Interactome Map Reveals Phospholipid Scramblase 1 as a Novel

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An Interactome Map Reveals Phospholipid Scramblase 1 as a Novel Regulator of Hepatitis B Virus X Protein Yanzhi Yuan, Chunyan Tian, Qiaoling Gong, Limin Shang, Yuehui Zhang, Chaozhi Jin, Fuchu He, and Jian Wang J. Proteome Res., Just Accepted Manuscript • Publication Date (Web): 03 Nov 2014 Downloaded from http://pubs.acs.org on November 4, 2014

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Journal of Proteome Research

An Interactome Map Reveals Phospholipid Scramblase 1 as a Novel Regulator of Hepatitis B Virus X Protein Yanzhi Yuan, Chunyan Tian, Qiaoling Gong, Limin Shang, Yuehui Zhang, Chaozhi Jin, Fuchu He*, Jian Wang* State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, Beijing 102206, China; National Engineering Research Center for Protein Drugs, Beijing 102206, China ABSTRACT HBV X protein plays crucial roles during viral infection and hepatocellular carcinoma (HCC) development through interaction with various host factors. Here, we mapped the interactome of HBx using a yeast two-hybrid screen. Nine human proteins were identified as novel interacting partners of HBx, one of which is phospholipid scramblase 1 (PLSCR1). PLSCR1 is an interferon-inducible protein that mediates antiviral activity against DNA and RNA viruses. However, the molecular mechanisms of PLSCR1 activity against HBV remain unclear. Here, we reported that PLSCR1 promotes HBx degradation by a proteasome- and ubiquitin-dependent mechanism. Furthermore, we found that PLSCR1 inhibits HBx-mediated cell proliferation. After HBV infection, the protein level of PLSCR1 in plasma is elevated, and chronic hepatitis B patients with low plasma levels of PLSCR1 have a high risk of developing HCC. These results suggest that the nuclear trafficking of PLSCR1 mediates the antiviral activity and anti-carcinogenesis against HBV by regulating HBx stability. KEYWORDS: hepatitis B virus, HBx, PLSCR1, ubiquitination, degradation, yeast two-hybrid

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INTRODUCTION Hepatitis B virus (HBV) infection is a significant global health problem. HBV infection causes acute or chronic liver inflammation1 and is considered to be a major etiological factor in the development of HCC, one of the leading causes of death induced by cancer in the modern world. 2-3

The gene encoding the HBV X (HBx) protein has the smallest open reading frame in the HBV

genome and is located at nucleotide position 1374 1838. The overall length of the HBx encoding gene is 435 to 462 bp, which is translated into a protein containing 154 amino acids.4 HBx is a multifunctional protein that modulates transcription, signal transduction, cell cycle progression, apoptosis, protein degradation pathways, and genetic stability through interaction with host factors.5-7 A number of reports have indicated that HBx is one of the most common viral ORFs that is integrated into the host genome, and its sequence variants play a crucial role in HCC.8 We have previously investigated HBV−host interactions using tandem affinity purification （TAP）under mild conditions to gain insight into the HBx complex in HepG2 cells. Using mass spectrometry, we identified 49 host factors that potentially associated with HBx. 9 However, the mechanisms by which direct binary interactions between HBx and host factors regulate HBx expression or contribute to HBV pathogenesis remains uninvestigated. In this study, we used HBx as bait to screen a human liver cDNA library using yeast two-hybrid (Y2H) screening. Nine host factors were identified as novel interacting partners of HBx, including PLSCR1. PLSCR1 is an α/β interferon-inducible protein that has been implicated in growth factor signaling pathway regulation, cell proliferation, and tumor growth and development.10-13 PLSCR1 mediates antiviral activity against DNA and RNA viruses, including HBV, vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV).14-15 However, the


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precise mechanisms involved in the antiviral effect of PLSCR1 against HBV have not been defined. We show that PLSCR1 interacts with the HBx protein in the nucleus and facilitates its degradation through a proteasome and ubiquitin-dependent mechanism. These results suggest that PLSCR1 mediates antiviral activity against HBV through attenuation of HBx levels.



