Hepatitis B virus (HBV) X protein-mediated regulation of hepatocyte metabolic pathways affects viral replication

Virology 498 (2016) 9–22

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Hepatitis B virus (HBV) X protein-mediated regulation of hepatocyte metabolic pathways affects viral replication Sumedha Bagga a, Siddhartha Rawat a,1, Marcia Ajenjo b,c, Michael J. Bouchard c,n a Graduate Program in Molecular and Cellular Biology and Genetics, Graduate School of Biomedical Sciences and Professional Studies, Drexel University College of Medicine, Philadelphia, PA, United States b Facultad de Ciencias Biosanitarias, Universidad Franscisco de Vitoria, Madrid, Spain c Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, 245 N. 15th Street, Philadelphia, PA, United States

art ic l e i nf o

a b s t r a c t

Article history: Received 2 June 2016 Returned to author for revisions 20 July 2016 Accepted 6 August 2016

Chronic HBV infection is a risk factor for hepatocellular carcinoma (HCC). The HBV HBx protein stimulates HBV replication and likely influences the development of HBV-associated HCC. Whether HBx affects regulators of metabolism in normal hepatocytes has not been addressed. We used an ex vivo, cultured primary rat hepatocyte system to assess the interplay between HBV replication and mechanistic target of rapamycin complex 1 (mTORC1) signaling. HBx activated mTORC1 signaling; however, inhibition of mTORC1 enhanced HBV replication. HBx also decreased ATP levels and activated the energy-sensing factor AMP-activated protein kinase (AMPK). Inhibition of AMPK decreased HBV replication. Inhibition of AMPK activates mTORC1, and we showed that activated mTORC1 is one factor that reduces HBV replication when AMPK is inhibited. HBx activation of both AMPK and mTORC1 suggests that these activities could provide a balancing mechanism to facilitate persistent HBV replication. HBx activation of mTORC1 and AMPK could also influence HCC development. & 2016 Published by Elsevier Inc.

Keywords: Hepatitis B Virus mTORC1 AMPK Primary hepatocyte Viral replication Viruses Metabolism Hepatocellular carcinoma

1. Introduction Viruses often encode proteins that subvert host cellular signaling pathways to achieve cellular conditions that are beneficial for viral replication and survival (reviewed in Condit (2013), Bagga and Bouchard (2014), Nascimento et al. (2012)). Virus-mediated deregulation of normal cellular signaling pathways can be detrimental to host cell physiology and can influence pathologies associated with viruses, including cell transformation and cancer progression (reviewed in McLaughlin-Drubin and Munger (2008),

Abbreviations: HBV, Hepatitis B virus; HCC, Hepatocellular carcinoma; HBx, HBV X protein; cccDNA, Covalently closed circular DNA; ORF, Open reading frame; mTORC1, Mammalian target of rapamycin (mTOR) complex 1; TSC2, Tuberoussclerosis complex-2; AMPK, AMP-activated protein kinase; S6K, p70 S6 Kinase; 4EBP1, Eukaryotic translation initiation factor 4E binding protein 1; AMPKK, AMPK-kinase; LKB1, Liver kinase B1; PKCζ, Protein kinase C ζ; ACC, Acetyl-CoA Carboxylase; ITS, Insulin-transferrin-selenium; EGF, Epidermal growth factor; GFP, Green fluorescent protein; PBS, Phosphate-buffered saline; SDS, Sodium dodecyl sulfate; PVDF, Polyvinylidene difluoride; TBST, Tris-buffered saline (TBS) tween n Corresponding author. E-mail address: [email protected] (M.J. Bouchard). 1 Current address: Baruch S. Blumberg Institute, Philadelphia, PA, United States of America. http://dx.doi.org/10.1016/j.virol.2016.08.006 0042-6822/& 2016 Published by Elsevier Inc.

Mesri et al. (2014)). Hepatitis B virus (HBV) is a DNA virus that predominately infects hepatocytes (reviewed in Seeger et al. (2007)). Worldwide, there are over 240 million people who are chronically infected with HBV, and a significant number of these individuals develop hepatocellular carcinoma (HCC), the second highest cause of cancer-related deaths in the world (Beasley et al., 1981; WHO, 2016) (and reviewed in Ferlay et al. (2013)). Globally, most HCC cases are linked to a chronic HBV infection (reviewed in Seeger et al. (2007), Block et al. (2003)). The mechanisms underlying the development of HBV-associated HCC are not entirely defined but are thought to involve recurrent immune-mediated destruction of HBV-infected hepatocytes and concomitant liver regeneration, potential consequences of HBV genome integration into the host genome, and the activities of HBV-encoded proteins such as the HBV X protein (HBx) (reviewed in Bouchard and Navas-Martin (2011), Neuveut et al. (2010), Koike (2009), Riviere et al. (2014), Chemin and Zoulim (2009), Guidotti and Chisari (2006), Casciano et al. (2012)), a multifunctional protein that stimulates HBV replication (reviewed in Koike (2009), Casciano et al. (2012), Bouchard and Schneider (2004), Benhenda et al. (2009), Wei et al. (2010)). Despite advances in therapy, the management of chronic HBV infections remains a clinical challenge (reviewed in Ghany and Doo (2009), Yang and Kao (2014), Zoulim (2004),

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Cheng et al. (2011), Zoulim (2007)). Elucidating the precise role of HBx in HBV replication will help define mechanisms that regulate HBV replication and facilitate the identification of potential therapeutic targets to treat individuals with a chronic HBV infection. HBx is a 154 amino acid, nonstructural, multifunctional protein encoded by the smallest open reading frame (ORF) of the HBV genome (reviewed in (Bouchard and Navas-Martin, 2011; Casciano et al., 2012; Bouchard and Schneider, 2004)). Studies of HBV replication in multiple model systems, including cultured primary rat hepatocytes, demonstrate that HBx strongly influences and is likely required for HBV replication (Clippinger et al., 2009; Bouchard et al., 2001; Keasler et al., 2007; Leupin et al., 2005; Lucifora et al., 2011; Tsuge et al., 2010; Melegari et al., 1998; Lim et al., 2010; Tang et al., 2005). Analyses performed in HBx-transgenic mice suggest that HBx has oncogenic potential (Kim et al., 1991; Koike et al., 1994; Slagle et al., 1996; Terradillos et al., 1997). HBx can modulate cytosolic calcium levels, regulate signal transduction and transcription pathways, and affect cell cycle progression, apoptosis, DNA repair, and protein degradation. These HBx activities provide potential mechanisms that link HBx expression to the development of HBV-associated HCC (Clippinger et al., 2009; Gearhart and Bouchard, 2010; Rawat and Bouchard, 2015; Yoo et al., 2003; Becker et al., 1998; Martin-Lluesma et al., 2008; Jung et al., 2007; Wang et al., 1998; Yang and Bouchard, 2012) (and reviewed in Bouchard and Navas-Martin (2011), Benhenda et al. (2009)). HBx effects are often dependent on the cell type and the method of HBx expression used in a study. Although studies in immortalized or transformed cell lines have provided valuable insights into the impact of HBx expression on signal transduction pathways, the effect of HBx expression on signal transduction pathways in normal hepatocytes, and in the context of HBV replication, remains incompletely understood (reviewed in Bouchard and Navas-Martin (2011), Casciano et al. (2012), Rawat et al. (2012)). Recent studies performed in cultured primary hepatocytes and in vivo models have begun to define HBx effects in normal, untransformed hepatocytes and determine how these HBx activities affect hepatocyte physiology and regulate HBV replication (Clippinger et al., 2009; Gearhart and Bouchard, 2010; Rawat and Bouchard, 2015; Gearhart and Bouchard, 2011; Gearhart and Bouchard, 2010; Clippinger and Bouchard, 2008). Mammalian target of rapamycin complex 1 (mTORC1) integrates diverse signals, including growth factors, energy status, nutrients, and stress, to regulate cell survival, growth, and metabolism, and mTORC1 signaling is upregulated in many types of cancer (reviewed in Laplante and Sabatini (2012), Dobashi et al. (2011)). Promotion of protein synthesis via phosphorylation of eukaryotic translation initiation factor 4E binding protein 1 (4EBP1) and p70 S6 Kinase (S6K) is a critical function of mTORC1. Growth factors activate mTORC1 through multiple signaling pathways. For example, the AKT signaling pathway is activated by growth factors, and AKT phosphorylates and inactivates tuberoussclerosis complex-2 (TSC2), a direct negative regulator of mTORC1 (reviewed in Laplante and Sabatini (2012), Dobashi et al. (2011), Laplante and Sabatini (2009), Hay and Sonenberg (2004)). In contrast, AMP-activated protein kinase (AMPK) activates TSC2 to reduce mTORC1 activity (Inoki et al., 2003). AMPK is a master sensor of intracellular energy; lowered intracellular ATP levels cause conformational changes in AMPK that make it susceptible to phosphorylation and activation by an AMPK-kinase (AMPKK). Liver kinase B1 (LKB1) is the major upstream AMPKK in the liver. Once activated, AMPK stimulates catabolic pathways and switches off anabolic processes (reviewed in Mihaylova and Shaw (2011), Hardie (2007), Luo et al. (2005)). Although a tumor suppressor role for AMPK has been proposed, recently, AMPK has been shown to promote cancer cell survival under stress (reviewed in Liang and

Mills (2013)). Interestingly, infection with Simian virus 40 (SV40) and human cytomegalovirus (HCMV) increases both AMPK and mTORC1 activity (reviewed in Brunton et al. (2013)). Although previous studies that were conducted in cell lines and in HBx-transgenic mice demonstrated that HBx activates mTORC1 signaling, those studies did not address the impact of mTORC1 signaling on HBV replication (Yen et al., 2012; Zhu et al., 2015). Alternatively, separate studies in immortalized or transformed cells that assessed the effect of mTORC1 signaling on HBV replication did not determine whether HBV replication or HBx expression affected mTORC1 signaling (Guo et al., 2007; Huang et al., 2014). Moreover, because signal transduction pathways are considerably altered in cancer, the cellular physiology of cell lines that are derived from tumors likely mimic the physiology of tumors rather than that of normal hepatocytes. Therefore, the results from studies that utilize transformed cell lines may be valid in a specific cellular context, but these studies might not identify effects of HBx and HBV on the physiology of normal hepatocytes, the target of an authentic HBV infection. Cultured primary rat hepatocytes are a biologically relevant model system that closely resembles normal hepatocytes in the liver and is used to study hepatocyte physiology (Bingham et al., 1998; Bour et al., 1996; Chitturi and Farrell, 2001; Cosgrove et al., 2008; Diot et al., 1992; James et al., 2003). Consequently, cultured primary rat hepatocytes can serve as a surrogate model for examining HBx and HBV effects in human hepatocytes (Rawat and Bouchard, 2015; Gearhart and Bouchard, 2011). We now demonstrate that HBx activates mTORC1 signaling in cultured primary rat hepatocytes, both when expressed in the absence of other HBV proteins and in the context of HBV replication. Surprisingly, we also observed that inhibition of mTORC1 signaling stimulates HBV replication and that inhibition of AMPK signaling, which results in activation of mTORC1, also inhibits HBV replication. Additionally, we show that HBx, both when expressed on its own and in the context of the HBV genome, decreases ATP levels and activates AMPK signaling. Although one study has assessed the impact of HBx on ATP levels in Huh 7 cells, a hepatoma cell line (Cho et al., 2011; Nakabayashi et al., 1982), the effect of HBV replication or HBx expression on AMPK signaling, and the role of AMPK signaling in HBV replication has not yet been addressed. Finally, we demonstrate that AMPK inhibition suppresses HBV replication because it leads to an activation of mTORC1 signaling. Our study provides a novel link between HBx expression and cellular metabolic pathways in normal hepatocytes. HBx activates both AMPK and mTORC1 signaling, which activate and inhibit HBV replication respectively. This suggests that HBx, by simultaneously activating pathways that stimulate and repress HBV replication, acts as a rheostat that controls the level of HBV replication and consequently, could facilitate the establishment of a persistent HBV infection. Cumulatively, our study has identified HBx regulation of the AMPK-mTORC1 signaling axis as a novel HBx function that modulates HBV replication in normal hepatocytes and could influence HCC development.

2. Materials and methods 2.1. Animal studies Animal surgery and hepatocyte isolation was approved by the Institutional Animal Care and Use Committee of the Drexel University College of Medicine (Protocol #20057) and complied with the Animal Welfare Act, the Public Health Service Policy on Humane Care and Use of Laboratory Animals (2002), and the NIH Guide for the Care and Use of Laboratory Animals (2011).

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2.2. Isolation and maintenance of primary rat hepatocytes Primary rat hepatocytes were isolated from male SpragueDawley rats using a two-step perfusion method, as previously described (Seglen, 1993). The hepatocytes were plated on 6-well (34.8-mm) collagen-coated tissue culture plates at approximately 1.5
106 cells/well (  80% confluent). The cells were maintained in Williams E medium supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 4 μg/ml insulin-transferrin-selenium (ITS), 5 ng/ ml epidermal growth factor (EGF), 5 μg/ml hydrocortisone and 10 μg/ml gentamicin at 37 °C in 5% CO2. Cultured primary rat hepatocytes were monitored for maintenance of hepatocyte morphology. Reverse transcriptase polymerase chain reaction was performed to assess the expression of hepatocyte-specific mRNAs throughout the time course of our experiments, as described previously (Clippinger et al., 2009; Gearhart and Bouchard, 2010). 2.3. Transfections and reagents Cultured primary rat hepatocytes were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer's directions. All transfections were performed 24 h after plating. Transfection efficiency was monitored by co-transfection with a green fluorescent protein (GFP)-expression plasmid. We consistently obtained a transfection efficiency of 30–40%. Rapamycin was purchased from Calbiochem (La Jolla, CA), and 6-[4(2-Piperidin-1-ylethoxy)phenyl] 3-pyridin-4-ylpyrazolo[1,5-a] pyrimidine (compound C) was purchased from Sigma-Aldrich (St. Louis, MO). 2.4. Antibodies The anti-p70 S6 Kinase, anti-phospho-p70 S6 Kinase (Thr389), anti-4E-BP1, anti-phospho-4E-BP1 (Ser65), anti-AMPKα, antiphospho-AMPKα (Thr172), anti-LKB1, anti-phospho-LKB1 (Ser428), anti-ACC, anti-phospho-ACC (Ser79), anti-raptor, and anti-phospho-raptor (Ser792) antibodies were purchased from Cell Signaling (Danvers, MA). The anti-HBx antibody was purchased from ViroStat, Inc. (Portland, ME). The anti-HBV core antibody was purchased from Dako (Carpinteria, CA). The anti-actin antibody was purchased from Sigma-Aldrich (St. Louis, MO).

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100% of hepatocytes were infected (Clippinger et al., 2009). HBV exhibits a narrow host range, and rat hepatocytes cannot be directly infected with HBV (reviewed in (Seeger et al., 2007)). HBV replication assays require that 100% of cultured primary rat hepatocytes express the HBV genome. Therefore, for these assays, we used AdGFP-HBV, a recombinant adenovirus that expresses a greater-than-unit-length copy of the HBV genome, to infect hepatocytes (Clippinger et al., 2009; Gearhart and Bouchard, 2010). 2.7. Cell collection and western blot analysis 48 h after transfection or recombinant adenovirus infection, cells were washed with cold phosphate-buffered saline (PBS), scraped into PBS, and pelleted at 2000 rpm for 5 min at 4 °C. Cells were then lysed in 0.8% sodium dodecyl sulfate (SDS) buffer (0.8% SDS, 240 mM Tris [pH 6.8], 10% glycerol). Protein concentrations were determined by using the Bio-Rad protein assay (Bio-Rad, Hercules, CA), according to the manufacturer's directions. Equal amounts of protein were loaded on a SDS-polyacrylamide gel. Proteins were then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad, Hercules, CA) and blocked for 1 h in 5% nonfat milk in Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). After incubation with primary antibody overnight at 4 °C, the membrane was washed 3 times with TBST and incubated with secondary antibody for 1 h at room temperature. Protein expression was detected using either horseradish peroxidaseconjugated secondary antibodies visualized by enhanced chemiluminescence or Alexa Fluor-conjugated secondary antibodies visualized using the Odyssey infrared imaging system (Licor Biosciences, Lincoln, NE). Protein expression was quantified using ImageJ software. 2.8. HBV replication assay To determine the effect of chemical inhibitors on HBV replication, cultured primary rat hepatocytes were infected with AdHBV and treated with rapamycin or compound C for 24 h. 48 h postinfection, hepatocytes were collected, and HBV core particles were isolated. Southern blot analyses was then performed to visualize core particle-associated viral DNA, as previously described (Bouchard et al., 2003).

2.5. Plasmids 2.9. ATP assay The FL-154HBx expression plasmid, which encodes an N-terminally FLAG epitope-tagged full-length HBx that is cloned into the pCDNA3.1(-) vector, was previously described (Clippinger and Bouchard, 2008). pGEMHBV (payw1.2) and pGEMHBV*7 (payw*7) were previously described (Melegari et al., 1998; Scaglioni et al., 1997; Scaglioni et al., 1997). pGEMHBV contains a greater-than-unit-length cDNA of the HBV genome, and all of the HBV genes in this construct are expressed under the control of endogenous HBV enhancers and promoters (Scaglioni et al., 1997; Scaglioni et al., 1997). pGEMHBV*7 is identical to pGEMHBV except for a point mutation in the seventh amino acid of HBx. This point mutation generates a stop codon so that the HBx protein is not produced; the overlapping HBV polymerase ORF is not disrupted (Melegari et al., 1998). 2.6. Recombinant adenovirus Recombinant adenoviruses used in this study were previously described (Clippinger et al., 2009). AdGFP is the control recombinant adenovirus, AdGFP-HBx expresses GFP and HBx, and Ad-GFP-HBV expresses GFP and HBV. GFP expression was used to monitor the efficiency of adenoviral infection and to ensure that

ATP levels were assessed using the ATP assay kit (Calbiochem, La Jolla, CA). 48 h following transfection, cultured primary rat hepatocytes were collected and washed with PBS. Cells were then dislodged with from the plate with trypsin and resuspended in PBS at a final concentration of 106 cells/ml. 10 μl of the cell suspension was then added to the 100 μl nucleotide releasing buffer in a luminometer plate (Black and White Isoplate, PerkinElmer, Waltham, MA). The plate was covered with aluminum foil and incubated at room temperature for 5 min with gentle shaking. 1 μl of ATP monitoring enzyme was then added to the cell lysate. The samples were read in a luminometer (1 min/well). ATP concentration of each sample was normalized to the protein concentration of that sample. The average ATP levels in pcDNA3.1(-), FL1–54HBx, pGEMHBV*7 and pGEMHBV-transfected cells were calculated. Fold differences were calculated by dividing the average ATP levels in pcDNA3.1(-)- or FL1–154HBx-transfected cells by the average ATP levels in pcDNA3.1(-)-transfected cells. Additionally, the fold differences for studies with pGEMHBV*7 and pGEMHBV were obtained by dividing the average ATP levels in pGEMHBV*7- or pGEMHBV-transfected cells by the average ATP levels in pGEMHBV *7-transfected cells. An average of the fold

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Fig. 1. HBx activates mTORC1 signaling in primary rat hepatocytes. (A and B) Primary rat hepatocytes were infected with AdHBx or AdGFP and collected at 48 h posttransfection. The levels of phosphorylated S6K (Thr389), total S6K, phosphorylated 4E-BP1 (Ser65), total 4E-BP1, and HBx were measured by western blot analyses. (C) Hepatocytes were transfected with the FL1–154HBx-expression plasmid or the pcDNA3.1(-) control vector and collected at 48 h post-transfection. Western blot analyses were conducted to assess the levels of phosphorylated 4E-BP1 (Ser65), total 4E-BP1, and HBx. (D) Hepatocytes were transfected with the pGEMHBV or pGEM*7 (HBxdeficient HBV) expression plasmid and collected at 48 h post-transfection. Western blot analyses were performed to check the levels of HBV core protein and actin. (E) Hepatocytes were transfected with the pGEMHBV or pGEM*7 expression plasmid and collected at 48 h post-transfection. The levels of phosphorylated S6K (Thr389), total S6K, phosphorylated 4E-BP1 (Ser65), and total 4E-BP1 were measured by western blot analyses. Results shown are representative samples from at least 2 independent experiments performed in duplicate. The band intensities of phosphorylated S6K and phosphorylated 4EBP1 were divided by the band intensities of total S6K and total 4EBP1 respectively, and the ratios are represented in terms of fold differences. The differences indicated below the western blots are average fold changes from at least 2 independent experiments performed 7 standard errors. An asterisk (*) represents a P value r0.05, determined by using Student's t test.

differences was calculated, and statistical significance of fold differences was determined using the Student's t test.

3. Results 3.1. HBx activates mTORC1 signaling in cultured primary rat hepatocytes We first assessed whether HBx regulates mTORC1 signaling in cultured primary rat hepatocytes. Because HBx expression has been observed in the absence of other HBV proteins in some HBVassociated tumors (Wang et al., 2004; Wollersheim et al., 1988), it is important to study HBx activities both when HBx is expressed in the absence of other HBV proteins and in the context of replicating HBV. Both these conditions could be relevant to the influence of HBx expression on hepatocyte physiology in various scenarios and may help identify mechanisms underlying HBV-associated HCC. mTORC1 kinase activity was analyzed by measuring the phosphorylation levels of S6K and 4EBP1, targets of mTORC1 (reviewed in Laplante and Sabatini (2012), Laplante and Sabatini (2009), Hay

and Sonenberg (2004)). 24 h after isolation and plating, cultured primary rat hepatocytes were infected with AdGFP-HBx or Ad-GFP replication-defective, recombinant adenoviruses. Primary rat hepatocytes were harvested at 48 h post-infection, and western blot analyses using phospho-specific antibodies were performed to determine the effect of HBx expression on mTORC1 signaling. Phosphorylation of S6K at threonine residue 389 (Thr389) is a marker of mTORC1 activation Pearson et al. (1995) (and reviewed in Dobashi et al. (2011), Dufner and Thomas (1999)). We observed that AdGFP-HBx-infected hepatocytes had higher levels of phosphorylated S6K (Thr389) as compared to AdGFP-infected hepatocytes, indicating that HBx activates mTORC1 signaling in primary rat hepatocytes (Fig. 1A). HBx expression was confirmed by western blot analyses (Fig. 1A). We next examined the effect of HBx expression on phosphorylation of 4E-BP1 at serine residue 65 (Ser65), which is another marker of mTORC1 activation (Gingras et al., 2001) (and reviewed in Hay and Sonenberg (2004)). AdGFPHBx-infected hepatocytes had higher levels of phosphorylated 4EBP1 (Ser65) than AdGFP-infected-hepatocytes (Fig. 1B). mTORC1 promotes protein synthesis by phosphorylating S6K and 4EBP1 (reviewed in Laplante and Sabatini (2012), Laplante and Sabatini

