E6AP Ubiquitin Ligase Mediates Ubiquitylation and Degradation of Hepatitis C Virus Core Protein

Abstract

Hepatitis C virus (HCV) core protein is a major component of viral nucleocapsid and a multifunctional protein involved in viral pathogenesis and hepatocarcinogenesis. We previously showed that the HCV core protein is degraded through the ubiquitin-proteasome pathway. However, the molecular machinery for core ubiquitylation is unknown. Using tandem affinity purification, we identified the ubiquitin ligase E6AP as an HCV core-binding protein. E6AP was found to bind to the core protein in vitro and in vivo and promote its degradation in hepatic and nonhepatic cells. Knockdown of endogenous E6AP by RNA interference increased the HCV core protein level. In vitro and in vivo ubiquitylation assays showed that E6AP promotes ubiquitylation of the core protein. Exogenous expression of E6AP decreased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected Huh-7 cells. Furthermore, knockdown of endogenous E6AP by RNA interference increased intracellular core protein levels and supernatant HCV infectivity titers in the HCV JFH1-infected cells. Taken together, our results provide evidence that E6AP mediates ubiquitylation and degradation of HCV core protein. We propose that the E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of viral nucleocapsid protein.

Hepatitis C virus (HCV; a single-stranded, positive-sense RNA virus that is classified in the family Flaviviridae) is the main cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (5, 26, 45). More than 170 million people worldwide are chronically infected with HCV (41). The approximately 9.6-kb HCV genome encodes a unique open reading frame that is translated into a polyprotein (5, 54). The polyprotein is cleaved cotranslationally into at least 10 proteins by viral proteases and cellular signalases (6, 10).

The HCV core protein represents the first 1 to 191 amino acids (aa) of the polyprotein and is followed by two glycoproteins, E1 and E2 (6). The core protein plays a central role in the packaging of viral RNA (25, 40); modulates various cellular processes, including signal transduction pathways, transcriptional control, cell cycle progression, apoptosis, lipid metabolism, and the immune response (9, 40); and has transforming potential in certain cells (43). Mice transgenic for the HCV core gene develop steatosis (32) and later hepatocellular carcinoma (31). These findings suggest that HCV core protein plays a crucial role in hepatocarcinogenesis.

Two major forms of the HCV core protein, p21 (mature form) and p23 (immature form), can be generated in cultured cells (60). Cellular signal peptidase cleaves at the junction of the core/E1, releasing the immature form of the core protein from the polypeptide (12, 46). Signal peptide peptidase cleaves just before the signal sequence, liberating the mature form of the HCV core protein at the cytoplasmic face of the endoplasmic reticulum (29). Several different sites have been proposed as potential cleavage sites of signal peptide peptidase, such as Leu-179 (15, 29), Phe-177 (36, 37), Leu-182 (15), and Ser-173 (46). Further processing of the HCV core protein yields a 17-kDa product with a C terminus at around amino acid 152. A truncated form of the core protein, p17, was found in transfected cells (42, 52) and liver tissues from humans with hepatocellular carcinoma (59). The majority of this protein translocates to the nucleus. The C terminus of the core protein is important for regulating the stability of the protein (20, 52).

We previously showed that the C-terminally truncated forms of the core protein are degraded through the ubiquitin-proteasome pathway (52). We found that the mature form of the core protein, p21, also links to a few ubiquitin moieties, suggesting that the ubiquitin-proteasome pathway involves proteolysis of heterologous species of the core protein (52). Overexpression of PA28γ (a REG family proteasome activator also known as REGγ or Ki antigen) enhances the proteasomal degradation of the HCV core protein (30). A recent study has shown that PA28γ is involved in the degradation of the steroid receptor coactivator 3 (SRC-3) in an ATP- and ubiquitin-independent manner (27). It is still unclear what E3 ubiquitin ligase is responsible for ubiquitylation of the HCV core protein.

E6AP was initially identified as the cellular factor that stimulates ubiquitin-mediated degradation of the tumor suppressor p53 in conjunction with the E6 protein of cancer-associated human papillomavirus types 16 and 18 (14, 48). The E6-E6AP complex functions as a E3 ubiquitin ligase in the ubiquitylation of p53 (49). E6AP is the prototype of a family of ubiquitin ligases called HECT domain ubiquitin ligases, all of which contain a domain homologous to the E6AP carboxyl terminus (13). Interestingly, E6AP is not involved in the regulation of p53 ubiquitylation in the absence of E6 (55). Several potential E6-independent substrates for E6AP have been identified, such as hHR23A, Blk, and Mcm7 (23, 24, 35). E6AP is also a candidate gene for Angelman syndrome, which is a severe neurological disorder characterized by mental retardation (21).

This study aimed to identify endogenous ubiquitin-proteasome pathway proteins that are associated with HCV core protein. Tandem affinity purification and mass spectrometry analysis identified E6AP as an HCV core-binding protein. Here we present evidence that E6AP associates with HCV core protein in vitro and in vivo and is involved in ubiquitylation and degradation of HCV core protein. We propose that an E6AP-mediated ubiquitin-proteasome pathway may affect the production of HCV particles through controlling the amounts of HCV core protein.

MATERIALS AND METHODS

Cell culture and transfection.

Human embryonic kidney 293T cells, human hepatoblastoma HepG2 cells, and human hepatoma Huh-7 cells were cultured in Dulbecco's modified Eagle's medium (Sigma) supplemented with 50 IU/ml penicillin, 50 μg/ml streptomycin (Invitrogen), and 10% (vol/vol) fetal bovine serum (JRH Biosciences) at 37°C in a 5% CO₂ incubator. 293T cells and HepG2 cells were transfected with plasmid DNA using FuGene 6 transfection reagents (Roche). Huh-7 cells were transfected with plasmid DNA using TransIT LT1 transfection reagents (Mirus).

Plasmids and recombinant baculoviruses.

MEF tag cassette (containing myc tag, the tobacco etch virus protease cleavage site, and FLAG tag) (16) was fused to the N terminus of the cDNA encoding core protein of HCV NIHJ1 (genotype 1b) (1). To express MEF-tagged core protein in mammalian cells, the genome coding for HCV core protein (amino acids 1 to 191) was amplified by PCR using pBR HCV NIHJ1 as a template. Sense oligonucleotide containing a Kozak consensus translation initiation codon and antisense oligonucleotide containing an in-frame translation stop codon were synthesized by PCR. The amplified PCR product was purified, digested with EcoRI and EcoRV, and then inserted into the EcoRI-EcoRV site of pcDNA3-MEF. FLAG-tagged HCV core expression plasmids based upon pCAGGS (34) were described previously (30). To express E6AP and the active-site cysteine-to-alanine mutant of E6AP in mammalian cells, pCMV4-HA-E6AP isoform II and pCMV4-HA-E6AP C-A were utilized (19). The C-A mutation was introduced at the site of E6AP C843. To express E6AP and E6AP C-A under the CAG promoter, the E6AP fragment and the E6AP C-A fragment were amplified by PCR, purified, digested with SmaI and NotI, and blunt ended using a DNA blunting kit (Takara). These PCR fragments were subcloned into pCAGGS.

To make a fusion protein consisting of glutathione S-transferase (GST) fused to the N terminus of E6AP in Escherichia coli, the E6AP fragment was amplified by PCR and the resultant product was cloned into the SmaI-NotI site of pGEX4T-1 vector (Amersham Biosciences). To express a series of E6AP truncation mutants as GST fusion proteins, each fragment was amplified by PCR and cloned into the SmaI-NotI site of pGEX4T-1. To purify GST core protein efficiently by two-step affinity purification, we fused hexahistidine (His) tag to the C terminus of GST fusion proteins. To bacterially express HCV core (aa 1 to 173) protein as a fusion protein containing N-terminal GST tag and C-terminal His tag, core fragment was amplified by PCR and the resultant product was cloned into the EcoRI-NotI site of pGEX4T-1 vector. The resultant plasmid was designated pGEX GST-C173HT. To express GST core (1-152)-His and GST-His in E. coli, pGEX core (1-152)-His and pGEX-His were constructed similarly. The resultant plasmids were designated pGEX GST-C152HT and pGEX GST-HT, respectively.

To generate recombinant baculoviruses expressing GST-E6AP, GST-E6AP fragment was excised from pGEX E6AP by digestion with SmaI and Tth111I and ligated into the SmaI-Tth111I site of pVL1392 (Invitrogen). To express GST-E6AP C-A, pVLGST-E6AP C-A was constructed similarly. To generate recombinant baculovirus expressing HCV core (aa 1 to 173) protein as a fusion protein containing N-terminal GST tag and C-terminal His tag, GST-C173HT fragment was amplified by PCR using pGEX GST-C173HT as a template, digested with BglII-XbaI, and subcloned into the BglII-XbaI site of pVL1392. To generate recombinant baculoviruses expressing GST-C152HT and GST-HT, cDNA fragments corresponding to GST-C152HT and GST-HT were amplified by PCR and subcloned into pVL1392, respectively. The resultant plasmids were designated pVLGST-C173HT, pVLGST-C152HT, and pVLGST-HT. To generate recombinant baculovirus expressing MEF-tagged E6AP, cDNA fragment encoding MEF-E6AP was subcloned into pVL1392. To express HCV core protein in the TNT-coupled wheat germ lysate system (Promega), HCV core cDNA was inserted in the EcoRI site of pCMVTNT (Promega). The primer sequences used in this study are available from the authors upon request. The sequences of the inserts were extensively verified using an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). Recombinant baculoviruses were recovered using a BaculoGold transfection kit (Pharmingen) according to the manufacturer's instructions.

Antibodies.

The mouse monoclonal antibodies (MAbs) used in this study were anti-hemagglutinin (anti-HA) MAb (12CA5; Roche), anti-FLAG (M2) MAb (Sigma), anti-c-myc MAb (9E10; Santa Cruz), anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) MAb (Chemicon), anti-GST MAb (Santa Cruz), anti-ubiquitin MAb (Chemicon), anti-E6AP MAb (E6AP-330) (Sigma), anticore MAb (B2; Anogen), and another anti-core MAb (2H9) (56). Polyclonal antibodies (PAbs) used in this study were anti-HA rabbit PAb (Y-11; Santa Cruz), anti-FLAG rabbit PAb (F7425; Sigma), anti-E6AP rabbit PAb (H-182; Santa Cruz), anti-DDX3 rabbit PAb (47), anti-PA28γ rabbit PAb (Affiniti), and anti-GST goat PAb (Amersham). Anticore rabbit PAb (TS1) was raised against the recombinant GST core protein.

MEF purification procedure.

293T cells were transfected with the plasmid expressing MEF core by the calcium phosphate precipitation method (4). After the cells were lysed, the expressed MEF core and its binding proteins were recovered following the procedure described previously (16). 293T cells transfected with pcDNA3-MEF core in four 10-cm dishes were lysed in 2 ml of lysis buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% (wt/vol) glycerol, 100 mM NaF, 1 mM Na₃VO₄, 1% (wt/vol) Triton X-100, 5 μM ZnCl₂, 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, and 1 μg/ml leupeptin. The lysate was centrifuged at 100,000 × g for 20 min at 4°C. The supernatant was passed through a 5-μm filter, incubated with 100 μl of Sepharose beads for 60 min at 4°C, and then passed through a 0.65-μm filter. The filtered supernatant was mixed with 100 μl of anti-myc-conjugated Sepharose beads for the first immunoprecipitation. After incubation for 90 min at 4°C, the beads were washed five times with 1 ml of TNTG buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% [wt/vol] glycerol, and 1% [wt/vol] Triton X-100), twice with 1 ml of buffer A (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 1% [wt/vol] Triton X-100), and finally once with 1 ml of TNT buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% [wt/vol] Triton X-100). The washed beads were incubated with 10 U of tobacco etch virus protease (Invitrogen) in TNT buffer (100 μl) to release bound protein complexes from the beads. After incubation for 60 min at room temperature, the supernatant was pooled and the beads were washed twice with 70 μl of buffer A. The resulting supernatants were combined and incubated with 12 μl of FLAG-Sepharose beads for the second immunoprecipitation. After incubation for 60 min at room temperature, the beads were washed three times with 240 μl of buffer A, and proteins bound to the immobilized HCV core protein on the FLAG beads were dissociated by incubation with 80 μg/ml FLAG peptide (NH₂-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys-COOH) (Sigma).

MS/MS.

Proteins were separated by 9% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by silver staining. The stained bands were excised and digested in the gel with lysylendoprotease-C (Lys-C), and the resulting peptide mixtures were analyzed using a direct nanoflow liquid chromatography-tandem mass spectrometry (MS/MS) system (33), equipped with an electrospray interface reversed-phase column, a nanoflow gradient device, a high-resolution Q-time of flight hybrid mass spectrometer (Q-TOF2; Micromass), and an automated data analysis system. All the MS/MS spectra were searched against the nonredundant protein sequence database maintained at the National Center for Biotechnology Information using the Mascot program (Matrixscience) to identify proteins. The MS/MS signal assignments were also confirmed manually.

Expression and purification of recombinant proteins.

E. coli BL21(DE3) cells were transformed with plasmids expressing GST fusion protein or His-tagged protein and grown at 37°C. Expression of the fusion protein was induced by 1 mM isopropyl-β-d-thiogalactopyranoside at 37°C for 4 h. Bacteria were harvested, suspended in lysis buffer (phosphate-buffered saline [PBS] containing 1% Triton X-100), and sonicated on ice.

Hi5 cells were infected with recombinant baculoviruses to produce GST-C173HT, GST-C152HT, GST-HT, MEF-E6AP, and His-tagged mouse E1 (17). GST and GST fusion proteins were purified on glutathione-Sepharose beads (Amersham Bioscience) according to the manufacturer's protocols. His-tagged proteins were purified on nickel-nitrilotriacetic acid beads (QIAGEN) according to the manufacturer's protocols. MEF-E6AP and MEF-E6AP C-A were purified on anti-FLAG M2 agarose beads (Sigma) according to the manufacturer's protocols.

Immunoblot analysis.

Immunoblot analysis was performed essentially as described previously (11). The membrane was visualized with SuperSignal West Pico chemiluminescent substrate (Pierce).

HCV core protein and E6AP binding assays.

To map the E6AP binding site on HCV core protein, 2.5 μg of purified recombinant GST-E6AP expressed in Hi5 cells was mixed with 1,000 μg of 293T cell lysates transfected with a series of FLAG-tagged HCV core deletion mutants as indicated. The protein concentration of the cells was determined using the bicinchoninic acid protein assay kit (Pierce). The mixtures were immunoprecipitated with anti-FLAG M2 agarose beads (Sigma), and proteins bound to the immobilized HCV core protein on anti-FLAG beads were dissociated with FLAG peptide (Sigma). The eluates were analyzed by immunoblotting with anti-GST PAb. To map the HCV core-binding site on E6AP, GST pull-down assays were performed as described previously (51).

In vivo ubiquitylation assay.

In vivo ubiquitylation assays were performed essentially as described previously (57). FLAG-core was immunoprecipitated with anti-FLAG beads. Immunoprecipitates were analyzed by immunoblotting, using either anti-HA PAb or anticore PAb (TS1) to detect ubiquitylated core proteins.

In vitro ubiquitylation assay.

For in vitro ubiquitylation of HCV core protein, purified GST-C173HT and GST-C152HT were used as substrates. Purified GST-HT was used as a negative control. Assays were done in 40-μl volumes containing 20 mM Tris-HCl, pH 7.6, 50 mM NaCl, 5 mM ATP, 10 mM MgCl₂, 8 μg of bovine ubiquitin (Sigma), 0.1 mM dithiothreitol, 200 ng mouse E1, 200 ng E2 (UbcH7), and 0.5 μg each of MEF-E6AP or MEF-E6AP C-A. The reaction mixtures were incubated at 37°C for 120 min followed by purification with glutathione-Sepharose beads and immunoblotting with the indicated antibodies.

siRNA transfection.

293T cells or Huh-7 cells at 3 × 10⁵ cells in a six-well plate were transfected with 40 pmol of either E6AP-specific short interfering RNA (siRNA; Sigma) or scramble negative-control siRNA duplexes (Sigma) using HiPerFect transfection reagent (QIAGEN) following the manufacturer's instructions. The siRNA target sequences were as follows: E6AP (sense), 5′-GGGUCUACACCAGAUUGCUTT-3′; scramble negative control (sense), 5′-UUGCGGGUCUAAUCACCGATT-3′.

CHX half-life experiments.

To examine the half-life of HCV core protein, transfected 293T cells were treated with 50 μg/ml cycloheximide (CHX) at 44 h posttransfection. The cells at zero time points were harvested immediately after treatment with CHX. Cells from subsequent time points were incubated in medium containing CHX at 37°C for 3, 6, and 9 h as indicated.

Infection of Huh-7 cells with secreted HCV.

Infectious HCV JFH1 was produced in Huh-7.5.1 cells (61) as described previously (56). Culture supernatant containing infectious HCV JFH1 was collected and passed through a 0.22-μm filter. Naïve Huh-7 cells were seeded 24 h before infection at a density of 1 × 10⁶ in a 10-cm dish. The cells were incubated with 2.5 ml of the inoculum (6.5 × 10³ 50% tissue culture infectious dose [TCID₅₀]/ml) for 3 h, washed three times with PBS, and supplemented with fresh complete Dulbecco's modified Eagle's medium. Then the cells were transfected with 6 μg each of pCAGGS, pCAG-HA-E6AP, or pCAG-HA-E6AP C-A by using TransIT LT1 (Mirus). The cells were trypsinized and replated in six-well plates at 1 day postinfection. The culture medium was changed every 2 days. The culture supernatants and the cells were collected at days 3 and 7 postinfection.

Quantitation of HCV RNA and core protein.

We quantitated HCV core protein in cell lysate using the HCV core antigen enzyme-linked immunosorbent assay (ELISA) (Ortho-Clinical Diagnostics). Total RNA was extracted from cells using TRIzol reagent (Invitrogen). To quantitate HCV RNAs, real-time reverse transcription-PCR was performed as described previously (53).

Infectivity assay.

The TCID₅₀ was calculated essentially based on the method described previously (28). Virus titration was performed by seeding Huh-7 cells in 96-well plates at 1 × 10⁴ cells/well. Samples were serially diluted fivefold in complete growth medium and used to infect the seeded cells (six wells per dilution). Following 3 days of incubation, the cells were immunostained for core with anticore MAb (2H9). Wells that expressed at least one core-expressing cell were counted as positive, and the TCID₅₀ was calculated.

Immunocytochemistry and fluorescence microscopy.

Cells on collagen-coated coverslips were washed with PBS, fixed with 4% paraformaldehyde for 30 min at 4°C, and permeabilized with PBS containing 0.2% Triton X-100. Cells were preincubated with BlockAce (Dainippon Pharmaceuticals), incubated with specific antibodies as primary antibodies, washed, and incubated with rhodamine-conjugated goat anti-rabbit immunoglobulin G (ICN Pharmaceuticals, Inc.) and Qdot 565-conjugated goat anti-mouse immunoglobulin G (Quantumdot) as secondary antibody. Then the cells were washed with PBS, counterstained with DAPI (4′,6′-diamidino-2-phenylindole) solution (Sigma) for 3 min, mounted on glass slides, and examined with a BZ-8000 microscope (Keyence).

Knockdown of endogenous E6AP in HCV JFH1-infected Huh-7 cells.

Naïve Huh-7 cells at 10⁶ cells/10-cm dish were inoculated with 2.5 ml of the inoculum including infectious HCV JFH1 (6.5 × 10³ TCID₅₀/ml) and cultured. The cells were replated in a six-well plate at 3 × 10⁵ cells/well at day 11 postinfection and transfected with 40 pmol of E6AP siRNA or control siRNA. The culture medium was changed at 24 h after transfection. The cells were harvested at day 2 after transfection, and the intracellular core protein levels were quantitated using the HCV core antigen ELISA. The culture supernatants were collected at day 2 after transfection and assayed for TCID₅₀ determinations.

RESULTS

Identification of E6AP as an HCV core-binding protein.

To identify the molecular machinery for HCV core ubiquitylation, we searched for endogenous ubiquitin-proteasome pathway proteins that associated with HCV core protein. HCV core-binding proteins (i.e., MEF core and its binding proteins, recovered from lysed cells) were purified by a tandem affinity purification procedure using a tandem tag (known as MEF tag) (16). Ten proteins were reproducibly detected (Fig. 1A, lane 2), but none were recovered from lysed control cells transfected with empty vector alone (Fig. 1A, lane 1).

FIG. 1.

HCV core protein associates with E6AP in vivo. (A) 293T cells were transfected with pcDNA3-MEF-core or empty plasmid, incubated for 48 h, and then harvested. The expressed MEF-core and binding proteins were recovered using the MEF purification procedure. Proteins bound to the MEF-core immobilized on anti-FLAG beads were dissociated with FLAG peptides, resolved by 9% SDS-PAGE, and visualized by silver staining. Control experiments were performed using 293T cells transfected with vector alone. The positions of E6AP, DDX3, and PA28γ are indicated by arrows. (B to E) The proteins detected in panel A were confirmed by immunoblotting with appropriate antibodies: E6AP (B), DDX3 (C), PA28γ (D), and MEF-core (E).

To identify the proteins, silver-stained bands were excised from the gel, digested by Lys-C, and analyzed using a direct nanoflow liquid chromatography-MS/MS system. Nine proteins were identified: two known HCV core-binding proteins, human DEAD box protein DDX3 (38) and proteasome activator PA28γ (30), and seven potential HCV core-binding proteins. E6AP was identified (Fig. 1A, lane 2) on the basis of five independent MS/MS spectra (Table 1). Immunoblot analyses confirmed the proteomic identification of E6AP, DDX3, PA28γ, and MEF-core (Fig. 1B to E).

TABLE 1.

Identification of E6AP by tandem mass spectrometry^a

Peptide m/z	Sequence determined	Residues
720.9	VFSSAEALVQSFR	156-168
922.4	AACSAAAMEEDSEASSSR	196-213
774.9	MMETFQQLITYK	339-350
1,053.1	ITVLYSLVQGQQLNPYLR	507-524
809.4	EFVISYSDYILNK	712-724

^aThe protein was ubiquitin protein ligase E3A (E6AP) isoform 2 (GenBank accession no. NP_000453).

E6AP binding domain for HCV core protein.

The E6AP binding domain for HCV core protein was investigated. Figure 2A is a schematic representation of E6AP and known motifs in E6AP. A series of deletion mutants of E6AP as GST fusion proteins were expressed in E. coli. GST pull-down assays found that the carboxyl-terminal deletion mutant E6AP (1-517), but not E6AP (1-418) (Fig. 2C, lanes C and D), and the amino-terminal deletion mutant E6AP (418-875), but not E6AP (517-875) (Fig. 2C, lanes J and K), were able to bind to the core protein. The signal was absent when unprogrammed wheat germ extracts (the negative control) were used as a source of proteins (data not shown). GST pull-down assays (Fig. 2B) found that the region from aa 418 to aa 517 is important for binding to the HCV core protein. An assay of the ability of GST-E6AP (418-517) to bind to the HCV core protein was confirmatory (Fig. 2C, lane N) and led to the conclusion that the HCV core-binding domain of E6AP was aa 418 to aa 517.

FIG. 2.

