Prolyl Isomerase Pin1 Regulates the Stability of Hepatitis B Virus Core Protein

Abstract

The dynamic interplay between virus and host proteins is critical for establishing efficient viral replication and virus-induced pathogenesis. Phosphorylation-dependent prolyl isomerization by Pin1 provides a unique mechanism of molecular switching to control both protein function and stability. We demonstrate here that Pin1 binds and stabilizes hepatitis B virus core protein (HBc) in a phosphorylation-dependent manner, and promotes the efficient viral propagation. Phos-tag gel electrophoresis with various site-directed mutants of HBc revealed that Thr160 and Ser162 residues within the C terminal arginine-rich domain are phosphorylated concomitantly. GST pull-down assay and co-immunoprecipitation analysis demonstrated that Pin1 associated with phosphorylated HBc at the Thr160-Pro and Ser162-Pro motifs. Chemical or genetic inhibition of Pin1 significantly accelerated the rapid degradation of HBc via a lysosome-dependent pathway. Furthermore, we found that the pyruvate dehydrogenase phosphatase catalytic subunit 2 (PDP2) could dephosphorylate HBc at the Pin1-binding sites, thereby suppressing Pin1-mediated HBc stabilization. Our findings reveal an important regulatory mechanism of HBc stability catalyzed by Pin1 and may facilitate the development of new antiviral therapeutics targeting Pin1 function.

Keywords: hepatitis B virus; lysosome; phosphorylation; prolyl isomerization; virus-host interaction.

FIGURE 1

Concomitant phosphorylation of HBc at Thr160 and Ser162. (A) Schematic representation of the HBc deletion mutants generated in this study. The sequence of the HBc CTD, with the four major phosphorylation sites (S155, T160, S162, and S170) and alanine substitutions, is shown. (B) Mobility shifts of HBc in Phos-tag Gel. HepG2 cells were transfected with plasmids encoding HA-HBc or its site-directed mutants. The transfected cells were harvested at 24 h post-transfection, and cell lysates were subsequently subjected to Phos-tag gel electrophoresis and analyzed by immunoblot analysis with anti-HA antibody. (C) Detection of phosphorylation of HBc by phospho-specific antibody. HepG2 cells were transfected with WT HBc or its site-directed (T160A/S162A) mutant for 48 h in the presence of protease inhibitors. Cell lysates were then subjected to immunoblot analysis with anti-phospho HBc (T160/S162), anti-HBc, or anti-α-tubulin antibodies. (D) Cell lysates from stably HBV-producing HepG2.2.15.7 cells were treated or not treated with calf intestine alkaline phosphatase (CIAP), and then subjected to immunoblotting with anti-phospho HBc (T160/S162), anti-HBc, and anti-α-tubulin antibodies.

FIGURE 2

Pin1 interacts with phosphorylated HBc. (A) HepG2 cells were transfected with plasmid encoding HBc. After 48 h, cell lysates were subjected to GST pull-down analysis with GST, GST-Pin1, or GST-Pin1W34A mutant followed by immunoblotting with anti-HA antibody. (B) Cell lysates derived from HepG2 cells transfected with HBc were treated or not treated with CIAP, followed by GST pull-down analysis as described in (A). (C) HepG2 cells were transfected with HA-HBc and FLAG-Pin1 expression vectors. After 48 h, cell lysates were subjected to immunoprecipitation (IP) analysis with anti-HA or non-immunized IgG, followed by immunoblotting analysis with anti-FLAG or anti-HA antibodies. (D) HepG2.2.15.7 cell lysates were subjected to IP analysis with anti-HBc or non-immunized IgG, followed by immunoblotting analysis with anti-Pin1 or anti-HBc antibodies. (E) Pin1 interacts with HBc via its Thr160-Pro and Ser162-Pro motifs. HepG2 cells were transfected with WT HBc or the indicated mutants for 48 h in the presence of lysosome inhibitors. Cell lysates were then subjected to GST pull-down followed by immunoblot analysis. (F) HepG2 cells were transfected with HA-HBc expression plasmid. At 24 h following transfection, cells were treated with roscovitine (Rosc, 50 μM). After 15 h, cell lysates were harvested and subjected to GST pull-down analysis as shown in (A). (G) HepG2 cells expressing HA-HBc were treated with roscovitine (Rosc, 25 or 50 μM). After 15 h, cell lysates were subjected to immunoblotting analysis with indicated antibodies.

FIGURE 3

Pin1 regulates HBc stability. (A) Lysates from HepG2.2.15.7 cells that were infected with retroviral vectors carrying control shRNA (shCtrl) or Pin1-specific shRNA (shPin1) were immunoblotted with anti-HBc, anti-Pin1, or anti-α-tubulin antibodies. (B) Total mRNA from indicated HepG2.2.15.7 cells were subjected to quantitative PCR for HBc mRNA. Data were normalized with the amounts of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). ns, not significant. (C) Pin1-depleted HepG2.2.15.7 cells were transfected with empty vector (EV), Pin1WT, or Pin1W34A mutant. After 48 h, cell lysates were subjected to immunoblotting with anti-HBc, anti-Pin1, or anti-α-tubulin antibodies. (D) HepG2.2.15.7 cells were treated with either DMSO or 5 μM juglone for 24 h. Cell lysates were then subjected to immunoblot analysis with anti-HBc or anti-α-tubulin antibodies. (E) HepG2.2.15.7 cells were treated with 100 μM cycloheximide (CHX) and harvested at the indicated time points, followed by immunoblotting analysis with anti-HBc, anti-Pin1, or anti-α-tubulin antibodies. Quantitative data are shown in the right panel. ^∗P < 0.05, two-tailed unpaired t-test. (F) HepG2 cells were transfected with WT HBc or the T160A/S162A mutant followed by CHX assay as shown in (D). Quantitative data are shown in the right panel. *P < 0.05, two-tailed unpaired t-test.

FIGURE 4

Pin1 inhibits lysosomal degradation of HBc. (A) HepG2.2.15.7 cells transduced with shCtrl or shPin1 were treated with the indicated inhibitors for 24 h. Cell lysates were then subjected to immunoblot analysis with anti-HBc or anti-α-tubulin antibodies. The final concentration of inhibitors as follows; BafilomycinA1, 100 nM; NH₄Cl, 4 mM; MG132, 10 μM. (B) HepG2 cells transduced with shCtrl or shPin1 cells were transfected with HA-HBc expression vector. After 24 h, cells were fixed with 3% formaldehyde and immunostained with anti-HA (green), LysoTracker (red), and DAPI (blue). Cells were then subjected to confocal microscopy. Scale bar, 10 μm. Line plots indicate the fluorescence intensity of the left images.

FIGURE 5

Screening of phosphatases for Pin1 binding sites within HBc. (A) NanoBRET-based screen to identify HBc-interacting proteins in living cells. Schematic representation of the NanoBRET-based screening method (left panel). HEK293 cells were co-transfected with NanoLuc-tagged HBc and HaloTag-conjugated phosphatase expression vectors, followed by Halotag-620 ligand and furimazine substrate addition to the cells. If two proteins were within 200 nm of each other, BRET signals were detected. Two candidates with high BRET ratios (>0.2; right panel) were also shown. (B) HEK293 cells were co-transfected with HA-HBc together with HT empty vector (EV), HT-SNAP23, or HT-PDP2 and cultured for 24 h in the presence of protease inhibitors. Cell lysates were then subjected to immunoprecipitation with anti-HA antibody, followed by immunoblot analysis with the indicated antibodies. (C) PDP2 decreases HBc-T160/S162 phosphorylation. HepG2 cells were co-transfected with the expression vector encoding HA-HBc and Halotag (HT)-PDP2. At 48 h post-transfection, cells were harvested and subjected to immunoblot analysis with the indicated antibodies. (D,E) PDP2 interferes with HBc-Pin1 interaction. HepG2 cells expressing HA-HBc and HT-PDP2 (D) or HepG2.2.15.7 cells expressing HT-PDP2 (E) were lysed and subjected to GST pull-down analysis with GST or GST-Pin1, followed by immunoblot analysis with indicated antibodies. (F,G) HepG2.2.15.7 cells were transfected with expression vector encoding HT-PDP2. At 48 h post-transfection, cell lysates were subjected to immunoblot analysis with indicated antibodies. The levels of HBV DNA in the culture supernatants were measured by real-time PCR.

FIGURE 6

The HBc–Pin1 interaction regulates HBV biosynthesis. (A) Cell viability analysis of HepG2.2.15.7 cells stably expressing shCtrl or shPin1. (B,C) The levels of HBV DNA (B) and HBcAg (C) in the culture supernatants of HepG2.2.15.7-shCtrl or HepG2.2.15.7-shPin1 cells were measured by real-time PCR and ELISA, respectively. **P < 0.01, two-tailed unpaired t-test. (D–F) HepG2 cells were transfected with an HBV molecular clone (pUC19-C_JPNAT) and its site-directed mutant (T160A/S162A). After 24 h, the levels of HBV DNA in the culture the culture supernatants were measured by real-time PCR (D), and the levels of HBcAg (E) or HBeAg (F) in the culture supernatants were measured by ELISA. ***P < 0.001, two-tailed unpaired t-test. (G) Schematic representation of the model proposed in this study. CDKs phosphorylate HBc to create Pin1-binding sites. Subsequently, Pin1 stabilizes HBc by preventing its lysosomal degradation, thereby promoting effective HBV biosynthesis. On the other hand, PDP2 dephosphorylates HBc to enhance its degradation.