Hepatitis C Virus Core Induces p53 Ser-15 Phosphorylation to Facilitate E6-Associated Protein-Mediated Proteasomal Degradation of p53

TL;DR

The hepatitis C virus Core protein manipulates p53 levels by promoting its degradation through a specific pathway, which may support viral replication and cancer development.

Contribution

The study reveals a novel mechanism by which HCV Core uses E6AP to degrade phosphorylated p53, bypassing MDM2.

Findings

HCV Core induces p53 phosphorylation at Ser-15, preventing MDM2-mediated degradation.

Phosphorylated p53 is targeted for E6AP-mediated proteasomal degradation by HCV Core.

E6AP's E3 ubiquitin ligase activity is essential for p53 degradation during HCV replication.

Abstract

What are the main findings?
HCV Core upregulates p53 levels by inhibiting MDM2-mediated degradation of unphosphorylated p53.HCV Core downregulates p53 levels by targeting phosphorylated p53 for E6AP-mediated degradation.

HCV Core upregulates p53 levels by inhibiting MDM2-mediated degradation of unphosphorylated p53.

HCV Core downregulates p53 levels by targeting phosphorylated p53 for E6AP-mediated degradation.

What are the implications of the main findings?
HCV Core adopts the E6AP-mediated host protein degradation system to counteract the antiviral strategies of p53.HCV Core fine-tunes p53 levels that support cell survival, viral replication, and potentially oncogenesis in human hepatocytes.

HCV Core adopts the E6AP-mediated host protein degradation system to counteract the antiviral strategies of p53.

HCV Core fine-tunes p53 levels that support cell survival, viral replication, and potentially oncogenesis in human hepatocytes.

The hepatitis C virus (HCV) Core activates the ATM-Chk2 pathway, leading to phosphorylation of p53 at Ser-15, which inhibits mouse double minute 2 (MDM2)-mediated proteasomal degradation. This study reveals that HCV Core also promotes E6-associated protein (E6AP)-mediated degradation of p53 during HCV replication. In the presence of HCV Core, E6AP expression induced p53 ubiquitination, reduced its stability, and decreased p53 levels, whereas E6AP knockdown increased p53 levels. The E3 ubiquitin ligase activity of E6AP was critical for this process, as demonstrated using the E6AP C833A mutant and the E3 ligase inhibitor Heclin. Proteasomal inhibition with MG132 confirmed that HCV Core and E6AP act together to regulate p53 levels via the proteasome. Importantly, HCV Core-induced p53 phosphorylation was essential for E6AP-mediated degradation, as shown by the impairment of degradation in the presence of the ATM inhibitor KU-55933. E6AP also targeted p53 phosphorylated at Ser-15 by etoposide, as well as phosphomimetic mutants such as p53 S15D, but not non-phosphorylatable mutants such as p53 S15A. These findings suggest that HCV Core-induced p53 phosphorylation enhances E6AP-mediated degradation while preventing MDM2 from targeting p53, thereby maintaining p53 levels that support cell survival, viral replication, and potentially oncogenesis in human hepatocytes.

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Full text

1. Introduction

Hepatitis C virus (HCV) is a leading cause of severe hepatic diseases, including hepatocellular carcinoma (HCC) [1,2]. As a species of the Flaviviridae family, HCV has a 9.6 kb positive-stranded RNA genome that encodes a large polyprotein of approximately 3000 amino acids [3]. This polyprotein is processed by viral and host proteases into structural and nonstructural proteins. HCV Core has garnered particular attention for its multifunctional roles in viral replication and disease progression. In addition to its essential role in viral replication as a nucleocapsid component [4], HCV Core also affects HCV pathogenesis by altering cytoplasmic signaling pathways and regulating nuclear gene transcription [2,5]. Notably, HCV Core can cooperate with Ras to transform rodent fibroblasts [6], promote host cell growth and division [7], immortalize primary human liver cells [8], and cause HCC in transgenic mice [9]. Although substantial evidence indicates that HCV Core contributes to HCV-related cancer development, the exact mechanisms remain unknown, especially regarding p53 regulation.

The tumor suppressor p53 plays a crucial role in maintaining cellular integrity in response to stress, such as DNA damage, by regulating genes involved in DNA repair, cell cycle progression, and apoptosis [10,11]. Under normal conditions, p53 is maintained at low levels because it is rapidly degraded, primarily by its E3 ubiquitin (Ub) ligase, mouse double minute 2 (MDM2) [12]. In response to stress signals, the ATM-Chk2 pathway is activated, leading to phosphorylation of p53 and MDM2. This phosphorylation disrupts the MDM2-p53 complex, stabilizing p53 by preventing MDM2-mediated ubiquitination [11,13,14]. Although it is well understood that phosphorylation stabilizes p53 by releasing it from MDM2, the vulnerability of phosphorylated p53 to degradation remains unclear. This issue is particularly relevant in cells exposed to viral infections, where p53 levels must remain within a specific range.

Oncogenic viral proteins often interact with p53 and alter its functions, enabling viruses to evade p53-mediated surveillance. For example, the human papillomavirus (HPV) E6 protein recruits E6-associated protein (E6AP) to target p53 for degradation [15,16]. E6AP also interacts with HCV Core, resulting in its ubiquitination and degradation [17]. Additionally, p53 interacts with HCV Core to promote E6AP-mediated proteasomal degradation [18]. These findings led to the hypothesis that HCV Core and E6AP may form a trimeric complex to induce p53 degradation, similar to the HPV E6-E6AP-p53 interaction [16]. Moreover, according to our recent report [19], E6AP can target p53 for ubiquitination if it is phosphorylated in response to genotoxic stress. Considering that HCV Core also induces p53 phosphorylation via activation of the ATM-Chk2 pathway [18,20], it is possible to hypothesize that HCV Core facilitates E6AP-mediated degradation of phosphorylated p53. This study addresses several key questions to explore these hypotheses. First, we examined whether HCV Core induces E6AP-mediated ubiquitination and proteasomal degradation of p53. Second, we compared the roles of MDM2 and E6AP in p53 degradation, both with and without HCV Core. Third, we investigated how HCV Core facilitates E6AP-mediated p53 degradation, emphasizing its ability to induce p53 phosphorylation. Fourth, we explored whether p53 phosphorylation is necessary and sufficient for E6AP-mediated ubiquitination of p53. Lastly, we aimed to identify the critical phosphorylation site(s) in p53 responsible for its susceptibility to E6AP-mediated ubiquitination. Understanding the fate of phosphorylated p53, especially under stress conditions such as HCV infection, is vital, as it could reveal how p53 levels are precisely regulated to support cell survival and virus replication. Furthermore, these insights may shed light on the mechanisms by which HCV manipulates the p53 pathway, potentially contributing to oncogenesis.

2. Materials and Methods

2.1. Plasmids

The plasmid pCMV-3 × HA1-Core [18] encodes the full-length HCV Core (genotype 1b) downstream of three copies of the influenza virus hemagglutinin (HA) epitope. Plasmids pCMVT N-HA-hE6AP encoding E6AP (#37601), p3869 HA-E6AP C833A (#8649) encoding E6AP with a Cys-to-Ala substitution at the active site [21], pcDNA3-MDM2 WT encoding MDM2 (#16233), pCH110 encoding the Escherichia coli β-galactosidase gene (β-gal; #27-4508-01), and Myc-p53 (#19930) were obtained from Addgene (Watertown, MA, USA). The pCMV p53-WT plasmid was kindly provided by Dr. C.-W. Lee (Sungkyunkwan University, Republic of Korea). Plasmids pcDNA3 p53 S15A (#69004) and pcDNA3 p53 S15D (#69005) were purchased from Addgene. Plasmids encoding p53 S20D, p53 S15A/S20D, and p53 S15D/S20D were generated by PCR-directed mutagenesis from pCMV p53-WT, pcDNA3 p53 S15A, and pcDNA3 p53 S15D, respectively. The pHA-Ub plasmid was obtained from Dr. Y. Xiong (University of North Carolina at Chapel Hill). Scrambled (SC) small hairpin RNA (shRNA; #sc-42964) and E6AP shRNA (#sc-43742) plasmids were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). To generate G4-E6AP and G4-MDM2, PCR fragments encoding E6AP (amino acids 262–853) and MDM2 (amino acids 1–491) from pCMVT N-HA-hE6AP and pcDNA3-MDM2 WT, respectively, were cloned into pSG424 in-frame and downstream of Gal4 (amino acids 1–147) [22]. Additionally, to construct pCMV p53-VP16, a PCR fragment encoding p53-WT (amino acids 1–393) from pCMV p53-WT was fused upstream of the VP16 activation domain (amino acids 423–490) in pCMV-VP16 [22]. The reporter plasmid G5E1b-luc [23] contains five copies of the Gal4 DNA-binding site upstream of an essential E1b promoter in pGL3 (Promega #E1751; Madison, WI, USA).

2.2. Cell Culture and Transfection

The HepG2 (#88065) and Hep3B (#88064) cell lines were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). The Huh7D cell line [24] was kindly provided by Dr. S.M. Feinstone (US FDA). The HepG2-Core stable cell line was established by transfecting HepG2 cells with pCMV-3 × HA1-Core and selecting with 500 μg/mL G418 sulfate (Sigma-Aldrich #A1720; Saint Louis, MO, USA). For transient expression, 4 × 105 cells per well in a 6-well plate were transfected with the designated plasmids using TurboFect transfection reagent (Thermo Fisher Scientific #R0532; Waltham, MA, USA). All cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; WelGENE #LM001-05; Gyeongsan, Republic of Korea) supplemented with 10% fetal bovine serum (FBS; Capricorn Scientific #FBS-22A; Ebsdorfergrund, Germany), 100 μg/mL streptomycin (United States Biological #21865; Salem, MA, USA), and 100 units/mL penicillin G (Sigma-Aldrich #P3032). Cells were treated with KU-55933 (Abcam #ab120637, Cambridge, UK), MG132 (Millipore #474790; Burlington, MA, USA), CHX (Sigma-Aldrich #C7698), etoposide (Sigma-Aldrich #E1383), Nutlin 3a (Sigma-Aldrich #SML0580), or Heclin (Sigma-Aldrich #SML1396), as necessary, under the specified conditions.

2.3. HCV Infection System

The plasmid pJFH-1 contains HCV cDNA from a Japanese patient with fulminant hepatitis, downstream of a T7 promoter [25]. The linearized DNA was used as a template for in vitro transcription with MEGAscript (Thermo Fisher Scientific #AM1333). Huh7D cells were transfected with 10 μg of in vitro-transcribed JFH1 RNA via electroporation and incubated for 48 h to generate initial viral seeds. To prepare HCV stocks, Huh7D cells were infected with the viral seeds at a multiplicity of infection (MOI) of 0.01 and incubated for 9 days to allow viral amplification, as previously described [26]. Quantitative RT-PCR assays were performed to determine HCV titers [27]. Huh7D cells were infected with HCV in serum-free DMEM at an MOI of 10 for 1 h. Cells were washed twice with serum-free DMEM and maintained for 47 h in DMEM supplemented with 2% FBS.

2.4. Co-Immunoprecipitation

Co-IP assays were performed using a Classic Magnetic IP/Co-IP Kit (Thermo Fisher Scientific, #88804). Approximately 1 × 10^6^ cells per 100 mm dish were transiently transfected with the indicated plasmids for 48 h or infected with HCV under the specified conditions. Whole-cell lysates (500 μg) were incubated with antibodies against p53 (Cell Signaling Technology #2527, Danvers, MA, USA), E6AP (Thermo Fisher Scientific #PA3-843), MDM2 (Santa Cruz Biotechnology #sc-965), c-Myc (Santa Cruz Biotechnology #sc-40y), and pSer-15 p53 (Cell Signaling Technology #9284) for 12 h at 4 °C to form immune complexes. After washing, the immune complexes were collected using Protein A/G magnetic beads (0.25 mg) by incubating for an additional 1 h with mixing. The beads were harvested using a magnetic stand (Pierce, Waltham, MA, USA), and the antigen/antibody complexes were subjected to Western blotting with anti-HA or other relevant antibodies.

2.5. Western Blot Analysis

Cell lysates were prepared using a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% SDS, and 1% NP-40, supplemented with protease inhibitors. Protein samples were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Cytiva #10600004, Little Chalfont, UK). After blocking, membranes were incubated with primary antibodies, including total p53 (Santa Cruz Biotechnology #sc-126, 1:1000), pSer-15 p53 (Cell Signaling Technology #9284, 1:1000), pSer-20 p53 (Cell Signaling Technology #9285, 1:1000), E6AP (Thermo Fisher Scientific #PA3-843, 1:2000), MDM2 (Santa Cruz Biotechnology #sc-965, 1:1000), pSer-1981 ATM (Abcam #ab5883, 1:1000), pThr-68 Chk2 (Abcam #ab2661, 1:1000), c-Myc (Santa Cruz Biotechnology #sc-40, 1:500), HCV Core (Abcam #ab58713, 1:500), HA (Santa Cruz Biotechnology #sc-7392, 1:500), and γ-tubulin (Santa Cruz Biotechnology #sc-17787, 1:500). After binding with primary antibodies, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies, including anti-mouse IgG (Bio-Rad #BR170-6516, 1:3000, Hercules, CA, USA), anti-rabbit IgG (Bio-Rad #BR170-6515, 1:3000), and anti-goat IgG (Thermo Fisher Scientific #31400, 1:10,000), for 1 h. Protein bands were visualized using an enhanced chemiluminescence kit (Advansta #K-12043-D20) and imaged on a ChemiDoc XRS system (Bio-Rad). The images of the target proteins were cropped from the original images to display the protein bands in the figure. Band intensities were quantified using ImageJ (version 2.1.0, NIH, Bethesda, MD, USA). Band intensities were normalized to the housekeeping protein γ-tubulin, thereby allowing determination of relative protein expression for each sample.

2.6. Luciferase Reporter Assay

Cells were seeded at 1 × 10^5^ cells per well in 12-well plates. The cells were transfected in triplicate with 0.2 μg of reporter plasmid along with the assigned plasmids under the indicated conditions. To control for variation in transfection efficiency, 0.1 μg of pCH110 was co-transfected as an internal control. Forty-eight hours after transfection, luciferase activity was measured using the Luciferase Reporter 1000 Assay System (Promega #E1910) and a microplate luminometer (LuBi, MicroDigital, Seongnam, Republic of Korea). β-gal activity was determined using a β-gal Assay kit (Thermo Fisher Scientific). Luciferase activity was normalized to the β-gal activity in the corresponding cell extracts.

2.7. Statistical Analysis

The data are presented as mean values ± standard deviation, derived from at least 3 independent experiments. Statistical analysis was conducted using a two-tailed Student’s t-test in SigmaPlot (version 12.5). Significance was assessed using p-values; p ≤ 0.05 indicates statistically significant differences between experimental groups.

3. Results

3.1. E6AP Reduces p53 Levels in a Manner Dependent on HCV Core

Initially, we examined how E6AP affects p53 levels in human hepatoma cells, both with and without HCV Core. Ectopic expression of E6AP alone had little effect on p53 levels in HepG2 and Hep3B cells (Figure 1a,b). Similarly, knocking down E6AP did not significantly change p53 levels in HepG2 cells without HCV Core (Figure 1c). These results are consistent with previous reports indicating that E6AP alone does not cause p53 degradation [28,29]. HCV Core increased p53 levels while decreasing E6AP levels (Figure 1a,b), as previously shown [18]. Interestingly, however, overexpression of E6AP reduced p53 levels, whereas knocking down E6AP increased p53 levels in a dose-dependent manner in the presence of HCV Core (Figure 1a–c). E6AP also lowered HCV Core levels (Figure 1a–c), aligning with earlier studies [17,20]. The E3 ligase activity of E6AP was crucial for downregulating both p53 and HCV Core, as shown by the use of the E6AP-C833A mutant [21] (Figure 1d). These results suggest that E6AP induces proteasomal degradation of p53 in a HCV Core-dependent manner.

3.2. HCV Core Facilitates E6AP-Mediated p53 Degradation While Inhibiting MDM2-Mediated Degradation

We next investigated how HCV Core affects the interaction between E6AP- and MDM2-mediated p53 degradation in human hepatoma cells. HCV Core decreased levels of both E6AP and MDM2 while increasing p53 levels in these cells (Figure 2a,b), as shown in Figure 1 and in a previous report [18]. Ectopic expression of E6AP significantly lowered p53 levels, but only in the presence of HCV Core, as shown in Figure 1. In contrast, MDM2 expression reduced p53 levels only when HCV Core was absent. The presence of E6AP did not influence MDM2 levels, and vice versa. Similar results were observed in HepG2 cells stably expressing HCV Core (Figure 2a). These findings suggest that HCV Core enhances E6AP-mediated p53 degradation while inhibiting MDM2-mediated p53 degradation.

To further examine the roles of E6AP and MDM2 in p53 degradation, we measured p53 stability in HepG2 cells treated with cycloheximide (CHX), with or without HCV Core (Figure 2c). Without HCV Core, the p53 half-life was about 49.5 min; with HCV Core, it increased to 163.8 min. E6AP expression, however, significantly shortened the p53 half-life to 28.8 min in the presence of HCV Core, whereas it reduced it only slightly to 47.1 min in its absence. In contrast, MDM2 expression decreased p53 stability to 29.4 min in the absence of HCV Core, but had little effect on p53 when HCV Core was present. These results suggest that E6AP decreases p53 stability only in the presence of HCV Core, whereas MDM2 reduces p53 stability in its absence.

To understand how HCV Core differentially influences E6AP- and MDM2-mediated p53 ubiquitination, we performed IP of ubiquitinated p53 in HepG2 cells with or without HCV Core. Proteasomal inhibition by MG132 almost completely abolished the effects of HCV Core, E6AP, and MDM2 on p53 levels (Figure 2d), confirming that proteasomal degradation is their primary mode of regulation [18,30]. In the presence of MG132, HCV Core no longer decreased MDM2 and E6AP levels, as these effects are also proteasome-dependent [18,20]. E6AP also failed to lower HCV Core levels in the presence of MG132. Co-IP experiments showed that both E6AP and MDM2 interact with p53, leading to its ubiquitination, as indicated by the presence of polyubiquitinated p53 bands (Figure 2d). Consistent with previous findings [31], HCV Core reduced polyubiquitinated p53 levels in HepG2 cells by decreasing MDM2 activity (Figure 2d, compare lanes 1 and 2), leading to p53 accumulation when HCV Core was present (Figure 2a). Ectopic E6AP expression caused strong p53 ubiquitination in the presence of HCV Core (Figure 2d, compare lanes 2 and 4), but this effect was weaker or absent when HCV Core was not present (Figure 2d, compare lanes 1 and 3). In contrast, ectopic MDM2 increased p53 ubiquitination without HCV Core (Figure 2d, compare lanes 5 and 7), but this effect was diminished when HCV Core was present (Figure 2d, compare lanes 6 and 8). These results show that HCV Core enhances E6AP-mediated p53 ubiquitination while blocking MDM2 from acting as a p53 E3 ligase. In other words, during HCV infection, E6AP becomes the primary E3 ligase responsible for p53 degradation, whereas MDM2 takes this role when HCV Core is absent.

3.3. HCV Core Enhances the Interaction Between E6AP and p53 While Interfering with MDM2 Binding to p53

We investigated how HCV Core affects the ubiquitination of p53 by E6AP and MDM2. Data from co-IP and mammalian two-hybrid assays showed that HCV Core consistently strengthened the interaction between E6AP and p53, while weakening MDM2 binding to p53 (Figure 2d,f). The effect of HCV Core on MDM2 appeared stronger than on E6AP, as shown by the decreased levels of ubiquitinated p53 in the presence of HCV Core (Figure 2d). Ectopic E6AP expression significantly increased the interaction between E6AP and p53, but only in the presence of HCV Core (Figure 2d). Treatment with the MDM2 inhibitor Nutlin 3a specifically inhibited MDM2 binding and subsequent p53 ubiquitination in the absence of HCV Core, without affecting E6AP effects in the presence of HCV Core (Figure 2e), confirming that E6AP, but not MDM2, induces p53 ubiquitination in the presence of HCV. Ectopic E6AP expression also increased the interaction between HCV Core and p53 (Figure 2d,e), suggesting cooperation between HCV Core and E6AP in p53 binding. In contrast, ectopic MDM2 expression enhanced the MDM2-p53 interaction when HCV Core was absent, but not in its presence (Figure 2d). Notably, MDM2 reduced HCV Core-p53 interaction, indicating mutual antagonism between HCV Core and MDM2 for p53 binding.

Data from co-immunoprecipitation (co-IP) and mammalian two-hybrid assays also indicated an antagonistic interaction between E6AP and MDM2 in their binding to p53 (Figure 2d,f). Ectopic MDM2 expression weakened the interaction between E6AP and p53 in the absence of HCV Core, but this effect was less pronounced when HCV Core was present. In contrast, ectopic E6AP expression reduced the interaction between MDM2 and p53 in the presence of HCV Core, whereas this effect was minimal or negligible when HCV Core was absent. These findings suggest that HCV Core promotes E6AP-mediated p53 ubiquitination while preventing MDM2 from inducing p53 ubiquitination by modulating their binding interactions with p53.

3.4. E6AP Targets Phosphorylated p53 When HCV Core Is Present, While MDM2 Targets Unphosphorylated p53 in Its Absence

Consistent with previous studies showing that HCV Core induces p53 upregulation through activation of the ATM-Chk2 pathway [18,20], HCV Core phosphorylated ATM at Ser-1981, which led to phosphorylation of p53 at Ser-15 (Figure 3a). Additionally, activated ATM phosphorylated Chk2 at Thr-68, resulting in phosphorylation of p53 at Ser-20. These modifications contributed to increased p53 levels. We investigated whether HCV Core-induced p53 phosphorylation affects its degradation by E6AP and MDM2. Interestingly, ectopic E6AP expression significantly decreased both phosphorylated and total p53 levels in the presence of HCV Core, but had little effect in its absence (Figure 3a). E6AP also reduced the levels of phosphorylated ATM and Chk2, likely by disrupting the positive feedback loop between p53 and the ATM-Chk2 pathway [32]. In contrast, ectopic MDM2 expression markedly decreased total p53 levels, especially when most p53 was unphosphorylated in the absence of HCV Core (Figure 3a). However, MDM2 had little impact on phosphorylated ATM, Chk2, and p53, particularly when HCV Core was present. These results suggest that HCV Core-driven p53 phosphorylation promotes E6AP-mediated degradation and inhibits MDM2’s action on p53.

To further explore whether E6AP and MDM2 promote the ubiquitination of phosphorylated p53 differently, we introduced E6AP or MDM2 into HepG2 cells with or without HCV Core expression and immunoprecipitated p53 phosphorylated at Ser-15 (pSer-15 p53). The results showed that E6AP, but not MDM2, interacts with pSer-15 p53 and triggers its ubiquitination, as indicated by multiple ubiquitinated pSer-15 p53 bands (Figure 3b). HCV Core increased the amount of E6AP bound to pSer-15 p53, mainly by inducing p53 phosphorylation at Ser-15, thereby raising levels of ubiquitinated pSer-15 p53. The interaction between HCV Core and pSer-15 p53 was also observed, consistent with prior findings for the interaction between HCV Core and total p53 (Figure 2d). Furthermore, ectopic E6AP expression strengthened the interaction between E6AP and pSer-15 p53, increased levels of ubiquitinated pSer-15 p53, and decreased pSer-15 p53 levels in the presence of HCV Core, but not without it (Figure 3b). In contrast, ectopic MDM2 expression had little effect on p53 interaction at pSer-15 or on p53 ubiquitination, regardless of HCV Core presence (Figure 3b). These findings suggest that E6AP, but not MDM2, mediates the ubiquitination of pSer-15 p53 when HCV Core is present.

Consistent with earlier findings (Figure 2d), treatment with the proteasome inhibitor MG132 prevented HCV Core from increasing total and phosphorylated p53 levels and from decreasing them in the presence of HCV Core (Figure 3c). However, MG132 did not affect HCV Core’s ability to increase pSer-15 p53 levels, as this effect is independent of proteasomal degradation and is linked to activation of the ATM-Chk2 pathway (Figure 3a). The differential roles of E6AP and MDM2 in regulating p53 levels were further confirmed by co-IP using antibodies against E6AP and MDM2. E6AP interacted with both total p53 and phosphorylated p53 at Ser-15 or Ser-20, even in the absence of HCV Core, likely due to low basal p53 phosphorylation (Figure 3c). HCV Core increased the interaction between E6AP and both total and phosphorylated p53. In contrast, MDM2 strongly interacted with total p53 but not with phosphorylated p53 at Ser-15 or Ser-20, nor with HCV Core, in HepG2 and Hep3B cells (Figure 3c). Additionally, HCV Core reduced the interaction between MDM2 and total p53, likely by promoting p53 phosphorylation, thereby preventing MDM2 from targeting p53. The effects of E6AP on total and phosphorylated p53 in the presence of HCV Core were abolished by treatment with MG132 (Figure 3d). Therefore, we conclude that E6AP promotes the ubiquitination of phosphorylated p53 in the presence of HCV Core, whereas MDM2 mainly targets unphosphorylated p53 for ubiquitination in the absence of HCV Core.

3.5. Phosphorylation of p53 Is Essential for E6AP-Mediated Ubiquitination in the Presence of HCV Core

To verify that E6AP specifically targets phosphorylated p53 for Ub-dependent proteasomal degradation, we used the ATM-specific inhibitor KU-55933, which blocks the ATM-Chk2 pathway [33]. Treatment with KU-55933 reduced total p53 and phosphorylated ATM, Chk2, and p53 levels in HepG2 cells (Figure 4a). It also prevented HCV Core from increasing p53 levels, confirming that HCV Core induces p53 upregulation by activating the ATM-Chk2 pathway [18,20]. In the presence of KU-55933, E6AP did not decrease total p53 levels, regardless of HCV Core expression (Figure 4a), indicating that p53 phosphorylation is necessary for E6AP to lower p53 levels in the presence of HCV Core.

Co-IP experiments using antibodies against p53 and E6AP showed that KU-55933 treatment prevented E6AP from binding to both total and phosphorylated p53, indicating that p53 phosphorylation is crucial for this interaction (Figure 4b,c). Ectopic expression of E6AP in the presence of KU-55933 did not increase the interaction between E6AP and p53, nor did it enhance p53 ubiquitination, regardless of HCV Core presence. In contrast, KU-55933 enhanced the interaction between MDM2 and p53, leading to a significant increase in polyubiquitinated p53 levels (Figure 4b). Interestingly, HCV Core bound either p53 or E6AP in the presence of KU-55933 (Figure 4b,c), indicating that these interactions do not depend on phosphorylated p53. Additionally, ectopic E6AP expression did not affect the interaction between Core and p53 in the presence of KU-55933, suggesting that this process is E6AP-independent (Figure 4b). Although ectopic E6AP increased its binding to HCV Core in the presence of KU-55933 (Figure 4c), it remains unclear whether this interaction results in ubiquitination of HCV Core.

The requirement for p53 phosphorylation in its interaction with E6AP was further studied using mammalian two-hybrid assays. Treatment with KU-55933 blocked the interaction between p53 and E6AP in the absence of HCV Core and prevented HCV Core from enhancing this interaction (Figure 4d). E6AP and MDM2 did not compete for binding to p53 in the presence of KU-55933 (Figure 4e,f), indicating that E6AP does not hinder MDM2 from binding to unphosphorylated p53, and vice versa. These findings suggest that p53 phosphorylation is crucial for E6AP-mediated ubiquitination and degradation of p53, while MDM2 mainly targets unphosphorylated p53.

3.6. Phosphorylation of p53 Is Enough for E6AP to Trigger Proteasomal Degradation

To determine whether p53 phosphorylation alone is sufficient for E6AP to induce its ubiquitination and subsequent degradation by the proteasome, we treated HepG2 cells with the topoisomerase II inhibitor etoposide, which generates double-stranded DNA breaks and stabilizes p53 through activation of the ATM-Chk2 pathway [34]. Etoposide treatment phosphorylated p53 at Ser-15 and Ser-20, even in the absence of HCV Core, increasing total p53 levels in HepG2 cells (Figure 5a). Ectopic expression of E6AP, even in the absence of HCV Core, reduced both total and phosphorylated p53 in cells treated with etoposide. Treatment with MG132 nearly completely prevented E6AP from decreasing total and phosphorylated p53 levels, confirming the role of the Ub-proteasome pathway.

The role of E6AP in mediating p53 degradation independently of HCV Core in etoposide-treated cells was further investigated by co-IP using antibodies against total p53, phosphorylated p53, and E6AP. Consistent with earlier results (Figure 2d and Figure 3b), E6AP showed minimal interaction with total and phosphorylated p53 in untreated HepG2 cells (Figure 5b–d). Additionally, MDM2 interacted with total p53 but not with phosphorylated. In these cells (Figure 5b,c), etoposide increased E6AP binding to total p53 but decreased the interaction between MDM2 and total p53, resulting in reduced ubiquitination (Figure 5b–d). It also increased E6AP binding to phosphorylated p53 without affecting MDM2 binding and increased the ubiquitination of phosphorylated p53 in the absence of HCV Core (Figure 5b,c). These results replicate observations made with HCV Core (Figure 2d and Figure 3b). Overexpressing E6AP in etoposide-treated cells enhanced its interaction with both total and phosphorylated p53 (Figure 5b–d), leading to higher ubiquitination (Figure 5b,c). These effects were more pronounced when MG132 stabilized pSer-15 p53 levels across all conditions (Figure 5b–d). Mammalian two-hybrid assays confirmed that etoposide enhances the interaction between E6AP and p53, regardless of the presence of HCV Core (Figure 5e). Moreover, etoposide had little effect on the interaction between MDM2 and p53, whether or not HCV Core was present (Figure 5f). Overexpression of MDM2 did not alter E6AP’s binding to p53 under these conditions (Figure 5g). These findings highlight that E6AP, rather than MDM2, is the primary E3 ligase responsible for p53 ubiquitination in cells treated with etoposide.

3.7. HCV Core Facilitates E6AP Binding to Phosphorylated p53 Through Direct Interactions

The ability of HCV Core to increase p53 levels was not observed in the presence of etoposide (Figure 5a). This is likely because both etoposide and HCV Core increase p53 levels through the same ATM-Chk2 pathway, which can be fully activated by etoposide alone. Interestingly, HCV Core decreased both total and phosphorylated p53 in etoposide-treated HepG2 cells, and this effect was enhanced by ectopic E6AP expression (Figure 5a). These results indicate that HCV Core has an additional mechanism, in addition to p53 phosphorylation, that promotes E6AP-mediated ubiquitination of p53.

Notably, HCV Core increases the amount of E6AP bound to both total and phosphorylated p53 without affecting MDM2, thereby enhancing their ubiquitination in etoposide-treated cells (Figure 5b–d). Data from the mammalian two-hybrid assay further confirm that HCV Core strengthens the interaction between p53 and E6AP in the presence of etoposide (Figure 5e). Overexpression of E6AP also enhances HCV Core binding to total p53, even at lower HCV Core levels (Figure 5b,d). However, this effect is observed with phosphorylated p53 only when MG132 is present (Figure 5c), likely because HCV Core binds phosphorylated p53, thereby increasing its susceptibility to E6AP-mediated degradation. These findings suggest that HCV Core and E6AP work together to bind phosphorylated p53 in human hepatoma cells. Overall, we conclude that, in addition to inducing p53 phosphorylation, HCV Core enhances E6AP binding to phosphorylated p53 through physical interactions, thereby facilitating proteasomal degradation.

3.8. Phosphorylation of p53 at Ser-15 Is Crucial for E6AP-Mediated Ubiquitination and Proteasomal Degradation

Figure 3a and Figure 5a demonstrate that both HCV Core expression and etoposide treatment trigger phosphorylation of p53 at Ser-15 and Ser-20 in human hepatoma cells. To identify which phosphorylation site is critical for E6AP-mediated p53 degradation, we employed several p53 mutants in which either Ser-15 or Ser-20 was replaced with a phosphomimetic amino acid (Asp, D) or a non-phosphorylatable amino acid (Ala, A). HCV Core induced phosphorylation of p53 S20D at Ser-15, similar to what was observed with WT p53 (Figure 6a). However, this phosphorylation was not observed, regardless of HCV Core presence, in p53 mutants with phosphomimetic or non-phosphorylatable substitutions at Ser-15. Additionally, HCV Core could not induce phosphorylation of Ser-20 in p53 mutants containing S15D or S15A substitutions, likely because it requires prior phosphorylation of Ser-15, as suggested by a sequential phosphorylation model [35,36,37,38,39]. Therefore, the p53 mutants used in this study provide an ideal system for examining the role of phosphorylation at Ser-15 and Ser-20 in E6AP-induced ubiquitination of p53.

HCV Core increased p53 S20D levels to levels comparable to WT p53, but it did not affect other p53 mutants in which the 15th amino acid cannot be phosphorylated (Figure 6a). This indicates that HCV Core’s ability to raise p53 levels depends primarily on its capacity to induce phosphorylation at Ser-15, which is crucial for protecting p53 from MDM2-mediated degradation [40]. The slightly higher baseline levels observed in the p53 mutants compared to WT p53 may result from their partial resistance to MDM2-mediated proteasomal degradation [12]. Notably, in the absence of HCV Core, ectopic E6AP expression decreased p53 S15D levels, while minimally affecting other p53 mutants or WT p53 (Figure 6b). Additionally, ectopic E6AP expression reduced p53 S20D and WT p53 levels only when HCV Core caused phosphorylation at Ser-15 (Figure 6a,b). Even in the presence of HCV Core, E6AP had minimal effect on p53 S15A because of its non-phosphorylatable Ala-15 residue (Figure 6b–d). These results show that E6AP can reduce p53 levels when Ser-15 is phosphorylated or replaced with a phosphomimetic residue, such as Asp.

We further aimed to demonstrate that phosphorylation of p53 at Ser-15 is sufficient for E6AP to induce p53 ubiquitination. In co-IP assays with an anti-p53 antibody, in the absence of HCV Core, only p53 S15D bound E6AP effectively, with affinity comparable to that of MDM2 in Hep3B cells (Figure 6c). In contrast, WT p53 and other mutants with S15A and S20D substitutions interacted strongly with MDM2, whereas they showed minimal interaction with E6AP (Figure 2d and Figure 6c). These results suggest that MDM2 and E6AP play distinct roles in p53 ubiquitination, depending on whether Ser-15 is phosphorylated.

We next examined whether HCV Core differentially influences E6AP- or MDM2-mediated proteasomal degradation of p53 depending on the phosphorylation status of Ser-15. HCV Core increased E6AP binding to p53 S15D but decreased MDM2-p53 S15D interaction (Figure 6c). The opposing effects of HCV Core on E6AP and MDM2 activities led to increased ubiquitination but decreased p53 S15D protein levels (Figure 6b,c), similar to what was observed with WT p53 in the presence of etoposide (Figure 5a,b). These effects were nearly abolished when ectopic E6AP expression balanced the amount of E6AP bound to p53 S15D in the presence and absence of HCV Core (Figure 6b,c). Although HCV Core also had opposite effects on the binding of E6AP and MDM2 to p53 S20D, it led to decreased ubiquitination and increased protein levels of p53 S20D (Figure 6b,c), similar to WT p53 (Figure 2a,d). Ectopic E6AP expression eliminated these effects, likely because it successfully countered the binding of E6AP and MDM2 to p53 S20D (Figure 6b,c). The effects of HCV Core on the binding of E6AP and MDM2 to p53 S15A, as well as on its ubiquitination and protein levels, were not observed, probably due to the non-phosphorylatable Ala-15 residue in this mutant. This indicates that HCV Core can promote the ubiquitination of phosphorylated p53 by enhancing its binding to E6AP, as seen in the presence of etoposide (Figure 5b).

3.9. Phosphorylation of p53 at Ser-15 Alone Does Not Prevent MDM2-Mediated p53 Degradation

Figure 3 and Figure 5 show that both HCV Core expression and etoposide treatment prevent MDM2-mediated p53 degradation. We examined whether p53 phosphorylation at Ser-15 is critical for this effect. p53 S15A interacted exclusively with MDM2 (Figure 6c). Both p53 S15D and S20D mutants also bound MDM2, but with lower affinity. HCV Core further weakened the binding of p53 S20D to MDM2, possibly by inducing phosphorylation at Ser-15 (Figure 6a,c). These results confirm a previous finding that phosphorylation at either site decreases p53’s affinity for MDM2 [12]. Although HCV Core also reduced p53 S15D binding to MDM2 (Figure 6e), this effect may involve a different mechanism, such as enhanced E6AP affinity, given that p53 S15D contains a phosphomimetic residue at position 15. Interestingly, ectopic MDM2 expression increased its binding to p53 S15D and S15D/S20D mutants, even in the presence of HCV Core, leading to increased ubiquitination but decreased protein levels, similar to what was seen with p53 S20D (Figure 6d,e). This may be because HCV Core cannot induce phosphorylation of Ser-20 and other sites (Figure 6a), which may be required to prevent MDM2-mediated degradation, as observed with WT p53 (Figure 3). Therefore, p53 phosphorylated at Ser-15 remains vulnerable to MDM2-mediated degradation, as previously described [12]. Overall, we conclude that p53 phosphorylation at Ser-15 plays a major role in HCV Core-induced resistance to MDM2-mediated degradation, although this effect also involves phosphorylation at other sites.

3.10. HCV Core Promotes E6AP-Mediated Ubiquitination of Phosphorylated p53 During HCV Replication in Huh7D Cells

We investigated whether HCV Core induces E6AP-mediated ubiquitination of p53 during HCV replication in Huh7D cells. The human hepatoma cell line Huh7 and its derivatives, including Huh7D, express a Y220C-mutated form of p53 [24]. We compared the effects of HCV Core on endogenous mutant p53 (p53-Y220C) and ectopically expressed WT p53 fused to a Myc tag (Myc-p53) during HCV replication in Huh7D cells. HCV, presumably through HCV Core, induced phosphorylation of both p53-Y220C and Myc-p53 at Ser-15 and Ser-20, and increased their levels during infection in Huh7D cells (Figure 7a). Additionally, HCV infection downregulated E6AP levels in Huh7D cells. Moreover, ectopic expression of E6AP reduced HCV Core levels in infected cells. These findings align with data obtained using the HCV Core expression system (Figure 1). Notably, unlike Myc-p53, p53-Y220C showed significant phosphorylation at Ser-15 and Ser-20 even in uninfected cells (Figure 7a). As a result, ectopic E6AP expression decreased both total and phosphorylated p53-Y220C levels in uninfected cells, confirming that E6AP promotes proteasomal degradation of phosphorylated p53 independently of HCV Core. Overexpression of E6AP caused a substantial decline in both p53-Y220C and Myc-p53 levels in HCV-infected cells (Figure 7a). Therefore, the functions of HCV Core in p53 phosphorylation and E6AP-mediated p53 degradation can be accurately reproduced using an in vitro HCV infection system.

To verify that E6AP’s E3 ligase activity is crucial for p53 degradation during HCV infection, we used a HECT E3 Ub ligase inhibitor, Heclin [41]. As expected, increasing Heclin doses progressively blocked E6AP’s ability to reduce Myc-p53, p53-Y220C, and HCV Core levels in infected cells (Figure 7b). Additionally, ectopic expression of the catalytically inactive E6AP C833A mutant failed to downregulate both Myc-p53 and Y220C-p53 in Huh7D cells, regardless of HCV infection (Figure 7c), consistent with data from the HCV Core expression system (Figure 1d). These findings confirm that E6AP’s E3 ligase activity is vital for downregulating p53 during HCV replication.

According to co-IP assays using an anti-Myc antibody, Myc-p53 showed lower affinity for E6AP than for MDM2 in uninfected cells, likely due to weak phosphorylation at Ser-15 in the absence of HCV Core, resulting in minimal ubiquitination of phosphorylated Myc-p53 (Figure 7d). Therefore, most ubiquitination observed on total Myc-p53 in uninfected cells is attributable to MDM2 activity. HCV infection increased E6AP binding to Myc-p53 while decreasing the interaction between MDM2 and Myc-p53 (Figure 7d), probably due to HCV Core-induced phosphorylation of Myc-p53 at Ser-15 (Figure 7a). Under these conditions, reduced MDM2 binding to Myc-p53 could outweigh the increased E6AP binding, as evidenced by lower levels of ubiquitinated total Myc-p53 in infected cells (Figure 7d). Ectopic E6AP expression minimally affected the binding of E6AP and MDM2 to total and phosphorylated Myc-p53, as well as their ubiquitination, in uninfected cells. In contrast, ectopic E6AP expression increased E6AP binding and decreased MDM2 binding to Myc-p53, thereby enhancing E6AP-mediated ubiquitination and degradation of both total and phosphorylated Myc-p53 in HCV-infected cells (Figure 7a,d). These findings suggest that during HCV infection in Huh7D cells, E6AP, rather than MDM2, exclusively mediates the ubiquitination of phosphorylated p53.

4. Discussion

The tumor suppressor protein p53 is known as the “guardian of the genome” because it plays a vital role in defending against viral infections and preventing cell transformation. In response, viruses have developed several strategies, often using specialized viral proteins to interfere with or manipulate the p53 pathway [42]. Some of the most effective viral oncoproteins cause rapid p53 degradation, effectively bypassing this cellular defense. For example, the E6 protein from HPV-16 and -18 triggers E6AP-mediated ubiquitination and proteasomal degradation of p53 in human cervical cancer cells [15,43]. The interaction of HPV E6 with E6AP and p53 to form a trimeric complex is essential for HPV-mediated p53 degradation [16,44]. In this complex, HPV E6 alters the conformation of E6AP, activating it to induce p53 ubiquitination. E6AP also induces Ub-dependent proteasomal degradation of HPV E6 in a p53-dependent manner [45]. Data from previous studies [18,46,47] and the present study (Figure 2d and Figure 3c) suggest that HCV Core also interacts with E6AP and p53 to form a trimeric complex. Additionally, HCV Core enhances the interaction between E6AP and p53 to induce p53 ubiquitination (Figure 2d, Figure 5b and Figure 6e, and 7e, step 9), while E6AP can induce HCV Core ubiquitination with the aid of p53 (Figure 7e, steps 8 and 10) [18]. Consequently, ectopic expression of E6AP lowered both p53 and HCV Core levels, whereas E6AP knockdown elevated their levels simultaneously (Figure 1a,c). This antagonistic interaction between HCV Core and p53, which depends on the E3 Ub ligase activity of E6AP, is likely crucial to their roles as a representative viral oncoprotein and a potent cellular tumor suppressor, respectively, in HCV replication and pathogenesis.

Although HCV Core and HPV E6 exploit a common strategy to induce p53 degradation, their mechanisms of action differ significantly, particularly in their dependence on p53 phosphorylation. Our recent report showed that E6AP can target p53 if it is phosphorylated in response to genotoxic stress [19]. The present study also shows that treatment with the ATM inhibitor KU-55933 nearly abolished HCV Core’s ability to induce p53 phosphorylation and impaired its capacity to facilitate E6AP interaction with p53 and subsequent ubiquitination (Figure 4a,b). Moreover, etoposide, a topoisomerase II inhibitor, induced p53 phosphorylation, enabling E6AP to interact with p53 and ubiquitinate it even in the absence of HCV Core (Figure 5a,b), as also demonstrated in our recent report [19]. The present study may extend the mechanism involving E6AP and phosphorylated p53 to the context of HCV Core-mediated regulation. Therefore, HCV Core does not need to directly interact with E6AP to potentiate E6AP’s enzyme activity in p53 ubiquitination. The ability of HCV Core to form a trimeric complex with E6AP and p53 may represent an additional mechanism that augments E6AP-mediated ubiquitination of phosphorylated p53. Indeed, HCV Core facilitated E6AP-mediated p53 ubiquitination by increasing the interaction between phosphorylated p53 and E6AP (Figure 5b,c and Figure 7e, step 9). These results indicate that HCV Core and E6AP cooperate to bind phosphorylated p53 in human hepatoma cells. In contrast, HPV E6 does not induce p53 phosphorylation and cannot enhance E6AP binding to phosphorylated p53, thereby failing to promote its ubiquitination and degradation. It is unknown whether HPV E6 adjusts E6AP and/or unphosphorylated p53 in a trimeric complex through physical interactions, which can fulfill the requirement of p53 phosphorylation. More studies are needed to examine structural differences in their interactions with p53 and E6AP.

Genotoxic stress and HCV infection induce a series of post-translational modifications of p53, including phosphorylation of its N-terminal transactivation domain (Figure 7e, steps 1 and 3) [31,48,49,50]. Our study confirms that HCV Core phosphorylates p53 at Ser-15 and Ser-20 via activation of the ATM-Chk2 pathway (Figure 3a and Figure 7e, steps 2 and 3). Phosphorylation at Ser-15 is particularly critical for p53 stabilization, as it alone can alter the interaction between p53 and MDM2 [51]. However, it typically induces additional phosphorylation events, further stabilizing and activating p53 by decreasing its association with MDM2 35–39]. HCV Core thus protects p53 from MDM2-mediated degradation primarily by inducing its phosphorylation at Ser-15. HCV Core may also exploit this activity by reducing cellular levels of MDM2 itself (Figure 2a,b and Figure 7e, step 4). It remains unclear whether HCV Core inhibits MDM2 activity either by direct interaction with MDM2 or by steric interference with MDM2 binding to p53. Unlike MDM2, E6AP notably interacts with pSer-15 p53 to induce its ubiquitination in both HCV Core-expressing and etoposide-treated cells (Figure 4b–d and Figure 7e, step 7). This role of E6AP was further confirmed using artificial p53 mutants: E6AP ubiquitinated and degraded p53 S15D, which contains a phosphomimetic Asp residue instead of Ser-15, but failed to do so with p53 S15A, which contains a non-phosphorylatable Ala residue (Figure 6). HCV Core thus induces p53 phosphorylation at Ser-15 to facilitate E6AP-mediated targeting of phosphorylated p53 for degradation. However, the present study does not fully exclude the possibility that additional phosphorylation sites are also involved. Although the p53 phosphorylation mutants provide supportive evidence, a more definitive conclusion can be drawn from experimental conditions that induce or inhibit phosphorylation of p53 at Ser-15 without affecting other sites. It is also possible that ATM-dependent cofactors, which are activated by etoposide or inactivated by the ATM inhibitor KU-55933, contribute to E6AP recruitment to p53. More detailed studies are needed to elucidate the mechanisms by which HCV Core regulates MDM2- and E6AP-mediated proteasomal degradation of p53. Our study demonstrates that both E6AP and MDM2 function as E3 ubiquitin ligases that target p53 for degradation, albeit through distinct mechanismsMDM2 is likely specialized for unphosphorylated p53, while E6AP targets phosphorylated p53 or p53 bound to a viral oncoprotein, such as HPV E6. The unexpected interference between MDM2 and E6AP in p53 ubiquitination observed in Figure 2d may not be due to direct competition for binding to p53. Instead, E6AP likely inhibits MDM2 by degrading phosphorylated p53, thereby triggering further p53 phosphorylation and reducing the pool of unphosphorylated p53. In addition, MDM2 may interfere with E6AP by degrading unphosphorylated p53, reducing the substrate available for phosphorylation. Indeed, the interference between MDM2 and E6AP was not detected in the presence of KU-55933 and etoposide (Figure 4 and Figure 5). This dynamic interplay between E6AP and MDM2 likely involves the HCV Core acting as a molecular switch, selectively enhancing or inhibiting each E3 ligase’s ability to target p53, depending on the cellular context. The possibility that E6AP and MDM2 compete for p53 ubiquitination under physiological conditions cannot be completely excluded, because p53 phosphorylation at Ser-15 alone cannot completely prevent MDM2 from binding to p53 [12]. More detailed structural studies of the interactions between E6AP, MDM2, and p53, depending on p53’s phosphorylation status, are required to clarify this issue.

Previous studies have shown that HCV Core inhibits E6AP expression through DNA methylation (Figure 7e, step 6) and induces proteasomal degradation of MDM2 (Figure 7e, steps 2 and 4) [18,31,52]. This study also demonstrated that HCV Core lowers E6AP and MDM2 levels while upregulating p53 levels (Figure 2a and Figure 7e, steps 4 and 6). Therefore, in the presence of HCV Core, p53 levels are primarily influenced by MDM2 inhibition rather than by E6AP activation (Figure 2a,d). However, E6AP-mediated p53 degradation remains effective under this condition, as evidenced by increased p53 levels following E6AP knockdown (Figure 1c). The recessive role of E6AP in this context appears to result from its reduced levels in the presence of HCV Core. Ectopic expression of E6AP shifts the balance, leading to downregulation of total and phosphorylated p53 in the presence of HCV Core (Figure 3a). The dominant role of E6AP in regulating p53 was also observed in cells expressing HCV Core in the presence of hydrogen peroxide or etoposide, both of which upregulate E6AP levels while downregulating MDM2 levels. Therefore, altering experimental conditions to modulate E6AP and MDM2 expression could influence their roles in p53 degradation. Additionally, the phosphorylation status of p53, particularly at Ser-15, plays a crucial role in determining the relative contributions of MDM2 and E6AP to p53 degradation. For example, E6AP predominates in p53 ubiquitination when p53 is phosphorylated at Ser-15 by either HCV Core or DNA-damaging agents such as etoposide (Figure 5b,c). Furthermore, p53 mutations can affect its susceptibility to degradation by MDM2 and E6AP. For instance, the p53-Y220C mutant, which is highly phosphorylated at Ser-15 even in the absence of HCV Core, resists MDM2-mediated degradation but remains vulnerable to E6AP-mediated degradation (Figure 7a), preventing its abnormal accumulation in Huh7D cells [24,53]. Similarly, the p53-S15D mutant, which mimics phosphorylation at Ser-15, is more susceptible to E6AP-mediated degradation than wild-type p53 (Figure 6), but is resistant to MDM2-mediated degradation [40]. These findings highlight the complex regulation of p53 degradation, which is influenced by phosphorylation and the relative expression levels of E6AP and MDM2. Additionally, mutations in MDM2, E6AP, and p53, as well as other genetic backgrounds, may affect the relative roles of E6AP and MDM2 in regulating p53 levels in response to HCV Core.

5. Conclusions

In this study, we claim that HCV Core induces phosphorylation of p53, particularly at Ser-15, thereby rendering it susceptible to E6AP-mediated but resistant to MDM2-mediated proteasomal degradation. In addition, we showed that HCV Core directly enhances E6AP’s E3 activity by facilitating its interaction with p53. Accordingly, HCV Core establishes a regulatory environment in which E6AP becomes the dominant E3 ligase for p53 ubiquitination, while MDM2’s role is diminished or blocked. This shift in E3 ligase activity could be crucial for maintaining a delicate balance of p53 levels during HCV infection, promoting cell survival and potentially contributing to oncogenesis. Although Huh7D in vitro infection data are included, most mechanistic data were obtained using artificial experimental techniques, such as HCV Core expression systems, pharmacological perturbation of the ATM–Chk2 pathway, and p53 phosphorylation mutants, which should be verified under more physiological conditions using primary human hepatocytes, humanized mice, and HCV-positive patients, to correctly evaluate their biological significance.