RNA-stimulated ATPase and RNA helicase activities and RNA binding domain of hepatitis G virus nonstructural protein 3

Hepatitis G virus (HGV) nonstructural protein 3 (NS3) contains amino acid s equence motifs typical of ATPase and RNA helicase proteins. In order to exa mine the RNA helicase activity of the HGV NS3 protein, the NS3 region (amin o acids 904 to 1580) was fused with maltose-binding protein (MBP), and the fusion protein was expressed in Escherichia coli and purified with amylose resin and anion-exchange chromatography. The purified MBP-HGV/NS3 protein p ossessed RNA-stimulated ATPase and RNA helicase activities. Characterizatio n of the ATPase and RNA helicase activities of MBP-HGV/NS3 showed that the optimal reaction conditions were similar to those of other Flaviviridae vir al NS3 proteins. However, the kinetic analysis of NTPase activity showed th at the MBP-HGV/NS3 protein had several unique properties compared to the ot her Flaviriridae NS3 proteins. The HGV NS3 helicase unwinds RNA-RNA duplexe s in a 3'-to-5' direction and can unwind RNA-DNA heteroduplexes and DNA-DNA duplexes as well. In a gel retardation assay, the MBP-HGV/ NS3 helicase bo und to RNA, RNA/DNA, and DNA duplexes with 5' and 3' overhangs but not to b lunt-ended RNA duplexes. We also found that the conserved motif VI was impo rtant for RNA binding. Further deletion mapping showed that the RNA binding domain was located between residues 1383 and 1395, QRRGRT-GRGRSGR, Our dat a showed that the MBP-HCV/NS3 protein also contains the RNA binding domain in the similar domain.