Nucleoside triphosphatase and RNA helicase activities associated with GB virus B nonstructural protein 3

GB virus B (GBV-B) is a positive-stranded RNA virus that belongs to the Fla viviridae family. This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species). Nonstructural pr otein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymat ic activities: serine protease, nucleoside triphosphatase (NTPase), and RNA helicase. The N-terminal serine protease has been characterized and shown to share similar substrate specificity with the HCV NS3 protease. In this r eport, a full-length GBV-B NS3 protein was expressed in Escherichia coil an d purified to homogeneity. This recombinant protein was shown to possess po lynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding ac tivities. Both activities were abolished by a single amino acid substitutio n, from the Lys (K) residue in the conserved walker motif A (or la) "AXXXXG K(210)S" to an Ala (A), confirming that they are intrinsic to GBV-B NS3. Ki netic parameters (K-m and k(cat)) for hydrolysis of various NTPs or dNTPs w ere obtained. The dsRNA unwinding activity depends on the presence of dival ent metal ions and ATP and requires an RNA duplex substrate with 3' unpaire d regions (RNAs with 5' unpaired regions only or with blunt ends are not su itable substrates for this enzyme). This indicates that GBV-B NS3 RNA helic ase unwinds dsRNA in the 3' to 5' direction. Direct interaction of the GBV- B NS3 protein with a single-stranded RNA was established using a gel-based RNA bandshift assay. Finally, a homology model of GBV-B NS3 RNA helicase do main based on the 3-dimensional structure of the HCV NS3 helicase that show s a great similarity in overall structure and surface charge distribution b etween the two proteins was proposed. (C) 1999 Academic Press.