Structural and Biological Identification of Residues on the Surface of NS3 Helicase Required for Optimal Replication of the Hepatitis C Virus

Abstract

The hepatitis C virus (HCV) nonstructural protein 3 (NS3) is a multifunctional enzyme with serine protease and DEXH/D-box helicase domains. A crystal structure of the NS3 helicase domain (NS3h) was generated in the presence of a single-stranded oligonucleotide long enough to accommodate binding of two molecules of enzyme. Several amino acid residues at the interface of the two NS3h molecules were identified that appear to mediate a proteinprotein interaction between domains 2 and 3 of adjacent molecules. Mutations were introduced into domain 3 to disrupt the putative interface and subsequently examined using an HCV subgenomic replicon, resulting in significant reduction in replication capacity. The mutations in domain 3 were then examined using recombinant NS3h in biochemical assays. The mutant enzyme showed RNA binding and RNA-stimulated ATPase activity that mirrored wild type NS3h. In DNA unwinding assays under single turnover conditions, the mutant NS3h exhibited a similar unwinding rate and only ϳ2-fold lower processivity than wild type NS3h. Overall biochemical activities of the mutant NS3h were similar to the wild type enzyme, which was not reflective of the large reduction in HCV replicative capacity observed in the biological experiment. Hence, the biological results suggest that the known biochemical properties associated with the helicase activity of NS3h do not reveal all of the likely biological roles of NS3 during HCV replication. Domain 3 of NS3 is implicated in protein-protein interactions that are necessary for HCV replication. Hepatitis C virus (HCV),5 a member of the family Flaviviridae, is a leading cause of liver cirrhosis and hepatocellular carcinoma and has infected more than 170 million people worldwide (1-3). Standard treatment with interferon-␣ and ribavirin is frequently ineffective or toxic, and no vaccine for HCV is currently available (4). Identification of a novel viral target for therapeutic intervention could lead to the development of a more effective treatment.The HCV genome is a 9.6-kb positive, single-stranded RNA that supports translation of a 3000-amino acid polyprotein, which is subsequently cleaved to produce both structural (C, E1, and E2) and nonstructural (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) viral proteins (5). Two viral proteases are encoded by the NS2 and NS3 genes (6, 7). In addition to its protease domain, the NS3 protein also contains a DEXH/ D-box helicase domain (8). NS4A encodes a peptide co-factor for NS3 protease (9), and NS5B encodes an RNA-dependent RNA polymerase (10). The functions of NS4B and NS5A have not been as well characterized, but NS5A is involved in viral interferon sensitivity (11) and in adaptation of subgenomic replicons for growth in cultured hepatoma cells (12).One obstacle to studying viral protein interactions is that HCV cannot be maintained efficiently in cell culture, although recent progress may overcome this difficulty (13). In order to circumvent this problem, selectable replicons carrying the neomycin phosphotransferase gene in add...

Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism

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“…Further analysis reveals different sugar puckers and base orientations among the bound DNA residues that had not been observed previously (7,8) (Fig. 5 and Figs.…”

“…It has polynucleotide-stimulated (d)NTPase activity (4) and can unwind both RNA and DNA in a 3′-5′ direction (5,6). Structures of NS3h in complex with single-stranded DNA (ssDNA) have shown that DNA binds in a groove between domain 3 and the RecA-like domains, with the bases stacked in a row between two "bookend" residues (V432 in the domain 2 β-hairpin and W501 in domain 3) (7,8). The structure of the substrate DNA remains ambiguous, however, as the pucker conformations assigned to the sugar groups differ between reports.…”

Flaviviral helicase: Insights into the mechanism of action of a motor protein

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