Structure-Based Mutagenesis Study of Hepatitis C Virus NS3 Helicase

Hepatitis C virus (HCV) is recognized as the main etiologic agent of parenterally transmitted non-A, non-B hepatitis (4, 24) and is responsible for a large proportion of cases of community-acquired hepatitis. The majority of HCV infections become persistent, leading to chronic hepatitis, liver cirrhosis, or even hepatocellular carcinoma (reviewed in reference 13). Development of an effective vaccine against HCV has been slow and difficult, in part due to the quasi-species nature of the virus. Approved therapies for viral hepatitis type C (hepatitis C) include alpha interferon and the combination of alpha interferon and ribavirin, which induce limited long-term response in hepatitis C patients and have relatively severe side effects. Therefore, it is of great interest to develop effective, HCV-specific therapeutic drugs. HCV is now classified in the Hepacivirus genus (reviewed in reference 13) of the Flaviviridae family (reviewed in reference 40), which includes two additional genera, Flavivirus and Pestivirus. The recently discovered hepatitis G virus (HGV or GBV-C) is closely related to HCV and has been proposed to be the fourth group in the Flaviviridae family. The majority of the 9.6-kb HCV genome encodes a large open reading frame corresponding to a polyprotein precursor of about 3,000 amino acids, which is flanked by 5′ and 3′ noncoding regions. The polyprotein precursor of the HCV H strain is proteolytically processed by cellular signal peptidase(s) and two HCV-encoded proteases into at least 10 distinct products, with the sequence NH2 - C - E1 - E2 - p7 - NS2 - NS3 - NS4A - NS4B - NS5A - NS5B - COOH (7–9, 27). The putative structural proteins include a core protein (C) and two envelope proteins (E1 and E2), whereas the nonstructural (NS) proteins, including two proteases, a combined helicase and nucleoside triphosphatase (NTPase), and an RNA-dependent RNA polymerase, are believed to be components of a complex responsible for viral RNA replication. The C-terminal 450 amino acids of the NS3 protein manifest a polynucleotide-stimulated NTPase activity (14, 45), a 3′-to-5′ unwinding activity (10, 14, 18, 46), and a single-stranded (ss) polynucleotide binding activity (10, 17, 46). Recently, elegant biochemical characterizations of this helicase have been presented by Porter and colleagues and Preugschat et al. (36–39). Helicases are enzymes that are able to separate the strands of duplex DNA or RNA by utilizing energy derived from hydrolysis of nucleoside 5′-triphosphates (NTPs, usually ATP) (reviewed in reference 30). Accordingly, the NTPase activity of helicases can be stimulated by polynucleotide. More than 200 proteins have been identified as putative helicases based on the presence of some or all of seven conserved amino acid motifs (for a recent review, see reference 6). Until now, only a handful of these putative helicases have been purified and shown to have duplex nucleic acid unwinding activity. Helicases have been shown to be involved in DNA metabolism (replication, repair, and genome recombination) (reviewed in references 30 and 32) and RNA metabolism (transcription, splicing or processing, transport, and translation initiation of RNA) (for a recent review, see reference 31). Mutations in several helicase genes have been associated with six human genetic disorders: Werner’s syndrome, Bloom’s syndrome, xeroderma pigmentosum, trichothiodystrophy, Cockayne’s syndrome, and α thalassemia-related mental retardation associated with the X chromosome (for a review, see reference 5). Helicases may also play critical roles in processes involved in the life cycles of many DNA and RNA viruses, including replication and recombination of viral DNA or RNA genome, RNA transcription, and translation (for a review, see references 16 and 21). Recently, four groups reported the X-ray crystallographic structures of three different helicases, i.e., the DNA helicases PcrA (44) and Rep (22) and the HCV NS3 RNA helicase (20, 48). These structures demonstrated that helicases have many common features in terms of protein folding and tertiary structure, although there is very limited homology in their primary amino acid sequences. For all three helicases, most of the six or seven conserved motifs (motifs I through VI) (see Fig. ​Fig.4A)4A) are located at similar positions within a cleft between two domains of the enzyme (see Fig. ​Fig.3A,3A, domains 1 and 2 of the HCV RNA helicase, and domains 1A and 2A of Rep or PcrA helicases (22, 44). The NTP-binding pocket, composed of residues of motifs I and II, is located on the side of domain 1 in this cleft. Our X-ray structural analysis of the HCV helicase bound to an ss (dU)8 oligonucleotide showed that the oligonucleotide binds in a groove that separates domain 3 from domains 1 and 2 (reference 20, and also see Fig. ​Fig.3A).3A). This nucleic acid-binding groove is oriented perpendicular to the NTP-binding cleft between domains 1 and 2. In this structure, most of the interactions between the enzyme and bound (dU)8 oligonucleotide involve hydrogen bonds with the phosphate backbone but not the bases of the oligonucleotide (20), which explains the sequence-independent nature of these helicases with regard to the duplex nucleic acid substrate. The most significant enzyme-base interaction involves a hydrophobic stacking interaction between Trp-501 of the HCV helicase and a base near the 3′ end of the bound (dU)8 oligonucleotide (see Fig. ​Fig.3B).3B). This structural analysis identifies several amino acids in this nucleic acid-binding groove that interact with the oligonucleotide (see Fig. ​Fig.3B).3B). Although some of these residues are conserved within various HCV strains, they are not a part of the previously reported conserved helicase motifs (motifs I through VI) (see Fig. ​Fig.4A).4A). In the present study, site-directed mutagenesis was used to investigate the role of these residues in the mechanism of action of the HCV NS3 helicase. FIG. 3 (A) Locations of conserved motifs in the ternary complex of HCV NS3 helicase, (dU)8 oligonucleotide, and ADP. The overall fold of the HCV NS3 helicase is illustrated by a green ribbon diagram. The HCV NS3 helicase consists of three domains: domain 1 (top ... FIG. 4 Alignment of functional conserved motifs or residues of selected helicases from SF1 and SF2. (A) Alignment of several conserved motifs and other functional conserved residues of two SF2 RNA helicases (HCV NS3 helicase and the eukaryotic translation factor ...