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 Flaviviridae family. This virus is closely related to hepatitis C virus (HCV) and causes acute hepatitis in tamarins (Saguinus species). Nonstructural protein 3 (NS3) of GBV-B contains sequence motifs predictive of three enzymatic 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 report, a full-length GBV-B NS3 protein was expressed in Escherichia coli and purified to homogeneity. This recombinant protein was shown to possess polynucleotide-stimulated NTPase and double-stranded RNA (dsRNA) unwinding activities. Both activities were abolished by a single amino acid substitution, from the Lys (K) residue in the conserved walker motif A (or Ia) "AXXXXGK(210)S" to an Ala (A), confirming that they are intrinsic to GBV-B NS3. Kinetic parameters (K(m) and k(cat)) for hydrolysis of various NTPs or dNTPs were obtained. The dsRNA unwinding activity depends on the presence of divalent metal ions and ATP and requires an RNA duplex substrate with 3' unpaired regions (RNAs with 5' unpaired regions only or with blunt ends are not suitable substrates for this enzyme). This indicates that GBV-B NS3 RNA helicase 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 domain based on the 3-dimensional structure of the HCV NS3 helicase that shows a great similarity in overall structure and surface charge distribution between the two proteins was proposed.
Zhong
W
W
Department of Antiviral Therapy, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033-0539, USA. weidong.zhong@spcorp.com
Ingravallo
P
P
Wright-Minogue
J
J
Skelton
A
A
Uss
A S
AS
Chase
R
R
Yao
N
N
Lau
J Y
JY
Hong
Z
Z
D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004789:Enzyme Activation; D018067:Flaviviridae / Q000737:chemistry / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D011487:Protein Conformation; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D017766:Acid Anhydride Hydrolases; D043583:Nucleoside-Triphosphatase; D020365:RNA Helicases; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C060028:NS3 protein, flavivirus; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases