Structurally Conserved Amino Acid W501 Is Required for RNA Helicase Activity but Is Not Essential for DNA Helicase Activity of Hepatitis C Virus NS3 Protein

Abstract

Hepatitis C virus (HCV) is a positive-strand RNA virus that encodes a helicase required for viral replication. Although HCV does not replicate through a DNA intermediate, HCV helicase unwinds both RNA and DNA duplexes. An X-ray crystal structure of the HCV helicase complexed with (dU) 8 has been solved, and the substrate-amino acids interactions within the catalytic pocket were shown. Among these, residues W501 and V432 were reported to have base stacking interactions and to be important for the unwinding function of HCV helicase. It has been hypothesized that specific interactions between the enzyme and substrate in the catalytic pocket are responsible for the substrate specificity phenotype. We therefore mutagenized W501 and V432 to investigate their role in substrate specificity in HCV helicase. Replacement of W501, but not V432, with nonaromatic residues resulted in complete loss of RNA unwinding activity, whereas DNA unwinding activity was largely unaffected. The loss of unwinding activity was fully restored in the W501F mutant, indicating that the aromatic ring is crucial for RNA helicase function. Analysis of ATPase and nucleic acid binding activities in the W501 mutant enzymes revealed that these activities are not directly responsible for the substrate specificity phenotype. Molecular modeling of the enzyme-substrate interaction at W501 revealed a putative -facial hydrogen bond between the 2-OH of ribose and the aromatic tryptophan ring. This evidence correlates with biochemical results suggesting that the -facial bond may play an important role in the RNA unwinding activity of the HCV NS3 protein.

Understanding Helicases as a Means of Virus Control

“…It was therefore somewhat surprising when a co-structure of a short oligonucleotide bound to HCV helicase showed the DNA bound to the cleft separating domain 3 from the RecA-like domains (Fig (2a)) [113]. Extensive sitedirected mutagenesis of HCV helicase has since confirmed the validity of the HCV helicase-DNA structure and demonstrated that the observed protein-nucleic acid contacts are needed for efficient unwinding [131][132][133][134][135][136]. Furthermore, a similar DNA binding cleft, which is perpendicular to the ATP binding cleft, has since been seen in co-structures of two other non-ring SF1 helicases with DNA [137,138].…”

“…Trp501 stacks against the base at the 3'-end of the HCV bound oligonucleotide, and is not in a RecA-like domain. Site-directed mutants with an alanine substituted at position 501 unwind RNA poorly [131][132][133][134][135]. Because it stacks with a base, Trp501 is believed to prevent DNA (or RNA) from sliding through the helicase in the absence of ATP.…”

The Hepatitis C Virus NS3 Protein: A Model RNA Helicase and Potential Drug Target

“…Most initial interest focused on the residues that act as bookends, or the teeth of the ratchet. Several groups have confirmed the importance of Trp 501 in both nucleic acid binding and unwinding (Lin and Kim, 1999;Paolini et al, 2000;Preugschat et al, 2000;Kim et al, 2003). Without a bulky aromatic amino acid at position 501, HCV helicase is unable to unwind RNA (Lin and Kim, 1999;Tai et al, 2001;Kim et al, 2003) but retains some ability to unwind DNA (Kim et al, 2003), albeit more slowly than wild type (Preugschat et al, 2000).…”

“…Kim et al propose that this residue is Val 432 in Domain 2 (Kim et al, 1998), but Paolini et al have suggested that Tyr 392 could play a similar role (Paolini et al, 2000). Some of these reports have suggested that mutation of these residues leads to decreases in helicase activity (Paolini et al, 2000;Preugschat et al, 2000;Tai et al, 2001;Kim et al, 2003).…”

“…Deletion of 97 amino acids from the C-terminus of NS3 results in an inactive helicase (Jin and Peterson, 1995;Kim et al, 1997a). Two key residues in domain 3 are Trp 501 , which stacks against a nucleic acid base to act like a bookend (Lin and Kim, 1999;Preugschat et al, 2000;Kim et al, 2003), and Glu 493 which helps repel nucleic acids from the binding cleft upon ATP binding (Frick et al, 2004a). …”