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(2009)). Recombinant adenoviruses expressing HBx can be utilized to achieve HBx expression in 100% of the cultured hepatocytes (Gearhart and Bouchard, 2010; Rawat and Bouchard, 2015; Gearhart and Bouchard, 2011; Gearhart and Bouchard, 2010). However, to confirm that our results with the recombinant adenoviruses were an effect of HBx expression and not an artifact of the adenovirus infection, we also assessed mTORC1 activation in HBxtransfected cultured primary rat hepatocytes. 24 h after plating, cultured primary rat hepatocytes were transfected with either a plasmid encoding HBx (FL1-154HBx) or a vector control [pcDNA3.1 (-)]. HBx-transfected hepatocytes had higher levels of phosphorylated 4E-BP1 (Ser65) in comparison to levels in controltransfected hepatocytes (Fig. 1C). Expression of HBx protein was confirmed by western blot analyses (Fig. 1C). These results indicate that the effects observed with the recombinant adenoviruses are an effect of HBx and not an artifact of the recombinant adenovirus infection. These observations are also consistent with our previously reported studies that compared HBx activities in hepatocytes infected with an HBx-expressing recombinant adenovirus to those in hepatocytes transfected with an HBx expression plasmid (Gearhart and Bouchard, 2010; Gearhart and Bouchard, 2010). We also examined the effect of HBx on mTORC1 signaling when HBx was expressed in the context of replicating HBV. Although it is likely that similar HBx activities will be observed in the context of HBV replication, it is also possible that other viral proteins could affect HBx regulation of mTORC1 signaling. Because hepadnavirus infections are species-specific, it is not possible to directly infect rat hepatocytes with HBV (reviewed in (Seeger et al., 2007)). To circumvent this problem, we transfected cultured primary rat hepatocytes with a plasmid encoding a greater-than-unit-length cDNA of the HBV genome (pGEMHBV). Previous studies have reported that when pGEMHBV is transfected into cells, all steps of HBV replication, excluding cell entry, are recapitulated (Scaglioni et al., 1997; Scaglioni et al., 1997; Lizzano et al., 2011). Expression of all HBV genes from the pGEMHBV expression plasmid, including HBx, is under the control of endogenous HBV promoters and enhancers (Scaglioni et al., 1997; Scaglioni et al., 1997). HBx is expressed at low levels during HBV replication (Jin et al., 2001). Similar to the observations during an authentic HBV infection, HBx is expressed at extremely low levels from pGEMHBV in cultured primary rat hepatocytes; however, we have previously demonstrated that HBx expression can be detected from pGEMHBV when a large number of pGEMHBV-transfected cells are combined and analyzed in a single western blot analysis (Clippinger and Bouchard, 2008; Jin et al., 2001). Cultured primary rat hepatocytes were transfected with either pGEMHBV or a mutant HBV-expression plasmid pGEMHBV*7, which expresses all HBV proteins except HBx (Melegari et al., 1998; Scaglioni et al., 1997; Scaglioni et al., 1997). Primary rat hepatocytes were harvested at 48 h postinfection, and expression of the HBV core protein (HBcAg) was examined by western blot analyses. The presence of HBcAg in both pGEMHBV- and pGEMHBV*7-expressing cells was used as a marker of expression of HBV proteins from the transfected plasmids (Fig. 1D). Similar to the results in AdGFP-HBx-infected and HBxtransfected hepatocytes, pGEMHBV-transfected hepatocytes had higher levels of phosphorylated S6K (Thr389) and 4E-BP1 (Ser65) than did pGEMHBV*7-transfected hepatocytes (Figs. 1E and 1F). These observations indicate that HBx, expressed in the context of the entire HBV genome, upregulates mTORC1 activity in cultured primary rat hepatocytes. Additionally, the studies with pGEMHBV and pGEMHBV*7 confirm that the results of the studies with AdGFP-HBx or FL1–154HBx were not an artifact of HBx over-expression. Overall, these results demonstrate that HBx, when expressed on its own and in the context of the HBV genome, activates mTORC1 signaling in cultured primary rat hepatocytes.

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3.2. HBx activates AMPK signaling in cultured primary rat hepatocytes Because we demonstrated that HBx activates mTORC1 signaling in cultured primary rat hepatocytes, we were next interested in determining whether HBx affects AMPK signaling, a negative regulator of mTORC1. Phosphorylation of AMPKα at Thr172 is critical for AMPK activation (reviewed in (Shackelford and Shaw, 2009; Viollet et al., 2010)). We therefore analyzed the levels of phosphorylated AMPKα (Thr172) in cultured primary rat hepatocytes that were infected with either AdGFP-HBx or AdGFP. Primary rat hepatocytes were harvested at 48 h post-infection, and the levels of phosphorylated AMPKα (Thr172) were analyzed by western blot analyses. Surprisingly, AdGFP-HBx-infected hepatocytes had higher levels of phosphorylated AMPKα (Thr172) than did AdGFP-infected cells, indicating that HBx activates AMPK in cultured primary rat hepatocytes (Fig. 2A). HBx expression was confirmed by western blot analyses (Fig. 2A). We next examined the effect of HBx expression on phosphorylation of liver kinase B1 (LKB1) at Ser428; LKB1 is an upstream AMPK kinase (reviewed in Shackelford and Shaw (2009)). Protein kinase C ζ (PKCζ) phosphorylates LKB1 at Ser428, which results in nuclear export of LKB1 and consequent AMPKα (Thr172) phosphorylation by LKB1 (Xie et al., 2008). We compared the levels of phosphorylated LKB1 (Ser428) in primary rat hepatocytes that were transfected with either FL1–154HBx or pcDNA3.1(-). Primary rat hepatocytes were harvested at 48 h post-infection, and the levels of phosphorylated LKB1 (Ser428) were determined by western blotting. HBx-transfected primary rat hepatocytes had higher levels of phosphorylated LKB1 (Ser428) than did control-transfected cells. These results suggest that HBx activates LKB1 to in turn activate AMPK (Fig. 2B). Expression of HBx was confirmed by western blotting (Fig. 2B). To further confirm the effect of HBx expression on AMPK activation, we analyzed the levels of phosphorylated acetyl-CoA carboxylase (ACC) (Ser79) and raptor (Ser792) in primary rat hepatocytes that were transfected with either FL1–154HBx or pcDNA3.1(-). Activated AMPK phosphorylates ACC at Ser79 and raptor at Ser792 (Gwinn et al., 2008; Ha et al., 1994) (and reviewed in (Shackelford and Shaw, 2009)). AMPK-mediated phosphorylation of raptor induces its binding to 14–3-3 and reduces mTORC1 activity (Gwinn et al., 2008). Moreover, AMPK inhibits fatty acid synthesis via phosphorylation of ACC1 (Ha et al., 1994) (and reviewed in (Hardie et al., 2012)). HBx-transfected primary rat hepatocytes had higher levels of phosphorylated ACC (Ser79) and raptor (Ser792), as compared to control-transfected hepatocytes, confirming that HBx activates AMPK signaling in primary rat hepatocytes (Figs. 2C and 2D). We next examined whether HBx expressed in the context of HBV replication affected AMPK signaling. Consistent with our previous observations when HBx was expressed by itself, pGEMHBV-transfected primary rat hepatocytes had higher levels of phosphorylated AMPKα (Thr172) and raptor (Ser792) than did pGEMHBV*7-transfected hepatocytes (Figs. 2E and 2F). Overall, these studies demonstrate that HBx, expressed either alone or in the context of replicating HBV, activates AMPK signaling in cultured primary rat hepatocytes. In addition, the observed activation of AMPK in pGEMHBV-transfected primary rat hepatocytes confirmed that the results of studies that used AdGFP-HBx or FL1– 154HBx were not a consequence of HBx overexpression. To our knowledge, this is the first study demonstrating that HBx expression can activate AMPK signaling. Finally, because our initial assessment of mTORC1 and AMPK activity was conducted in separate preparations of hepatocytes, we assessed phosphorylated S6K, ACC, and raptor in the same cell lysates and confirmed that HBx simultaneously activated mTORC1 and AMPK in hepatocytes (data not shown).

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Fig. 2. HBx activates AMPK signaling in primary rat hepatocytes. (A) Primary rat hepatocytes were infected with AdHBx or AdGFP and collected at 48 h post-infection. The levels of phosphorylated AMPKα (Thr172), total AMPK, and HBx were measured by western blot analyses. (B-D) Hepatocytes were transfected with the FL1–154HBxexpression plasmid or the pcDNA3.1(-) control vector and collected at 48 h post-transfection. Western blot analyses were conducted to assess the levels of phosphorylated LKB1 (Ser428), total LKB1, phosphorylated ACC (Ser79), total ACC, phosphorylated raptor (Ser792), total raptor, and HBx. (E-F) Hepatocytes were transfected with the pGEMHBV or pGEM*7 expression plasmid and collected at 48 h post-transfection. The levels of phosphorylated AMPKα (Thr172), total AMPK, phosphorylated raptor (Ser792), and total raptor were assessed by western blot analyses. Results shown are representative samples from at least 2 independent experiments performed in duplicate. The band intensities of phosphorylated AMPK, phosphorylated LKB1, phosphorylated ACC, and phosphorylated raptor were divided by the band intensities of total AMPK, total LKB1, total ACC, and total raptor respectively, and the ratios are represented in terms of fold differences. The differences indicated below the western blots are average fold changes from at least 2 independent experiments performed 7 standard errors. An asterisk (*) represents a P value r 0.05, determined by using Student's t test.

3.3. HBx decreases ATP levels in cultured primary rat hepatocytes Since we observed that HBx activated AMPK in primary rat hepatocytes, we next examined whether HBx affected ATP levels. Cultured primary rat hepatocytes were transfected with FL1–154HBx or pcDNA3.1(-). At 48 h post-transfection, hepatocytes were lysed, and intracellular ATP levels were measured using an ATP bioluminescence assay. Interestingly, and consistent with the activation of AMPK, ATP levels were lower in HBx-transfected hepatocytes as compared to control-transfected hepatocytes (Fig. 3A). Similar to our observations with FL-154HBx, pGEMHBV-transfected hepatocytes also had lower levels of ATP than did pGEMHBV*7-transfected hepatocytes (Fig. 3B). Taken together, these results demonstrate that HBx, when expressed on its own or in the context of HBV replication, decreases ATP levels in cultured primary rat hepatocytes. 3.4. mTORC1 and AMPK signaling regulate HBV replication in cultured primary rat hepatocytes Hepadnaviruses have a narrow host range, and HBV naturally infects only human hepatocytes (reviewed in Seeger et al. (2007)).

Chimpanzees and, to a certain extent, the Northern tree shrew Tupaia belangeri, a primate like animal, are two species other than humans that can be infected by HBV; each have been used for experimental HBV infection studies (Sureau et al., 1988; Walter et al., 1996; Yan et al., 1996) (and reviewed in Dandri et al. (2005), Inuzuka et al. (2014), Seeger et al. (2013), Wieland (2015)). However, rat hepatocytes cannot be directly infected with HBV. For studies assessing HBV replication in cultured primary rat hepatocytes, the initial steps required for HBV entry into hepatocytes can be bypassed by transfecting cells with pGEMHBV or infecting cells with a replication-defective, recombinant adenovirus that contains a copy of the HBV genome (AdHBV). Although we have optimized protocols for transfecting cultured primary rat hepatocytes and routinely achieve 30–40% transfection efficiency (Clippinger et al., 2009; Gearhart and Bouchard, 2010), transfection of primary rat hepatocytes with the pGEMHBV expression plasmid results in low levels of HBV replication (Clippinger et al., 2009). In contrast, recombinant adenoviruses can infect 100% of the cultured primary rat hepatocytes (Clippinger et al., 2009; Gearhart and Bouchard, 2010), and higher levels of HBV replication are obtained when cultured primary rat hepatocytes are infected with AdHBV,

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Fig. 3. HBx decreases ATP levels in primary rat hepatocytes. (A) Primary rat hepatocytes were transfected with the FL1–154HBx expression plasmid or the pcDNA3.1(-) control vector and collected at 48 h post-transfection. ATP levels were measured by using the ATP-assay kit, as described in materials and methods. (B) Hepatocytes were transfected with the pGEMHBV or pGEM*7 expression plasmid and collected at 48 h post-transfection. The levels of ATP in pGEMHBV- and pGEM*7-expressing hepatocytes were ascertained by using the ATP-assay kit, as described in materials and methods. The graph represents averages of data from 3 independent experiments, each with triplicate samples. Statistical analysis was conducted by using Student's t test, where the asterisk represents a P value of o 0.01.

facilitating easy detection of HBV replication (Clippinger et al., 2009). Because the focus of the studies described here was to determine how mTORC1 and AMPK affect HBV replication, we focused on specific effects on HBV replication in AdHBV-infected hepatocytes. However, we first confirmed that mTORC1 and AMPK

were activated in AdHBV-infected as compared to AdGFP-infected primary rat hepatocytes. Phosphorylation of 4E-BP1 at Ser65 was increased in AdHBV-infected hepatocytes compared to AdGFP-infected hepatocytes, indicating an upregulation of mTORC1 signaling in AdHBV-infected cultured primary rat hepatocytes (Fig. 4A).

Fig. 4. HBV activates mTORC1 and AMPK signaling in primary rat hepatocytes. Hepatocytes were infected with AdHBV or AdGFP and collected at 48 h post-transfection. Western blot analyses were performed to assess the levels of phosphorylated 4E-BP1 (Ser65), total 4E-BP1, and HBV core. Results shown are representative samples from at least 2 independent experiments performed in duplicate. The numbers indicated below the western blots are average fold changes from at least 2 independent experiments performed 7 standard errors. An asterisk (*) represents a P value r 0.05, determined by using Student's t test. (B and C) Hepatocytes were infected with AdHBV and AdGFP and collected at 48 h post transfection. Western blot analyses were performed to analyze the levels of phosphorylated AMPK (Thr172), total AMPK, phosphorylated ACC (Ser79), total ACC, and core. Results shown are representative samples from at least 2 independent experiments performed in duplicate. The band intensites of phosphorylated 4EBP1, phosphorylated AMPK, and phosphorylated ACC were divided by the band intensities of total 4EBP1, total AMPK, and total ACC respectively, and the ratios are represented in terms of fold differences. The differences indicated below the western blots are average fold changes from at least 2 independent experiments performed 7 standard errors. An asterisk (*) represents a P value r0.05, determined by using Student's t test.

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Fig. 5. mTORC1 signaling reduces HBV replication in primary rat hepatocytes. (A) Primary rat hepatocytes were treated with either rapamycin (mTORC1 inhibitor) or the vehicle control. Cells were harvested at 24 h post-treatment and western blot analyses were performed to assess the levels of phosphorylated S6K (Thr389), total S6K, phosphorylated 4E-BP1 (Ser65), total 4E-BP1, and actin. (B) Hepatocytes were infected with AdHBV, and the cells were treated with either rapamycin or the vehicle control. Hepatocytes were collected at 48 h post–infection, and HBV replication was assessed via Southern blot analysis of core particles that were isolated from the infected hepatocytes. RC, relaxed circular; DL, double-stranded linear; SS, single stranded. (C) Western blot analyses were performed on the samples from panel B to measure the levels of HBV core protein and actin. Southern and western blots shown here are one representative result from three independent experiments, each with duplicate samples. The numbers indicated in panel B are the average fold changes þ /- standard errors. An asterisk (*) represents a P value r 0.05, determined by using Student's t test. The Southern blot data shown is from the same Southern blot and from the same Southern blot exposure.

Phosphorylation of AMPKα at Thr172 and ACC at Ser79 was also increased in AdHBV-infected hepatocytes compared to AdGFP-infected hepatocytes, indicating an upregulation of AMPK signaling in AdHBV-infected primary rat hepatocytes (Figs. 4B and 4C). Expression of HBcAg was confirmed by western blot analyses (Fig. 4A and C). Taken together, these results indicate that HBV simultaneously activates mTORC1 and AMPK signaling in cultured primary rat hepatocytes and is consistent with similar HBV effects that we observed in pGEMHBV transfected hepatocytes (Figs. 1 and 2). We next analyzed the effect of mTORC1 signaling on HBV replication in primary rat hepatocytes; for these studies we treated AdHBV-infected primary rat hepatocyte with 50 nM rapamycin, an mTORC1 inhibitor (reviewed in Laplante and Sabatini (2012), Laplante and Sabatini (2009)), or dimethyl sulfoxide (DMSO), the vehicle control. The hepatocytes were harvested 24 h after treatment, and phosphorylation of S6K and 4EBP1 was analyzed by western blot analyses. Rapamycin-treated hepatocytes had lower levels of phosphorylated S6K (Thr389) and 4E-BP1 (Ser65) in comparison to the levels for DMSO-treated hepatocytes (Fig. 5A). We observed that rapamycin-induced inhibition of S6K phosphorylation was more evident than rapamycin-mediated downregulation of 4E-BP1 phosphorylation (Fig. 5A). This is consistent with previous reports, which showed that rapamycin is only

partially effective at inhibiting the phosphorylation of 4E-BP1 (Thoreen and Sabatini, 2009) (and reviewed in Laplante and Sabatini (2012)). Although rapamycin-treated hepatocytes had slightly lower levels of total S6K as compared to DMSO-treated hepatocytes, phosphorylation of S6K at Thr389 was completely inhibited in rapamycin-treated hepatocytes, indicating that the effect of rapamycin on phosphorylated S6K (Thr389) was stronger than its effect on total S6K (Fig. 5A). Surprisingly, rapamycintreated hepatocytes also had lower levels of total 4EBP1 than did DMSO-treated hepatocytes (Fig. 5A). Although rapamycin treatment affected the levels of total S6K and 4EBP1 in rat hepatocytes, we can still conclude that rapamycin inhibits mTORC1 signaling in cultured primary rat hepatocytes. 24 h after plating, cultured primary rat hepatocytes were infected with AdHBV. At 24 h postinfection, AdHBV-infected hepatocytes were treated with either rapamycin or the DMSO vehicle control. AdHBV-infected hepatocytes were collected at 48 h post-infection, and HBV replication was analyzed by Southern blotting of cytosolic, encapsidated HBV replicative intermediates. Because HBV has a partially doublestranded DNA genome, HBV replication intermediates appears as a smear in Southern blot analyses with three distinct bands that represent three viral replication intermediates: relaxed circular (RC), double-stranded linear (DL), and single-stranded linear (SS) (Bouchard et al., 2001) (and reviewed in (Bouchard and Navas-

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Fig. 6. AMPK signaling enhances HBV replication in primary rat hepatocytes. (A and B) Primary rat hepatocytes were treated with either the AMPK inhibitor compound C (10 μM/50 μM) or the vehicle control. Cells were harvested at 24 h post-treatment and western blot analyses were performed to assess the levels of phosphorylated AMPKα (Thr172), total AMPKα, phosphorylated S6K (Thr389), total S6K, phosphorylated 4E-BP1 (Ser65), total 4E-BP1, and actin. (C) Hepatocytes were infected with AdHBV, and the cells were treated with compound C (10 μM/50 μM) or the vehicle control. Cells were collected at 48 h post-infection and Southern blot analysis was performed to ascertain the levels of HBV replication. (D) Western blot analyses were conducted on the samples from panel C to analyze the levels of HBV core protein and actin. Southern and western blots shown here are one representative result from three independent experiments, each with duplicate samples. The differences indicated in panel C are the average fold changes þ/  standard errors. An asterisk (*) represents a P value r 0.05, determined by using Student's t test. The Southern blot data shown is from the same Southern blot and from the same Southern blot exposure.

Martin, 2011; Rawat et al., 2012; Seeger and Mason, 2000)). Interestingly, we observed that rapamycin increased HBV replication (Fig. 5B). Rapamycin did not affect the level of HBV core protein, suggesting that increased replication is likely not a consequence of rapamycin up-regulating the expression of HBV proteins (Fig. 5C). Overall, these results indicate that inhibition of mTORC1 signaling in cultured primary rat hepatocytes stimulates HBV replication. We next examined the effect of AMPK signaling, which can inhibit mTORC1 signaling, on HBV replication. To assess the effect of AMPK signaling on HBV replication, we utilized compound C, a pharmacological inhibitor of AMPK (Zhou et al., 2001; McCullough et al., 2005). Cultured primary rat hepatocytes treated with 10 mm or 50 mm of compound C had lower levels of phosphorylated AMPKα (Thr172) than did DMSO-treated hepatocytes, indicating that compound C inhibits AMPK activation in cultured primary rat hepatocytes (Fig. 6A). To confirm that AMPK inhibition activates mTORC1 signaling in primary rat hepatocytes, we examined the phosphorylation of mTORC1 substrates in hepatocytes treated with DMSO and compound C. Compound C–treated hepatocytes had higher levels of phosphorylated 4E-BP1 (Ser65) and S6K (Thr389) than did DMSO-treated hepatocytes, indicating activation of mTORC1 signaling (Fig. 6B). Total 4E-BP1 levels were also elevated in compound C-treated hepatocytes, as compared to DMSOtreated hepatocytes (Fig. 6B). In contrast, total S6K levels remained unaffected by compound C treatment (Fig. 6B). Although compound C treatment affected the levels of total 4E-BP1 in rat hepatocytes, overall we can conclude that compound C activates

mTORC1 signaling in cultured primary rat hepatocytes. Cultured primary rat hepatocytes were infected with AdHBV, and at 24 h post-infection, AdHBV-infected hepatocytes were treated with compound C or the DMSO vehicle control. 24 h after treatment, Ad-HBV infected hepatocytes were harvested, and HBV replication was analyzed by Southern blotting. Consistent with our previous observations regarding the regulation of HBV replication by mTORC1 signaling, we observed that compound C, which leads to an activation of mTORC1 signaling, reduced HBV replication (Fig. 6C). The level of HBV core protein was also decreased when AMPK signaling was inhibited by compound C (Fig. 6D). Overall, these results indicate that inhibition of the AMPK pathway in cultured primary rat hepatocytes decreases HBV core protein levels and reduces HBV replication. To our knowledge, this is the first evidence for a role of AMPK signaling in promoting HBV replication. 3.5. When AMPK is inhibited, concomitant activation of mTORC1 is one factor that decreases the level of HBV replication While mTORC1 decreased HBV replication in cultured primary rat hepatocytes, AMPK signaling promoted HBV replication. Because AMPK can suppress mTORC1 activity, we next analyzed whether diminished HBV replication when AMPK was inhibited was at least partially related to concomitant activation of mTORC1. AdHBV-infected cultured primary rat hepatocytes were treated with a combination of 10 μM compound C and 50 nm rapamycin,

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Fig. 7. Activated mTORC1 signaling inhibits HBV replication in primary rat hepatocytes when AMPK is inhibited. (A) Primary rat hepatocytes were infected with AdHBV, and the cells were treated with a combination of rapamycin and compound C, compound C alone, or the vehicle control. Hepatocytes were collected at 48 h post-infection and Southern blot analysis was performed to assess the levels of HBV replication. (B) Western blot analyses were conducted on the samples from panel A to check the levels of HBV core protein and actin. Southern and western blots shown here are one representative result from two independent experiments, each with triplicate samples. The numbers indicated in panel A are the average fold changes þ/  standard errors. An asterisk (*) represents a P value r 0.05, determined by using Student's t test. The Southern blot data shown is from the same Southern blot and from the same Southern blot exposure.

10 μM compound C alone, or the DMSO vehicle control. 24 h posttreatment, primary rat hepatocytes were harvested, and HBV DNA replication was analyzed by Southern blotting. We observed that AdHBV-infected hepatocytes treated with both compound C and rapamycin had higher levels of HBV replication as compared to the compound C-treated, AdHBV-infected hepatocytes, indicating that rapamycin could partially rescue HBV replication in compound C-treated, AdHBV-infected cells (Fig. 7A). Inhibition of AMPK signaling decreased HBV replication, and the observation that rapamycin partially rescued HBV replication in compound C-treated cells suggests that increased activation of the mTORC1 pathway in compound C-treated, AdHBV-infected hepatocytes inhibits HBV replication. Since inhibition of the mTORC1 pathway in compound C-treated, AdHBV-infected cells did not completely rescue viral replication to the same levels as in DMSO-treated cells, it is likely that there is more than one factor that affects HBV replication in compound C-treated, AdHBV-infected primary rat hepatocytes. Expression of the HBV core protein was analyzed by western blotting (Fig. 7B). Overall, the results of these studies suggest that activated mTORC1 signaling is one factor that can decrease the level of HBV replication in primary rat hepatocytes when AMPK is inhibited.

4. Discussion Chronic infection with the hepatitis B virus (HBV) is associated with the development of hepatocellular carcinoma (HCC). Alteration of hepatocyte physiology by HBV proteins such as the HBV X protein (HBx) has been invoked as a contributor to the development of HBV-associated HCC (reviewed in Bouchard and NavasMartin (2011), Neuveut et al. (2010), Koike (2009) Riviere et al. (2014), Chemin and Zoulim (2009), Guidotti and Chisari (2006),

Casciano et al. (2012)). While certain HBx activities, such as modulation of cytosolic calcium signaling, regulation of the cell cycle, and activation of Pyk2 and Src kinases can stimulate HBV replication, others, such as HBx-induced stimulation of the AKT signaling pathway, decrease HBV replication (Bouchard et al., 2001; Gearhart and Bouchard, 2010; Rawat and Bouchard, 2015; Gearhart and Bouchard, 2010; Bouchard et al., 2003; Klein et al., 1999). Because of the extremely compact nature of the HBV genome, which encodes a limited number of proteins, it is not surprising that HBx, the major HBV regulatory protein, can affect multiple cellular processes that regulate HBV replication and affect hepatocyte physiology. An unfortunate consequence of HBxmediated deregulation of normal hepatocyte physiology is that some of these HBx effects may also contribute to processes that can influence hepatocyte transformation during a chronic HBV infection (reviewed in Bouchard and Navas-Martin (2011), Koike (2009), Casciano et al. (2012), Benhenda et al. (2009), Rawat et al. (2012)). The liver is a metabolic hub of the human body, and whether HBx affects metabolic pathways in normal hepatocytes has not been previously addressed. We used an ex vivo cultured primary rat hepatocyte model system to examine the interplay between HBV replication and the mammalian target of rapamycin complex 1 (mTORC1) signaling axis, a master regulator of cell metabolism. We previously demonstrated that HBx activities are similar in cultured primary rat and human hepatocytes. Since HBV naturally infects normal, differentiated hepatocytes, our ex vivo hepatocyte model system is a biologically relevant model for assessing the impact of HBx and HBV on hepatocyte signal transduction pathways and is likely to generate results that are more analogous to an authentic HBV infection than similar types of studies in established cell lines (Clippinger et al., 2009; Gearhart and Bouchard, 2010; Rawat and Bouchard, 2015; Gearhart and Bouchard, 2010;

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Fig. 8. Model of regulation of HBV replication by HBx activation of the AMPK and mTORC1 signaling pathways.

Clippinger and Bouchard, 2008). An intrinsic feature of transformed and immortalized cells is that normal signal transduction pathways have been altered. These alterations may change the ultimate outcome of HBx expression in a particular cell line. In the studies described here, we demonstrated that HBx, when expressed on its own or in the context of HBV replication in normal hepatocytes, activates mTORC1 signaling. Surprisingly, inhibition of mTORC1 signaling increased HBV replication, showing that HBx activation of mTORC1 actually reduces HBV replication in normal hepatocytes. We also observed that HBx, when expressed on its own and in the context of replicating HBV, decreased ATP levels. HBx also activated both LKB1 and AMPK, crucial sensors of the energy status of a cell. Inhibition of AMP-activated protein kinase (AMPK) signaling, which stimulates mTORC1 activity, decreased HBV replication. To our knowledge, this is the first evidence that HBx activates AMPK to stimulate HBV replication. We observed that HBV replication was higher in AdHBV-infected hepatocytes treated with both rapamycin and compound C in comparison to the levels of replication obtained in AdHBV-infected cells treated with compound C alone, suggesting that increased activation of mTORC1 is at least partially responsible for reducing HBV replication in hepatocytes when AMPK is inhibited (Fig. 8). Our study is the first comprehensive analysis of the effects of HBx on major regulators of metabolism, the AMPK and mTORC1 signaling pathways, in a primary hepatocyte model system. Since HBx stimulates HBV replication (reviewed in (Bouchard and Navas-Martin, 2011)), pGEMHBV-transfected hepatocytes will have elevated HBV replication as compared to pGEMHBV*7 transfected hepatocytes. Although we have not ruled out the possibility that the enhanced AMPK and mTORC1 signaling observed in pGEMHBV-transfected hepatocytes as compared to pGEMHBV*7-transfected hepatocytes could also be influenced by elevated replication of the wildtype virus, because we observed the same effect when we expressed HBx alone or in the context of HBV replication, we expect that much of the observed effect on mTORC1 and AMPK signaling in the context of HBV replication is a consequence of HBx expression. Our results suggest that the HBxinduced decrease in ATP levels leads to AMPK activation; a drop in ATP levels in HBx- and HBV-expressing cells could facilitate LKB1mediated activation of AMPK. Although it is well known that activation of AMPK inhibits mTORC1, we did not observe an inhibition of mTORC1 in HBx-expressing cells. Active AMPK can phosphorylate tuberous-sclerosis complex-2 (TSC2), which in turn inhibits mTORC1. On the other hand, AKT can activate mTORC1 by phosphorylating and inhibiting TSC2, directly opposing the effect of AMPK on mTORC1 (reviewed in Laplante and Sabatini (2012), Laplante and Sabatini (2009)). Ectopic expression of a constitutively active form of AKT in pancreatic beta cells restored phosphorylation of the mTORC1 targets, ribosomal protein S6 and eIF4E-binding protein (4E-BP) in cells cultured in low glucose and also restored 4E-BP phosphorylation in cells treated with the AMPK stimulator, AICAR, despite AMPK activation in these

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conditions (Cai et al., 2008). It is possible that HBx-mediated activation of signaling pathways that activate mTORC1 dominates over the AMPK-mediated inhibition of mTORC1, resulting in simultaneous activation of both AMPK and mTORC1. The mechanism(s) that underlie HBx activation of AMPK and mTORC1 in primary hepatocytes is under investigation. To our knowledge, our study is the first in which HBx has been shown in the same study to activate two signaling pathways that have opposing effects on viral replication, one stimulating HBV replication and the other inhibiting it. Interestingly, simultaneous activation of mTORC1 and AMPK is not a unique feature of HBV; a number of viruses have evolved mechanisms that permit the activation of both mTORC1 and AMPK. For example, the human cytomegalovirus (HCMV) can circumvent AMPK-mediated inhibition of mTORC1: HCMV activates AMPK but also produces the pUL38 protein, which binds and inhibits TSC1/2 function, thus blocking the negative regulation of mTORC1 by AMPK (Moorman et al., 2008) (and reviewed in Brunton et al. (2013)). It is possible that HBV has also devised a mechanism to circumvent the inhibition of mTORC1, which will be the focus of future studies. The observation that HBx can activate a cellular signaling pathway that inhibits HBV replication may seem counterintuitive considering its well-established role in stimulating HBV replication. However, we recently reported that HBx activates AKT in hepatocytes and that this reduces HBV replication (Rawat and Bouchard, 2015) but also inhibits hepatocyte apoptosis, an established consequence of AKT activation (Rawat and Bouchard, 2015) (and reviewed in Rawat et al. (2012)). This study suggested that HBx can function as a rheostat that balances HBV replication and cell survival, which may be necessary for a persistent, noncytopathic HBV infection (Rawat and Bouchard, 2015). It is possible that HBx-mediated activation of both mTORC1 and AMPK also constitutes a balancing mechanism that helps facilitate persistent viral replication. For example, by reducing the level of HBV replication, HBx activation of mTORC1 may allow immune evasion of HBV-infected cells and help in the establishment of a chronic HBV infection. HBV replicates well in cultured primary rat hepatocytes, suggesting that the stimulatory effects of HBx on HBV replication, such as activation of AMPK, are dominant over the inhibitory effects. Although we did not specifically address the mechanisms underlying AMPK promotion of HBV replication, potential mechanisms could involve AMPK-mediated activation of PGC1α; PGC1α has been show to stimulate HBV replication (Shlomai et al., 2006) (and reviewed in (Fernandez-Marcos and Auwerx, 2011; Scarpulla, 2011; Canto and Auwerx, 2009; Lira et al., 2010)). Moreover, early stages of autophagy are activated in HBx-expressing cultured primary rat hepatocytes (S. Bagga and M.J. Bouchard, unpublished observations). Our observations are consistent with previous studies that showed that HBx can induce an incomplete autophagic response in transformed cells, HBV transgenic mice, and human liver tissues (Sir et al., 2010; Liu et al., 2014). AMPK can activate autophagy independent of mTORC1 inhibition (reviewed in (Roach, 2011)). Therefore, it is possible that AMPK mediates activation of autophagy in HBx-expressing cells, which may be essential for HBV replication. Studies conducted in HBx-transgenic mice suggest that HBx contributes to the development of HBV-associated HCC. However, whether HBx is directly oncogenic or functions more as a cofactor in cancer progression is unclear (Kim et al., 1991; Koike et al., 1994; Slagle et al., 1996; Terradillos et al., 1997). The notion that HBx is strongly oncogenic does not reflect the biology of HCC development in chronically HBV-infected individuals, which involves decades of chronic HBV infection. Instead, it is likely that HBx has a cofactor role in the development of HBV-associated HCC, and that subtle HBx activities, such as modulation of cellular signal transduction pathways and alteration of hepatocyte metabolism,

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regulate viral replication while sensitizing cells to other oncogenic signals (reviewed in Seeger et al. (2007), Bouchard and NavasMartin (2011), Casciano et al. (2012)). The subtleness of HBx effects and the cell-specific consequences of certain HBx activities have sometimes made it challenging to define the effects of HBx in an authentic HBV infection and directly link these activities to hepatocyte transformation. However, the subtleness of many HBx effects, such as the 2–3 fold activation of AMPK and mTORC1 by HBx observed in our analyses in primary hepatocytes, are, in fact, consistent with both a) the biology of an HBV infection, which is persistent and non-cytopathic, and b) the decades long time span required for progression from a chronic HBV infection to the development of HCC in a chronically HBV-infected individual (reviewed in (Bouchard and Navas-Martin, 2011; Neuveut et al., 2010; Rawat et al., 2012; Nguyen et al., 2008)). HBx activation of mTORC1 could influence oncogenic pathways that affect the development of HBV-associated HCC. AMPK, usually presumed to be a tumor suppressor, is also thought to be oncogenic in certain cellular contexts (reviewed in Liang and Mills (2013)). Cumulatively, our results demonstrate a novel HBx function, regulation of the AMPK-mTORC1 axis by HBx expression, and the effect of this metabolic axis on HBV replication. Our studies also demonstrate that HBx functions as a rheostat that controls the level of HBV replication by simultaneously activating signals that both elevate and decrease the levels of HBV replication. Therapeutic options for chronic HBV infections are limited, and mTORC1- and AMPK-regulated factors may provide novel targets for inhibiting HBx activities, HBV replication, and HBV-associated diseases. Our study also cautions against the use of mTORC1 inhibitors in individuals with a chronic HBV infection since the administration of mTORC1 inhibitors such as rapamycin might elevate in vivo HBV replication and associated liver disease. Overall, our study identifies novel functions of HBx that regulate HBV replication in normal hepatocytes and could influence the development of HBV-associated HCC.

Funding This project was supported by NIH, United States, grant AI064844 to M.J.B.

Competing interests The authors declare no conflict of interests.

Author contributions Conceived and designed the experiments: SB, MJB. Performed the experiments and data collection: SB. Contributed to data collection: SR, MA. Analyzed and interpreted the data: SB, MJB. Statistical analysis: SB. Wrote the paper: SB, MJB. This manuscript has been read and approved in its final form by all the authors.

Acknowledgments The authors would like to thank members of the Bouchard lab as well as Dr. Jane Clifford, Dr. Mauricio Reginato, Dr. Brad Jameson, and Dr. Robert Schneider for advice and many helpful discussions.

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