Plasmids.

HBV expression plasmids, pUC19 HBV Ae_US, pUC19 HBV Bj_JPN35, and pUC19 HBV_JPNAT (48), were used for amplification of the regions of HBx. PCR products were cloned into pEF FLAG pGK puro (49), pGEX6P-1 (GE Healthcare), and pGBKT7 (Clontech) vectors. cDNA of JMJD5 was cloned into pEF HA pGK hygro (where HA is hemagglutinin and hygro is hygromycin) and pGADT7 (Clontech) vectors. pEF OSF-HBx was generated by insertion of a One-Strep-FLAG (OSF) tag and HBx cDNA into pEF pGK puro (where puro is puromycin) vector. cDNAs of DDB1, STUB1, SMC4, PSME3, and TIF1B were amplified by using cDNA derived from Huh7 cells and cloned into pGADT7 vector. A cDNA of HNF4A was amplified by using cDNA derived from Huh7 cells and cloned into pEF FLAG pGK puro vector. To express HBV pregenomic RNA (pgRNA), full-length HBV DNA (C_JPNAT strain) was amplified by PCR and cloned into pcDNA3.1(+), with the resulting construct named pCMV pgRNA (where CMV is cytomegalovirus). A lentiviral vector that expresses a short hairpin RNA (shRNA) was generated by using pFTRE3G_pGK_GFP (where GFP is green fluorescent protein) (50). Briefly, pFTRE3G_pGK_GFP was cut by PacI/AscI and cloned into the U6 promoter and pGK_puro cassette, with the resulting product designated FU6_pGKpuro. The shRNA target sequence of JMJD5 was 5′-GCCTGACATGCAGACAGCATT-3′ and was located in the 5′ noncoding region. The shRNA sequence of LacZ was 5′-CTACACAAATCAGCGATTTC-3′. The reporter plasmids of the nucleocapsid promoter of HBV were generated by insertion of a DNA fragment of the HBV nucleocapsid promoter (Ae_US strain). DNA fragments of the core upstream regulatory sequence (CURS) and basic core promoter (BCP) were cloned into pGL4.18 (Promega). The mutations of JMJD5, HBx, and pHBVΔX (C_JPNAT strain) were generated by the method of splicing by overlap extension. For the CRISPR/Cas9 system, the plasmids of pX330 (catalog number 42230) and pCAG EGxxFP (catalog number 50716) were obtained from Addgene. The target sequence of the JMJD5 gene was 5′-CCACTGAGCTCTTCTACGAGGGC-3′. Genomic DNAs of Huh7 were amplified by using the primer set 5′-CAACCACTGAGGATCCCTGCCCAACAGAGAAGACTGC-3′ and 5′-TGCCGATATCGAATTCCACTACTGCACTCCAGCCTG-3′ and cloned into pCAG EGxxFP by using an In-Fusion HD cloning kit (TaKaRa). For lentivirus production, pCMV δR8.2 and pCMV-VSV-G (where VSV-G is vesicular stomatitis virus G protein) were obtained from Addgene. The plasmids used in this study were confirmed by sequencing with an ABI Prism 3130 genetic analyzer (Applied Biosystems).

Immunoprecipitation and immunoblotting.

Cells were lysed with lysis buffer consisting of 20 mM Tris-HCl (pH 7.4), 135 mM NaCl, 1% Triton X-100, 1% glycerol, and protease inhibitor cocktail tablets (Roche), incubated for 30 min at 4°C, and subjected to centrifugation at 14,000 × g for 15 min at 4°C. The supernatants were boiled at 95°C for 5 min and then incubated with anti-FLAG antibody at 4°C for 90 min. After incubation with protein G-Sepharose 4B (GE Healthcare) at 4°C for 90 min, the beads were washed three times by lysis buffer and boiled at 95°C for 5 min. The proteins were resolved by SDS-PAGE (Novex gels; Invitrogen), transferred onto nitrocellulose membranes (iBlot; Life Technologies), blocked with phosphate-buffered saline (PBS) containing 0.05% Tween 20 and 5% skim milk, incubated with primary antibody at room temperature for 12 h, and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. The immune complexes were visualized with Super Signal West Femto substrate (Pierce) and detected by an LAS-3000 image analyzer system (Fujifilm).

Mass spectrometry analysis.

293T cells transfected with pEF OSF-HBx or empty vector were incubated for 48 h and lysed by lysis buffer. The cell lysates were incubated with Strep-Tactin Sepharose (IBM GmbH) for 90 min and washed with lysis buffer three times, and then the precipitants were boiled in sample buffer at 95°C for 5 min. Copurified proteins were subjected to SDS-PAGE, stained with Coomassie brilliant blue, and digested by trypsin. The extracted peptides from the SDS-PAGE gel were directly analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using an L-column (C₁₈, 0.1 by 150 mm; CERI) on a Nanoflow LC system (Advance UHPLC; Bruker Daltonics) coupled to an linear trap quadrupole (LTQ) Orbitrap Velos mass spectrometer (Thermo Scientific). Tandem mass spectra were acquired automatically and then searched against a human database from Swiss-Prot with the Mascot server (Matrix Science).

Antibodies.

pGEX6P-1 JMJD5 was transformed into Escherichia coli [E. coli BL21(DE3)/pLysS; Novagen] and glutathione S-transferase (GST) fused with JMJD5 was purified by glutathione-Sepharose 4B (GE Healthcare). Recombinant JMJD5 proteins were immunized into BALB/c mice (4-week-old females; SLC Japan), and hybridomas were generated by fusion with the myeloma cell line PAI. We successfully obtained a clone, named AI6, as an anti-JMJD5 monoclonal antibody. Antibodies to the following proteins were purchased from the indicated manufacturers: anti-FLAG mouse monoclonal antibody (clone M2; Sigma), anti-HA rat monoclonal antibody (clone 3F10; Roche), horseradish peroxidase (HRP)-conjugated anti-FLAG mouse monoclonal antibody (clone M2; Sigma), and anti-β-actin mouse monoclonal antibody (A2228; Sigma).

Yeast two-hybrid assays.

The interaction of HBx and host proteins was analyzed by using a Matchmaker 3 two-hybrid system (Clontech) according to the manufacturer's protocol. Saccharomyces cerevisiae (AH109) cells were transformed with pGBKT7 HBx and pGADT7 host proteins and plated onto yeast medium (synthetic dextrose [SD]) depleted of Leu and Trp (SD/−LW) or depleted of Leu, Trp, Ade, His (SD/−LWAH).

Production of lentiviruses.

HEK293T cells (2 × 10⁶) were seeded on a 10-cm dish and incubated at 37°C for 1 day. A lentiviral transfer vector (FU6_pGKpuro; 1.5 μg), 2.5 μg of pCMV-dR8.2dvpr, and 1 μg of pCMV-VSV-G were mixed with 500 μl of Opti-MEM and 40 μl of polyethylenimine ([PEI], 1 mg/ml; MW, 25,000) (Polysciences, Inc.) and incubated for 15 min. DNA complex was inoculated into HEK293T cells, and the culture medium was changed at 4 h posttransfection. Culture supernatants at 3 days posttransfection were passed through a 0.45-μm-pore-size filter. For the infection of lentivirus, 2 × 10⁵ HepG2.2.15-7 cells seeded on a six-well plate were incubated for 1 day, and the virus-containing culture supernatants (2 ml) and hexadimethrine bromide (polybrene; 4 mg/ml; Sigma) were inoculated onto cells and centrifuged at 2,500 rpm for 45 min at 32°C. Puromycin was added at 2 days postinfection to select stable cell lines.

Purification of intracellular and supernatant HBV relaxed circular DNA (rcDNA) and cellular RNA.

Extracellular and intracellular HBV DNA was extracted as reported previously (48). Briefly, the cell pellets were lysed by using lysis buffer (50 mM Tris-HCl [pH 7.4], 1 mM EDTA, 1% NP-40) at 4°C for 15 min. After centrifugation at 15,000 rpm for 5 min, supernatants were incubated with 7 mM magnesium acetate (MgOAc), 0.2 mg/ml of DNase I (Roche), and 0.1 mg/ml of RNase A (Sigma) at 37°C for 3 h. After the addition of 10 mM EDTA, the lysates were digested by proteinase K (0.3 mg/ml; Life Technologies) and 2% SDS at 37°C for 12 h. Extracted HBV DNA was purified by phenol-chloroform-isoamyl alcohol, precipitated with ethanol, and resolved in pure water. Total RNA was extracted by using IsogenII (Nippon Gene) according to the manufacturer's protocol.

qPCR.

Quantitative PCR (qPCR) for HBV DNA was performed by using Fast SYBR green master mix (Applies Biosystems). The following primers were used for detection of HBV DNA: 5′-GGAGGGATACATAGAGGTTCCTTGA-3′ and 5′-GTTGCCCGTTTGTCCTCTAATTC-3′. Total RNA was used for quantification of mRNA expression by using a Power SYBR green RNA-to-Ct 1-Step kit (Applied Biosystems). The PCR was performed by using a ViiA7 real-time PCR system (Life Technologies). The following primers were used: for HNF4A, 5′-GCAGTGCGTGGTGGACAAAG-3′ and 5′-CTTCGAGTGCTGATCCGGTC-3′; for FOXA3, 5′-GTGAAGATGGAGGCCCATGAC-3′ and 5′-GAGGATTCAGGGTCATGTAGGAGTT-3′; for CEBPA, 5′-GAAGTCGGTGGACAAGAACAG-3′ and 5′-GTCATTGTCACTGGTCAGCTC-3′; for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5′-TGTAGTTGAGGTCAATGAAGGG-3′ and 5′-ACATCGCTCAGACACCATG-3′. The expression level of each gene was determined by the ΔΔC_T (where C_Tis threshold cycle) method using GAPDH as an internal control.

HBcAg measurement.

The supernatants from HBV-infected HepG2-hNTCP-C4 cells were collected at 10 days postinfection, and the amounts of HBV core antigen (HBcAg) were determined by Lumipulse assay (Fujirebio, Inc.) according to the manufacturer's protocol.

Immunofluorescence microscopy.

HepG2 cells cultured on glass slides for 1 day were transfected with plasmids for the expression of HBx and JMJD5 and fixed with 4% paraformaldehyde in PBS for 2 h at 1 day posttransfection; they were then washed with PBS three times and permeabilized by incubation with 0.2% Triton X-100 in PBS for 15 min. After cells were washed with PBS three times, they were incubated with anti-HA and anti-FLAG antibodies (1/1,000) diluted by 2% FBS in PBS at room temperature for 1 h. The cells were again washed with PBS three times and then incubated with Alexa Fluor 488 (AF488)-conjugated anti-mouse antibody (1/2,000) and AF488-conjugated anti-rat antibody (1/2,000) diluted by 2% FBS in PBS at room temperature for 1 h. The stained cells were covered with Prolong Gold AntiFade reagent with 4′,6′-diamidino-2-phenylindole (DAPI; Life Technologies) and observed under a FluoView FV1000 confocal microscope (Olympus).

Generation of JMJD5-knockout Huh7 cells.

The gene knockout Huh7 cell line was constructed by using CRISPR/Cas9 as described previously (51, 52). Huh7 cells were transfected with pX330 and pCAG EGxxFP and incubated for 1 week. GFP-positive Huh7 cells were sorted by fluorescence-activated cell sorting (FACS) and formed single colonies. Gene deficiency was confirmed by sequencing and Western blotting.

Reporter assay.

Huh7 cells were transfected with either CURS BCP-Luc or BCP-Luc together with pRL-SV40 (where SV40 is simian virus 40), and luciferase activity in the cell lysate was assayed by using a Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's protocol at 24 h posttransfection.

Southern blotting.

HBV core-incorporated DNA was electrophoresed in 1% agarose with Tris-acetate EDTA (TAE) buffer and transferred onto a nylon membrane (Hybond-N+; GE Healthcare). The pUC19 HBV (C_JPNAT) was digested by AatII and AclI, and the DNA fragment was extracted and purified by using a QIAquick gel extraction kit (Qiagen). Labeling probes, hybridization, and detection were performed by using a Gene Images AlkPhos Direct Labeling and Detection system (Amersham), according to the manufacturer's protocol.

Northern blotting.

Total RNA (5 μg) was subjected to electrophoresis in 1.2% agarose-formaldehyde gel and morpholinepropanesulfonic acid (MOPS) buffer. rRNA was visualized by ethidium bromide staining, and electrophoresed RNA was transferred onto a positively charged nylon membrane (Roche). A pBluscriptII (SK−) HBV-chloramphenicol acetyltransferase (CAT) construct (53) was linearized by HindIII, an RNA probe was synthesized by a digoxigenin (DIG) RNA labeling kit (Roche), and hybridization and detection were performed by using a DIG Northern Starter kit (Roche) according to the manufacturer's protocols. The signals were detected by an LAS-3000 image analyzer system (Fujifilm).

Microarray analyses of Huh7 and JMJD5KO cells.

Total RNA was extracted from Huh7 cells and from JMJD5KO Huh7 cells and purified with an miRNeasy kit (Qiagen) according to the manufacturer's instructions. Microarray analysis was performed as described previously (54). Briefly, 200 ng of total RNA was reverse transcribed, and the resulting cDNAs were used for in vitro transcription. Each cRNA sample was hybridized using a SurePrint G3 human gene expression (8 by 60,000) microarray kit, version 2.0 (model 039494; Agilent Technologies). Raw data were imported into a Subio platform, version 1.18 (Subio), for database management and quality control. Gene expression profiling was compared between Huh7 and JMJD5KO cells.

Random mutagenesis assay.

Random mutagenesis against JMJD5 was introduced by using a GeneMorph II random mutagenesis kit (Agilent Technologies) according to the manufacturer's protocol. The primers used were 5′-ATCGATACGGGATCCATATGGCTGGAGACACCCACTG-3′ and 5′-TCATCTGCAGCTCGAGCTACGACCACCAGAAGCTGA-3′. PCR products were electrophoresed, purified by using a QIAquick gel extraction kit (Qiagen), and cloned into pGADT7 vector by using an In-Fusion HD cloning kit (TaKaRa). After transformation, the bacterial colonies were picked up, and their plasmids were extracted and used to transform yeasts into yeasts expressing HBx, which were then plated on SD/−LW or SD/−LWAH medium.

JMJD5 structural modeling.

The JMJD5 secondary structure and the disordered regions were predicted from the amino acid sequence using the programs PSIPRED (55) and DISOPRED (56), respectively. The putative structure of the N-terminal region (residues 1 to 150) of JMJD5 was modeled using I-TASSER (57). The electrostatic potential was calculated by the APBS tool, and figures were rendered by PyMol (The PyMol Molecular Graphics System, version 1.7.6, Schrödinger, LLC).

JMJD5 participates in HBV replication.

To examine the roles of JMJD5 in HBV replication, shRNA against JMJD5 was lentivirally transduced in HepG2.2.15-7 cells, which produce HBV particles (41, 61), resulting in JMJD5-knockdown (JMJD5KD) HepG2.2.15-7 cells. We also prepared a monoclonal antibody against JMJD5 (AI6) after immunization of mice with recombinant JMJD5 proteins. Endogenous expression of JMJD5 detected by AI6 antibody was reduced in JMJD5KD HepG2.2.15-7 cells (Fig. 2A). No significant difference in cell growth was observed between control and JMJD5KD HepG2.2.15-7 cells by MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide] assay (Fig. 2B). To examine the roles of JMJD5 on HBV replication, JMJD5KD HepG2.2.15-7 cells were incubated for 6 days. The levels of both intracellular and extracellular HBV DNAs in JMJD5KD HepG2.2.15-7 cells were lower than those of cells transfected with control shRNA (Fig. 2C). Southern blot analysis revealed a reduction of HBV DNA in JMJD5KD HepG2.2.15-7 cells (Fig. 2D). Northern blot analysis showed no significant difference in transcription levels of HBV RNA between control and JMJD5KD HepG2.2.15-7 cells (Fig. 2E). In addition, the reductions of HBV DNA by reverse transcriptase inhibitor treatment were comparable between control and JMJD5KD HepG2.2.15-7 cells (Fig. 2F), suggesting that susceptibility to treatment with the reverse transcriptase inhibitor is not affected by suppression of JMJD5 expression. To further confirm the roles of JMJD5 in HBV replication, we established JMJD5KD HepG2.2.15-7 cells expressing HA-tagged RNA interference (RNAi)-resistant JMJD5. The amounts of intracellular HBV DNA were significantly enhanced by the expression of RNAi-resistant JMJD5 in JMJD5KD HepG2.2.15-7 cells (Fig. 2G). In addition, the reduction of HBcAg expression in JMJD5KD HepG2.2.15-7 cells was rescued by the exogenous expression of RNAi-resistant JMJD5 (Fig. 2H). Recently, HepG2 cells expressing sodium taurocholate cotransporting polypeptide (NTCP) were shown to be susceptible to HBV infection (46). Next, therefore, we established the JMJD5KD cell line upon transfection with shRNA into HepG2-hNTCP-C4 cells stably expressing NTCP (47) (Fig. 2I) and inoculated with HBV derived from the supernatants of HepAD38.7 cells (41). Although the amounts of HBV DNA were comparable between the HepG2-hNTCP-C4 cell lines established by transfection with either an shRNA targeting LacZ (shLacZ) or shJMJD5 at 2 days postinfection, HBV DNA in JMJD5KD HepG2-hNTCP-C4 cells was significantly decreased at 10 days postinfection (Fig. 2J). The amounts of HBcAg in the culture supernatants were also decreased in the JMJD5KD HepG2-hNTCP-C4 cells (Fig. 2K). Finally, Northern blot analysis revealed that HBV RNA was significantly reduced in JMJD5KD HepG2-hNTCP-C4 dells (Fig. 2L). Taken together, these results suggest that JMJD5 participates in HBV replication.

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FIG 2

JMJD5 participates in HBV replication. (A) The control and JMJD5KD HepG2.2.15-7 cell lines were lysed and subjected to Western blotting. (B) Control and JMJD5KD HepG2.2.15-7 cells were seeded onto 24-well plates and incubated for 3 days. The cell growth of each cell line was analyzed by MTT assay. Data represent the means ± standard deviations of two independent experiments. (C) Control and JMJD5KD HepG2.2.15-7 cells (2 × 10⁵) were treated with DNase and RNase, and core-incorporated HBV DNA resistant to the treatment was precipitated by ethanol after proteinase K digestion and quantified by qPCR (left). Culture supernatants of the control and JMJD5KD HepG2.2.15-7 cells were collected after 3 days of incubation, and the core-incorporated HBV DNA was extracted and quantified by qPCR (right). Data represent the means ± standard deviations of two independent experiments. (D) Core-incorporated HBV DNA was extracted from the control and JMJD5KD HepG2.2.15-7 (2 × 10⁶) cells and subjected to Southern blotting. ssDNA, single-stranded DNA. (E) The total RNA was extracted from the control and JMJD5KD HepG2.2.15-7 cells and subjected to Northern blotting. (F) The control and JMJD5KD HepG2.2.15-7 cells were treated with 10 μM lamivudine for 6 days, and intracellular HBV DNA was quantified by qPCR. Data represent the means ± standard deviations of two independent experiments. NTC, no-treatment control. (G) Core-incorporated HBV DNA was extracted from control HepG2.2.15-7 cells, JMJD5KD HepG2.2.15-7 cells, and JMJD5KD HepG2.2.15-7 cells stably expressing shRNA-resistant HA-JMJD5 and quantified by qPCR. Data represent the means ± standard deviations of two independent experiments. (H) Western blotting of the control, JMJD5KD, and JMJD5KD stably expressing shRNA-resistant HA-JMJD5 HepG2.2.15-7 cells. (I) Western blotting of the control and JMJD5KD HepG2-hNTCP-C4 cells. (J) The viral DNA levels of control and JMJD5KD HepG2-hNTCP-C4 cells infected with HBV (1000 GEq/cell) were determined by Southern blotting at 2 days (left) and 10 days (right) postinfection. (K) HBcAg in the culture supernatants of the control and JMJD5KD HepG2-hNTCP-C4 cells infected with HBV (1,000 GEq/cell) was quantified at 10 days postinfection. Data represent the means ± standard deviations of two independent experiments. (L) The viral RNA levels of control and JMJD5KD HepG2-hNTCP-C4 cells infected with HBV (1,000 GEq/cell) were determined by Northern blotting at 10 days postinfection.

Establishment of JMJD5-knockout (KO) Huh7 cells by the CRISPR/Cas9 system.

Next, to examine the roles of JMJD5 on viral replication in a human hepatoma cell line, Huh7, we established two JMJD5-deficient Huh7 cell lines (JMJD5KO Huh7 lines 45 and 47) by the CRISPR/Cas9 system (Fig. 3A) and JMJD5-deficient Huh7 cell lines complemented with HA-JMJD5 (Fig. 3B). The growth of JMJD5KO Huh7 cells was similar to that of parental Huh7 cells (Fig. 3C). Although complementation with HA-JMJD5 in JMJD5-deficient Huh7 cells exhibited a slight enhancement of HSV-1 propagation, there was no effect on the propagation of JEV and HCV (Fig. 3D), suggesting that deficiency of JMJD5 in Huh7 cells has only a moderate or no effect on the propagation of HSV-1, JEV and HCV.

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FIG 3

Establishment of JMJD5-knockout Huh7 cells by a CRISPR/Cas9 system. (A) Capitalized letters indicate the sequences of the CRISPR/Cas9 system targeting JMJD5. Gene knockout by sequence modification in the JMJD5 gene is shown. The dotted lines and the characters in red indicate deletion and insertion of the sequences, respectively. WT, wild type. (B) Western blotting of Huh7 cells, JMJD5-knockout Huh7 cell lines (JMJD5KO Huh7 lines 45 and 47) and JMJD5KO Huh7 cells complemented with HA-JMJD5. (C) Growth kinetics of Huh7 cells, and JMJD5KO Huh7 lines 45 and 47 cells. Cells seeded onto 24-well plates were cultured for 3 days, and cell growth was analyzed by MTT assay. Data represent the means ± standard deviations of two independent experiments. (D) Huh7 cells, JMJD5KO Huh7 (line 45) cells, and JMJD5KO Huh7 cells complemented with HA-JMJD5 were infected with either HSV-1 (multiplicity of infection of 0.1), JEV (multiplicity of infection of 1), or HCV (multiplicity of infection of 3), and infectious titers in the culture supernatants were determined at 2 to 3 days postinfection. Data represent the means ± standard deviations of two independent experiments. FFU, focus-forming units.

Deficiency of JMJD5 downregulates several host factors involved in liver homeostasis.

Although JMJD5 has been shown to be a tumor suppressor against lymphoma (62), JMJD5 has also been suggested to function as an oncogene by regulating hypoxia-inducible factor 1α (HIF-1α) through its interaction with pyruvate kinase muscle isozyme (PKM2) (40) or cyclin A1 (63) and to be involved in colon carcinogenesis (64). Therefore, the function of JMJD5, especially in hepatocytes, remains unclear. To examine the roles of JMJD5 in hepatocytes, we performed DNA microarray analysis by using parental and JMJD5KO Huh7 cells and showed that numbers of transcription factors involved in the hepatocyte development and differentiation listed by Qiagen's Ingenuity Pathway Analysis (IPA) were downregulated in JMJD5KO Huh7 cells (see Table S1 in the supplemental material). Among them, HNF4A, FOXA1, FOXA2, and FOXA3 have been shown to convert primary mouse embryonic fibroblasts into hepatocyte-like cells (65). Therefore, we next examined the roles of JMJD5 on the expression of HNF4A, CEBPA, and FOXA3 by qPCR. Deficiency of JMJD5 significantly reduced the expression of HNF4A, and exogenous expression of JMJD5 in JMJD5KO Huh7 cells recovered the expression (Fig. 4A). Similar to HNF4A expression, the expression levels of CEBPA and FOXA3 were downregulated in JMJD5KO Huh7 cells and then rescued by exogenous expression of JMJD5 (Fig. 4B and ​andC).C). The jumonji C (JmjC) domain of JMJD5 has been predicted to possess activity of hydroxylase rather than demethylase (37, 38). To determine the enzymatic activity of JMJD5 on HNF4A expression, an enzymatically deficient mutant, JMJD5 H321A, that possessed a substitution of Ala for His³²¹ in the JmjC domain, was expressed in JMJD5KO Huh7 cells, but no significant enhancement of HNF4A expression was observed by the expression of this mutant (Fig. 4D), suggesting that JMJD5 regulates the expression of several transcriptional factors involved in the maintenance of hepatocyte function through its enzymatic activity. The HBV nucleocapsid promoter region contains binding sites for HNF4 and CEBPA (Fig. 4E, top) (66), and HNF4A has been suggested to be involved in the expression of the HBV nucleocapsid (67,–69). Therefore, we considered that downregulation of HNF4A in JMJD5KO Huh7 cells likely suppresses the promoter activity of HBV nucleocapsid. To test this, we constructed a luciferase reporter plasmid to express firefly luciferase under the control of the HBV nucleocapsid promoter. The region of the basic core promoter (BCP) contains an HNF4-binding site. The region of the core upstream regulatory sequence (CURS) contains one HNF4A- and two CEBP-binding sites (Fig. 4E, top). The luciferase assay revealed that there was no significant difference in the promoter activities of the HBV nucleocapsid between JMJD5KO Huh7 cells and Huh7 cells with restored JMJD5 expression (Fig. 4E, bottom). Previous studies showed that the activity of the HBV nucleocapsid promoter was enhanced by the overexpression of HNF4A. Therefore, we examined the effect of overexpression of HNF4A on the nucleocapsid promoter. In spite of an 80-fold increase in HNF4A expression by transfection (Fig. 4F), only marginal and 2-fold increases in luciferase activity were observed in the BCP and CURS promoters, respectively (Fig. 4G). These results suggest that a 50% reduction of HNF4A expression by the knockdown of JMJD5 has no effect on the promoter activity of the HBV nucleocapsid, but JMJD5 might play some roles in the maintenance of hepatocytes. Further studies are needed to clarify the biological significance of JMJD5 in liver homeostasis.

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FIG 4

Deficiency of JMJD5 downregulates several host factors involved in liver homeostasis. (A to C) The expression levels of HNF4A, CEBPA, and FOXA1 in Huh7 cells, JMJD5KO Huh7 cells (lines 45 and 47), and JMJD5KO Huh7 cells complemented with HA-JMJD5 were determined by qPCR and expressed as the relative expression after normalization with expression of GAPDH. Data represent the means ± standard deviations of two independent analyses. Significant differences are indicated by asterisks (*, P < 0.05). (D) Expression levels of HNF4A in Huh7 cells, JMJD5KO Huh7 cells (line 45), and JMJD5KO Huh7 cells complemented with either the wild-type or the H321A mutant of HA-JMJD5 were determined by qPCR and expressed as the relative expression after normalization with expression of GAPDH. Data represent the means ± standard deviations of two independent analyses. Significant differences are indicated by asterisks (*, P < 0.05). (E) Reporter plasmids carrying a firefly luciferase gene under either the basic core promoter (BCP-Luc) or the core upstream regulatory sequence and BCP (CURS BCP-Luc) of the HBV nucleocapsid were transfected with pRL-SV40 into JMJD5KO Huh7 cells (line 45) and JMJD5KO Huh7 cells complemented with HA-JMJD5. Relative luciferase activities were determined by using a dual-luciferase kit (Promega) at 24 h posttransfection. Data represent the means ± standard deviations of two independent analyses. (F) Levels of mRNA of HNF4A were determined by qPCR upon overexpression of HNF4A. Data are shown as relative expression levels normalized by the expression of GAPDH. (G) The reporter plasmids (BCP-Luc and CURS BCP-Luc) were transfected with pRL-SV40 into Huh7 cells and those overexpressing HNF4A. Relative luciferase activities were determined by using a dual-luciferase kit (Promega) at 24 h posttransfection. Data represent the means ± standard deviations of two independent analyses. Significant differences are indicated by asterisks (*, P < 0.05).

JMJD5 participates in HBx-mediated HBV replication.

Next, to determine the roles of JMJD5 in HBV replication, Huh7 cells, JMJD5KO Huh7 cells, and Huh7 cells with restored JMJD5 expression were transfected with an HBV expression plasmid (pHBV C_JPNAT), and the levels of intracellular HBV DNA were determined by qPCR and Southern blotting at 3 days posttransfection (Fig. 5A and ​andB).B). To examine the effects of interaction between JMJD5 and HBx on transcription of HBV pgRNA, we constructed the expression vector of pgRNA under the CMV promoter (pCMV pgRNA) and transfected it into parental, JMJD5KO, and JMJD5-restored JMJD5KO Huh7 cells. Intracellular core-incorporated HBV DNA was reduced in JMJD5KO Huh7 cells but was restored by the exogenous expression of JMJD5 (Fig. 5C). In addition, Southern blot analysis also confirmed the reduction of HBV DNA replication by the knockout of JMJD5 in Huh7 cells (Fig. 5D). These data suggest that interaction of JMJD5 and HBx has no effect on the transcription of HBV RNA from the viral genome but participates in the posttranscriptional steps. Intracellular HBV core-incorporated DNA was significantly reduced in JMJD5KO Huh7 cells but was restored by the exogenous expression of JMJD5, suggesting that JMJD5 regulates HBV replication. To examine the role of JMJD5 on HBx-mediated HBV replication, JMJD5KO Huh7 cells were transfected with a plasmid encoding an HBV genome lacking the HBx region (pHBVΔX) (21). Although exogenous expression of HBx enhanced the replication of HBVΔX in a dose-dependent manner in Huh7 cells (Fig. 5E), rescue of HBVΔX replication by the expression of HBx was not observed in JMJD5KO Huh7 cells, in contrast to Huh7 and JMJD5KO Huh7 cells complemented with JMJD5 (Fig. 5F), suggesting that the interaction between HBx and JMJD5 participates in HBx-mediated HBV replication.

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FIG 5

JMJD5 participates in HBx-mediated HBV replication. (A and B) JMJD5KO Huh7 cell lines (lines 45 and 47) and those complemented with HA-JMJD5 were transfected with pHBV plasmids (genotype C, C_JPNAT strain), and intracellular core-incorporated HBV DNA was quantified by qPCR (A) and Southern blotting (B) at 3 days posttransfection. Data of qPCR represent the means ± standard deviations of two independent experiments. (C) JMJD5KO Huh7 cells (line 45) and JMJD5KO Huh7 cells complemented with HA-JMJD5 were transfected with pCMV HBV pgRNA plasmids, and intracellular core-incorporated HBV DNA was quantified by qPCR. Data represent the means ± standard deviations of two independent analyses. (D) JMJD5KO Huh7 cells (line 45) and JMJD5KO Huh7 cells complemented with HA-JMJD5 were transfected with pCMV HBV pgRNA plasmids, and intracellular core-incorporated HBV DNA was examined by Southern blotting. (E) Huh7 cells were transfected with pHBVΔX together with various amounts of HBx expression vector (0, 0.1, 0.2, and 0.3 μg/well), and intracellular core-incorporated HBV DNA was quantified by qPCR at 3 days posttransfection. Data represent the means ± standard deviations of two independent experiments. (F) Huh7 cells, JMJD5KO Huh7 cells (lines 45 and 47), and JMJD5KO Huh7 cells complemented with HA-JMJD5 were transfected with pHBVΔX together with/without HBx expression vector (0.3 μg/well), and the level of intracellular core-incorporated HBV DNA was determined by qPCR at 3 days posttransfection. Data represent the means ± standard deviations of two independent experiments. Significant differences are indicated by asterisks (*, P < 0.05).

We are grateful to M. Tomiyama for secretarial work and to M. Ishibashi and M. Nagao for their technical assistance. We also thank D. C. S. Huang, A. Ryo, and T. Suzuki for providing experimental materials. The Core Instrumentation Facility of Osaka University conducted the FACS sorting.

This work was supported by the Research Program on Hepatitis from the Japan Agency for Medical Research and Development, the Japanese Ministry of Education, Culture, Sports, Science, and Technology, the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Uehara Memorial Foundation.

T. Kouwaki, T. Okamoto, and Y. Matsuura designed the research; T. Kouwaki, T. Okamoto, A. Ito, Y. Sugiyama T. Suzuki, S. Kusakabe, J. Hirano, and T. Fukuhara performed most of the experiments; K. Saito performed the mass analysis; D. Okuzaki performed the DNA microarray analysis; K. Yamashita and D. M. Standley performed the prediction of the JMJD5 structure. A. Yamashita, K. Watashi, M. Sugiyama, S. Yoshio, T. Kanto, K. Moriishi, and M. Mizokami assisted with the experiments; and T. Kouwaki, T. Okamoto, and Y. Matsuura wrote the manuscript.