An Amino-terminal Amphipathic α-Helix Mediates Membrane Association of the Hepatitis C Virus Nonstructural Protein 5A

Volker Brass, Elke Bieck, Roland Montserret, Benno Wölk, Jan Albert Hellings, Hubert E. Blum, François Penin, Darius Moradpour
2001 Journal of Biological Chemistry  
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A), a phosphoprotein of unknown function, is believed to be a component of a membrane-associated viral replication complex. The determinants for membrane association of NS5A, however, have not been defined. By double label immunofluorescence analyses, NS5A was found to be associated with the endoplasmic reticulum (ER) or an ER-derived modified compartment both when expressed alone or in the context of the entire HCV polyprotein. Systematic
more » ... tein. Systematic deletion and green fluorescent protein fusion analyses allowed us to map the membrane anchor to the amino-terminal 30 amino acid residues of NS5A. Membrane association occurred by a posttranslational mechanism and resulted in properties of an integral membrane protein. Circular dichroism structural studies of a synthetic peptide corresponding to the NS5A membrane anchor, designated NS5A(1-31), demonstrated the presence of an amphipathic ␣-helix that was found to be highly conserved among 280 HCV isolates of various genotypes. The detergent-binding properties of this helical peptide together with the nature and location of its amino acids suggest a mechanism of membrane insertion via the helix hydrophobic side, yielding a topology parallel to the lipid bilayer in the cytoplasmic leaflet of the ER membrane. These findings have important implications for the structural and functional organization of the HCV replication complex and may define novel targets for antiviral intervention. Hepatitis C virus (HCV) 1 infection is a major cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma world-wide (1). A protective vaccine does not exist to date, and therapeutic options are still limited (2, 3). HCV has been classified in the Hepacivirus genus within the Flaviviridae family which includes the classical flaviviruses, such as yellow fever virus, and the animal pestiviruses, such as bovine viral diarrhea virus (BVDV) (4). The structure and replication cycle of HCV are incompletely understood due to the low viral titers found in sera and livers of HCV-infected individuals and the lack of an efficient cell culture system or small animal model permissive for HCV infection. Nevertheless, considerable progress has been made using heterologous expression systems, functional cDNA clones, and more recently, selectable subgenomic replicons (see Refs. 5 and 6 for recent reviews). HCV contains a single-stranded RNA genome of positive polarity and ϳ9600 nucleotides (nt) length that encodes a polyprotein precursor of about 3000 amino acids (aa) (Fig. 1A) . The polyprotein precursor is co-and posttranslationally processed by cellular and viral proteases to yield the mature structural and nonstructural proteins. The structural proteins include the core protein, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The non-structural proteins NS2 through NS5B include the NS2-3 autoprotease and the NS3 serine protease, an RNA helicase located in the carboxyl-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 viral nonstructural proteins are believed to form a membrane-associated replication complex together with as yet unidentified host cell components. However, the determinants for membrane association and the protein-protein interactions involved in formation of the HCV replication complex are poorly understood. The aim of this study was to investigate the subcellular localization and mechanism of membrane association of the HCV NS5A protein. NS5A is a phosphoprotein of unknown function (7, 8) . It is found in a basally phosphorylated form of 56 kDa and in a hyperphosphorylated form of 58 kDa. NS5A of HCV and BVDV as well as NS5 of yellow fever virus are phosphorylated by as yet unidentified serine/threonine kinases, suggesting that these proteins share a common function related to their phosphorylation state (9). NS5A has attracted considerable interest because of its potential role in modulating the interferon response (reviewed in Ref. 10). Numerous additional potential functions have been described recently in heterologous expression systems (11-13). However, the relevance of these observations for the natural course and pathogenesis of hepatitis C remains to be established. Interestingly, adaptive mutations have been found in NS5A in the context of selectable subgenomic HCV replicons, suggesting that NS5A is involved, either directly or by interaction with cellular proteins and pathways, in the viral replication process. This observation,
doi:10.1074/jbc.m111289200 pmid:11744739 fatcat:sgqwfwsubvfu3gdqmu3taklhai