Comparative characterization of two DEAD-box RNA helicases in superfamily II: human translation-initiation factor 4A and hepatitis C virus non-structural protein 3 (NS3) helicase.
Eukaryotic initiation factor 4A (eIF4A) is an ATP-dependent RNA helicase and is homologous to the non-structural protein 3 (NS3) helicase domain encoded by hepatitis C virus (HCV). Reported here is the comparative characterization of human eIF4A and HCV NS3 helicase in an effort to better understand viral and cellular helicases of superfamily II and to assist in designing specific inhibitors against HCV infections. Both eIF4A and HCV NS3 helicase domain were expressed in bacterial cells as histidine-tagged proteins and purified to homogeneity. Purified eIF4A exhibited RNA-unwinding activity and acted on RNA or RNA/DNA but not DNA duplexes. In the absence of cellular cofactors, eIF4A operated unwinding in both the 3' to 5' and 5' to 3' directions, and was able to unwind blunt-ended RNA duplex, suggesting that bidirectionality is an intrinsic property of eIF4A. In contrast, HCV NS3 helicase showed unidirectional 3' to 5' unwinding of RNA and RNA/DNA, as well as of DNA duplexes. With respect to NTPase activity, eIF4A hydrolysed only ATP or dATP in the presence of RNAs, whereas HCV NS3 helicase could hydrolyse all ribo- and deoxyribo-NTPs in an RNA-independent manner. In parallel, only ATP or dATP could drive the unwinding activity of eIF4A whereas HCV NS3 could function with all eight standard NTPs and dNTPs. The observed differences in their substrate specificity may prove to be useful in designing specific inhibitors targeting HCV NS3 helicase but not human eIF4A.
Du
Mark X
MX
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Johnson
Robert B
RB
Sun
Xin-Lai
XL
Staschke
Kirk A
KA
Colacino
Joseph
J
Wang
Q May
QM
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D017931:DNA Primers / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A; D016174:Hepacivirus / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000251:Adenosine Triphosphatases; D000255:Adenosine Triphosphate; D017931:DNA Primers; D018076:DNA, Complementary; D039601:Eukaryotic Initiation Factor-4A; D010448:Peptide Initiation Factors; D012313:RNA; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases