Enhanced nucleic acid binding to ATP-bound hepatitis C virus NS3 helicase at low pH activates RNA unwinding.
The molecular basis of the low-pH activation of the helicase encoded by the hepatitis C virus (HCV) was examined using either a full-length NS3 protein/NS4A cofactor complex or truncated NS3 proteins lacking the protease domain, which were isolated from three different viral genotypes. All proteins unwound RNA and DNA best at pH 6.5, which demonstrate that conserved NS3 helicase domain amino acids are responsible for low-pH enzyme activation. DNA unwinding was less sensitive to pH changes than RNA unwinding. Both the turnover rate of ATP hydrolysis and the K(m) of ATP were similar between pH 6 and 10, but the concentration of nucleic acid needed to stimulate ATP hydrolysis decreased almost 50-fold when the pH was lowered from 7.5 to 6.5. In direct-binding experiments, HCV helicase bound DNA weakly at high pH only in the presence of the non-hydrolyzable ATP analog, ADP(BeF3). These data suggest that a low-pH environment might be required for efficient HCV RNA translation or replication, and support a model in which an acidic residue rotates toward the RNA backbone upon ATP binding repelling nucleic acid from the binding cleft.
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
Angela M I
AM
Lam
Ryan S
RS
Rypma
David N
DN
Frick
D000244:Adenosine Diphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004265:DNA Helicases / Q000378:metabolism; D004789:Enzyme Activation; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D020365:RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000378:metabolism*; D062367:ortho-Aminobenzoates / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D004265:DNA Helicases; D008274:Magnesium; D012313:RNA; D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; D062367:ortho-Aminobenzoates; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C081523:3'-O-(N-methylanthraniloyl)adenosine 5'-diphosphate