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Nonstructural protein 4B (NS4B) plays an essential role in the formation of the hepatitis C virus (HCV) replication complex. It is an integral membrane protein that has been only poorly characterized to date. It is believed to comprise a cytosolic N-terminal part, a central part harboring four transmembrane passages, and a cytosolic C-terminal part. Here, we describe an amphipathic α-helix at the C terminus of NS4B (amino acid residues 229 to 253) that mediates membrane association and is involved in the formation of a functional HCV replication complex.
Hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively poorly characterized 27-kDa integral membrane protein (14, 23). It plays a central role in the formation of the membranous web, a likely endoplasmic reticulum (ER)-derived membrane alteration that harbors the HCV replication complex (7, 11). In addition, it has been reported to possess NTPase (8, 30) and RNA binding activities (9).
HCV NS4B comprises an N-terminal part (amino acids [aa] 1 to ~69), a central part harboring four putative transmembrane passages (aa ~70 to 194), and a C-terminal part (aa ~195 to 261) (14, 18, 19, 26). Both the N and the C termini of NS4B are believed to be oriented toward the cytosol. An amphipathic α-helix in the N-terminal part, represented by amino acid residues 42 to 66, has recently been shown to mediate membrane association (12) and to be translocated through the membrane at least partially (12, 19). However, a precise membrane topology of NS4B is thus far elusive. Here, we describe an amphipathic α-helix at the C terminus of NS4B that mediates membrane association and is involved in the formation of a functional HCV replication complex.
Sequence analyses and predictions of secondary structure and lipotropic properties were performed to assess the degree of conservation of the C-terminal part of NS4B and to identify motifs potentially involved in membrane association. The amino acid repertoire deduced from the analysis of 27 reference sequences representative for the major HCV genotypes and subtypes (Fig. (Fig.1A)1A) coupled with secondary structure predictions revealed two putative α-helices, represented by residues ~201 to 212 and ~228 to 253, respectively (Fig. (Fig.1B).1B). The second predicted α-helix displayed marked hydrophobicity, as calculated using the algorithm developed by Wimley and White (32), indicating a propensity for this segment to partition into a lipid bilayer (Fig. (Fig.1C).1C). From these analyses, we concluded that the segment comprising NS4B aa ~228 to 253 could potentially fold into a membrane-associated α-helix.
A panel of green fluorescent protein (GFP) fusion constructs harboring NS4B segments derived from the HCV H77 consensus clone (16) (kindly provided by Charles M. Rice, The Rockefeller University, New York, NY) was prepared as described previously (12) and analyzed by fluorescence microscopy and differential membrane extraction to validate the above-mentioned predictions. As shown in Fig. Fig.2A2A and as described previously (10, 12, 13, 18, 19), fluorescence microscopy of U-2 OS cells transiently transfected with full-length NS4B-GFP revealed a fluorescence pattern that included the nuclear membrane, was strongest in the perinuclear region, and extended in a reticular fashion throughout the cytoplasm. This pattern has previously been shown to correspond to that of ER and likely to those of ER-derived modified membranes. The GFP fusion construct comprising the entire C-terminal part of NS4B (aa 195 to 261) revealed a membrane-associated fluorescence pattern corresponding primarily to mitochondria, as confirmed by colocalization with mitofilin (data not shown). Interestingly, a similar construct (aa 195 to 254), which lacks the recently described palmitoylation sites Cys 257 and Cys 261 (34), displayed the same pattern, indicating that C-terminal palmitoylation is dispensable for membrane association of the C-terminal part of NS4B. The construct harboring the first predicted α-helix (aa 198 to 219) showed a diffuse fluorescence pattern indistinguishable from that of GFP alone, indicating a lack of membrane association for this segment. In contrast, fusion of aa 227 to 254, comprising the second predicted α-helix, to GFP resulted in a membrane-associated fluorescence pattern which partially colocalized with both protein disulfide isomerase and mitofilin as markers of ER and mitochondrial membranes, respectively (Fig. (Fig.2B).2B). These results demonstrate the presence of a novel determinant for membrane association between NS4B residues 227 and 254.
Differential membrane extractions followed by flotation assays were performed as described previously (21) to further characterize the membrane association mediated by this segment. Briefly, membrane fractions were isolated by ultracentrifugation of hypotonic lysates from U-2 OS cells transiently transfected with construct NS4B aa 227 to 254-GFP (NS4B227-254-GFP), followed by differential extraction of the pellet fraction and equilibrium centrifugation in Nycodenz gradients. NTE (100 mM NaCl, 10 mM Tris·HCl [pH 8.0], 1 mM EDTA) was used as a physiologic buffer, while high-salt (1 M NaCl) and alkaline (100 mM sodium carbonate, pH 11.5) extractions were used to release peripheral proteins from membranes. As shown in Fig. Fig.2C,2C, the bulk of the GFP fusion construct comprising the segment containing NS4B aa 227 to 254 remained in the membrane fraction under physiological conditions as well as after high-salt and alkaline extractions. Only detergent extraction (1% Triton X-100) disrupted membrane association. Taken together, these observations indicate that NS4B aa 227 to 254 mediate tight membrane association.