Mapping of the HCV core-binding domain for E6AP. (A) Structure of E6AP. Shown is a schematic representation of the regions of E6AP isoform II that mediate E6 binding (aa 401 to 418), E6-dependent association with p53 (aa 290 to 791), and the HECT catalytic domain (aa 525 to 875). The catalytic cysteine residue is located at aa 843. (B) Schematic representation of GST-E6AP proteins. GST proteins A through N contain the E6AP amino acids indicated to the right. The shaded region of each represents the GST sequence. Closed boxes represent proteins that are bound specifically to HCV core protein, and open boxes represent those that are not bound. (C) Binding of HCV core protein to GST-E6AP proteins A through N. In vitro-translated core protein (aa 1 to 173) was assayed for association with GST (-) or the GST-E6AP fusion proteins A through N. Association of core protein was detected by immunoblotting with anti-core MAb.

The HCV core-binding domain for E6AP.

By use of a panel of HCV core deletion mutants (Fig. 3A), GST-E6AP was found to coimmunoprecipitate with all of the FLAG-core proteins (Fig. 3A, lanes A to H) except FLAG-core (72-191) or FLAG-core (92-191) (Fig. 3A, lanes I and J). No association of control GST protein with any FLAG-core proteins was observed (data not shown). These data suggest that the aa-58-to-aa-71 segment of the HCV core binds to E6AP. The ability of GST-core (58-71) to associate with purified MEF-E6AP confirmed that the core (aa 58-71) was the site for E6AP binding on the HCV core protein (Fig. 3B).

FIG. 3.

Mapping of the E6AP binding domain for HCV core protein. (A) In vitro binding of E6AP to HCV core protein. 293T cells were transfected with each plasmid indicated in the upper panel. At 48 h posttransfection, cell lysates were mixed with purified GST-E6AP, immunoprecipitated with anti-FLAG beads, and then immunoblotted with anti-GST PAb (middle panel) or anti-FLAG MAb (bottom panel). The last lane (input) represents GST-E6AP used in this assay (middle panel). (B) Binding of GST-core (aa 58 to aa 71) to purified MEF-E6AP. GST served as a negative control for binding. Upper panel, Coomassie blue-stained SDS-PAGE of GST and GST-core (58-71). Lower panel, results of the GST pull-down assay. MEF-E6AP was detected by anti-myc MAb. CBB, Coomassie brilliant blue; IB, immunoblot.

E6AP decreases steady-state levels of HCV core protein in 293T cells and HepG2 cells.

One of the features of HECT domain ubiquitin ligases is direct association with their substrates (50). Thus, we hypothesized that E6AP would function as an E3 ubiquitin ligase for the HCV core protein. We assessed the effects of E6AP on the HCV core protein in 293T cells. FLAG-core (1-191) together with HA-tagged wild-type E6AP, catalytically inactive mutant E6AP, E6AP C-A (19), or WWP1 (another HECT domain ubiquitin ligase) (22) was introduced into 293T cells, and the levels of the core protein were examined by immunoblotting. The steady-state levels of the core protein decreased with an increase in the amount of E6AP plasmids (Fig. 4A and B). However, neither E6AP C-A mutant nor WWP1 decreased the steady-state levels of the core protein, suggesting that E6AP enhances degradation of the core protein.

FIG. 4.

E6AP decreases steady-state levels of HCV core protein in 293T cells and in HepG2 cells. (A) 293T cells (1 × 10⁶ cells/10-cm dish) were transfected with 1 μg of pCAG FLAG-core (1-191) along with either pCAG-HA-E6AP, pCAG-HA-E6AP C-A, or pCAG-HA-WWP1 as indicated. At 48 h posttransfection, protein extracts were separated by SDS-PAGE and analyzed by immunoblotting with anti-HA PAb (top panel), anti-FLAG MAb (middle panel), and anti-GAPDH MAb (bottom panel). (B) Quantitation of data shown in panel A. Intensities of the gel bands were quantitated using the NIH Image 1.62 program. The level of GAPDH served as a loading control. Circles, E6AP; triangles, E6AP C-A; squares, WWP1. (C) Knockdown of endogenous E6AP by siRNA inhibits degradation of HCV core protein in 293T cells. 293T cells (3 × 10⁵ cells/six-well plate) were transfected with 40 pmol of E6AP-specific duplex siRNA (or control siRNA) as described in Materials and Methods. The cells were transfected with 2 μg of FLAG-core (1-191) expression plasmid and cultured for 24 h, harvested, and analyzed by immunoblotting. Shown is immunoblot detection of FLAG-tagged core protein (top panel), E6AP protein (middle panel), and GAPDH (bottom panel) in control siRNA-treated 293T cells or E6AP-siRNA-treated 293T cells. The relative levels of protein expression were quantitated by densitometry and indicated below in the respective lanes. GAPDH served as a loading control. (D) HepG2 cells (2 × 10⁵ cells/six-well plate) were transfected with pCAG FLAG-core (1-152) along with either empty vector or pCMV E6AP as indicated. The cells were harvested at 44 h posttransfection. Where indicated, cells were treated with 25 μM MG132 or with dimethyl sulfoxide control 14 h prior to collection. Equivalent amounts of the whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-FLAG MAb (upper panel) or anti-GAPDH MAb (lower panel).

To verify the critical need for endogenous E6AP in the core degradation, expression of E6AP was knocked down by siRNA and the expression of the core protein and E6AP was assayed by immunoblotting. Transfection of the E6AP-specific siRNA duplex reduced the protein level of E6AP by 90% at 48 h posttransfection (Fig. 4C, middle panel). Immunoblotting revealed a 4.1-fold increase in the level of the core protein in the cells transfected with E6AP siRNA (Fig. 4C, top panel), suggesting that endogenous E6AP plays a role in the proteolysis of the HCV core protein.

Then we examined whether E6AP reduces the steady-state levels of the core protein in hepatic cells as well as in 293T cells. Exogenous expression of E6AP resulted in reduction of the core protein in human hepatoblastoma HepG2 cells (Fig. 4D). Treatment of the cells with the proteasome inhibitor MG132 increased the core protein level, suggesting that the core protein was degraded through the ubiquitin-proteasome pathway. These results indicate that E6AP enhances proteasomal degradation of the HCV core protein in both hepatic cells and nonhepatic cells.

Kinetic analysis of E6AP-dependent degradation of HCV core protein.

To determine whether the E6AP-induced reduction of the core protein is due to an increase in the rate of core degradation, we performed kinetic analysis using the protein synthesis inhibitor CHX. HCV core protein together with wild-type E6AP or inactive mutant E6AP C-A was expressed in 293T cells. At 44 h after transfection, cells were treated with either 50 μg/ml CHX alone or 50 μg/ml CHX plus 25 μM MG132 to inhibit proteasome function. Cells were collected at 0, 3, 6, and 9 h following treatment and analyzed by immunoblotting (Fig. 5A). Overexpression of E6AP resulted in rapid degradation of the core protein, whereas inactive mutant E6AP C-A increased the half-life of the core protein (Fig. 5B), suggesting that the inactive E6AP inhibited degradation of the core protein in a dominant-negative manner, which is in agreement with previous studies (19, 55). Treatment of the cells with MG132 inhibited the degradation of the core protein (Fig. 5B). Reverse transcription-PCR to determine mRNA levels of the HCV core gene and GAPDH gene found that neither wild-type E6AP nor inactive E6AP changed mRNA levels of the HCV core gene and GAPDH gene (data not shown). These results indicate that E6AP enhances proteasomal degradation of the core protein.

FIG. 5.

Kinetic analysis of E6AP-dependent degradation of HCV core protein. (A) 293T cells (1 × 10⁶ cells/10-cm dish) were transfected with 1 μg of pCAG-FLAG core (1-152) plus 4 μg of empty vector, pCMV-HA-E6AP, or pCMV-HA-E6AP C-A. The cells were treated with 50 μg/ml CHX at 44 h after transfection. Cell extracts were collected at 0, 3, 6, and 9 h after treatment with CHX, followed by immunoblotting. (B) Specific signals were quantitated by densitometry, and the percent remaining core at each time was compared with that at the starting point. The level of GAPDH served as a loading control. Open circles, E6AP; closed circles, empty plasmid; closed triangles, E6AP C-A; closed squares, E6AP with MG132 treatment. Data are representative of three independent experimental determinations.

E6AP promotes degradation of the full-length core protein in Huh-7 cells.

To determine whether the full-length HCV core protein expressed in hepatic cells is degraded through an E6AP-dependent pathway, human hepatoma Huh-7 cells were transfected with pCAG HCV core (1-191) along with either E6AP or E6AP C-A. To rule out the effects of N-terminal FLAG tag on the core degradation, HCV core protein was expressed as untagged protein. Expression of wild-type E6AP resulted in reduction of the core protein (Fig. 6). On the other hand, HCV core protein was not decreased after transfection of inactive E6AP, indicating that the full-length core protein expressed in Huh-7 cells is also degraded through an E6AP-dependent pathway.

FIG. 6.

E6AP promotes degradation of full-length HCV core protein in Huh-7 cells. Huh-7 cells (2 × 10⁵ cells/six-well plate) were transfected with 0.5 μg of pCAG-core (1-191) together with 2 μg of pCMV-HA-E6AP or pCMV-HA-E6AP C-A. At 48 h posttransfection, cells were harvested and analyzed by immunoblotting with anticore MAb (top panel), anti-E6AP PAb (middle panel), or anti-GAPDH MAb (bottom panel).

E6AP mediates ubiquitylation of HCV core protein in vivo.

To determine whether E6AP can induce ubiquitylation of HCV core protein in cells, we performed in vivo ubiquitylation assays. 293T cells were cotransfected with FLAG-core (1-191) and either E6AP or empty plasmid, together with a plasmid encoding HA-tagged ubiquitin to facilitate detection of ubiquitylated core protein. Cell lysates were immunoprecipitated with anti-FLAG MAb and immunoblotted with anti-HA PAb to detect ubiquitylated core protein (Fig. 7A). Only a little ubiquitin signal was observed on the core protein in the absence of cotransfected E6AP (Fig. 7A, lane 3). In contrast, coexpression of E6AP led to readily detectable ubiquitylated forms of the core protein as a ladder and a smear of higher-molecular-weight bands (Fig. 7A, compare lane 3 with lane 4). Immunoblot analysis with anticore PAb confirmed that FLAG-core proteins were immunoprecipitated (Fig. 7B, lanes 2 to 4, short exposure) and that higher-molecular-weight bands conjugated with HA-ubiquitin were indeed ubiquitylated forms of the core protein (Fig. 7B, lanes 3 and 4, long exposure).

FIG. 7.

E6AP-dependent ubiquitylation of HCV core protein in vivo. 293T cells (1 × 10⁶ cells/10-cm dish) were transfected with 1 μg of pCAG FLAG-core (1-191) together with 2 μg of plasmid encoding E6AP as indicated. Each transfection also included 2 μg of plasmid encoding HA-ubiquitin. The cell lysates were immunoprecipitated with FLAG beads and analyzed by immunoblotting with anti-HA PAb (A) or anticore PAb (B). A shorter exposure of the core blot shows immunoprecipitated FLAG-core protein (B, right panel). A longer exposure of the core blot shows the presence of a ubiquitin smear (B, left panel). Asterisks indicate cross-reacting immunoglobulin light chain or heavy chain. Arrows indicate FLAG-core. IB, immunoblot; IP, immunoprecipitation.

E6AP mediates ubiquitylation of HCV core protein in vitro.

To rule out the possibility that E6AP contributes to core protein degradation by inducing degradation of inhibitors of core turnover, we determined whether E6AP functions directly as a ubiquitin ligase by testing the ability of purified MEF-E6AP to mediate in vitro ubiquitylation of the purified recombinant HCV core protein. HCV core protein was expressed as a fusion protein containing N-terminal GST tag and C-terminal His tag and purified as described in Materials and Methods. GST-C173HT (aa 1-173) and GST-C152HT (aa 1-152) (see Materials and Methods) were used to determine whether the mature core protein and the C-terminally truncated core protein are targeted for ubiquitylation in vitro. The validity of this assay was established by demonstrating that E6AP but not E6AP C-A induced ATP-dependent ubiquitylation of GST-core protein. When in vitro ubiquitylation reactions were carried out either in the absence of MEF-E6AP or in the presence of MEF-E6AP C-A, no ubiquitylation signal was detected (Fig. 8A, lanes 4 and 5). However, inclusion of purified MEF-E6AP in the reaction mixture resulted in marked ubiquitylation of GST-C173HT (Fig. 8A, lane 6), while no ubiquitylation was observed in the absence of ATP (Fig. 8A, lane 7). No signal was detected when GST-HT was used as a substrate (Fig. 8A, lane 8). The higher-molecular-weight species of GST-core proteins were reactive with both anti-ubiquitin MAb (Fig. 8B, right panel, lanes 2 and 4) and anti-GST MAb (Fig. 8B, left panel, lanes 2 and 4). Both GST-C152HT and GST-C173HT were polyubiquitylated by E6AP in vitro (Fig. 8B), indicating that both the C-terminally truncated core and the mature core are polyubiquitylated by E6AP in vitro. These results revealed that E6AP directly mediated ubiquitylation of HCV core proteins in an ATP-dependent manner.

FIG. 8.

In vitro ubiquitylation of HCV core protein by recombinant E6AP. For in vitro ubiquitylation of HCV core protein, purified GST-C173HT and GST-C152HT were used as substrates. Purified GST-HT was used as a negative control. Assays were done in 40-μl volumes containing each component as indicated. The reaction mixture is described in Materials and Methods. The reaction was carried out at 37°C for 120 min followed by purification with glutathione-Sepharose beads and analysis by immunoblotting with the indicated antibodies. Arrows indicate GST-C173HT, GST-C152HT, and GST-HT, respectively. Ubiquitylated species of GST-core proteins are marked by brackets. IB, immunoblot.

Exogenous expression of E6AP reduces intracellular HCV core protein levels and supernatant infectivity titers in HCV-infected Huh-7 cells.

We used a recently developed system for the production of infectious HCV particles using the HCV JFH1 strain (28, 56, 61) to examine whether E6AP can promote degradation of HCV core protein expressed from infectious HCV. E6AP-dependent core degradation was assessed in Huh-7 cells inoculated with the culture supernatant containing HCV JFH1. Levels of HCV core protein were detectable at day 3 postinfection and increased with time. Immunofluorescence staining for the core protein indicated that the percentage of HCV core-positive cells in the Huh-7 cells was almost 100 at day 7 postinfection. Transfection efficiency was 50 to 60% as measured with GFP-expressing plasmid. At day 7 postinfection, exogenous expression of E6AP reduced the intracellular core protein level by about 60% compared to the empty plasmid-transfected control cells (Fig. 9A). Inactive E6AP had little effect on the core protein levels. Total protein levels in the cells (Fig. 9B) and intracellular HCV RNA levels (Fig. 9C) did not change after transfection of wild-type E6AP or inactive E6AP. The immunofluorescence study revealed that HCV core protein was variably detected and the intensity of core staining was reduced in the cells staining positive for wild-type E6AP compared with neighboring cells staining negative for E6AP (Fig. 9E). Using inactive E6AP revealed colocalization of the core protein and E6AP in the perinuclear region (Fig. 9F) of HCV-infected cells. These results suggest that E6AP enhanced degradation of HCV core protein expressed from infectious HCV. Then we titrated HCV infectivity in the culture supernatant at day 7 postinfection by limiting dilution assays. Exogenous expression of E6AP reduced the supernatant infectivity titer, whereas inactive E6AP had no effect on its infectivity titer (Fig. 9D), suggesting that the E6AP-dependent ubiquitin proteasome pathway affects the production of HCV particles through downregulation of the core protein.

FIG. 9.

Exogenous expression of E6AP reduces intracellular HCV core protein levels and supernatant infectivity titers in HCV-infected Huh-7 cells. Naïve Huh-7 cells were seeded as described in Materials and Methods; inoculated with 2.5 ml of the inoculum including infectious HCV JFH1 (6.5 × 10³ TCID₅₀/ml); and transfected with 6 μg of empty plasmid, pCAG-HA-E6AP, or pCAG-HA-E6AP C-A. The culture supernatant and the cells were collected at days 3 and 7 postinfection. (A) Intracellular HCV core protein levels. (B) Levels of total protein. (C) Levels of intracellular HCV RNA in HCV-infected Huh-7 cells. Data represent the averages of three experiments with error bars. (D) Supernatant infectivity titers. At day 7 postinfection, culture supernatants were collected and assayed for TCID₅₀ determinations. The difference between empty vector and E6AP or between E6AP and E6AP C-A was significant (*, P < 0.05, Student's t test). (E and F) HCV JFH1-infected Huh-7 cells were transfected with either MEF-E6AP plasmid or MEF-E6AP C-A plasmid, grown on coverslips, fixed, and processed for double-label immunofluorescence for HCV core and MEF-E6AP (E) or MEF-E6AP C-A (F). Anticore MAb (2H9) and anti-FLAG PAb were used as primary antibodies. Nuclei were visualized by staining the cells with DAPI. All the samples were examined with a BZ-8000 microscope. Representative images of individual cells are shown with merge images. emp, empty vector.

E6AP silencing increases the levels of intracellular HCV core protein and supernatant infectivity titers in HCV-infected Huh-7 cells.

Finally, to further validate the role of E6AP in HCV production, expression of endogenous E6AP was knocked down by siRNA and the HCV infectivity titers released from HCV JFH1-infected cells were examined. Knockdown of E6AP by siRNA led to an increase in intracellular core protein levels (Fig. 10A) and supernatant HCV infectivity titers (Fig. 10B). Taken together, our results suggest that E6AP mediates ubiquitylation and degradation of HCV core protein in HCV-infected cells, thereby affecting the production of HCV particles.

FIG. 10.

E6AP silencing leads to an increase in the level of intracellular HCV core protein and supernatant infectivity titer in HCV-infected Huh-7 cells. (A) HCV JFH1-infected cells were replated in a six-well plate at 3 × 10⁵ cells/well and transfected with 40 pmol of E6AP siRNA or control siRNA. The culture medium was changed at 24 h after transfection. The cells were harvested at day 2 after transfection, and the intracellular core protein levels were quantitated using the HCV core antigen ELISA. Equivalent amounts of the whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting with anti-E6AP MAb or anti-GAPDH MAb. (B) Culture supernatants were collected at day 2 after transfection and assayed for TCID₅₀ determinations. For both panels, the difference between E6AP siRNA and control siRNA was significant (*, P < 0.05, Student's t test).

DISCUSSION

HCV core protein is a major component of viral nucleocapsid, plays a central role in viral assembly (25, 40), and contributes to viral pathogenesis and hepatocarcinogenesis (9). Therefore, it is important to clarify the molecular mechanisms that govern the cellular stability of this viral protein. We have previously reported that processing at the C-terminal hydrophobic domain of the core protein leads to efficient polyubiquitylation of the core protein (52). In this study, we identified E6AP as an HCV core-binding protein and showed that HCV core protein interacts with E6AP in vivo and in vitro, that E6AP enhances ubiquitylation and degradation of the mature core protein as well as the C-terminally truncated core protein, and that HCV core protein expressed from infectious HCV is degraded via E6AP-dependent proteolysis. HCV core protein and E6AP were found to colocalize in the cytoplasm, especially in the perinuclear region. Moreover, exogenous expression of E6AP reduces intracellular core protein levels and supernatant HCV infectivity titers in HCV-infected Huh-7 cells. Knockdown of endogenous E6AP by siRNA increases intracellular core protein levels and supernatant infectivity titers in HCV-infected cells. These findings suggest that E6AP mediates ubiquitylation and degradation of HCV core protein, thereby affecting the production of HCV particles.

HCV core protein interacts with E6AP through the region of the core protein between aa 58 and aa 71. These 14 amino acids are highly conserved, with the first nine amino acids (PRGRRQPIP) present in the core protein of all the HCV genotypes (3). This result suggests that E6AP-dependent degradation of HCV core protein is common to all HCV genotypes and plays an important role in the HCV life cycle or viral pathogenesis. Our data indicated that HCV core proteins of genotypes 1b and 2a are subjected to proteolysis through an E6AP-mediated degradation pathway. We are currently examining whether E6AP promotes degradation of HCV core proteins of other genotypes.

Studies in addition to ours have reported that other HCV proteins, such as NS5B (8), the unglycosylated cytosolic form of E2 (39), NS2 (7), and F protein (58), are degraded through the ubiquitin-proteasome pathway. These studies suggest that the ubiquitin-proteasome pathway plays a role in the HCV life cycle or viral pathogenesis. To our knowledge, the present study is the first to demonstrate that the ubiquitin-proteasome pathway affects the HCV life cycle.

PA28γ was found to interact with HCV core protein in hepatocytes and promote proteasomal degradation of HCV core protein (30). PA28γ, however, has been shown to function in a ubiquitin-independent, ATP-independent, and 20S proteasome-dependent pathway (27). There have been reports that several cellular factors, such as p53 (2), p73 (2), and RPN4 (18), are degraded through two alternative pathways, the ubiquitin-dependent 26S proteasome-dependent pathway and the ubiquitin-independent 20S proteasome-dependent pathway. Here we provide evidence that E6AP mediates ubiquitylation of HCV core protein. Still unclear is whether the PA28γ-dependent pathway requires polyubiquitylation of HCV core protein. HCV core protein is predominantly localized in the cytoplasm, especially at the endoplasmic reticulum membrane, on the surface of lipid droplets, and on mitochondria and mitochondrion-associated membranes (51). In HCV JFH1-infected cells, HCV core was found to localize in the cytoplasm and frequently to accumulate in the perinuclear region and the lipid droplets (44). Our results indicated that E6AP colocalized with HCV core protein especially in the perinuclear region. PA28γ was found to colocalize with HCV core protein in the nucleus. Functional differences may exist between the E6AP-dependent pathway and the PA28γ-dependent pathway in the stability control of HCV core protein. The functional role of the E6AP-dependent pathway and the PA28γ-dependent pathway remains to be elucidated.

The HCV core-binding region of E6AP was mapped to the region between aa 418 and aa 517. The multicopy maintenance protein 7, Mcm7, interacts with E6AP through a short motif, termed the L2G box (aa 412 to 414), that lies within the E6 binding site of E6AP (23). Our data indicated that the E6 binding region containing the L2G motif is not required for interaction between HCV core protein and E6AP (Fig. 2C, lane M).

We propose here that E6AP may affect the production of HCV particles through controlling the amounts of HCV core protein. This mechanism may contribute to persistent infection. The E6AP binding domain of the core protein resides in the RNA-binding domain and binding domains for many host factors (40). These factors may affect the binding between E6AP and HCV core protein, resulting in control of E6AP-dependent core degradation. Another possibility is that HCV core protein may affect the normal function of E6AP, thereby contributing to pathogenesis. It will be intriguing to investigate whether HCV core protein has any effect on E6AP-dependent degradation of host factors. The other intriguing possibility is that HCV core-E6AP complex may function as an E3 ligase-like E6-E6AP complex to target host factors for proteasomal degradation and contribute to viral pathogenesis.

In conclusion, we have demonstrated that E6AP interacts with HCV core protein in vitro and in vivo and mediates ubiquitin-dependent degradation of the core protein, leading to downregulation of HCV particles. We propose that the E6AP-mediated ubiquitin-proteasome pathway may play a role in affecting the production of HCV particles through controlling the amounts of viral nucleocapsid protein. Identification of the specific E3 ubiquitin ligase may contribute to gaining a better understanding of the biology of the HCV life cycle as well as molecular details of the ubiquitin-dependent degradation of HCV core protein.