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

Abstract

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 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 worldwide (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 ...

Conserved Determinants for Membrane Association of Nonstructural Protein 5A fromHepatitis C Virus and Related Viruses

“…2A). These features are very reminiscent of the previously reported characteristics of an HCV NS5A 1-31 peptide (8) and suggest that the GBV-C NS5A 1-27 peptide forms micelle-like oligomers that interact by hydrophobic interactions. In contrast, at neutral pH in water, the amplitude of the CD signal strongly decreased and the spectral shape indicated a mixture of poorly defined conformations, including alpha helix structures, suggesting some aggregation.…”

“…5, the staining pattern of C-terminally tagged NS5A proteins from GBV-C and BVDV was similar to that of the GFP fusion constructs (Fig. 3) and to that previously described for HCV NS5A (8). Unexpectedly, immunofluorescent staining of the C-terminally tagged GBV-B NS5A construct revealed in most cells a diffuse pattern (data not shown), conflicting with the data obtained with the GFP fusion proteins described above.…”

“…We recently reported that the N terminus of HCV NS5A comprises a unique membrane anchor domain that is folded into an amphipathic alpha helix embedded in plane into the cytoplasmic leaflet of the membrane bilayer (8,46). We also reported that the N terminus of BVDV NS5A as well includes an amphipathic alpha-helix sharing similar membrane interaction features (50).…”

“…In this context, we and others have shown that an N-terminal amphipathic alpha helix mediates the membrane association of HCV NS5A (8,19). Targeting to the endoplasmic reticulum (ER) or an ER-derived modified compartment occurred via a posttranslational mechanism (8). Analysis of the three-dimensional structure of the membrane segment by nuclear magnetic resonance (NMR) spectroscopy revealed an amphipathic alpha helix that is embedded in plane into the cytosolic leaflet of the membrane bilayer (46).…”

“…In addition, the determinants for membrane association of the nonstructural proteins involved in HCV RNA replication have been mapped (reviewed in reference 41). In this context, we and others have shown that an N-terminal amphipathic alpha helix mediates the membrane association of HCV NS5A (8,19). Targeting to the endoplasmic reticulum (ER) or an ER-derived modified compartment occurred via a posttranslational mechanism (8).…”