ME
Cameron
Craig E
CE
Raney
Kevin D
KD
D000251:Adenosine Triphosphatases / Q000378:metabolism; D020029:Base Pairing; D002384:Catalysis; D004247:DNA / Q000378:metabolism; D016174:Hepacivirus / Q000201:enzymology*; D011072:Poly U / Q000378:metabolism; D011485:Protein Binding; D012313:RNA / Q000737:chemistry* / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D013379:Substrate Specificity; D020349:Surface Plasmon Resonance; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D000251:Adenosine Triphosphatases; D004247:DNA; D011072:Poly U; D012313:RNA; D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase