Crystal Structure of a Novel Dimeric Form of NS5A Domain I Protein from Hepatitis C Virus

Abstract

Hepatitis C virus (HCV) is a member of the Flaviviridae family of enveloped, positive-strand RNA viruses (23). It is responsible for persistent infections in humans, with associated risk of chronic liver diseases, including cirrhosis and hepatocellular carcinoma. Nearly 3% of the global population is chronically infected with HCV, and there are no clinically proven vaccines. Antiviral therapeutic agents are at an early stage of clinical evaluation, and standard treatments (interferon and ribavirin combinations) are associated with suboptimal response rates and/or high incidence of side effects. Complicating the discovery of new therapies is the highly complex and incompletely understood nature of the viral life cycle. The HCV genome consists of a single strand of RNA of about 9,600 nucleotides encoding a polypeptide precursor of about 3,000 amino acids (26). Co-and posttranslational proteolytic cleavage of this precursor by cellular and viral enzymes yields structural proteins involved in viral assembly, along with nonstructural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B, which are required for membrane-associated RNA replication (14).Nonstructural protein NS5A is a critical component of HCV replication and is involved in several cellular processes, such as interferon resistance (3, 13) and apoptotic regulation (9). It is a phosphoprotein of 447 residues with three domains (35), and while no clear enzymatic functions have been assigned, it appears to function through interactions with other HCV proteins and host cell factors (17). Domain I (residues 1 to 213) contains a zinc-binding motif (35) and an amphipathic N-terminal helix which promotes membrane association (4, 12, 30), possibly through specific interaction of the helix with target membrane proteins (8). Domain II (residues 250 to 342) has regulatory functions, such as interactions with protein kinase PKR and PI3K (13), as well as NS5B (32); contains the interferon sensitivity-determining region (13); and appears to lack major elements of secondary structure (22). Recent studies have demonstrated that domain III (residues 356 to 447) plays a critical role in infectious virion assembly but not in RNA replication (1,34) and that the former role is modulated by phosphorylation within the domain (33). High-throughput screening of small-molecule inhibitors using HCV replicon cell systems has identified NS5A as a promising therapeutic target (31).A crystal structure of domain I lacking the amphipathic helix and spanning residues 25 to 215 showed two subdomains and a homodimeric association and was interpreted as having a potential role in RNA binding (36). Although specific binding to domain I was not described, RNA binding to full-length NS5A has been reported, using, for example, the 3Ј nontranslated region of HCV (15). Efforts in our laboratory to study the structure of NS5A have yielded an alternative arrangement of the domain I homodimer (residues 33 to 202) that differs substantially from that previously described. The observation that the NS5A do...

HCV NS5A Inhibitors Disrupt Replication Factory Formation: A Novel Mechanism of Antiviral Action

“…24 As NS5A expression alone induces DMV formation, 22 it may also participate more directly in membrane rearrangements beyond roles in enrichment of PI4P and cholesterol. It is possible that the alternative dimer forms of NS5A coexist and form a superhelical polymer, as suggested by Love et al, 10 such that membrane bending during DMV formation is the result of membrane anchorage of NS5A by its N-terminal amphipathic helix and NS5A oligomerization. The presence of inhibitor-bound NS5A molecules could "lock" a particular dimer form, prevent NS5A oligomerization, and/or distort NS5A oligomers to disrupt DMV formation and ultimately prevent de novo formation of functional RCs.…”

“…In this context, the class-defining resistance site Y93 is located at opposing, membrane-proximal surfaces of the dimer interface for both "back-to-back" and "clam-like" alternative domain I (genotype 1b) crystal structures. 10,11 Third, biotin-tagged DCV derivatives enable precipitation of NS5A from pretreated HCV replicon-harboring cells, 6,12 although interestingly fail to precipitate NS5A from replicon lysates or pretreated NS5A-overexpressing cells. 12 In this context, it is noteworthy that preformed replication complexes (RCs) are refractory to inhibition of HCV RNA replication by NS5A inhibitors.…”

Correlation between NS5A Dimerization and Hepatitis C Virus Replication

“…We hypothesize that HCV RNA replication is impeded by the prevention of NS5A dimer formation. The N-terminal Domain I of NS5A has been shown in crystallography structural models to be dimeric in nature (29,31). In addition, this domain has been shown to be most conserved among genotypes with Zn 2ϩ binding and RNA binding activities (28 -30).…”

“…However, the ability to bind specifically to uridine-and guanosine-rich RNA resides in domain I and the adjacent linker region (33). An alternative structure of Domain I has been determined that is also dimeric in nature, although the interface lacks the large groove or any obvious RNA interaction surface (31). The most advanced inhibitor of NS5A, BMS 790052, seems to bind NS5A at its dimer interface based on current models of NS5A structure and analysis of drug resistance mutants (32).…”

Inhibitors of HCV NS5A: From Iminothiazolidinones to Symmetrical Stilbenes

“…5 Interestingly, the discovery of symmetry within HCV NS5A inhibitors coincides well with X-ray crystallographic data subsequently published for the amino terminus of the NS5A protein. 30,31 In one report, NS5A residues 36-198 crystallized as a dimer forming a U-shaped structure with the inner surface lined with a preponderance of basic amino acids proposed to form a binding site for viral RNA. 30 The residues conferring resistance to inhibitor 55 are proximal to the amino terminus of NS5A and are located between the protein and the membrane, suggesting that the symmetrical compounds bind across the dimer interface.…”