Analysis of aptamer binding site for HCV-NS3 protease by alanine scanning mutagenesis.
Nonstructural protein 3 (NS3) of Hepatitis C virus (HCV) is a multifunctional protein and possesses protease, nucleotide triphosphatase and helicase activities. The N-terminal domain of NS3 (amino acids 1027-1218; delta NS3) has a trypsin-like protease activity and is essential for processing of viral polyprotein. Accordingly it is a potential target for anti-HCV drugs and we isolated RNA aptamers (Kd = 10 nM, Ki = 100 nM) using in vitro selection strategy. To study the interaction between delta NS3 and its aptamer, we applied alanine scanning mutagenesis and constructed seven mutant proteins at positive amino acid residues on the surface of delta NS3. Binding and inhibitory activities of the NS3 aptamer against mutant proteins were kinetically analyzed. These results clarified that especially Arg161 and Arg130 are important for interaction with the NS3 aptamer.
Hwang
J
J
National Institute of Bioscience and Human Technology, AIST, Institute of Applied Biochemistry, University of Tsukuba.
Fauzi
H
H
Fukuda
K
K
Sekiya
S
S
Kakiuchi
N
N
Taira
K
K
Kusakabe
I
I
Nishikawa
S
S
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D016174:Hepacivirus / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011480:Protease Inhibitors / Q000737:chemistry / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D011480:Protease Inhibitors; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases