Two Novel Conserved Motifs in the Hepatitis C Virus NS3 Protein Critical for Helicase Action

Abstract

The hepatitis C virus (HCV) NS3 helicase shares several conserved motifs with other superfamily 2 (SF2) helicases. Besides these sequences, several additional helicase motifs are conserved among the various HCV genotypes and quasispecies. The hepatitis C virus (HCV)1 has caused a global crisis by infecting more than 2% of the world's population (1). Combination therapies using interferon and the antiviral drug ribavirin eliminate HCV in a majority of patients but such treatments are costly and cause debilitating side effects. Vaccine and drug development has been difficult because the only natural hosts for HCV are humans and chimpanzees, and the virus cannot be conventionally cultivated in vitro. As a result, the enzymes encoded by the virus have been intensely studied for rational drug design (2). Several groups have reported potent compounds that inhibit the HCV RNA-dependent RNA polymerase (3) and its serine protease (4). However, few inhibitors of the HCV helicase have been explored as antivirals because less is known about the mechanism of action of the HCV helicase and if or how it differs from similar cellular enzymes.HCV helicase is part of the viral non-structural protein 3 (NS3). The N-terminal one-third of NS3 is a chymotrypsin-like serine protease, and the C-terminal NS3 portion is a helicase that is fueled by the hydrolysis of ATP. Although the HCV helicase only unwinds duplex RNA during viral replication, the purified protein unwinds DNA dramatically more efficiently than RNA (5). In these reactions, HCV helicase requires a 3Ј single-stranded (ss) DNA or RNA overhang to initiate unwinding and is therefore classified as a 3Ј-5Ј helicase. The structure of the HCV helicase has been determined four times using x-ray crystallography: twice as an apoenzyme (6, 7), once with a bound ssDNA (8) and once as part of the full-length NS3 protein (9). NMR structures of parts of the HCV helicase have also been reported (10, 11). HCV helicase consists of 3 domains, with ATP likely binding between the structurally similar domains 1 and 2. One strand of the unwound duplex binds in the cleft that separates domain 3 from domains 1 and 2 (8). Gorbalenya and Koonin (12) have classified the HCV helicase as a member of helicase superfamily 2, which contains several large protein families that all share several signature sequences. The SF2 helicase signature sequence motifs line the "ATP-binding" cleft between domains 1 and 2. Numerous studies have elucidated the roles of the SF2 motifs in HCV helicase function, and structure-based mutagenesis has revealed a few other critical conserved residues (reviewed in Refs. 2 and 13). Little is known about the function of residues outside these conserved motifs.In the present study, sequence alignments and structurebased site-directed mutagenesis were used to identify two additional conserved motifs in domain 2 of the HCV helicase that are critical for helicase action. After introducing individual point mutations into these regions of the enzyme, ATPase, unwinding, translocation r...

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

“…The Protease Domain Aids RNA Unwinding-Previous studies using Hel-His have shown that it unwinds RNA poorly relative to DNA (15,16), and when excess DNA is added upon initiation to reactions containing Hel-His, virtually no RNA unwinding is detected (15). Although a preference for DNA has been long established for HCV helicase (32), the absence of RNA helicase activity in the presence of an enzyme trap (such as excess DNA) starkly contrasted results published previously by other authors (25,31).…”

“…Cloning, expression, and purification of Hel-His (which was previously designated Hel-1a) have been described (15). A detailed enzymatic characterization of Hel-1a was reported recently (16).…”

“…The truncated helicase proteins lacking protease function (Fig. 1A) were based on those previously analyzed and include versions with N-terminal (10 -12) and C-terminal (7,(13)(14)(15)(16) polyhistidine tags (His tags) and a version in which the protease is replaced with a glutathione Stransferase (GST) protein (17). The presence of an intact protease domain dramatically enhances the ability of the protein to unwind RNA.…”

Crystal Structure of Quinone Reductase 2 in Complex with Resveratrol,

“…The fluorescence of the samples with QR2 subtracted by that of the samples without QR2 is used as the fluorescence of the QR2-polyphenol complex. The obtained values were globally fitted to an exhaustive binding model (25) with the software Prism.…”

Erratum to: Structure and Mechanisms of SF1 DNA Helicases

“…Dda contains a long -hairpin which is anchored at its tip by electrostatic interactions to domain 2B, allowing it to function not only directly in splitting the dsDNA but also in coupling ATP hydrolysis to unwinding (He et al, in press). Like Dda, the hepatitis C virus NS3 helicase (SF2) contains an extended pin [100], and mutation of conserved residues with this pin also uncouples ATP hydrolysis from unwinding [130].…”