Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus

The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepat itis C virus (HCV) consists of three domains with highly conserved NTP bind ing motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and i t was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364, of the HCV polyprotein, was co nfirmed by N- and C-terminal sequencing and by its capability to bind 5'-fl uorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of bind ing site with the K-d Of 43.6 mu M. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as r egulators of the ATP-binding capacity of the domain. Polynucleotides bind t o the domain and enhance its affinity for ATP. In addition, ATP enhances th e affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding si tes of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents beha ved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50=22 mu M), and trifluoperazine, which inhibits the ATP binding by a n oncompetitive mechanism (IC50 = 98 mu M). Kinetic experiments with the NTPa se/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.