Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Mar 5;93(6):e01840-18.
doi: 10.1128/JVI.01840-18. Print 2019 Mar 15.

Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner

Umar Saeed  1   2 , Jumi Kim  1   2 , Zahra Zahid Piracha  1   2 , Hyeonjoong Kwon  1   2 , Jaesung Jung  1 , Yong-Joon Chwae  1   2 , Sun Park  1   2 , Ho-Joon Shin  1   2 , Kyongmin Kim  3   2
Affiliations

Parvulin 14 and Parvulin 17 Bind to HBx and cccDNA and Upregulate Hepatitis B Virus Replication from cccDNA to Virion in an HBx-Dependent Manner

Umar Saeed et al. J Virol. .

Abstract

The parvulin 14 (Par14) and parvulin 17 (Par17) proteins, which are both encoded by the PIN4 gene, play roles in protein folding, chromatin remodeling, DNA binding, ribosome biogenesis, and cell cycle progression. However, the effects of Par14 and Par17 on viral replication have never been explored. In this study, we found that, in the presence of HBx, either Par14 or Par17 could upregulate hepatitis B virus (HBV) replication, whereas in the absence of HBx, neither Par14 nor Par17 had any effect on replication. Overexpression of Par14/Par17 markedly increased the formation of covalently closed circular DNA (cccDNA), synthesis of HBV RNA and DNA, and virion secretion. Conversely, PIN4 knockdown significantly decreased HBV replication in HBV-transfected and -infected cells. Coimmunoprecipitation revealed that Par14/Par17 engaged in direct physical interactions with HBx in the cytoplasm, nucleus, and mitochondria, possibly mediated through substrate-binding residues on Par14/Par17 (E46/D74 and E71/D99, respectively) and conserved 19R20P-28R29P motifs on HBx. Furthermore, these interactions enhanced HBx stability, promoted HBx translocation to the nuclear and mitochondrial fractions, and increased HBV replication. Chromatin immunoprecipitation assays revealed that, in the presence of HBx, Par14/Par17 were efficiently recruited to cccDNA and promoted transcriptional activation via specific DNA-binding residues (S19/44). In contrast, in the absence of HBx, Par14/Par17 bound cccDNA only at the basal level and did not promote transcriptional activation. Taken together, our results demonstrate that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA-Par14/17-HBx complex.IMPORTANCE The HBx protein plays an essential regulatory role in HBV replication. We found that substrate-binding residues on the human parvulin peptidylprolyl cis/trans isomerase proteins Par14 and Par17 bound to conserved arginine-proline (RP) motifs on HBx in the cytoplasm, nucleus, and mitochondria. The HBx-Par14/Par17 interaction stabilized HBx; promoted its translocation to the nucleus and mitochondria; and stimulated multiple steps of HBV replication, including cccDNA formation, HBV RNA and DNA synthesis, and virion secretion. In addition, in the presence of HBx, the Par14 and Par17 proteins bound to cccDNA and promoted its transcriptional activation. Our results suggest that inhibition or knockdown of Par14 and Par17 may represent a novel therapeutic option against HBV infection.

Keywords: HBV; HBV replication; HBx; cccDNA; parvulin 14; parvulin 17.

PubMed Disclaimer

Figures

FIG 1
FIG 1
Inhibition or KD of PIN4 reduces HBV replication. (A) Juglone decreases HBV replication. The tetracycline (TC)-containing medium of HepAD38 cells (lane 1) was replaced with fresh medium without TC 24 h postseeding (lanes 2 to 4), and the cells were mock treated (none) (lane 2) or treated with ethanol (juglone solvent) (lane 3) or 20 μM juglone (lane 4) for 72 h. (B) PiB decreases HBV replication. HepAD38 (lane 1) and TC-depleted HepAD38 cells were mock treated (lane 2) or treated with DMSO (PiB solvent) (lane 3) or 20 μM PiB (lane 4) for 72 h. (C) Juglone and PiB, parvulin inhibitors, decreased HBV replication in infected cells. HepG2 and HepG2-hNTCP-C9 cells in collagen-coated 6-well plates were mock infected (lane 2) or infected with 1.7 × 103 GEq of HBV per cell (lanes 1 and 3 to 6). Infected cells were treated with ethanol (lane 3) or 20 μM juglone (lane 4) or with DMSO (lane 5) or 20 μM PiB (lane 6) for 9 days. (D) HBV replication decreased when PIN4 was knocked down. HepAD38 cells were transduced with lentivirus-like particles containing control shRNA (shControl) (lane 3) or PIN4-targeting shRNAs (shPIN4-1, shPIN4-2, shPIN4-3, shPIN4-4, and shPIN4-5) (lanes 4 to 8). Nontransduced (lane 1) and TC-depleted (lane 2) HepAD38 cells served as negative and positive controls, respectively. Lysates were prepared and subjected to SDS-13.5% PAGE or 1% native agarose gel electrophoresis, followed by immunoblotting, to detect proteins and core particles, respectively. Southern blot analysis was performed to detect HBV DNA synthesis. Endogenous PIN4, HBc, and GAPDH proteins were detected using rabbit monoclonal anti-PIN4 (1:1,000; Abcam; ab155283), rabbit polyclonal anti-HBc (1:1,000) (52), and mouse monoclonal anti-GAPDH (1:5,000; Santa Cruz; sc-32233) antibodies, respectively. GAPDH was used as a loading control. hNTCP-C9 was detected using mouse monoclonal anti-rhodopsin C9 (1:1,000; Millipore; MAB5356) antibody. HBV replication intermediate, partially double-stranded relaxed circular, and double-stranded linear DNAs are marked as HBV RI, RC, and DL DNA, respectively. Endogenous Par17 is marked with arrows. Relative levels of HBV core particle and HBV DNA were measured using ImageJ v.1.46r. Data are presented as means from four (panels A, B, and D) and three (panel C) independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05, and **, P < 0.005 relative to the corresponding control.
FIG 2
FIG 2
Overexpression of Par14 or Par17 increases HBV replication. (A) Schematic diagram and amino acid sequences of Par14 and Par17. The N-terminal basic and C-terminal PPIase domains of the proteins are indicated. The additional N-terminal 25 amino acids of Par17 are depicted as a barrel shape. Important amino acids are shown in italics and underlined. Mutants of important residues are indicated on the diagram. (B) HepAD38 cells stably expressing empty pCDH vector, Par14, or Par17 were seeded in TC-containing medium (lanes 1 to 4), and HBV DNA replication was induced by TC removal. The cells were incubated for the indicated times (day 1 [lanes 5 to 7], day 2 [lanes 8 to 10], and day 3 [lanes 11 to 13]), and then lysates were prepared. (C) HepG2.2.15 cells were mock transfected (lane 2) or transfected with pCMV-3×FLAG (lane 3), pCMV-3×FLAG-Par14 (lane 4), or pCMV-3×FLAG-Par17 (lane 5). HepG2 cells were used as a negative control (lane 1). Lysates were prepared 72 h after transfection. (D) Par14 and Par17 overexpression increased HBV replication in HBV-infected HepG2-hNTCP-C9 cells. HepG2 cells (lane 1) and mock-transduced (lane 2), vector-transduced (lane 3), Par14-transduced (lane 4), or Par17-transduced (lane 5) HepG2-hNTCP-C9 cells were grown in collagen-coated 6-well plates, infected with 1.7 × 103 GEq of HBV per cell (lanes 1 and 3 to 5), and lysed at 5 (for total RNA) or 9 days p.i. Lane 2 is a mock-infected control. SDS-PAGE, native agarose gel electrophoresis and immunoblotting of core particles, and Southern blotting were performed as described in the legend to Fig. 1. For Northern blotting, 20 μg of total RNA was loaded per lane. The 3.5-kb pgRNA, 2.1- and 2.4-kb S mRNAs, 0.7-kb X mRNA, and 28S and 18S rRNAs are indicated. Endogenous and overexpressed Par14 are marked with arrows, and overexpressed Par14 or Par17 is marked with double arrowheads or open arrowheads, respectively. Relative levels were calculated using ImageJ v.1.46r. Data are presented as means of the results from five (B and C) and three (D) independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05 and **, P < 0.005 relative to the corresponding control.
FIG 3
FIG 3
HBV RNA transcription increases upon overexpression of Par14 or Par17, possibly due to an increase in the cccDNA level. (A) Luciferase reporter assays to detect HBV enhancer and promoter activities upon overexpression of Par14 or Par17. HepG2 cells were transiently transfected with the indicated luciferase reporter vectors and/or expression plasmid for Par14 or Par17. (B) Northern blotting showing elevated expression of HBV mRNAs upon overexpression of Par14 or Par17. TC was removed from cultures of HepAD38 cells stably overexpressing vector alone (lane 2), Par14 (lane 3), or Par17 (lane 4). HepAD38 cells in the presence of TC were used as a negative control (lane 1). Northern blotting was performed as described in the legend to Fig. 2. (C) HBV cccDNA levels increased upon overexpression of Par14 or Par17. HepAD38 cells stably overexpressing vector alone (lanes 2 and 6), Par14 (lanes 3 and 7), or Par17 (lanes 4 and 8) were used in the experiment. (D) HBV cccDNA levels decreased upon PIN4 KD. HepAD38 stable cells transduced with shControl (lanes 2 and 6), shPIN4-1 (lanes 3 and 7), or shPIN4-5 transduced (lanes 4 and 8) were seeded as described above. (C and D, lanes 1 and 5) Nontransduced HepAD38 cells grown in TC-containing medium until day 10 were used as a negative control. (C and D) Cells were grown to 100% confluence; cccDNA was extracted on day 10 by the Hirt DNA extraction procedure with minor modifications (56) and subjected to Southern blotting without linearization (lanes 1 to 4) or following linearization with EcoRI (lanes 5 to 8). (E) PIN4 knockdown decreased HBV replication in infected cells. HepG2 (lane 1), HepG2-hNTCP-C9 (lane 2), HepG2-hNTCP-C9-shControl (lane 3), HepG2-hNTCP-C9-shPIN4-1 (lane 4), and HepG2-hNTCP-C9-shPIN4-5 (lane 5) cells in collagen-coated 6-well plates were infected with 1.7 × 103 GEq of HBV per cell (lanes 1 and 3 to 5). Lane 2 is a mock-infected control. HBV cccDNA was extracted, and subjected to Southern blotting as described above. SDS-PAGE and immunoblotting, Northern blotting, native agarose gel electrophoresis and immunoblotting of core particles, and Southern blotting were performed as described in the legends to Fig. 1 and 2. Relative levels of luciferase activity, HBV pgRNA and S and X mRNAs, cccDNAs, core particles, and HBV RI DNA were calculated using ImageJ v.1.46r. The viral RNA level was normalized to the cccDNA level. Data are expressed as means and SD of the results from three (A), six (B), five (C), three (D), or four (E) independent experiments. Statistical significance was evaluated using Student's t test. **, P < 0.00, and ***, P < 0.0005 relative to the corresponding control. Exact P values are shown for some conditions.
FIG 4
FIG 4
Par14 and Par17 upregulate HBV virion secretion. (A) The levels of extracellular HBV virions were increased by overexpression of Par14 or Par17. Pellets containing virions, HBs subviral particles, and naked core particles from culture supernatants of nontransduced (lanes 1), vector-transduced (lanes 2), Par14-overexpressing (lanes 3), or Par17-overexpressing (lanes 4) HepAD38 cells were prepared by ultracentrifugation with a 20% sucrose cushion. (Right, top three gels) Pellets dissolved in TNE buffer were subjected to 1% native agarose gel electrophoresis and immunoblotting to detect virions and/or HBs subviral particles and naked core particles using anti-HBs (1:1,000; Virostat; GF528), anti-PIN4, and anti-HBc (52) antibodies, respectively. (Bottom gel) HBV DNAs inside virions and naked core particles were detected by in situ nucleic acid blotting. (Left, top three gels) SDS-PAGE and immunoblotting to detect HBs, PIN4, and HBc proteins using rabbit polyclonal anti-HBs (1:1,000; Virostat; GF528), anti-PIN4, and anti-HBc (52) antibodies, respectively. (Bottom gel) Southern blotting for HBV DNA was performed as described in the legend to Fig. 1. (B) Intracellular HBV replication is augmented by Par14 or Par17 overexpression. Lysates of nontransduced (lanes 1), vector-transduced (lanes 2), Par14-overexpressing (lanes 3), or Par17-overexpressing (lanes 4) cells were analyzed as described above. Relative levels of HBc protein, HBV DNA, core particles, subviral HBs particles and virions, and in situ virion DNA were calculated using ImageJ v.1.46r. Data are presented as the means of the results from three independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; and ***, P < 0.0005 relative to the corresponding control.
FIG 5
FIG 5
S19, E46, and D74 of Par14 and S44, E71, and D99 of Par17 are important for Par14/Par17-mediated upregulation of HBV replication. (A and B) Overexpression of Par14 and Par17 mutants failed to rescue HBV replication in PIN4-KD HepAD38 cells. The effects of Par14 WT or mutants (A) and Par17 WT or mutants (B) on HBV replication were examined. Nontransduced and mock-transfected HepAD38 cells grown in TC-containing (lane 1) or TC-depleted (lane 2) medium were used as negative controls, and control shRNA-transduced HepAD38 cells were used as positive controls (lane 3). shPIN4-5-transduced PIN4-KD HepAD38 cells in 6-cm plates were mock transfected (lane 4) or transfected with 3×FLAG (lane 5), 3×FLAG-Par14 WT or 3×FLAG-Par17 WT (lane 6), or the respective mutants (lanes 7 to 11). TC depletion (lanes 2 to 11) and transfection (lanes 5 to 11) were performed at the same time. (C and D) Transcriptional activity is not enhanced by dephosphorylation-mimetic (Par14-S19A and Par17-S44A) and phosphorylation-mimetic (Par14-S19E and Par17-S44E) mutants. HepG2-hNTCP-C9 cells were infected, and Northern blotting was performed, as described in the legend to Fig. 2. Data are presented as means of the results from three independent experiments. Statistical significance was evaluated using Student's t test. ns, not significant; *, P < 0.05; and **, P < 0.005 relative to the corresponding control. (E) HBV replication in HepAD38 cells was decreased by the PPIase-deficient Par17 mutant. (Left) HepG2 cells were cotransfected with 1.3-mer HBV WT (lanes 2 to 6) plus 3×FLAG (lane 2), 3×FLAG-Par14 (lane 3), 3×FLAG-Par14-D74A (lane 4), 3×FLAG-Par17 WT (lane 5), or 3×FLAG-Par17-D99A (lane 6). Lane 1 contained mock-transfected HepG2 cells. (Right) TC-depleted HepAD38 cells were mock transfected (lane 8) or transfected with 3×FLAG (lane 9), 3×FLAG-Par14 WT (lane 10), 3×FLAG-Par14-D74A (lane 11), 3×FLAG-Par17 WT (lane 12), or 3×FLAG-Par17-D99A (lane 13). HepAD38 cells in TC-containing medium in 10-cm plates were used as a negative control (lane 7). TC depletion (lanes 8 to 13) and transfection (lanes 9 to 11) were performed at the same time. At 72 h posttransfection, lysates were prepared and subjected to analyses as described in the legend to Fig. 1. The 3×FLAG-tagged proteins were detected using mouse monoclonal anti-FLAG (1:1,000; Sigma; F1804) antibody. Relative levels of core particle and HBV DNA were calculated using ImageJ v.1.46r. Data are presented as means from four (A, B, and E) or three (C and D) independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 relative to the corresponding control.
