Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a membrane-associated, essential component of the viral replication complex. Here, we report the three-dimensional structure of the membrane anchor domain of NS5A as determined by NMR spectroscopy. An ␣-helix extending from amino acid residue 5 to 25 was observed in the presence of different membrane mimetic media. This helix exhibited a hydrophobic, Trprich side embedded in detergent micelles, while the polar, charged side was exposed to the solvent. Thus, the NS5A membrane anchor domain forms an in-plane amphipathic ␣-helix embedded in the cytosolic leaflet of the membrane bilayer. Interestingly, mutations affecting the positioning of fully conserved residues located at the cytosolic surface of the helix impaired HCV RNA replication without interfering with the membrane association of NS5A. In conclusion, the NS5A membrane anchor domain constitutes a unique platform that is likely involved in specific interactions essential for the assembly of the HCV replication complex and that may represent a novel target for antiviral intervention.
Hepatitis C virus (HCV)1 infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma worldwide (1). HCV contains a 9.6-kb single-stranded RNA genome of positive polarity that encodes a polyprotein precursor of about 3,000 amino acids (for review, see Refs. 2-4). The polyprotein precursor is co-and post-translationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. The structural proteins include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins include the NS2-3 autoprotease and the NS3 serine protease, an RNA helicase located in the C-terminal region of NS3, the NS4A polypeptide, the NS4B and NS5A proteins, and the NS5B RNA-dependent RNA polymerase. As in all positive-strand RNA viruses, the nonstructural proteins form a membrane-associated replication complex together with replicating viral RNA, altered membranes, and additional as yet unidentified host cell components (5-8). Determinants for membrane association of the HCV nonstructural proteins have been mapped (for review, see Ref. 9), but the protein-protein interactions involved in formation of a functional HCV replication complex are poorly understood.HCV NS5A is a phosphoprotein of unknown structure and function (10, 11). It is found in a basally phosphorylated form of 56 kDa and in a hyperphosphorylated form of 58 kDa. NS5A has attracted considerable interest because of its potential role in modulating the interferon response (for review, see Ref. 12), and numerous additional functions have recently been ascribed to this protein (for review, see Ref. 13). Interestingly, cell culture-adaptive changes cluster in the central portion of NS5A in the context of selectable subgenomic HCV replicons (14, 15), indicating that NS5A is involved, either directly or by interaction with cellular proteins, in the viral replication process. This observati...
