Molecular Determinants for Subcellular Localizatio
http://www.100md.com
病菌学杂志 2005年第2期
Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku
Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Minato-ku
Department of Pathology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo
Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita-shi, Osaka
Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki-shi, Nagasaki, Japan
ABSTRACT
Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic -helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.
INTRODUCTION
Hepatitis C virus (HCV), the most important causative agent of posttransfusion and sporadic non-A, non-B hepatitis, is a positive-stranded RNA virus belonging to the family Flaviviridae (7). A precursor polyprotein of about 3,000 amino acids is encoded by a large open reading frame of the genome and undergoes cellular and viral protease-mediated posttranslational modification to produce a series of structural and nonstructural proteins (8, 13, 16).
HCV core protein, which is derived from the N terminus of the viral polyprotein, forms multimers and interacts physically with the viral RNA to constitute the nucleocapsid (28, 47, 50). Tissue transglutaminase is responsible for stabilizing the core protein by cross-linking it into a dimeric form (26). In addition, the core viral protein has properties which enable it to modulate a number of cellular processes, including transcription, inhibition or stimulation of apoptosis, and suppression of host immunity, as reviewed previously (21, 29, 51, 52). Several studies suggest that expression of the core protein affects mitochondrial function and lipid metabolism. The core protein increases the cellular production of reactive oxygen species with subsequent increases in lipid peroxidation (35, 39). The viral protein also colocalizes with human apolipoprotein AII, associates with lipid droplets, and has the capacity to influence metabolic events involving lipid storage (2, 17, 30, 36, 44). In addition, the core protein reduces microsomal triglyceride transfer, leading to defects in very low density lipoprotein assembly and secretion (40). Furthermore, the HCV core protein has transforming potential in some cells under certain conditions (5, 42). Transgenic mice expressing this protein in the liver develop hepatic steatosis due to increased oxidative stress in the absence of inflammation, with subsequent development of hepatocellular carcinoma (34, 36). These results suggest that the HCV core protein might play a pivotal role in the pathogenesis of hepatitis C in addition to its role as a structural component of the viral capsid.
The amino acid sequence of the core protein is well conserved among different HCV isolates and genotypes compared to other HCV proteins. The N-terminal domain of the HCV core protein is highly basic, while its C terminus is hydrophobic. Although several core proteins of various sizes exist (17 to 23 kDa) (15, 23, 25, 49, 56), two processing events result in the predominant production of a 21-kDa core protein. Both of these events utilize the endoplasmic reticulum (ER). The first one is to be cleaved from downstream envelope protein E1 at position 191, where the C-terminal hydrophobic domain serves as a putative signal peptide sequence. Subsequently, the signal sequence of 13 or 18 residues is processed by signal peptide peptidase (19, 23, 56).
The HCV core protein is found primarily within the membranes of cytoplasmic organelles, but it is also found in the nucleus (23, 48, 56). Immunofluorescence studies show a punctate pattern, consistent with ER localization, as well as perinuclear localization (15, 24, 32, 46, 56). Some studies suggest direct effects of the core protein on mitochondrial function. In fact, the core protein localizes to the mitochondria (34, 39). The N-terminal domain of the core protein contains three stretches of arginine- and lysine-rich sequences. Translocation of the core protein to the nucleus, mediated by these basic-residue stretches which function as nuclear localization signals (NLSs), is observed (6, 48). In addition, Moriishi et al. demonstrated that the N-terminal region of the core protein is also essential for nuclear retention through its interaction with the proteasome activator PA28 (33).
In this study, we found a region that is important for localization of the mature core protein to the ER and to the mitochondrial outer membrane. We also identified a novel bipartite NLS responsible for nuclear targeting of the core protein, presumably via an importin-dependent pathway.
MATERIALS AND METHODS
Plasmid construction. The construction of a plasmid expressing the full-length core protein of 191 amino acids, pCAGC191, was described previously (49). pGFP, a construct expressing green fluorescent protein (GFP) with a C-terminal Myc epitope tag sequences, was prepared as follows. pCMV/Myc/mito/GFP (Invitrogen Corp., Carlsbad, Calif.) was digested with PmlI, followed by treatment with the Klenow fragment of DNA polymerase I. The resultant linear fragment was ligated to a PstI linker (GCTGCAGC) and digested with PstI to remove the mitochondrial targeting signal sequence, followed by self-ligation. A series of HCV core-GFP fusion constructs were made by amplifying the core gene fragments with PCR with primers containing Flag epitope tag sequences (sense) and a PstI site (both). After digestion with PstI, the segments were inserted into the PstI site of pGFP. A series of GFP-core-E1 fusion constructs were made by amplifying core and E1 gene fragments with PCR with primers containing a NotI site. After digestion with NotI, the segments were inserted into the NotI site of pGFP.
pGEX-4T-1 (Amersham Bioscience Corp., Piscataway, N.J.) was used to express core protein fused with glutathione S-transferase (GST) in Escherichia coli. Core cDNA fragments encoding amino acids 1 to 71 were inserted into the EcoRI site of pGEX-4T-1. Alanine substitutions were introduced into the core protein by PCR mutagenesis with primers containing base alterations. The PCR products were then cloned into pCR2.1 (Invitrogen Corp.) and verified by DNA sequencing. Individual cDNAs were excised and inserted separately into pGFP or pGEX-4T-1. The primer sequences used in this study are available from the authors upon request.
Plasmid pRSET-hSRP1 (54), containing importin- cDNA under the control of a T7 promoter, was kindly provided by Karsten Weis (University of California, Berkeley). A cDNA clone of importin- possessing 14 residues (MYPYDVPDYGGGGS), derived in part from the hemagglutinin (HA) tag at the N terminus, was constructed by PCR. The resultant linear fragment was inserted under the control of a CAG promoter of pCAGGS and designated pCAG-HA-imp.
Cell culture and transfection. Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units of penicillin per ml, 100 μg of streptomycin per ml, and 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Monolayers of 293T cells were transfected with plasmid DNA in the presence of Lipofectamine (Gibco-BRL, Life Technologies, Gaithersburg, Md.) according to the manufacturer's instructions.
Confocal immunofluorescence microscopy. Transfected cells were grown on glass coverslips. Two days after transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. Intracellular localization of HCV core-GFP fusion proteins was visualized in cells transfected with a variety of GFP fusion constructs.
In order to detect the HCV core protein by immunofluorescence, fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 3 min at room temperature, followed by blocking with a nonfat milk solution (Block Ace; Snow Brand Milk Products Co., Sapporo, Japan). The cells were then incubated with anticore monoclonal antibody B2 (Anogen, Mississauga, Canada) for 60 min at room temperature, followed by incubation with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin G (IgG) (ICN Pharmaceuticals, Aurora, Ohio) for 45 min. To visualize mitochondria, MitoTracker Red CM-H2XRos (Molecular Probes, Eugene, Oreg.) was added to the culture medium to a final concentration of 100 nM and incubated for 120 min at 37°C prior to fixation. To visualize the ER, goat anticalregulin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) and rhodamine-conjugated rabbit anti-goat IgG (ICN Pharmaceuticals) were used as the first and second antibodies, respectively. To visualize HA-importin-, mouse anti-HA antibody (Roche Molecular Biochemicals, Indianapolis, Ind.) and rhodamine-conjugated goat anti-mouse IgG (ICN Pharmaceuticals) were used as the first and second antibodies, respectively. All specimens were examined with an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
Immunoelectron microscopy. Cells were transfected as described above. After 2 days, cells were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PBS (pH 7.4). Free aldehyde groups were quenched with 50 mM NH4Cl in PBS. The cell pellets were embedded at progressively lower temperatures (down to –35°C) in Lowicryl k4M according to an established protocol (43). Ultrathin sections were prepared and mounted on carbon-coated nickel grids. To perform electron microscopy, Lowicryl k4M ultrathin sections, mounted on grids, were floated on a droplet of PBS containing 1% bovine serum albumin, 0.1% Triton X-100, and 0.1% Tween 20 for 10 min, after which they were exposed to droplets of mouse anticore monoclonal antibody (Anogen) diluted in PBS for 45 min. Following this, they were rinsed twice for 5 min each in PBS and incubated with anti-mouse IgG-coated 10-nm immunogold particles (British Biocell, Cardiff, United Kingdom) for 45 min. After rinsing with PBS and distilled water, the grids and embedded sections were air dried and exposed to uranyl and lead acetate contrast agents.
Subcellular fractionation. All steps were performed at 4°C in the presence of a protease inhibitor cocktail called Complete (Roche Molecular Biochemicals). To isolate the ER fraction, transfected cells were washed with PBS, lysed in homogenization buffer A (50 mM Tris-HCl [pH 8.0], 1 mM ?-mercaptoethanol, 1 mM EDTA, and 0.32 M sucrose), and then centrifuged at 5,000 x g for 10 min. The supernatant was then collected and centrifuged at 105,000 x g for 1 h. The pellet was disrupted in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and 10 mM sodium fluoride), after which it was centrifuged at 15,000 x g for 20 min. The resulting supernatant was used as the ER fraction.
To isolate the mitochondrial fraction, transfected cells were washed with PBS and homogenized in ice-cold homogenization buffer B (200 mM mannitol, 50 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl) at pH 7.4. The supernatant was then centrifuged at 1,000 x g for 10 min to remove large debris and nuclei. The resulting supernatant was then centrifuged at 20,000 x g for 20 min to obtain crude mitochondria. The crude mitochondria pellet was subfractionated in Nycodenz gradients for further purification of mitochondria. Nycodenz (Axis-Shield PoC AS, Oslo, Norway) solution at 50% (wt/vol) was prepared in buffer containing 5 mM Tris-HCl and 1 mM EDTA at pH 7.4. This stock solution was then diluted with buffer containing 0.25 M sucrose, 5 mM Tris-HCl, and 1 mM EDTA at pH 7.4 before use. The crude mitochondrial pellets was suspended in 4 ml of 25% Nycodenz solution and overlaid onto the following discontinuous Nycodenz gradients: 1 ml of 40%, 1 ml of 34%, and 2 ml of 30%. The samples were topped off with 2 ml of 23% Nycodenz solution after placement onto the discontinuous gradients. The tubes were then centrifuged at 52,000 x g for 90 min. The dense band seen after centrifugation at the 25 to 30% interface was recovered as the purified mitochondrial fraction.
To determine the submitochondrial localization pattern of the core protein, mitochondria were resuspended in SH buffer (0.6 M sorbitol and 20 mM HEPES-KOH [pH 7.2]) in the absence or presence of 30 μg of proteinase K per ml after purification by Nycodenz density gradient centrifugation. Samples were incubated for 30 min at 0°C, after which protease digestion was halted by the addition of p-aminophenyl methanesulfonyl fluoride hydrochloride (p-APMSF) (5 mM). Proteins lysed in sodium dodecyl sulfate (SDS) sample buffer were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted as described below.
Immunoblot analysis. The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Tokyo, Japan) after separation by SDS-PAGE. After blocking, the membranes were probed with monoclonal- or polyclonal-antibody against core protein (Anogen), prohibitin (Neo Markers, Fremont, Calif.), ribophorin I (Santa Cruz Biotechnology), translocase of the outer membrane (Tom) 20 (Santa Cruz Biotechnology), translocase of the inner membrane (Tim) 17 (Santa Cruz Biotechnology), or GFP (Santa Cruz Biotechnology). Immunoblots were developed as previously described (15).
GST pulldown assay. Escherichia coli BL21 cells were transformed with GST-core fusion plasmids and grown at 37°C. Expression of the fusion protein was induced by 1 mM isopropyl-?-D-thiogalactopyranoside at 37°C for 3 h. Bacteria were harvested, suspended in lysis buffer (1% Triton X-100 in PBS), and sonicated on ice. GST and GST fusion proteins were purified from bacterial lysates with glutathione-Sepharose beads (Amersham Bioscience Corp.). The beads were washed four times with lysis buffer. Approximately equal amounts of purified protein, as estimated by Coomassie brilliant blue staining, were used for the binding assays. For pulldown assays, in vitro transcription and translation of importin- was done with pRSET-hSRP1 and the TNT-coupled reticulocyte lysate system (Promega Corp., Madison, Wis.) with T7 RNA polymerase. The reaction was carried out at 30°C for 4 h in the presence of [35S]methionine/cysteine (ICN Pharmaceuticals). The translation product was then incubated with glutathione-Sepharose beads bound to GST fusion proteins in 1 ml of binding buffer (40 mM HEPES [pH 7.5], 100 mM KCl, 0.1% NP-40, and 20 mM 2-mercaptoethanol) at 4°C for 1 h. The beads were washed four times with binding buffer, and the pulldown complexes were separated by SDS-PAGE on 15% polyacrylamide gels. The gels were then fixed, dried, and analyzed with autoradiography.
RESULTS
Subcellular localization of HCV core protein. To assess the subcellular localization of HCV core protein, we first analyzed cells transfected with a full-length core-expressing construct by confocal microscopy. In accordance with previous observations (2, 15, 32, 45, 56), a granular cytoplasmic staining pattern of the core protein was observed in 293T (Fig. 1) and human hepatoblastoma HepG2 (data not shown) cells. Dual staining of transfected cells with antibody against the ER protein calregulin along with anticore antibody confirmed the ER localization of the core protein (Fig. 1a, b, and c show the core, calregulin, and a merged image, respectively). The pattern of subcellular localization of the core protein (Fig. 1d) was compared to the distribution of mitochondria, as revealed by MitoTracker staining (Fig. 1e). Although distribution of the core protein was not completely identical with that of the mitochondrion-selective dye, overlapping staining was observed, particularly in the perinuclear region (Fig. 1f).
Intracellular localization of the core protein was further examined in 293T cells by subcellular fractionation and Western blotting. The core protein was present in both the ER and mitochondrial fractions (Fig. 2), while it was not detected in the cytosol fraction (data not shown). The purity of the ER and mitochondrial fractions was confirmed with antibodies against ribophorin I as an ER marker and prohibitin as a mitochondrial marker.
It is generally difficult to identify the nuclear distribution of proteins of interest due to contamination of the nuclear preparation with unbroken, intact cells. Thus, to investigate whether the core protein localizes to the nucleus, we examined transfected cells by immunoelectron microscopy. Although gold particles were primarily observed within cytoplasmic membranes, perhaps highlighting the ER, immunoreactivity to anticore antibody was also observed in the nucleus (Fig. 3A, arrows). In contrast, no antibody labeling was observed in cells transfected with an empty vector (Fig. 3B).
Thus, HCV core protein predominates in the cytoplasm in a membrane-associated form(s) with ER and mitochondria, but nuclear localization is also observed.
Regions responsible for directing core protein to the ER and mitochondria. Given the tendency of the core protein to localize to the ER and to mitochondria, we next investigated whether specific sequences might be responsible for transporting the core protein to these organelles. Fusion proteins between different regions of the core protein and GFP were developed, with specific emphasis on the region downstream of amino acid 72 because this region contains clusters of hydrophobic amino acids and the N-terminal 71 residues of the core are known to play a role in nuclear targeting (6, 48).
Western blotting of subcellular fractions with anti-GFP antibody revealed the localization of a core (72-173)-GFP fusion protein to the ER and to mitochondria (Fig. 4). Fusion proteins containing GFP and core proteins with N- or C-terminal deletions (72-152-GFP and 112-152-GFP) were likewise identified within the ER and mitochondrial fractions. In contrast, the ER and mitochondrial fractions did not contain GFP fusion proteins containing core protein amino acids 124 to 152, 124 to 173, 152 to 173, or 72 to 111. These fusion proteins demonstrated distribution profiles similar to that of GFP alone. We also tested GFP-core-E1 fusions, which are processed at the C terminus of the core by signal peptidase and signal peptide peptidase (19, 30). GFP-core fusions expressed from pGFP/72-382 and pGFP/112-382 were detected in the ER and mitochondrial fractions. The fusion expressed from pGFP/152-382 was not identified in these fractions.
We further analyzed subcellular localization of the fusion proteins by confocal immunofluorescence microscopy (Fig. 5). As expected, fusions of (72-173)-GFP and (112-152)-GFP exhibited localization to the ER and mitochondria. The patterns of subcellular localization of these fusions are indistinguishable from that of the full-length core protein, as shown in Fig. 1. Expression of (124-152)-GFP or (112-123)-GFP resulted in widespread diffusion of the fusion in the cell. Thus, these results indicate that the region spanning amino acids 112 to 152 can mediate association of the core protein not only to the ER but also to the mitochondria.
We subsequently examined the submitochondrial localization of the core protein with a protease protection assay. As shown in Fig. 6A, HCV core protein localized in the mitochondria was completely digested upon treatment with proteinase K for 30 min at 0°C. Under identical conditions, a marker specific for the mitochondrial outer membrane, Tom20, was also observed to disappear, whereas digestion of a mitochondrial inner membrane marker, Tim17, was not observed. These findings confirm that HCV core protein is localized to the mitochondrial outer membrane.
The predicted secondary structure of the region, amino acids 72 to 173, is shown in Fig. 6B. The presence of a long helical segment, lying between amino acids 116 and 134, and two short -helices (amino acids 146 to 152 and amino acids 155 to 159) were predicted. The results of the cell fractionation assay and confocal microscopy with a series of deletion mutants shown in Fig. 4 and 5 suggest that an -helix between amino acids 116 and 134 may be required for associating the core protein with the ER and the mitochondrial outer membrane. When amino acids 117 to 134 are portrayed as a helical wheel, we found an amphipathic structure with hydrophobic residues on one side and polar residues on the other side of the -helix (Fig. 6C), which is often observed in membrane-associated proteins. This helical conformation might be important for directing the core protein to the ER and mitochondrial outer membranes.
Nuclear localization of the HCV core protein is mediated by a bipartite NLS, possibly via an importin-dependent pathway. Although HCV core protein is mainly localized within the cytoplasm, it is also found in the nucleus, as shown in Fig. 3. The results of previous studies demonstrate that the N-terminal region of the core protein is responsible for nuclear targeting. It contains three clusters of basic amino acid residues that represent putative consensus motifs for NLS sequences PKPQRKTKR (amino acids 5 to 13), PRRGPR (amino acids 38 to 43), and PRGRRQPIPKARRP (amino acids 58 to 71) (6, 48). Nuclear targeting is generally governed by a family of transporters or cytosolic receptor proteins, known as importins or karyopherins, which function in concert with a guanine nucleotide-binding protein named Ran and other regulatory proteins such as NTF2/p10. Conventional NLS-dependent nuclear targeting occurs when importin- recognizes the NLS sequence, mediating binding to importin-?1, after which the trimeric complex translocates to the nucleus (12).
In order to determine whether the putative NLS motifs identified within the core protein sequence are capable of binding to importin-, we examined the in vitro interaction between bacterially expressed GST-fused core protein and 35S-labeled importin- with a GST pulldown assay. We then substituted lysine and arginine residues of one or more of the putative NLS motifs of the core protein (all contained within the first 71 amino acids of the N terminus) with alanine and fused the resultant constructs with GST, as shown schematically in Fig. 7A. As shown in Fig. 7B (upper panel), importin- was pulled down by a GST fusion protein containing wild-type core (amino acids 1 to 71) protein but not with GST alone, suggesting that direct binding occurs between the core protein and importin-. Importin- was also pulled down by GST-core fusion proteins containing substitutions in one or two NLS motifs (NLS/m1, NLS/m2, NLS/m3, NLS/m4, NLS/m5, and NLS/m6). However, importin- was not pulled down by GST-core fusion proteins containing alanine substitutions in all three NLS motifs (NLS/m7). It should be noted that similar amounts of GST fusion proteins were used for each of the in vitro pulldown assays, followed by SDS-PAGE and Coomassie brilliant blue staining (Fig. 7B, lower panel). These results demonstrated that all three putative NLS motifs of the N-terminal region of the core protein can mediate binding to importin-, which suggests that nuclear translocation of the core protein occurs via an importin-dependent pathway (12).
The interaction between the core and importin- was further analyzed by a colocalization assay (Fig. 7C). The GFP fusion containing the wild-type core (amino acids 1 to 71) was well colocalized with HA-importin-; distribution of the two proteins showed similar nuclear staining patterns, confirming the presence of a functional NLS sequence(s) within the core protein. In contrast, NLS/m4, with substitutions in two NLS motifs, was partly colocalized with HA-importin- near or around the nuclear membrane, suggesting that NLS motif double mutants bind to importin- but their binding efficiency is lower than that of wild-type core protein.
Finally, we examined the subcellular localization of core protein expressed by the wild-type and NLS mutants (Fig. 7D). As expected, a fusion protein containing wild-type core protein (amino acids 1 to 71) and GFP was localized exclusively to the nucleus. Core proteins from three fusion proteins containing substitutions in each NLS motif (NLS/m1, NLS/m2, and NLS/m3) were detected primarily in the nucleus. Weak fluorescence was also observed in the cytoplasm, suggesting that these mutations caused a slight reduction in the efficiency of nuclear translocation. On the other hand, two or three NLS motif substitution mutations (NLS/m4, NLS/m5, NLS/m6, and NLS/m7) completely abolished nuclear translocation, resulting in a diffuse distribution of core protein, similar to that of GFP alone. Although it is likely that all three putative NLS motifs play a role, the above results suggest that at least two of the three putative NLS motifs are prerequisite for efficient nuclear translocation of the core protein.
DISCUSSION
HCV core protein is released from the viral polyprotein by a host protease(s) within the ER membrane at a signal peptide sequence lying between the core and envelope (E1) proteins (16, 41). Subsequently, the signal peptide is further processed by an intramembranous protease called signal peptide peptidase (38, 53). This mature form of the core protein is then released and undergoes subcellular trafficking (30, 53). The core protein localizes mainly to the ER, mitochondria, and lipid droplets. Some reports also describe localization of the core protein to the nuclei of hepatocytes in HCV-infected patients (10), transgenic mice (34), and cultured cells expressing viral polyproteins (56). Although it has been reported which sequence motifs are responsible for localization of the HCV core protein to lipid droplets and nuclei, it is uncertain which sequences target the core protein to the ER and to mitochondria. In this study, we identified sequences related to localization of the mature core protein to the ER and to mitochondria.
Through heterologous expression of core-GFP fusion proteins containing a series of deletions, we determined that a sequence extending from amino acids 112 to 152 of the core protein is required for its localization at the mitochondrial outer membrane. Translocation of nucleus-encoded mitochondrial proteins is usually dependent on N-terminal sequences, referred to as mitochondrial targeting sequences (37). However, it is also true that a significant proportion of mitochondrial proteins lack these N-terminal mitochondrial targeting sequences. Specifically, a number of outer membrane proteins do not have cleavable sequences at their N termini; rather, they are targeted to mitochondria by means of internal or C-terminal signals (31).
Since it has been reported that amino acid sequences required for targeting to the outer mitochondrial membrane form a highly hydrophobic -helical wheel, as seen in A-kinase associated protein 84/12 (4) and NADH-cytochrome b reductase (14), a predicted structure of an amphipathic -helix present between amino acids 116 and 134 (Fig. 6B and C) possibly plays a role in directing the core protein to the mitochondrial outer membrane. Sequence comparisons demonstrate conservation of the amino acid sequence and secondary structure of the region, amino acids 112 to 152, among a variety of HCV isolates, including the infectious H77c clone (55), as well as a full-length adaptive replicon (3). To gain insight into the significance of the secondary structure of the region in targeting to the mitochondria, further structural and biochemical analyses are needed.
The association of HCV core protein with the mitochondrial membrane suggests that the core protein has the ability to modulate mitochondrial function, possibly by altering the permeability of the mitochondrial membrane. The core protein induces the production of cellular reactive oxygen species in the livers of core-transgenic mice and in core-expressing cell lines (35). Reactive oxygen species, predominantly generated in mitochondria, induce genetic mutations and act as secondary messengers to regulate a variety of cellular functions, including gene expression and proliferation (1). Although the molecular mechanism by which core protein induces reactive oxygen species production is still unclear, HCV core protein is known to impair the mitochondrial electron transfer system (35). The core protein may also modulate apoptosis, since mitochondria play a major role in regulating programmed cell death. Expression of HCV proteins, including the core protein, suppresses the release of cytochrome c from mitochondria to the cytoplasm in HCV-transgenic mice, thus inhibiting Fas-mediated apoptosis (27).
Okamoto et al. recently reported that not only the C-terminal signal sequence but also amino acids 128 to 151 are required for ER retention of the core protein by using a series of N-terminally truncated core protein constructs (38). Here, in this study, we further showed that amino acids 112 to 152 mediate association of the core protein with the ER in the absence of the C-terminal signal sequence. Hope and McLauchlan demonstrated that the central domain of the core protein, amino acids 119 to 174, is important for association with lipid droplets (17). They also showed that this corresponding domain is shared with GB virus B, which is most closely related to HCV, but not with either pestiviruses or flaviviruses (18). It appears that the 41 residues identified as the sequence mediating association with the ER membrane in the present study are crucial for directing the core protein to lipid droplets, since the surface of lipid droplets must derive from the cytoplasmic side of the ER membrane.
The HCV core protein contains NLS sequences which are composed of three stretches of sequences rich in basic residues. These sequences were originally identified by experiments with fused forms of wild-type and mutated core proteins with ?-galactosidase (6, 48). C-terminally truncated versions of the core protein localize exclusively to the nucleus (48). A fraction of the core protein is detected in the nucleus even when full-length HCV core gene is expressed (Fig. 2) and as described (34, 56). However, it is difficult to demonstrate clearly the nuclear localization of the core protein by immunofluorescence, presumably because of the instability of nuclearly localized core protein (49, 33). We only observed a nuclear staining pattern of the matured core protein after adding proteasome inhibitors to the culture (33).
Generally, NLS sequences fall into two categories; (i) monopartite NLSs, which contain a single cluster of basic residues, and (ii) bipartite NLSs, which contain two clusters of basic residues separated by an unconserved linker sequence of variable length (reviewed in reference 12). Nuclear translocation of an NLS-containing cargo protein is initiated when the soluble import receptor (importin) recognizes the NLS-containing protein within the cytoplasm. Importin- contains an NLS-binding site(s), and importin-? docks importin-cargo complexes to the cytoplasmic filaments of a nuclear pore complex, after which translocation occurs through the nuclear pore. Thus, importin- functions as an adaptor between the bona fide import receptor and the NLS-containing protein.
We further characterized the NLS of the core protein and found that each of the NLS motifs of the core protein is able to bind to importin- and that at least two NLS motifs are required for efficient nuclear distribution of the core protein in cells. It appears that double mutations among three NLS motifs decrease the ability of the core protein to bind importin-. These observations suggest that the binding of the double mutants with importin- leads to no or little active translocation of the core protein into the nucleus. The double mutations may also block subsequent interactions with importin-?1, GTPase Ran, and/or NTF2/p10, which are required for translocation through the nuclear pore complexes.
The findings obtained in this study suggest that HCV core protein NLS motifs have a bipartite function. Crystallographic studies of monopartite (e.g., simian virus 40 large T antigen) and bipartite (e.g., nucleoplasmin) NLSs show that the basic residue clusters of bipartite NLSs occupy separate binding sites on importin-. In contrast, while monopartite NLSs can bind to the same sites as bipartate NLSs on importin-, they mainly bind to the N-terminal binding site, which is referred to as the major binding site on importin- (9, 11). A recent report describes an importin- variant with a mutation in the major site which results in decreased ability to bind both monopartite and bipartite NLSs. Another variant with a mutation in the minor site exhibits decreased binding only to bipartite NLS-containing proteins, making importin- nonfunctional in vivo (22). Thus, we favor a model in which the core protein bipartite NLS, composed of any two of the three basic clusters, occupies both major and minor binding sites on importin-, resulting in efficient nuclear translocation. Importin- may be equally accessible to all clusters, given their close proximity to one another, as well as the distinct conformational flexibility of the 70-residue N-terminal region of the core protein.
With regard to the molecular mechanisms participating in nuclear localization of the core protein, Moriishi et al. found that PA28 is involved in nuclear localization of the core protein. Interaction of the core protein with PA28 plays an important role in retention of the core protein in the nucleus (33). Furthermore, in yeast cells, nuclear transport of the core protein requires the activity of the small GTPase Ran/Gsp1p and is mediated by Kap123p, but neither importin- nor importin-? is involved (20). Differences in nucleocytoplasmic transport between yeast and mammalian cells might explain the inconsistencies observed in the present study. Further experiments are required to characterize the exact nature of the interaction between the core protein and components of the nuclear import machinery, particularly in cells where HCV is replicating.
In conclusion, the mature HCV core protein has an internal 41-amino-acid sequence mediating association of the viral protein with the ER and mitochondria. We also provide evidence for a novel class of bipartite NLS contained within the core protein, which comprises two of three basic motifs, thus enabling efficient nuclear targeting. Multiple functional domains influence the subcellular localization of the core protein, which ultimately depends on the balance of the respective signals.
ACKNOWLEDGMENTS
We thank colleagues in the laboratories of the Department of Virology II at the National Institute of Infectious Diseases of Japan for providing advice and help. We especially thank Mami Matsuda and Makiko Yahata for assistance in sequencing and the preparation of experimental reagents and Tomoko Mizoguchi for secretarial work. We are grateful to Karsten Weis for providing us with the plasmid containing importin- cDNA.
This work was supported in part by Second Term Comprehensive 10-Year Strategy for Cancer Control and Research on Emerging and Reemerging Infectious Diseases, Health Sciences Research Grants of the Ministry of Health, Labor and Welfare, and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan (ID:01-3). This work was also supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology to R.S. (15790244).
REFERENCES
Adler, V., Z. Yin, K. D. Tew, and Z. Ronai. 1999. Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18:6104-6111.
Barba, G., F. Harper, T. Harada, M. Kohara, S. Goulinet, Y. Matsuura, G. Eder, Z. Schaff, M. J. Chapman, T. Miyamura, and C. Bréchot. 1997. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc. Natl. Acad. Sci. USA 94:1200-1205.
Bukh, J., T. Pietschmann, V. Lohmann, N. Krieger, K. Faulk, R. E. Engle, S. Govindarajan, M. Shapiro, M. St Claire, and R. Bartenschlager. 2002. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. USA 99:14416-14421.
Cardone, L., T. de Cristofaro, A. Affaitati, C. Garbi, M. D. Ginsberg, M. Saviano, S. Varrone, C. S. Rubin, M. E. Gottesman, E. V. Avvedimento, and A. Feliciello. 2002. A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs. J. Mol. Biol. 320:663-675.
Chang, J., S. H. Yang, Y. G. Cho, S. B. Hwang, Y. S. Hahn, and Y. C. Sung. 1998. Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. J. Virol. 72:3060-3065.
Chang, S. C., J. H. Yen, H. Y. Kang, M. H. Jang, and M. F. Chang. 1994. Nuclear localization signals in the core protein of hepatitis C virus. Biochem. Biophys. Res. Commun. 205:1284-1290.
Choo, Q. L., G. Kuo, A. J. Weiner, L. R. Overby, D. W. Bradley, and M. Houghton. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359-362.
Choo, Q. L., K. H. Richman, J. H. Han, K. Berger, C. Lee, C. Dong, C. Gallegos, D. Coit, R. Medina-Selby, P. J. Barr, et al. 1991. Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 88:2451-2455.
Conti, E., M. Uy, L. Leighton, G. Blobel, and J. Kuriyan. 1998. Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. Cell 94:193-204.
Falcon, V., N. Acosta-Rivero, G. Chinea, M. C. de la Rosa, I. Menendez, S. Duenas-Carrera, B. Gra, A. Rodriguez, V. Tsutsumi, M. Shibayama, J. Luna-Munoz, M. M. Miranda-Sanchez, J. Morales-Grillo, and J. Kouri. 2003. Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient. Biochem. Biophys. Res. Commun. 310:54-58.
Fontes, M. R., T. Teh, and B. Kobe. 2000. Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha. J. Mol. Biol. 297:1183-1194.
G?rlich, D., and U. Kutay. 1999. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell. Dev. Biol. 15:607-660.
Grakoui, A., D. W. McCourt, C. Wychowski, S. M. Feinstone, and C. M. Rice. 1993. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J. Virol. 67:2832-2843.
Hahne, K., V. Haucke, L. Ramage, and G. Schatz. 1994. Incomplete arrest in the outer membrane sorts NADH-cytochrome b5 reductase to two different submitochondrial compartments. Cell 79:829-839.
Harada, S., Y. Watanabe, K. Takeuchi, T. Suzuki, T. Katayama, Y. Takebe, I. Saito, and T. Miyamura. 1991. Expression of processed core protein of hepatitis C virus in mammalian cells. J. Virol. 65:3015-3021.
Hijikata, M., N. Kato, Y. Ootsuyama, M. Nakagawa, and K. Shimotohno. 1991. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc. Natl. Acad. Sci. USA 88:5547-5551.
Hope, R. G., and J. McLauchlan. 2000. Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein. J. Gen. Virol. 81:1913-1925.
Hope, R. G., D. J. Murphy, and J. McLauchlan. 2002. The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins. J. Biol. Chem. 277:4261-4270.
Hüssy, P., H. Langen, J. Mous, and H. Jacobsen. 1996. Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224:93-104.
Isoyama, T., S. Kuge, and A. Nomoto. 2002. The core protein of hepatitis C virus is imported into the nucleus by transport receptor Kap123p but inhibits Kap121p-dependent nuclear import of yeast AP1-like transcription factor in yeast cells. J. Biol. Chem. 277:39634-39641.
Lai, M. M., and C. F. Ware. 2000. Hepatitis C virus core protein: possible roles in viral pathogenesis. Curr. Top. Microbiol. Immunol. 242:117-134.
Leung, S. W., M. T. Harreman, M. R. Hodel, A. E. Hodel, and A. H. Corbett. 2003. Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding. J. Biol. Chem. 278:41947-41953.
Liu, Q., C. Tackney, R. A. Bhat, A. M. Prince, and P. Zhang. 1997. Regulated processing of hepatitis C virus core protein is linked to subcellular localization. J. Virol. 71:657-662.
Lo, S. Y., F. Masiarz, S. B. Hwang, M. M. Lai, and J. H. Ou. 1995. Differential subcellular localization of hepatitis C virus core gene products. Virology 213:455-461.
Lo, S. Y., M. Selby, M. Tong, and J. H. Ou. 1994. Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution. Virology 199:124-131.
Lu, W., A. Strohecker, and J. H. Ou. 2001. Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase. J. Biol. Chem. 276:47993-47999.
Machida, K., K. Tsukiyama-Kohara, E. Seike, S. Tone, F. Shibasaki, M. Shimizu, H. Takahashi, Y. Hayashi, N. Funata, C. Taya, H. Yonekawa, and M. Kohara. 2001. Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins. J. Biol. Chem. 276:12140-12146.
Matsumoto, M., S. B. Hwang, K. S. Jeng, N. Zhu, and M. M. Lai. 1996. Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 218:43-51.
McLauchlan, J. 2000. Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J. Viral Hepatitis 7:2-14.
McLauchlan, J., M. K. Lemberg, G. Hope, and B. Martoglio. 2002. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J. 21:3980-3988.
Mihara, K. 2000. Targeting and insertion of nuclear-encoded preproteins into the mitochondrial outer membrane. Bioessays 22:364-371.
Moradpour, D., C. Englert, T. Wakita, and J. R. Wands. 1996. Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 222:51-63.
Moriishi, K., T. Okabayashi, K. Nakai, K. Moriya, K. Koike, S. Murata, T. Chiba, K. Tanaka, R. Suzuki, T. Suzuki, T. Miyamura, and Y. Matsuura. 2003. Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein. J. Virol. 77:10237-10249.
Moriya, K., H. Fujie, Y. Shintani, H. Yotsuyanagi, T. Tsutsumi, K. Ishibashi, Y. Matsuura, S. Kimura, T. Miyamura, and K. Koike. 1998. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat. Med. 4:1065-1067.
Moriya, K., K. Nakagawa, T. Santa, Y. Shintani, H. Fujie, H. Miyoshi, T. Tsutsumi, T. Miyazawa, K. Ishibashi, T. Horie, K. Imai, T. Todoroki, S. Kimura, and K. Koike. 2001. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res. 61:4365-4370.
Moriya, K., H. Yotsuyanagi, Y. Shintani, H. Fujie, K. Ishibashi, Y. Matsuura, T. Miyamura, and K. Koike. 1997. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J. Gen. Virol. 78:1527-1531.
Neupert, W. 1997. Protein import into mitochondria. Annu. Rev. Biochem. 66:863-917.
Okamoto, K., K. Moriishi, T. Miyamura, and Y. Matsuura. 2004. Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein. J. Virol. 78:6370-6380.
Okuda, M., K. Li, M. R. Beard, L. A. Showalter, F. Scholle, S. M. Lemon, and S. A. Weinman. 2002. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 122:366-375.
Perlemuter, G., A. Sabile, P. Letteron, G. Vona, A. Topilco, Y. Chretien, K. Koike, D. Pessayre, J. Chapman, G. Barba, and C. Bréchot. 2002. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis. FASEB J. 16:185-194.
Ralston, R., K. Thudium, K. Berger, C. Kuo, B. Gervase, J. Hall, M. Selby, G. Kuo, M. Houghton, and Q. L. Choo. 1993. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J. Virol. 67:6753-6761.
Ray, R. B., L. M. Lagging, K. Meyer, and R. Ray. 1996. Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J. Virol. 70:4438-4443.
Roth, J., D. J. Taatjes, and M. J. Warhol. 1989. Prevention of non-specific interactions of gold-labeled reagents on tissue sections. Histochemistry 92:47-56.
Sabile, A., G. Perlemuter, F. Bono, K. Kohara, F. Demaugre, M. Kohara, Y. Matsuura, T. Miyamura, C. Brechot, and G. Barba. 1999. Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates. Hepatology 30:1064-1076.
Santolini, E., G. Migliaccio, and N. La Monica. 1994. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J. Virol. 68:3631-3641.
Selby, M. J., Q. L. Choo, K. Berger, G. Kuo, E. Glazer, M. Eckart, C. Lee, D. Chien, C. Kuo, and M. Houghton. 1993. Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J. Gen. Virol. 74:1103-1113.
Shimoike, T., S. Mimori, H. Tani, Y. Matsuura, and T. Miyamura. 1999. Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation. J. Virol. 73:9718-9725.
Suzuki, R., Y. Matsuura, T. Suzuki, A. Ando, J. Chiba, S. Harada, I. Saito, and T. Miyamura. 1995. Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted. J. Gen. Virol. 76:53-61.
Suzuki, R., K. Tamura, J. Li, K. Ishii, Y. Matsuura, T. Miyamura, and T. Suzuki. 2001. Ubiquitin-mediated degradation of hepatitis C virus core protein is regulated by processing at its carboxyl terminus. Virology 280:301-309.
Tanaka, Y., T. Shimoike, K. Ishii, R. Suzuki, T. Suzuki, H. Ushijima, Y. Matsuura, and T. Miyamura. 2000. Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome. Virology 270:229-236.
Tellinghuisen, T. L., and C. M. Rice. 2002. Interaction between hepatitis C virus proteins and host cell factors. Curr. Opin. Microbiol. 5:419-427.
Thomson, M., and T. J. Liang. 2000. Molecular biology of hepatitis C virus, p. 1-23. In T. J. Liang and J. H. Hoofnagle (ed.), Hepatitis C. Academic Press, San Diego, Calif.
Weihofen, A., K. Binns, M. K. Lemberg, K. Ashman, and B. Martoglio. 2002. Identification of signal peptide peptidase, a presenilin-type aspartic protease. Science 296:2215-2218.
Weis, K., I. W. Mattaj, and A. I. Lamond. 1995. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science 268:1049-1053.
Yanagi, M., R. H. Purcell, S. U. Emerson, and J. Bukh. 1997. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc. Natl. Acad. Sci. USA 94:8738-8743.
Yasui, K., T. Wakita, K. Tsukiyama-Kohara, S. I. Funahashi, M. Ichikawa, T. Kajita, D. Moradpour, J. R. Wands, and M. Kohara. 1998. The native form and maturation process of hepatitis C virus core protein. J. Virol. 72:6048-6055.(Ryosuke Suzuki, Shinichir)
Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Minato-ku
Department of Pathology, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo
Research Center for Emerging Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Suita-shi, Osaka
Department of Pathology, Institute of Tropical Medicine, Nagasaki University, Nagasaki-shi, Nagasaki, Japan
ABSTRACT
Hepatitis C virus (HCV) core protein is a putative nucleocapsid protein with a number of regulatory functions. In tissue culture cells, HCV core protein is mainly located at the endoplasmic reticulum as well as mitochondria and lipid droplets within the cytoplasm. However, it is also detected in the nucleus in some cells. To elucidate the mechanisms by which cellular trafficking of the protein is controlled, we performed subcellular fractionation experiments and used confocal microscopy to examine the distribution of heterologously expressed fusion proteins involving various deletions and point mutations of the HCV core combined with green fluorescent proteins. We demonstrated that a region spanning amino acids 112 to 152 can mediate association of the core protein not only with the ER but also with the mitochondrial outer membrane. This region contains an 18-amino-acid motif which is predicted to form an amphipathic -helix structure. With regard to the nuclear targeting of the core protein, we identified a novel bipartite nuclear localization signal, which requires two out of three basic-residue clusters for efficient nuclear translocation, possibly by occupying binding sites on importin-. Differences in the cellular trafficking of HCV core protein, achieved and maintained by multiple targeting functions as mentioned above, may in part regulate the diverse range of biological roles of the core protein.
INTRODUCTION
Hepatitis C virus (HCV), the most important causative agent of posttransfusion and sporadic non-A, non-B hepatitis, is a positive-stranded RNA virus belonging to the family Flaviviridae (7). A precursor polyprotein of about 3,000 amino acids is encoded by a large open reading frame of the genome and undergoes cellular and viral protease-mediated posttranslational modification to produce a series of structural and nonstructural proteins (8, 13, 16).
HCV core protein, which is derived from the N terminus of the viral polyprotein, forms multimers and interacts physically with the viral RNA to constitute the nucleocapsid (28, 47, 50). Tissue transglutaminase is responsible for stabilizing the core protein by cross-linking it into a dimeric form (26). In addition, the core viral protein has properties which enable it to modulate a number of cellular processes, including transcription, inhibition or stimulation of apoptosis, and suppression of host immunity, as reviewed previously (21, 29, 51, 52). Several studies suggest that expression of the core protein affects mitochondrial function and lipid metabolism. The core protein increases the cellular production of reactive oxygen species with subsequent increases in lipid peroxidation (35, 39). The viral protein also colocalizes with human apolipoprotein AII, associates with lipid droplets, and has the capacity to influence metabolic events involving lipid storage (2, 17, 30, 36, 44). In addition, the core protein reduces microsomal triglyceride transfer, leading to defects in very low density lipoprotein assembly and secretion (40). Furthermore, the HCV core protein has transforming potential in some cells under certain conditions (5, 42). Transgenic mice expressing this protein in the liver develop hepatic steatosis due to increased oxidative stress in the absence of inflammation, with subsequent development of hepatocellular carcinoma (34, 36). These results suggest that the HCV core protein might play a pivotal role in the pathogenesis of hepatitis C in addition to its role as a structural component of the viral capsid.
The amino acid sequence of the core protein is well conserved among different HCV isolates and genotypes compared to other HCV proteins. The N-terminal domain of the HCV core protein is highly basic, while its C terminus is hydrophobic. Although several core proteins of various sizes exist (17 to 23 kDa) (15, 23, 25, 49, 56), two processing events result in the predominant production of a 21-kDa core protein. Both of these events utilize the endoplasmic reticulum (ER). The first one is to be cleaved from downstream envelope protein E1 at position 191, where the C-terminal hydrophobic domain serves as a putative signal peptide sequence. Subsequently, the signal sequence of 13 or 18 residues is processed by signal peptide peptidase (19, 23, 56).
The HCV core protein is found primarily within the membranes of cytoplasmic organelles, but it is also found in the nucleus (23, 48, 56). Immunofluorescence studies show a punctate pattern, consistent with ER localization, as well as perinuclear localization (15, 24, 32, 46, 56). Some studies suggest direct effects of the core protein on mitochondrial function. In fact, the core protein localizes to the mitochondria (34, 39). The N-terminal domain of the core protein contains three stretches of arginine- and lysine-rich sequences. Translocation of the core protein to the nucleus, mediated by these basic-residue stretches which function as nuclear localization signals (NLSs), is observed (6, 48). In addition, Moriishi et al. demonstrated that the N-terminal region of the core protein is also essential for nuclear retention through its interaction with the proteasome activator PA28 (33).
In this study, we found a region that is important for localization of the mature core protein to the ER and to the mitochondrial outer membrane. We also identified a novel bipartite NLS responsible for nuclear targeting of the core protein, presumably via an importin-dependent pathway.
MATERIALS AND METHODS
Plasmid construction. The construction of a plasmid expressing the full-length core protein of 191 amino acids, pCAGC191, was described previously (49). pGFP, a construct expressing green fluorescent protein (GFP) with a C-terminal Myc epitope tag sequences, was prepared as follows. pCMV/Myc/mito/GFP (Invitrogen Corp., Carlsbad, Calif.) was digested with PmlI, followed by treatment with the Klenow fragment of DNA polymerase I. The resultant linear fragment was ligated to a PstI linker (GCTGCAGC) and digested with PstI to remove the mitochondrial targeting signal sequence, followed by self-ligation. A series of HCV core-GFP fusion constructs were made by amplifying the core gene fragments with PCR with primers containing Flag epitope tag sequences (sense) and a PstI site (both). After digestion with PstI, the segments were inserted into the PstI site of pGFP. A series of GFP-core-E1 fusion constructs were made by amplifying core and E1 gene fragments with PCR with primers containing a NotI site. After digestion with NotI, the segments were inserted into the NotI site of pGFP.
pGEX-4T-1 (Amersham Bioscience Corp., Piscataway, N.J.) was used to express core protein fused with glutathione S-transferase (GST) in Escherichia coli. Core cDNA fragments encoding amino acids 1 to 71 were inserted into the EcoRI site of pGEX-4T-1. Alanine substitutions were introduced into the core protein by PCR mutagenesis with primers containing base alterations. The PCR products were then cloned into pCR2.1 (Invitrogen Corp.) and verified by DNA sequencing. Individual cDNAs were excised and inserted separately into pGFP or pGEX-4T-1. The primer sequences used in this study are available from the authors upon request.
Plasmid pRSET-hSRP1 (54), containing importin- cDNA under the control of a T7 promoter, was kindly provided by Karsten Weis (University of California, Berkeley). A cDNA clone of importin- possessing 14 residues (MYPYDVPDYGGGGS), derived in part from the hemagglutinin (HA) tag at the N terminus, was constructed by PCR. The resultant linear fragment was inserted under the control of a CAG promoter of pCAGGS and designated pCAG-HA-imp.
Cell culture and transfection. Human embryonic kidney 293T cells were maintained in Dulbecco's modified Eagle's medium supplemented with 100 units of penicillin per ml, 100 μg of streptomycin per ml, and 10% fetal bovine serum at 37°C in a 5% CO2 incubator. Monolayers of 293T cells were transfected with plasmid DNA in the presence of Lipofectamine (Gibco-BRL, Life Technologies, Gaithersburg, Md.) according to the manufacturer's instructions.
Confocal immunofluorescence microscopy. Transfected cells were grown on glass coverslips. Two days after transfection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. Intracellular localization of HCV core-GFP fusion proteins was visualized in cells transfected with a variety of GFP fusion constructs.
In order to detect the HCV core protein by immunofluorescence, fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 3 min at room temperature, followed by blocking with a nonfat milk solution (Block Ace; Snow Brand Milk Products Co., Sapporo, Japan). The cells were then incubated with anticore monoclonal antibody B2 (Anogen, Mississauga, Canada) for 60 min at room temperature, followed by incubation with fluorescein isothiocyanate-conjugated rabbit anti-mouse immunoglobulin G (IgG) (ICN Pharmaceuticals, Aurora, Ohio) for 45 min. To visualize mitochondria, MitoTracker Red CM-H2XRos (Molecular Probes, Eugene, Oreg.) was added to the culture medium to a final concentration of 100 nM and incubated for 120 min at 37°C prior to fixation. To visualize the ER, goat anticalregulin antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) and rhodamine-conjugated rabbit anti-goat IgG (ICN Pharmaceuticals) were used as the first and second antibodies, respectively. To visualize HA-importin-, mouse anti-HA antibody (Roche Molecular Biochemicals, Indianapolis, Ind.) and rhodamine-conjugated goat anti-mouse IgG (ICN Pharmaceuticals) were used as the first and second antibodies, respectively. All specimens were examined with an LSM510 laser scanning confocal microscope (Carl Zeiss, Oberkochen, Germany).
Immunoelectron microscopy. Cells were transfected as described above. After 2 days, cells were fixed with 3% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PBS (pH 7.4). Free aldehyde groups were quenched with 50 mM NH4Cl in PBS. The cell pellets were embedded at progressively lower temperatures (down to –35°C) in Lowicryl k4M according to an established protocol (43). Ultrathin sections were prepared and mounted on carbon-coated nickel grids. To perform electron microscopy, Lowicryl k4M ultrathin sections, mounted on grids, were floated on a droplet of PBS containing 1% bovine serum albumin, 0.1% Triton X-100, and 0.1% Tween 20 for 10 min, after which they were exposed to droplets of mouse anticore monoclonal antibody (Anogen) diluted in PBS for 45 min. Following this, they were rinsed twice for 5 min each in PBS and incubated with anti-mouse IgG-coated 10-nm immunogold particles (British Biocell, Cardiff, United Kingdom) for 45 min. After rinsing with PBS and distilled water, the grids and embedded sections were air dried and exposed to uranyl and lead acetate contrast agents.
Subcellular fractionation. All steps were performed at 4°C in the presence of a protease inhibitor cocktail called Complete (Roche Molecular Biochemicals). To isolate the ER fraction, transfected cells were washed with PBS, lysed in homogenization buffer A (50 mM Tris-HCl [pH 8.0], 1 mM ?-mercaptoethanol, 1 mM EDTA, and 0.32 M sucrose), and then centrifuged at 5,000 x g for 10 min. The supernatant was then collected and centrifuged at 105,000 x g for 1 h. The pellet was disrupted in lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM dithiothreitol, 1 mM sodium orthovanadate, and 10 mM sodium fluoride), after which it was centrifuged at 15,000 x g for 20 min. The resulting supernatant was used as the ER fraction.
To isolate the mitochondrial fraction, transfected cells were washed with PBS and homogenized in ice-cold homogenization buffer B (200 mM mannitol, 50 mM sucrose, 1 mM EDTA, and 10 mM Tris-HCl) at pH 7.4. The supernatant was then centrifuged at 1,000 x g for 10 min to remove large debris and nuclei. The resulting supernatant was then centrifuged at 20,000 x g for 20 min to obtain crude mitochondria. The crude mitochondria pellet was subfractionated in Nycodenz gradients for further purification of mitochondria. Nycodenz (Axis-Shield PoC AS, Oslo, Norway) solution at 50% (wt/vol) was prepared in buffer containing 5 mM Tris-HCl and 1 mM EDTA at pH 7.4. This stock solution was then diluted with buffer containing 0.25 M sucrose, 5 mM Tris-HCl, and 1 mM EDTA at pH 7.4 before use. The crude mitochondrial pellets was suspended in 4 ml of 25% Nycodenz solution and overlaid onto the following discontinuous Nycodenz gradients: 1 ml of 40%, 1 ml of 34%, and 2 ml of 30%. The samples were topped off with 2 ml of 23% Nycodenz solution after placement onto the discontinuous gradients. The tubes were then centrifuged at 52,000 x g for 90 min. The dense band seen after centrifugation at the 25 to 30% interface was recovered as the purified mitochondrial fraction.
To determine the submitochondrial localization pattern of the core protein, mitochondria were resuspended in SH buffer (0.6 M sorbitol and 20 mM HEPES-KOH [pH 7.2]) in the absence or presence of 30 μg of proteinase K per ml after purification by Nycodenz density gradient centrifugation. Samples were incubated for 30 min at 0°C, after which protease digestion was halted by the addition of p-aminophenyl methanesulfonyl fluoride hydrochloride (p-APMSF) (5 mM). Proteins lysed in sodium dodecyl sulfate (SDS) sample buffer were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotted as described below.
Immunoblot analysis. The proteins were transferred to a polyvinylidene difluoride membrane (Immobilon; Millipore, Tokyo, Japan) after separation by SDS-PAGE. After blocking, the membranes were probed with monoclonal- or polyclonal-antibody against core protein (Anogen), prohibitin (Neo Markers, Fremont, Calif.), ribophorin I (Santa Cruz Biotechnology), translocase of the outer membrane (Tom) 20 (Santa Cruz Biotechnology), translocase of the inner membrane (Tim) 17 (Santa Cruz Biotechnology), or GFP (Santa Cruz Biotechnology). Immunoblots were developed as previously described (15).
GST pulldown assay. Escherichia coli BL21 cells were transformed with GST-core fusion plasmids and grown at 37°C. Expression of the fusion protein was induced by 1 mM isopropyl-?-D-thiogalactopyranoside at 37°C for 3 h. Bacteria were harvested, suspended in lysis buffer (1% Triton X-100 in PBS), and sonicated on ice. GST and GST fusion proteins were purified from bacterial lysates with glutathione-Sepharose beads (Amersham Bioscience Corp.). The beads were washed four times with lysis buffer. Approximately equal amounts of purified protein, as estimated by Coomassie brilliant blue staining, were used for the binding assays. For pulldown assays, in vitro transcription and translation of importin- was done with pRSET-hSRP1 and the TNT-coupled reticulocyte lysate system (Promega Corp., Madison, Wis.) with T7 RNA polymerase. The reaction was carried out at 30°C for 4 h in the presence of [35S]methionine/cysteine (ICN Pharmaceuticals). The translation product was then incubated with glutathione-Sepharose beads bound to GST fusion proteins in 1 ml of binding buffer (40 mM HEPES [pH 7.5], 100 mM KCl, 0.1% NP-40, and 20 mM 2-mercaptoethanol) at 4°C for 1 h. The beads were washed four times with binding buffer, and the pulldown complexes were separated by SDS-PAGE on 15% polyacrylamide gels. The gels were then fixed, dried, and analyzed with autoradiography.
RESULTS
Subcellular localization of HCV core protein. To assess the subcellular localization of HCV core protein, we first analyzed cells transfected with a full-length core-expressing construct by confocal microscopy. In accordance with previous observations (2, 15, 32, 45, 56), a granular cytoplasmic staining pattern of the core protein was observed in 293T (Fig. 1) and human hepatoblastoma HepG2 (data not shown) cells. Dual staining of transfected cells with antibody against the ER protein calregulin along with anticore antibody confirmed the ER localization of the core protein (Fig. 1a, b, and c show the core, calregulin, and a merged image, respectively). The pattern of subcellular localization of the core protein (Fig. 1d) was compared to the distribution of mitochondria, as revealed by MitoTracker staining (Fig. 1e). Although distribution of the core protein was not completely identical with that of the mitochondrion-selective dye, overlapping staining was observed, particularly in the perinuclear region (Fig. 1f).
Intracellular localization of the core protein was further examined in 293T cells by subcellular fractionation and Western blotting. The core protein was present in both the ER and mitochondrial fractions (Fig. 2), while it was not detected in the cytosol fraction (data not shown). The purity of the ER and mitochondrial fractions was confirmed with antibodies against ribophorin I as an ER marker and prohibitin as a mitochondrial marker.
It is generally difficult to identify the nuclear distribution of proteins of interest due to contamination of the nuclear preparation with unbroken, intact cells. Thus, to investigate whether the core protein localizes to the nucleus, we examined transfected cells by immunoelectron microscopy. Although gold particles were primarily observed within cytoplasmic membranes, perhaps highlighting the ER, immunoreactivity to anticore antibody was also observed in the nucleus (Fig. 3A, arrows). In contrast, no antibody labeling was observed in cells transfected with an empty vector (Fig. 3B).
Thus, HCV core protein predominates in the cytoplasm in a membrane-associated form(s) with ER and mitochondria, but nuclear localization is also observed.
Regions responsible for directing core protein to the ER and mitochondria. Given the tendency of the core protein to localize to the ER and to mitochondria, we next investigated whether specific sequences might be responsible for transporting the core protein to these organelles. Fusion proteins between different regions of the core protein and GFP were developed, with specific emphasis on the region downstream of amino acid 72 because this region contains clusters of hydrophobic amino acids and the N-terminal 71 residues of the core are known to play a role in nuclear targeting (6, 48).
Western blotting of subcellular fractions with anti-GFP antibody revealed the localization of a core (72-173)-GFP fusion protein to the ER and to mitochondria (Fig. 4). Fusion proteins containing GFP and core proteins with N- or C-terminal deletions (72-152-GFP and 112-152-GFP) were likewise identified within the ER and mitochondrial fractions. In contrast, the ER and mitochondrial fractions did not contain GFP fusion proteins containing core protein amino acids 124 to 152, 124 to 173, 152 to 173, or 72 to 111. These fusion proteins demonstrated distribution profiles similar to that of GFP alone. We also tested GFP-core-E1 fusions, which are processed at the C terminus of the core by signal peptidase and signal peptide peptidase (19, 30). GFP-core fusions expressed from pGFP/72-382 and pGFP/112-382 were detected in the ER and mitochondrial fractions. The fusion expressed from pGFP/152-382 was not identified in these fractions.
We further analyzed subcellular localization of the fusion proteins by confocal immunofluorescence microscopy (Fig. 5). As expected, fusions of (72-173)-GFP and (112-152)-GFP exhibited localization to the ER and mitochondria. The patterns of subcellular localization of these fusions are indistinguishable from that of the full-length core protein, as shown in Fig. 1. Expression of (124-152)-GFP or (112-123)-GFP resulted in widespread diffusion of the fusion in the cell. Thus, these results indicate that the region spanning amino acids 112 to 152 can mediate association of the core protein not only to the ER but also to the mitochondria.
We subsequently examined the submitochondrial localization of the core protein with a protease protection assay. As shown in Fig. 6A, HCV core protein localized in the mitochondria was completely digested upon treatment with proteinase K for 30 min at 0°C. Under identical conditions, a marker specific for the mitochondrial outer membrane, Tom20, was also observed to disappear, whereas digestion of a mitochondrial inner membrane marker, Tim17, was not observed. These findings confirm that HCV core protein is localized to the mitochondrial outer membrane.
The predicted secondary structure of the region, amino acids 72 to 173, is shown in Fig. 6B. The presence of a long helical segment, lying between amino acids 116 and 134, and two short -helices (amino acids 146 to 152 and amino acids 155 to 159) were predicted. The results of the cell fractionation assay and confocal microscopy with a series of deletion mutants shown in Fig. 4 and 5 suggest that an -helix between amino acids 116 and 134 may be required for associating the core protein with the ER and the mitochondrial outer membrane. When amino acids 117 to 134 are portrayed as a helical wheel, we found an amphipathic structure with hydrophobic residues on one side and polar residues on the other side of the -helix (Fig. 6C), which is often observed in membrane-associated proteins. This helical conformation might be important for directing the core protein to the ER and mitochondrial outer membranes.
Nuclear localization of the HCV core protein is mediated by a bipartite NLS, possibly via an importin-dependent pathway. Although HCV core protein is mainly localized within the cytoplasm, it is also found in the nucleus, as shown in Fig. 3. The results of previous studies demonstrate that the N-terminal region of the core protein is responsible for nuclear targeting. It contains three clusters of basic amino acid residues that represent putative consensus motifs for NLS sequences PKPQRKTKR (amino acids 5 to 13), PRRGPR (amino acids 38 to 43), and PRGRRQPIPKARRP (amino acids 58 to 71) (6, 48). Nuclear targeting is generally governed by a family of transporters or cytosolic receptor proteins, known as importins or karyopherins, which function in concert with a guanine nucleotide-binding protein named Ran and other regulatory proteins such as NTF2/p10. Conventional NLS-dependent nuclear targeting occurs when importin- recognizes the NLS sequence, mediating binding to importin-?1, after which the trimeric complex translocates to the nucleus (12).
In order to determine whether the putative NLS motifs identified within the core protein sequence are capable of binding to importin-, we examined the in vitro interaction between bacterially expressed GST-fused core protein and 35S-labeled importin- with a GST pulldown assay. We then substituted lysine and arginine residues of one or more of the putative NLS motifs of the core protein (all contained within the first 71 amino acids of the N terminus) with alanine and fused the resultant constructs with GST, as shown schematically in Fig. 7A. As shown in Fig. 7B (upper panel), importin- was pulled down by a GST fusion protein containing wild-type core (amino acids 1 to 71) protein but not with GST alone, suggesting that direct binding occurs between the core protein and importin-. Importin- was also pulled down by GST-core fusion proteins containing substitutions in one or two NLS motifs (NLS/m1, NLS/m2, NLS/m3, NLS/m4, NLS/m5, and NLS/m6). However, importin- was not pulled down by GST-core fusion proteins containing alanine substitutions in all three NLS motifs (NLS/m7). It should be noted that similar amounts of GST fusion proteins were used for each of the in vitro pulldown assays, followed by SDS-PAGE and Coomassie brilliant blue staining (Fig. 7B, lower panel). These results demonstrated that all three putative NLS motifs of the N-terminal region of the core protein can mediate binding to importin-, which suggests that nuclear translocation of the core protein occurs via an importin-dependent pathway (12).
The interaction between the core and importin- was further analyzed by a colocalization assay (Fig. 7C). The GFP fusion containing the wild-type core (amino acids 1 to 71) was well colocalized with HA-importin-; distribution of the two proteins showed similar nuclear staining patterns, confirming the presence of a functional NLS sequence(s) within the core protein. In contrast, NLS/m4, with substitutions in two NLS motifs, was partly colocalized with HA-importin- near or around the nuclear membrane, suggesting that NLS motif double mutants bind to importin- but their binding efficiency is lower than that of wild-type core protein.
Finally, we examined the subcellular localization of core protein expressed by the wild-type and NLS mutants (Fig. 7D). As expected, a fusion protein containing wild-type core protein (amino acids 1 to 71) and GFP was localized exclusively to the nucleus. Core proteins from three fusion proteins containing substitutions in each NLS motif (NLS/m1, NLS/m2, and NLS/m3) were detected primarily in the nucleus. Weak fluorescence was also observed in the cytoplasm, suggesting that these mutations caused a slight reduction in the efficiency of nuclear translocation. On the other hand, two or three NLS motif substitution mutations (NLS/m4, NLS/m5, NLS/m6, and NLS/m7) completely abolished nuclear translocation, resulting in a diffuse distribution of core protein, similar to that of GFP alone. Although it is likely that all three putative NLS motifs play a role, the above results suggest that at least two of the three putative NLS motifs are prerequisite for efficient nuclear translocation of the core protein.
DISCUSSION
HCV core protein is released from the viral polyprotein by a host protease(s) within the ER membrane at a signal peptide sequence lying between the core and envelope (E1) proteins (16, 41). Subsequently, the signal peptide is further processed by an intramembranous protease called signal peptide peptidase (38, 53). This mature form of the core protein is then released and undergoes subcellular trafficking (30, 53). The core protein localizes mainly to the ER, mitochondria, and lipid droplets. Some reports also describe localization of the core protein to the nuclei of hepatocytes in HCV-infected patients (10), transgenic mice (34), and cultured cells expressing viral polyproteins (56). Although it has been reported which sequence motifs are responsible for localization of the HCV core protein to lipid droplets and nuclei, it is uncertain which sequences target the core protein to the ER and to mitochondria. In this study, we identified sequences related to localization of the mature core protein to the ER and to mitochondria.
Through heterologous expression of core-GFP fusion proteins containing a series of deletions, we determined that a sequence extending from amino acids 112 to 152 of the core protein is required for its localization at the mitochondrial outer membrane. Translocation of nucleus-encoded mitochondrial proteins is usually dependent on N-terminal sequences, referred to as mitochondrial targeting sequences (37). However, it is also true that a significant proportion of mitochondrial proteins lack these N-terminal mitochondrial targeting sequences. Specifically, a number of outer membrane proteins do not have cleavable sequences at their N termini; rather, they are targeted to mitochondria by means of internal or C-terminal signals (31).
Since it has been reported that amino acid sequences required for targeting to the outer mitochondrial membrane form a highly hydrophobic -helical wheel, as seen in A-kinase associated protein 84/12 (4) and NADH-cytochrome b reductase (14), a predicted structure of an amphipathic -helix present between amino acids 116 and 134 (Fig. 6B and C) possibly plays a role in directing the core protein to the mitochondrial outer membrane. Sequence comparisons demonstrate conservation of the amino acid sequence and secondary structure of the region, amino acids 112 to 152, among a variety of HCV isolates, including the infectious H77c clone (55), as well as a full-length adaptive replicon (3). To gain insight into the significance of the secondary structure of the region in targeting to the mitochondria, further structural and biochemical analyses are needed.
The association of HCV core protein with the mitochondrial membrane suggests that the core protein has the ability to modulate mitochondrial function, possibly by altering the permeability of the mitochondrial membrane. The core protein induces the production of cellular reactive oxygen species in the livers of core-transgenic mice and in core-expressing cell lines (35). Reactive oxygen species, predominantly generated in mitochondria, induce genetic mutations and act as secondary messengers to regulate a variety of cellular functions, including gene expression and proliferation (1). Although the molecular mechanism by which core protein induces reactive oxygen species production is still unclear, HCV core protein is known to impair the mitochondrial electron transfer system (35). The core protein may also modulate apoptosis, since mitochondria play a major role in regulating programmed cell death. Expression of HCV proteins, including the core protein, suppresses the release of cytochrome c from mitochondria to the cytoplasm in HCV-transgenic mice, thus inhibiting Fas-mediated apoptosis (27).
Okamoto et al. recently reported that not only the C-terminal signal sequence but also amino acids 128 to 151 are required for ER retention of the core protein by using a series of N-terminally truncated core protein constructs (38). Here, in this study, we further showed that amino acids 112 to 152 mediate association of the core protein with the ER in the absence of the C-terminal signal sequence. Hope and McLauchlan demonstrated that the central domain of the core protein, amino acids 119 to 174, is important for association with lipid droplets (17). They also showed that this corresponding domain is shared with GB virus B, which is most closely related to HCV, but not with either pestiviruses or flaviviruses (18). It appears that the 41 residues identified as the sequence mediating association with the ER membrane in the present study are crucial for directing the core protein to lipid droplets, since the surface of lipid droplets must derive from the cytoplasmic side of the ER membrane.
The HCV core protein contains NLS sequences which are composed of three stretches of sequences rich in basic residues. These sequences were originally identified by experiments with fused forms of wild-type and mutated core proteins with ?-galactosidase (6, 48). C-terminally truncated versions of the core protein localize exclusively to the nucleus (48). A fraction of the core protein is detected in the nucleus even when full-length HCV core gene is expressed (Fig. 2) and as described (34, 56). However, it is difficult to demonstrate clearly the nuclear localization of the core protein by immunofluorescence, presumably because of the instability of nuclearly localized core protein (49, 33). We only observed a nuclear staining pattern of the matured core protein after adding proteasome inhibitors to the culture (33).
Generally, NLS sequences fall into two categories; (i) monopartite NLSs, which contain a single cluster of basic residues, and (ii) bipartite NLSs, which contain two clusters of basic residues separated by an unconserved linker sequence of variable length (reviewed in reference 12). Nuclear translocation of an NLS-containing cargo protein is initiated when the soluble import receptor (importin) recognizes the NLS-containing protein within the cytoplasm. Importin- contains an NLS-binding site(s), and importin-? docks importin-cargo complexes to the cytoplasmic filaments of a nuclear pore complex, after which translocation occurs through the nuclear pore. Thus, importin- functions as an adaptor between the bona fide import receptor and the NLS-containing protein.
We further characterized the NLS of the core protein and found that each of the NLS motifs of the core protein is able to bind to importin- and that at least two NLS motifs are required for efficient nuclear distribution of the core protein in cells. It appears that double mutations among three NLS motifs decrease the ability of the core protein to bind importin-. These observations suggest that the binding of the double mutants with importin- leads to no or little active translocation of the core protein into the nucleus. The double mutations may also block subsequent interactions with importin-?1, GTPase Ran, and/or NTF2/p10, which are required for translocation through the nuclear pore complexes.
The findings obtained in this study suggest that HCV core protein NLS motifs have a bipartite function. Crystallographic studies of monopartite (e.g., simian virus 40 large T antigen) and bipartite (e.g., nucleoplasmin) NLSs show that the basic residue clusters of bipartite NLSs occupy separate binding sites on importin-. In contrast, while monopartite NLSs can bind to the same sites as bipartate NLSs on importin-, they mainly bind to the N-terminal binding site, which is referred to as the major binding site on importin- (9, 11). A recent report describes an importin- variant with a mutation in the major site which results in decreased ability to bind both monopartite and bipartite NLSs. Another variant with a mutation in the minor site exhibits decreased binding only to bipartite NLS-containing proteins, making importin- nonfunctional in vivo (22). Thus, we favor a model in which the core protein bipartite NLS, composed of any two of the three basic clusters, occupies both major and minor binding sites on importin-, resulting in efficient nuclear translocation. Importin- may be equally accessible to all clusters, given their close proximity to one another, as well as the distinct conformational flexibility of the 70-residue N-terminal region of the core protein.
With regard to the molecular mechanisms participating in nuclear localization of the core protein, Moriishi et al. found that PA28 is involved in nuclear localization of the core protein. Interaction of the core protein with PA28 plays an important role in retention of the core protein in the nucleus (33). Furthermore, in yeast cells, nuclear transport of the core protein requires the activity of the small GTPase Ran/Gsp1p and is mediated by Kap123p, but neither importin- nor importin-? is involved (20). Differences in nucleocytoplasmic transport between yeast and mammalian cells might explain the inconsistencies observed in the present study. Further experiments are required to characterize the exact nature of the interaction between the core protein and components of the nuclear import machinery, particularly in cells where HCV is replicating.
In conclusion, the mature HCV core protein has an internal 41-amino-acid sequence mediating association of the viral protein with the ER and mitochondria. We also provide evidence for a novel class of bipartite NLS contained within the core protein, which comprises two of three basic motifs, thus enabling efficient nuclear targeting. Multiple functional domains influence the subcellular localization of the core protein, which ultimately depends on the balance of the respective signals.
ACKNOWLEDGMENTS
We thank colleagues in the laboratories of the Department of Virology II at the National Institute of Infectious Diseases of Japan for providing advice and help. We especially thank Mami Matsuda and Makiko Yahata for assistance in sequencing and the preparation of experimental reagents and Tomoko Mizoguchi for secretarial work. We are grateful to Karsten Weis for providing us with the plasmid containing importin- cDNA.
This work was supported in part by Second Term Comprehensive 10-Year Strategy for Cancer Control and Research on Emerging and Reemerging Infectious Diseases, Health Sciences Research Grants of the Ministry of Health, Labor and Welfare, and by the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan (ID:01-3). This work was also supported in part by a Grant-in-Aid for Young Scientists from the Ministry of Education, Culture, Sports, Science and Technology to R.S. (15790244).
REFERENCES
Adler, V., Z. Yin, K. D. Tew, and Z. Ronai. 1999. Role of redox potential and reactive oxygen species in stress signaling. Oncogene 18:6104-6111.
Barba, G., F. Harper, T. Harada, M. Kohara, S. Goulinet, Y. Matsuura, G. Eder, Z. Schaff, M. J. Chapman, T. Miyamura, and C. Bréchot. 1997. Hepatitis C virus core protein shows a cytoplasmic localization and associates to cellular lipid storage droplets. Proc. Natl. Acad. Sci. USA 94:1200-1205.
Bukh, J., T. Pietschmann, V. Lohmann, N. Krieger, K. Faulk, R. E. Engle, S. Govindarajan, M. Shapiro, M. St Claire, and R. Bartenschlager. 2002. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees. Proc. Natl. Acad. Sci. USA 99:14416-14421.
Cardone, L., T. de Cristofaro, A. Affaitati, C. Garbi, M. D. Ginsberg, M. Saviano, S. Varrone, C. S. Rubin, M. E. Gottesman, E. V. Avvedimento, and A. Feliciello. 2002. A-kinase anchor protein 84/121 are targeted to mitochondria and mitotic spindles by overlapping amino-terminal motifs. J. Mol. Biol. 320:663-675.
Chang, J., S. H. Yang, Y. G. Cho, S. B. Hwang, Y. S. Hahn, and Y. C. Sung. 1998. Hepatitis C virus core from two different genotypes has an oncogenic potential but is not sufficient for transforming primary rat embryo fibroblasts in cooperation with the H-ras oncogene. J. Virol. 72:3060-3065.
Chang, S. C., J. H. Yen, H. Y. Kang, M. H. Jang, and M. F. Chang. 1994. Nuclear localization signals in the core protein of hepatitis C virus. Biochem. Biophys. Res. Commun. 205:1284-1290.
Choo, Q. L., G. Kuo, A. J. Weiner, L. R. Overby, D. W. Bradley, and M. Houghton. 1989. Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome. Science 244:359-362.
Choo, Q. L., K. H. Richman, J. H. Han, K. Berger, C. Lee, C. Dong, C. Gallegos, D. Coit, R. Medina-Selby, P. J. Barr, et al. 1991. Genetic organization and diversity of the hepatitis C virus. Proc. Natl. Acad. Sci. USA 88:2451-2455.
Conti, E., M. Uy, L. Leighton, G. Blobel, and J. Kuriyan. 1998. Crystallographic analysis of the recognition of a nuclear localization signal by the nuclear import factor karyopherin alpha. Cell 94:193-204.
Falcon, V., N. Acosta-Rivero, G. Chinea, M. C. de la Rosa, I. Menendez, S. Duenas-Carrera, B. Gra, A. Rodriguez, V. Tsutsumi, M. Shibayama, J. Luna-Munoz, M. M. Miranda-Sanchez, J. Morales-Grillo, and J. Kouri. 2003. Nuclear localization of nucleocapsid-like particles and HCV core protein in hepatocytes of a chronically HCV-infected patient. Biochem. Biophys. Res. Commun. 310:54-58.
Fontes, M. R., T. Teh, and B. Kobe. 2000. Structural basis of recognition of monopartite and bipartite nuclear localization sequences by mammalian importin-alpha. J. Mol. Biol. 297:1183-1194.
G?rlich, D., and U. Kutay. 1999. Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell. Dev. Biol. 15:607-660.
Grakoui, A., D. W. McCourt, C. Wychowski, S. M. Feinstone, and C. M. Rice. 1993. Characterization of the hepatitis C virus-encoded serine proteinase: determination of proteinase-dependent polyprotein cleavage sites. J. Virol. 67:2832-2843.
Hahne, K., V. Haucke, L. Ramage, and G. Schatz. 1994. Incomplete arrest in the outer membrane sorts NADH-cytochrome b5 reductase to two different submitochondrial compartments. Cell 79:829-839.
Harada, S., Y. Watanabe, K. Takeuchi, T. Suzuki, T. Katayama, Y. Takebe, I. Saito, and T. Miyamura. 1991. Expression of processed core protein of hepatitis C virus in mammalian cells. J. Virol. 65:3015-3021.
Hijikata, M., N. Kato, Y. Ootsuyama, M. Nakagawa, and K. Shimotohno. 1991. Gene mapping of the putative structural region of the hepatitis C virus genome by in vitro processing analysis. Proc. Natl. Acad. Sci. USA 88:5547-5551.
Hope, R. G., and J. McLauchlan. 2000. Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein. J. Gen. Virol. 81:1913-1925.
Hope, R. G., D. J. Murphy, and J. McLauchlan. 2002. The domains required to direct core proteins of hepatitis C virus and GB virus-B to lipid droplets share common features with plant oleosin proteins. J. Biol. Chem. 277:4261-4270.
Hüssy, P., H. Langen, J. Mous, and H. Jacobsen. 1996. Hepatitis C virus core protein: carboxy-terminal boundaries of two processed species suggest cleavage by a signal peptide peptidase. Virology 224:93-104.
Isoyama, T., S. Kuge, and A. Nomoto. 2002. The core protein of hepatitis C virus is imported into the nucleus by transport receptor Kap123p but inhibits Kap121p-dependent nuclear import of yeast AP1-like transcription factor in yeast cells. J. Biol. Chem. 277:39634-39641.
Lai, M. M., and C. F. Ware. 2000. Hepatitis C virus core protein: possible roles in viral pathogenesis. Curr. Top. Microbiol. Immunol. 242:117-134.
Leung, S. W., M. T. Harreman, M. R. Hodel, A. E. Hodel, and A. H. Corbett. 2003. Dissection of the karyopherin alpha nuclear localization signal (NLS)-binding groove: functional requirements for NLS binding. J. Biol. Chem. 278:41947-41953.
Liu, Q., C. Tackney, R. A. Bhat, A. M. Prince, and P. Zhang. 1997. Regulated processing of hepatitis C virus core protein is linked to subcellular localization. J. Virol. 71:657-662.
Lo, S. Y., F. Masiarz, S. B. Hwang, M. M. Lai, and J. H. Ou. 1995. Differential subcellular localization of hepatitis C virus core gene products. Virology 213:455-461.
Lo, S. Y., M. Selby, M. Tong, and J. H. Ou. 1994. Comparative studies of the core gene products of two different hepatitis C virus isolates: two alternative forms determined by a single amino acid substitution. Virology 199:124-131.
Lu, W., A. Strohecker, and J. H. Ou. 2001. Post-translational modification of the hepatitis C virus core protein by tissue transglutaminase. J. Biol. Chem. 276:47993-47999.
Machida, K., K. Tsukiyama-Kohara, E. Seike, S. Tone, F. Shibasaki, M. Shimizu, H. Takahashi, Y. Hayashi, N. Funata, C. Taya, H. Yonekawa, and M. Kohara. 2001. Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins. J. Biol. Chem. 276:12140-12146.
Matsumoto, M., S. B. Hwang, K. S. Jeng, N. Zhu, and M. M. Lai. 1996. Homotypic interaction and multimerization of hepatitis C virus core protein. Virology 218:43-51.
McLauchlan, J. 2000. Properties of the hepatitis C virus core protein: a structural protein that modulates cellular processes. J. Viral Hepatitis 7:2-14.
McLauchlan, J., M. K. Lemberg, G. Hope, and B. Martoglio. 2002. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets. EMBO J. 21:3980-3988.
Mihara, K. 2000. Targeting and insertion of nuclear-encoded preproteins into the mitochondrial outer membrane. Bioessays 22:364-371.
Moradpour, D., C. Englert, T. Wakita, and J. R. Wands. 1996. Characterization of cell lines allowing tightly regulated expression of hepatitis C virus core protein. Virology 222:51-63.
Moriishi, K., T. Okabayashi, K. Nakai, K. Moriya, K. Koike, S. Murata, T. Chiba, K. Tanaka, R. Suzuki, T. Suzuki, T. Miyamura, and Y. Matsuura. 2003. Proteasome activator PA28gamma-dependent nuclear retention and degradation of hepatitis C virus core protein. J. Virol. 77:10237-10249.
Moriya, K., H. Fujie, Y. Shintani, H. Yotsuyanagi, T. Tsutsumi, K. Ishibashi, Y. Matsuura, S. Kimura, T. Miyamura, and K. Koike. 1998. The core protein of hepatitis C virus induces hepatocellular carcinoma in transgenic mice. Nat. Med. 4:1065-1067.
Moriya, K., K. Nakagawa, T. Santa, Y. Shintani, H. Fujie, H. Miyoshi, T. Tsutsumi, T. Miyazawa, K. Ishibashi, T. Horie, K. Imai, T. Todoroki, S. Kimura, and K. Koike. 2001. Oxidative stress in the absence of inflammation in a mouse model for hepatitis C virus-associated hepatocarcinogenesis. Cancer Res. 61:4365-4370.
Moriya, K., H. Yotsuyanagi, Y. Shintani, H. Fujie, K. Ishibashi, Y. Matsuura, T. Miyamura, and K. Koike. 1997. Hepatitis C virus core protein induces hepatic steatosis in transgenic mice. J. Gen. Virol. 78:1527-1531.
Neupert, W. 1997. Protein import into mitochondria. Annu. Rev. Biochem. 66:863-917.
Okamoto, K., K. Moriishi, T. Miyamura, and Y. Matsuura. 2004. Intramembrane proteolysis and endoplasmic reticulum retention of hepatitis C virus core protein. J. Virol. 78:6370-6380.
Okuda, M., K. Li, M. R. Beard, L. A. Showalter, F. Scholle, S. M. Lemon, and S. A. Weinman. 2002. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 122:366-375.
Perlemuter, G., A. Sabile, P. Letteron, G. Vona, A. Topilco, Y. Chretien, K. Koike, D. Pessayre, J. Chapman, G. Barba, and C. Bréchot. 2002. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis. FASEB J. 16:185-194.
Ralston, R., K. Thudium, K. Berger, C. Kuo, B. Gervase, J. Hall, M. Selby, G. Kuo, M. Houghton, and Q. L. Choo. 1993. Characterization of hepatitis C virus envelope glycoprotein complexes expressed by recombinant vaccinia viruses. J. Virol. 67:6753-6761.
Ray, R. B., L. M. Lagging, K. Meyer, and R. Ray. 1996. Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype. J. Virol. 70:4438-4443.
Roth, J., D. J. Taatjes, and M. J. Warhol. 1989. Prevention of non-specific interactions of gold-labeled reagents on tissue sections. Histochemistry 92:47-56.
Sabile, A., G. Perlemuter, F. Bono, K. Kohara, F. Demaugre, M. Kohara, Y. Matsuura, T. Miyamura, C. Brechot, and G. Barba. 1999. Hepatitis C virus core protein binds to apolipoprotein AII and its secretion is modulated by fibrates. Hepatology 30:1064-1076.
Santolini, E., G. Migliaccio, and N. La Monica. 1994. Biosynthesis and biochemical properties of the hepatitis C virus core protein. J. Virol. 68:3631-3641.
Selby, M. J., Q. L. Choo, K. Berger, G. Kuo, E. Glazer, M. Eckart, C. Lee, D. Chien, C. Kuo, and M. Houghton. 1993. Expression, identification and subcellular localization of the proteins encoded by the hepatitis C viral genome. J. Gen. Virol. 74:1103-1113.
Shimoike, T., S. Mimori, H. Tani, Y. Matsuura, and T. Miyamura. 1999. Interaction of hepatitis C virus core protein with viral sense RNA and suppression of its translation. J. Virol. 73:9718-9725.
Suzuki, R., Y. Matsuura, T. Suzuki, A. Ando, J. Chiba, S. Harada, I. Saito, and T. Miyamura. 1995. Nuclear localization of the truncated hepatitis C virus core protein with its hydrophobic C terminus deleted. J. Gen. Virol. 76:53-61.
Suzuki, R., K. Tamura, J. Li, K. Ishii, Y. Matsuura, T. Miyamura, and T. Suzuki. 2001. Ubiquitin-mediated degradation of hepatitis C virus core protein is regulated by processing at its carboxyl terminus. Virology 280:301-309.
Tanaka, Y., T. Shimoike, K. Ishii, R. Suzuki, T. Suzuki, H. Ushijima, Y. Matsuura, and T. Miyamura. 2000. Selective binding of hepatitis C virus core protein to synthetic oligonucleotides corresponding to the 5' untranslated region of the viral genome. Virology 270:229-236.
Tellinghuisen, T. L., and C. M. Rice. 2002. Interaction between hepatitis C virus proteins and host cell factors. Curr. Opin. Microbiol. 5:419-427.
Thomson, M., and T. J. Liang. 2000. Molecular biology of hepatitis C virus, p. 1-23. In T. J. Liang and J. H. Hoofnagle (ed.), Hepatitis C. Academic Press, San Diego, Calif.
Weihofen, A., K. Binns, M. K. Lemberg, K. Ashman, and B. Martoglio. 2002. Identification of signal peptide peptidase, a presenilin-type aspartic protease. Science 296:2215-2218.
Weis, K., I. W. Mattaj, and A. I. Lamond. 1995. Identification of hSRP1 alpha as a functional receptor for nuclear localization sequences. Science 268:1049-1053.
Yanagi, M., R. H. Purcell, S. U. Emerson, and J. Bukh. 1997. Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proc. Natl. Acad. Sci. USA 94:8738-8743.
Yasui, K., T. Wakita, K. Tsukiyama-Kohara, S. I. Funahashi, M. Ichikawa, T. Kajita, D. Moradpour, J. R. Wands, and M. Kohara. 1998. The native form and maturation process of hepatitis C virus core protein. J. Virol. 72:6048-6055.(Ryosuke Suzuki, Shinichir)