PreS1BP mediates inhibition of Hepatitis B virus replication by promoting HBx protein degradation

Abstract

Background: PreS1-binding protein (PreS1BP), recognized as a nucleolar protein and tumor suppressor, influences the replication of various viruses, including vesicular stomatitis virus (VSV) and herpes simplex virus type 1 (HSV-1). Its role in hepatitis B virus (HBV) replication and the underlying mechanisms, however, remain elusive.

Methods: We investigated PreS1BP expression levels in an HBV-replicating cell and animal model and analyzed the impact of its overexpression on viral replication metrics. HBV DNA, covalently closed circular DNA (cccDNA), hepatitis B surface antigen (HBsAg), hepatitis B core antigen (HBcAg), and HBV RNA levels were assessed in HBV-expressing stable cell lines under varying PreS1BP conditions. Furthermore, co-immunoprecipitation and ubiquitination assays were used to detect PreS1BP- hepatitis B virus X protein (HBx) interactions and HBx stability modulated by PreS1BP.

Results: Our study revealed a marked decrease in PreS1BP expression in the presence of active HBV replication. Functional assays showed that PreS1BP overexpression significantly inhibited HBV replication and transcription, evidenced by the reduction in HBV DNA, cccDNA, HBsAg, HBcAg, and HBV RNA levels. At the molecular level, PreS1BP facilitated the degradation of HBx in a dose-dependent fashion, whereas siRNA-mediated knockdown of PreS1BP led to an increase in HBx levels. Subsequent investigations uncovered that PreS1BP accelerated HBx protein degradation via K63-linked ubiquitination in a ubiquitin-proteasome system-dependent manner. Co-immunoprecipitation assays further established that PreS1BP enhances the recruitment of the proteasome 20S subunit alpha 3 (PSMA3) for interaction with HBx, thereby fostering its degradation.

Conclusions: These findings unveil a previously unidentified mechanism wherein PreS1BP mediates HBx protein degradation through the ubiquitin-proteasome system, consequentially inhibiting HBV replication. This insight positions PreS1BP as a promising therapeutic target for future HBV interventions. Further studies are warranted to explore the clinical applicability of modulating PreS1BP in HBV therapy.

Keywords: HBx; Hepatitis B virus; PreS1BP; Ubiquitination.

Fig 1

PreS1BP expression levels decreased in HBV replicating cell and mouse model (A) Western blot analysis of HepG2 cells and HBV replicating cells (HepG2 transfected with pHBV1.3, HepG2.2.15). (B) Western blot analysis of HepAD38 cells cultured with doxycycline and then removed (Dox-) at indicated days. (C) Immunohistochemical staining for PreS1BP in liver of healthy mice and HBV mouse model, scale bar = 50 μm. (D–I) Huh7 cells were transfected with pHBx (D), pHBc (E), pPre-S1 (F), pLHBs (G), pMHBs (H), pSHBs (I), and then PreS1BP levels were detected by Western blotting. Quantification of the PreS1BP level of each group shown in the right graph was normalized to the corresponding GAPDH (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 2

PreS1BP expression inhibited HBV DNA replication and RNA transcription (A) HepAD38 cells were cultured with doxycycline, and then, the cells were cultured without doxycycline (Dox-) at the indicated time. And then HepAD38 cells were transfected with PreS1BP-siRNA or overexpressed plasmids for 48 h, and intracellular HBV DNA was extracted and detected by Southern blot. (B–D) HepG2, HepG2.2.15, and HepAD38 cells were transfected with PreS1BP-siRNA or overexpressed plasmids for 48 h, then cellular supernatant HBsAg levels were detected by ELISA; (E–G) and HBcAg, HBx levels were detected by Western blotting (n = 3). (H, K) HepG2 cells were transfected with pHBV1.3 minus PreS1BP-siRNA or overexpressed plasmids for 48 h, and then, the HBV RNA level was detected by RT-qPCR (n = 3). (I, L) HepG2.2.15 cells were transfected with PreS1BP-siRNA or overexpressed plasmids for 48 h, and then, the HBV RNA level was measured by RT-qPCR (n = 3). (J, M) HepAD38 cells were transfected with PreS1BP-siRNA or overexpressed plasmids for 48 h, and then, the HBV RNA level was identified by RT-qPCR (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3

PreS1BP decreases HBx expression (A, B) Huh7 cells were co-transfected with pHBx-His-and PreS1BP-siRNA or 0.5 μg, 1 μg, 2 μg PreS1BP plasmids, then Western blot analysis was detected. Values represent percentages of the HBx proteins normalized against GAPDH and compared to the signal of the HBx alone. (C) Co-IP. Western blot analysis of Huh7 cells transfected with pHBx-Flag. (D) Co-IP. Western blot analysis of Huh7 cells co-transfected with pHBx-flag and pPreS1BP-His. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 4

PreS1BP promotes HBx degradation (A) Huh7 cells were transfected with pHBx-His-or in combination with siRNA-mediated knockdown of PreS1BP. (B) Huh7 cells were transfected with pHBx-His-or in combination with PreS1BP plasmids. 48 h post transfection, cells were incubated with 100 μg/μl cycloheximide (CHX). Cells were lysed at the time points indicated and the HBx protein levels were analyzed with anti-His-antibody. The HBx expression intensity for each time point was quantified by densitometry and plotted. Quantification of the remaining HBx level of each group shown in the right graph was normalized to the corresponding GAPDH and then to the signal at 0 min (n = 3).

Fig 5

PreS1BP promotes HBx degradation through ubiquitin-proteasome pathway (A, B) Huh7 cells were co-transfected with pHBx and siRNA of PreS1BP (A) or pPreS1BP (B). 42 h after transfection, cells were treated with 20 μM MG132 for 6 h and harvested for Western blot analysis. Quantification of the HBx level of each group shown in the right graph was normalized to the corresponding GAPDH (n = 3). (C–F) Huh7 cells were co-transfected with pHBx and pUb-HA in combination with siRNA of PreS1BP (C) or pPreS1BP (D–F). 42 h after transfection, cells were treated with 20 μM MG132 for 6 h. Total cell extracts were first precipitated by anti-Flag magnetic antibody and then the immunocomplex was detected by Western blot. Corresponding ubiquitin antibody was used to detect the ubiquitination level of HBx. (G) Co-IP. Huh7 cells were transfected with pHBx-flag alone or in combination with pPreS1BP-His, cell extracts were first precipitated by anti-Flag magnetic antibody and then the immunocomplex was detected by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 6

PreS1BP-recruited PSMA3 destabilized the HBx protein (A) Huh7 cells were cotransfected with pHBx-His, pPreS1BP, and siPSMA3 targeting endogenous PSMA3, or siCtrl as the control. Then, the cells were lysed and harvested for Western blotting with anti-His-antibody to analyze the presence of HBx. Quantification of the HBx level of each group shown in the right graph was normalized to the corresponding GAPDH (n = 3). (B) Huh7 cells were cotransfected with pHBV1.3, pPreS1BP, and siPSMA3 targeting endogenous PSMA3, or siCtrl as the control. Then, the cells were harvested for Western blotting with antibody to assess HBcAg expression. Quantification of the HBcAg level of each group shown in the right graph was normalized to the corresponding GAPDH (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.