To gain insights into the structure and lipotropic properties of the segment comprising NS4B aa 227 to 254, the corresponding peptide, designated NS4B[227-254], was chemically synthesized, purified, and analyzed by circular dichroism (CD) and nuclear magnetic resonance (NMR), as described previously (see reference 12 and references therein). The CD spectrum of this peptide that is poorly soluble in water indicates mainly a random coil conformation (~60%), with the presence of some undefined secondary structures (Fig. (Fig.3A).3A). However, solubilization in membrane mimetic media, including detergents (n-dodecyl phosphocholine [DPC], n-dodecyl β-d-maltoside, and sodium dodecyl sulfate [SDS]), a lysophospholipid (α-lysophosphatidyl choline [LPC]), or a 2,2,2-trifluoroethanol (TFE)-water mixture, resulted in spectra typical of an α-helix. An α-helix content of 86% was calculated in detergents and LPC and of 92% in 50% TFE. These results indicate a high propensity of NS4B[227-254] to interact with lipids and to adopt an α-helical structure upon lipid binding.
NS4B[227-254] samples prepared in deuterated SDS and DPC micelles displayed broad, poorly resolved NMR spectra, possibly as a consequence of peptide oligomerization. Therefore, the NMR structure was solved in 50% TFE-d2, which yielded well-resolved homo- and heteronuclear multidimensional spectra (data not shown). Sequential attribution of all spin systems was complete, and an overview of the sequential and medium range nuclear Overhauser enhancement (NOE) connectivities as well as the deviation of 1Hα and 13Cα chemical shifts from random coil values are shown in Fig. 3B, C, and D, respectively. These data clearly demonstrate that the main body of the peptide, comprising residues 229 to 253, is folded as an α-helix. On the basis of NOE-derived interproton distance constraints and of dihedral angle constraints calculated with TALOS (5) from 1Hα and 13Cα chemical shifts, a set of 50 structural models of NS4B[227-254] was calculated. Superimposition of the 27 structures that fully satisfied the experimental NMR data shows that the main part of the α-helix is well defined, with a root mean square deviation of 0.92 Å (Fig. (Fig.3E;3E; see also Table S1 in the supplemental material).
This α-helix displays several remarkable features, including two highly hydrophobic patches constituted by conserved Leu residues in amino acid positions 237 and 240 as well as 246 and 249, an amphipathic, positively charged C-terminal portion (residues 246 to 253) (Fig. 3F to H), and an unusually high number of conserved Thr and Ser residues in the center of the helix (residues 238 to 243). The amphipathic character, the positively charged residues, and the typical Trp interface residue (33), all of which are absolutely conserved among different HCV genotypes (Fig. (Fig.1A),1A), suggest that the C-terminal half of the α-helix represents the main determinant for membrane association and likely interacts with the membrane interface in an in-plane topology, at least transiently. Further studies, including molecular dynamics simulations, will be necessary to determine the precise positioning and topology of this helix in the membrane. Also, it is tempting to speculate that the conserved Thr and Ser residues in the center of the helix may play a role in the sensing of membrane curvature (6) and, thereby, in membranous web formation.
To explore the role of the conserved Leu residues in the membrane association and function of NS4B α-helix residues 229 to 253, they were replaced by Ala, as illustrated in Fig. Fig.4A.4A. These mutations are expected to abrogate the hydrophobic patches while preserving the overall α-helical fold, as described previously (2, 12). Their impact on membrane association of the segment comprising NS4B aa 227 to 254 fused to GFP was examined by fluorescence microscopy and membrane flotation analyses, as shown in Fig. 4B and C, respectively. Note that, in contrast to the assay shown in Fig. Fig.2B,2B, which represents a differential extraction starting from a membrane preparation, the assay shown in Fig. Fig.4C4C assesses the global distribution of the fusion proteins. Under these conditions, full-length NS4B fused to GFP is found entirely in the upper fractions (representing membrane-associated material), whereas GFP alone is found entirely in the lower fractions (representing non-membrane-associated material) (12). Interestingly, pairwise Ala mutagenesis (constructs AALL and LLAA) only slightly reduced membrane association, while Ala replacement of the four Leu residues (construct AAAA) resulted in almost complete loss of membrane association. Thus, these Leu residues play an important role in membrane association of α-helix residues 229 to 253.