FIG 6
FIG 6
Upregulation of HBV replication by Par14 or Par17 is HBx dependent. (A) In the absence of HBx, neither Par14 nor Par17 can upregulate HBV replication. HepG2 cells were mock transfected (lane 1) or cotransfected with 1.3-mer HBV WT plus 3×FLAG (lane 2), 1.3-mer HBV WT plus 3×FLAG-Par14 WT (lane 3), 1.3-mer HBV WT plus 3×FLAG-17 WT (lane 4), 1.3-mer HBx-deficient mutant plus 3×FLAG (lane 5), 1.3-mer HBx-deficient mutant plus 3×FLAG-Par14 WT (lane 6), or 1.3-mer HBx-deficient mutant plus 3×FLAG-17 WT (lane 7). (B) HBV replication is upregulated by Par14 or Par17 when HBx is supplied in trans to HBx-deficient-mutant-transfected cells. HepG2 cells were mock transfected (lane 1) or cotransfected (lanes 1 to 7) as described in the legend to Fig. 6A. Myc-HBx (lanes 8 to 10) was transfected into HepG2 cells cotransfected with 1.3-mer HBx-deficient mutant plus 3×FLAG, 3×FLAG-Par14 WT, or 3×FLAG-17 WT. The amount of transfecting DNA was adjusted with pCMV-3×FLAG. At 72 h posttransfection, lysates were prepared and subjected to analyses as described in the legends to Fig. 1 and 3. Myc-tagged protein was detected with rabbit polyclonal anti-Myc (1:1,000; Santa Cruz Biotechnology; sc-789) antibody. The relative levels of mRNAs, core particles, and HBV DNA were calculated using ImageJ v.1.46r. Data are presented as means of the results from five independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 relative to the corresponding control.
FIG 7
FIG 7
Par14 and Par17 are novel binding partners of HBx. (A and B) Par14 or Par17 directly interacts with HBx. HEK293T cells were cotransfected with 3×FLAG plus Myc-HBx WT (lanes 1, 4, and 7), 3×FLAG-Par14 WT plus Myc-HBx WT (lanes 2, 5, and 8), or 3×FLAG-Par17 WT plus Myc-HBx WT (lanes 3, 6, and 9). (Input) At 72 h posttransfection, whole-cell lysates were prepared and subjected to SDS-PAGE and immunoblotting (lanes 1 to 3). (IP) Lysates were immunoprecipitated with anti-Myc antibody and immunoblotted with anti-FLAG antibody (A) or vice versa (B) (lanes 4 to 6). (IgG) As a negative control, lysates were immunoprecipitated with normal mouse IgG (Merck Millipore; 12-371) (lanes 7 to 9). (C) Colocalizations of HBx WT and Par14 WT or HBx WT and Par17 WT in HEK293T cells. HEK293T cells were cotransfected with Myc-HBx WT plus 3×FLAG-Par14 WT (a to h) or 3×FLAG-Par17 WT (i to p). Confocal images of nuclei using DAPI (a and i) and of mitochondria using MitoTracker Orange CMTMRos (b and j) and of Par14 or Par17 using FITC (c and k) and of HBx using Alexa Fluor 647 (e and m) are shown. Dual (d, f, g, l, n, and o) and triple (h and p) merged images are indicated. Digital images of stained cells were captured through confocal microscopy (A1R-A1; Nikon, Japan). Representative data from four independent experiments are presented. (D) HBx-Par14 and HBx-Par17 interactions in the cytoplasm, nucleus, and mitochondria. HEK293T cells were mock transfected (lanes 1, 5, and 9) or cotransfected as described above. At 72 h posttransfection, cytoplasmic, nuclear, and mitochondrial fractions were prepared from whole-cell lysates by differential centrifugation. (IP) Total cell lysates and the indicated fractions were immunoprecipitated with anti-FLAG antibody (lanes 5 to 8). (IgG) As a negative control, lysates were immunoprecipitated with normal IgG (lanes 9 to 12). (Input) Total cell lysates and the corresponding fractions were prepared (lanes 1 to 4). The lysates and immunoprecipitants were subjected to SDS-PAGE and immunoblotting with anti-Myc, anti-FLAG, anti-GAPDH, anti-H3 (1:5,000; Abcam; ab1791), and anti-VDAC (1:1,000; Calbiochem; 529532) antibodies. Representative results from four independent experiments are shown.
FIG 8
FIG 8
The HBx 19R20P motif is completely conserved, and the 28R29P motif is highly conserved, among isolates from 10 HBV genotypes. The N-terminal 50 amino acids of HBx proteins from 10 genotypes were aligned using the CLC Main Workbench 8 software. The HBV genotype of each isolate is indicated on the left, followed by accession numbers. Conserved RP motifs are shown in boldface and italicized. Consensus sequences and conservation percentages are shown at the bottom.
FIG 9
FIG 9
HBx RP motifs are important for Par14- or Par17-mediated upregulation of HBV replication. (A) Schematic diagram of HBx protein. HBx RP (19R20P and 28R29P) motif sequences and alanine-substituted RP mutants are indicated. (B) Par14 cannot upregulate HBx-deficient mutant HBV replication when the HBx RP motif mutant is supplied in trans, unlike HBx WT. HepG2 cells were mock transfected (lane 1) or (co)transfected with 1.3-mer HBx-deficient mutant plus 3×FLAG (lane 2) or 1.3-mer HBx-deficient mutant plus 3×FLAG-Par14 (lane 3). For triple transfections, HepG2 cells were transfected with 1.3-mer HBx-deficient mutant plus 3×FLAG-Par14 plus Myc-HBx WT (lane 4) or the corresponding RP motif mutants (lanes 5 to 11). (C) Par17 cannot upregulate HBx-deficient mutant HBV replication when the HBx RP motif mutant is supplied in trans, unlike HBx WT. HepG2 cells were transfected as described in the legend to panel B, except that 3×FLAG-Par17 was used. The amount of transfected DNA was adjusted with pCMV-Myc vector. At 72 h posttransfection, lysates were prepared and subjected to analyses as described in the legend to Fig. 1. Relative levels of core particles and HBV DNA were calculated using ImageJ v.1.46r. Data are presented as means from four independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 relative to the corresponding control.
FIG 10
FIG 10
HBx RP motifs and Par14/Par17 substrate-binding residues are important for HBx-Par14/Par17 interactions. (A and B) HBx RP motifs are crucial for binding of Par14 and Par17. HEK293T cells were mock transfected (lane 1) or cotransfected with Myc-HBx WT plus 3×FLAG (lane 2). For experimental groups, 3×FLAG-Par14 (A) or 3×FLAG-Par17 (B) was cotransfected along with Myc-tagged HBx WT (lane 3) or the HBx AARP (lane 4), RPAA (lane 5), or AAAA (lane 6) mutant. (C and D) Substrate-binding residues (E46 and D74 of Par14 and E71 and D99 of Par 17) are important for binding to HBx. HEK293T cells were mock transfected (lane 1) or cotransfected with Myc-HBx WT plus 3×FLAG (lane 2). For experimental groups, 3×FLAG-Par14 WT or its mutants (E46A, D74A, or E46A/D74A) (C) or 3×FLAG-Par17 or its mutants (E71A, D99A, or E71A/D99A) (D) were cotransfected along with Myc-tagged HBx WT (lanes 3 to 6). At 72 h posttransfection, lysates were prepared; immunoprecipitated with anti-FLAG antibody; and immunoblotted with anti-Myc, anti-FLAG, and anti-GAPDH antibodies, as described in the legend to Fig. 7. Normal mouse IgG was used as a negative control. A representative result from four independent experiments is shown.
FIG 11
FIG 11
RP motifs of HBx and substrate-binding residues of Par14 and Par17 are important for HBx stability. (A) HBx stability is decreased in PIN4-KD HepG2 stable cells. Control shRNA-transduced (lanes 3, 4, and 7 to 10) and stable PIN4-KD (lanes 5, 6, and 11 to 14) HepG2 cells were transfected with Myc-HBx (lanes 2 to 14). As controls, HepG2 cells were mock transfected (lane 1) or transfected with Myc-HBx (lane 2). At 24 h posttransfection, the cells were treated with nothing (lanes 1 to 6) or 100 μg/ml cycloheximide (lanes 7 to 14). (B and C) Substrate-binding residues E46 and D74 of Par14 (B) and E71 and D99 of Par17 (C) are important for HBx stability. Control shRNA-transduced (lanes 2 and 3) and stable PIN4-KD (lanes 4 to 15) HepG2 cells were transfected with Myc-HBx WT (lanes 2 to 7) or cotransfected with Myc-HBx WT plus 3×FLAG-Par14/Par17 WT (lanes 8 to 11) or 3×FLAG-Par14/Par17 mutants (lanes 12 to 15). (D and E) HBx RP motifs are important for stabilization by Par14 (D) or Par17 (E). Control shRNA-HepG2 (lanes 2 and 3) and stable PIN4-KD HepG2 (lanes 4 to 15) cells were transfected with Myc-HBx WT (lanes 2 to 7) or cotransfected with 3×FLAG-Par14/Par17 WT plus Myc-HBx WT (lanes 8 to 11) or Myc-HBx-AAAA (lanes 12 to 15). Mock-transfected HepG2 cells served as a negative control (lane 1). (B to D) At 24 h posttransfection, cells were treated with nothing (lanes 1 to 3) or 100 μg/ml cycloheximide (lanes 4 to 15). Cells were harvested at the indicated times and analyzed by SDS-PAGE and immunoblotting with anti-Myc, anti-FLAG, anti-PIN4, and anti-GAPDH antibodies. Representative results from three independent experiments are shown. Data are presented as mean HBx level ± SD.
FIG 12
FIG 12
Par14 and Par17 promote translocation of HBx to the nucleus and mitochondria via HBx RP motifs. (A) Par14 WT, but not the Par14-E46A/D74A mutant, promotes HBx translocation to the nucleus and mitochondria via HBx RP motifs. (B) Par17 WT, but not the Par17-E71A/D99A mutant, promotes HBx translocation to the nucleus and mitochondria via HBx RP motifs. (A and B) Nontransduced and mock-transfected HepG2 cells (lane 1) and control shRNA-transduced HepG2 cells (lane 2) were used as controls. shPIN4-5-transduced stable PIN4-KD HepG2 cells (lanes 3 to 11) were mock transfected (lane 3); transfected with 3×FLAG-Par14/Par17 WT (lane 4), Par14/Par17 mutants (lane 5), Myc-HBx WT (lane 6), or Myc-HBx-AAAA (lane 7); or cotransfected with Myc-HBx WT plus 3×FLAG-Par14/Par17 WT (lane 8), Myc-HBx WT plus Par14/Par17 mutant (lane 9), Myc-HBx-AAAA plus 3×FLAG-Par17 WT (lane 10), or Myc-HBx-AAAA plus Par14/Par17 mutant (lane 11). At 72 h posttransfection, total cell lysates and the cytoplasmic, nuclear, and mitochondrial fractions were prepared and analyzed by SDS-PAGE and immunoblotting. Relative levels of Myc-HBx protein were calculated using ImageJ v.1.46r. Representative data from four independent experiments are shown. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.005; ***, P < 0.0005 relative to the corresponding control.
FIG 13
FIG 13
Par14 and Par17 are recruited to HBV cccDNA, possibly via S19 and S44, respectively. (A and B) Par14 and Par17 bind cccDNA. Vector-transduced (lanes 2, 6, and 10), Par14-transduced (lanes 3, 7, and 11), or Par17-transduced (lane 4, 8, and 12) HepAD38 cells were grown in TC-depleted medium. Nontransduced HepAD38 cells grown in TC-containing medium were used as a negative control (lanes 1, 5, and 9). (C) Par14 and Par17 binding to cccDNA was decreased when PIN4 was knocked down in infected cells. HepG2 (lane 1), HepG2-hNTCP-C9 (lane 2), HepG2-hNTCP-C9-shControl (lane 3), HepG2-hNTCP-C9-shPIN4-1 (lane 4), and HepG2-hNTCP-C9-shPIN4-5 (lane 5) cells were infected as described in the legend to Fig. 3. (D) Serines 19 and 44 of Par14 and Par17, respectively, bind HBV cccDNA. HepAD38 cells were plated as described above and mock transfected (lane 1) or transfected with 3×FLAG (lane 2), 3×FLAG-Par14 WT (lane 3), 3×FLAG-Par14-S19A (lane 4), 3×FLAG-Par14-S19E (lane 5), 3×FLAG-Par17 WT (lane 6), 3×FLAG-Par17-S44A (lane 7), or 3×FLAG-Par17-S44E (lane 8). (E) Binding of Par14 and Par17 to HBV cccDNA is promoted by HBx. HepG2 cells were mock transfected (lane 1); cotransfected with 1.3-mer HBV WT plus 3×FLAG (lane 2), 3×FLAG-Par14 WT (lane 3), or 3×FLAG-17 WT (lane 4); cotransfected with 1.3-mer HBx-deficient mutant plus 3×FLAG (lane 5), 3×FLAG-Par14 WT (lane 6), or 3×FLAG-17 WT (lane 7); or triple-transfected with 1.3-mer HBx-deficient mutant, Myc-HBx, and 3×FLAG (lane 8), 3×FLAG-Par14 WT (lane 9), or 3×FLAG-17 WT (lane 10). (F) HepG2 cells were mock transfected (lanes 1, 8, and 15); cotransfected with 1.3-mer HBV WT plus 3×FLAG (lanes 2, 9, and 16), 3×FLAG-Par14 WT (lanes 3, 10, and 17), or 3×FLAG-Par17 WT (lanes 4, 11, and 18); or cotransfected with 1.3-mer HBx-deficient mutant plus 3×FLAG (lanes 5, 12, and 19), 3×FLAG-Par14 WT (lanes 6, 13, and 20), or 3×FLAG-Par17 WT (lanes 7, 14, and 21). (A to F) The amount of transfected DNA was adjusted with pCMV-3×FLAG. Eight days after TC removal (A, B, and D), after transfection (E and F), or after infection (C), the chromatin solutions were prepared as described in Materials and Methods and subjected to immunoprecipitation with anti-PIN4, anti-acetyl H3 (Merck Millipore; 06-599), anti-RNA Pol II (Abcam; ab817), anti-H3 antibody (positive control), or normal rabbit polyclonal IgG (negative control). (A to G) Immunoprecipitated chromatins was analyzed by PCR (A and C to E) or qPCR (B, F, and G). Data for panels B, F, and G are expressed as means and SD of the results from three independent experiments. Statistical significance was evaluated using Student's t test. Exact P values relative to the corresponding control are shown.
FIG 14
FIG 14
Model of HBV cccDNA-Par14 and -Par17 interactions in the presence of HBx. HBV cccDNA is associated with histone H2A, H2B, H3, and H4 and nonhistone cellular and viral proteins (such as HBc and HBx proteins). We demonstrated that Par14 and Par17 bind to HBV cccDNA via serines 19 and 44, respectively. Also, E46 and D74 of Par14, and E71 and D99 of Par17, are critical for the corresponding interactions with HBx. In addition, the HBx RP motifs are essential for the formation of these complexes. Based on our findings, we propose that Par14 and Par17 upregulate HBV RNA transcription and DNA synthesis, thereby increasing the HBV cccDNA level, through formation of the cccDNA-Par14/17-HBx complex.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Seeger C, Mason WS. 2015. Molecular biology of hepatitis B virus infection. Virology 479–480:672–686. doi:10.1016/j.virol.2015.02.031. - DOI - PMC - PubMed
    1. World Health Organization. 2017. Global hepatitis report 2017. World Health Organization, Geneva, Switzerland. http://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/. Accessed 20 August 2018.
    1. Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. 2012. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1:e00049. doi:10.7554/eLife.00049. - DOI - PMC - PubMed
    1. Nassal M. 2015. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 64:1972–1984. doi:10.1136/gutjnl-2015-309809. - DOI - PubMed
    1. Slagle BL, Bouchard MJ. 2016. Hepatitis B virus X and regulation of viral gene expression. Cold Spring Harb Perspect Med 6:a021402. doi:10.1101/cshperspect.a021402. - DOI - PMC - PubMed

Publication types

MeSH terms