We previously characterized cell lines inducibly expressing the entire HCV polyprotein derived from the HCV H77 prototype and consensus clones (22, 27). In these cells, HCV nonstructural proteins accumulate in dot-like structures which at the ultrastructural level correspond to membranous webs (7). The membranous webs formed in these cells are very similar to the ones observed in Huh-7 cells harboring HCV replicons and represent viral replication complexes (11). To explore the impact of the Ala substitutions in NS4B α-helix residues 229 to 253 on dot-like structure formation as a correlate of membranous web formation, the mutants were expressed in the context of the entire H77 consensus clone polyprotein, as described previously (22, 27). Immunoblotting confirmed that polyprotein processing was intact in the different mutant constructs (data not shown). Pools of stably transfected U-2 OS cells inducibly expressing the different mutants in the context of the HCV polyprotein were analyzed by double-label immunofluorescence microscopy 48 h after tetracycline withdrawal. As shown in Fig. Fig.4D4D and as previously described for the wild-type construct (2, 7, 11, 12), NS4B and NS5A colocalized in cytoplasmic dot-like structures. Interestingly, the dot-like structures appeared much smaller in the AALL and LLAA mutants, and they were no longer detectable in the AAAA mutant. Similar results were obtained when J6/JFH-1 (Jc1)-derived HCV polyproteins harboring these Ala substitutions were expressed in Huh-7 cells (data not shown). However, the targeting of core and NS5A to lipid droplets, which are abundant in Huh-7 cells, rendered the identification of dot-like structures as a correlate of membranous web more difficult in this system. While a detailed ultrastructural analysis of the Ala substitution and additional mutants will be required, electron microscopy confirmed the lack of membranous web formation in cells expressing the AAAA mutant (Philippe Roingeard, J. Gouttenoire, and D. Moradpour, unpublished data). Taken together, these results suggest that NS4B α-helix residues 229 to 253 play an important role in the formation of the HCV replication complex.
The impact of these mutations on HCV RNA replication was examined using a JFH-1-derived subgenomic replicon harboring a firefly luciferase reporter gene (1). As shown in Fig. Fig.4E,4E, the LLAA mutant was only slightly impaired, while the AALL and AAAA mutants showed a severe replication defect. In fact, the latter mutants did not yield any viable clones when introduced into a selectable subgenomic replicon (data not shown). While these results do not prove that membrane association of NS4B α-helix residues 229 to 253 is essential for HCV RNA replication, they suggest that Leu residues 237 and 240 may be implicated in additional processes besides membrane association, e.g., in protein-protein (34) or protein-RNA (9) interactions required for replication. This is consistent with our observations reported previously for the membrane segments of HCV NS4A, NS5A, and NS5B, indicating that these segments have overlapping and essential functions in the assembly of a functional replication complex (2, 21, 24).
In conclusion, membrane association of HCV NS4B is mediated not only by transmembrane domains in its central part but also by determinants for membrane association in the N- (12) and C-terminal (this report; a very recent report by Liefhebber et al. indicating a role for electrostatic interactions in membrane association of this segment ) parts. Conserved hydrophobic residues in α-helix residues 229 to 253 are required for membrane association of this segment and the formation of a functional replication complex. Further studies are required to elucidate the mechanistic role of the NS4B C-terminal α-helix in HCV RNA replication and to resolve the complete membrane topology and structure of NS4B.
The atomic coordinates of NS4B[227-254] and the NMR restraints in 50% TFE are available in the Research Collaboratory for Structural Bioinformatics Protein Data Bank under accession code 2KDR. The proton chemical shifts of all residues have been deposited in the BioMagResBank under accession number 16122.
We gratefully acknowledge Philippe Roingeard for sharing unpublished data, Pantxika Bellecave for discussions, Ralf Bartenschlager, Jan Albert Hellings, and Charles M. Rice for reagents, and Eric Diesis for peptide synthesis. CD experiments were performed on the platform Production et Analyse de Protéines of the IFR 128 BioSciences Gerland-Lyon Sud.
This work was supported by the Swiss National Science Foundation (grants 3100A0-107831 and 3100A0-122447), the Swiss Cancer League/Oncosuisse (grant OCS-01762-08-2005), the French Centre National de la Recherche Scientifique (CNRS), the Agence Nationale pour la Recherche sur le SIDA et les Hépatites Virales (ANRS), the program Biotherapeutics of Lyon Biopole, and the European Commission through the EMBRACE project (LHSG-CT-2004-512092).
Published ahead of print on 19 August 2009.
†Supplemental material for this article may be found at http://jvi.asm.org/.