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EXPERIMENTAL SECTION Cell Culture HEK293, HepG2 and Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum. HBx-TAP-HepG2 and TAP-HepG2 cell lines were established by stably transfecting cells with either TAP-HBx or an empty vector as a control, respectively.9 Huh7 cells stably transfected with either a PLSCR1 shRNA cassette targeting PLSCR1 or an empty vector as a control were established as described previously.16 The cells were transfected with Thermo Scientific TurboFect Transfection Reagent according to the manufacturer’s instructions (Thermo Fisher Scientific, San Jose, CA). Plasmids and Reagents HBx was amplified by PCR using full-length HBV as a template and inserted into the pDBLeu vector. The HBx gene was then cloned into pFLAG-CMV-2 (Sigma, St. Louis, MO) and pEGFP-C3 (Clontech, Mountain View, CA), respectively, to produce the Flag-HBx and pEGFP-HBx expression vectors. pcDNA3/poly-HA-tagged ubiquitin were kindly provided by Dr. Yue Xiong (University of North Carolina, Chapel Hill, NC). The cDNA encoding human PLSCR1 was PCR amplified from the human liver marathon cDNA library and subcloned into the pCMV-Myc and pDsRed1-C1 vectors (Clontech, Mountain View, CA). Various constructs of PLSCR1 were generated according to standard molecular techniques. All deletion mutants were created by PCR to obtain DNA fragments and subjected to sequencing verification. The details of the primer sequences used for deletion mutations are available upon request. The proteasome inhibitor MG132, anti-Flag antibody, and anti-Flag-HRP antibody were from Sigma (St. Louis, MO). PLSCR1 antibody was from Proteintech (Proteintech Group, Chicago, IL); anti-HA-HRP


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antibody was from Cali-Bio (Coachella, CA); anti-Myc and anti-Myc-HRP antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); IFN-α2a was from Peprotech (Rocky Hill, NJ). The Integrated Network of HBx The reported interactions of HBx were collected from the VirHostNet (Virus-Host Network) database

(http://pbildb1.univ-lyon1.fr/virhostnet/),

the

VirusMINT

database

(http://mint.bio.uniroma2.it/virusmint) and related literature (Supporting Information, Table S1). The HBx network was visualized using the Cytoscape software (3.1.1) plugin GeneMANIA,17 which includes physical interactions, genetic interactions and pathway information. Yeast Two-hybrid Screening The yeast two-hybrid screen was performed with full length HBx cloned in-frame with the GAL4 DNA binding domain in vector pDBLeu. MaV203 yeast cells were obtained from the ProQuest two-hybrid system (Invitrogen, Carlsbad, CA). The yeast cells were transformed with pDBLeu-HBx and the human liver cDNA library. A total of 1×106 independent transformants were analyzed, and clones were selected for positive interactions based on screening for expression of the reporter genes His3, LacZ and URA3. To eliminate nonspecific interactions, only those interactions that activated all three reporter genes were considered. The positives were subsequently retested in fresh yeast cells, and the identities of prey were determined with interaction sequence tags (ISTs) obtained by DNA sequencing. The reading frame was verified. Co-immunoprecipitation (Co-IP), Immunoblotting and GST Pull-down Assay Co-IP, immunoblotting and GST pull-down assays were performed as previously described. 16 In vivo ubiquitination assay Huh7 cells (60% confluence, 100-mm diameter plate) were transfected with plasmids expressing



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HA-ubiquitin (2 µg), Flag-HBx (4 µg), Myc-PLSCR1 (1 µg) or a Myc-PLSCR1 (5C→5A, K→A) mutant (1 µg) in various combinations. After 24 h, the cells were treated with proteasome inhibitor MG132 (20 µM) for 6 h and then lysed in lysis buffer and incubated with Flag antibody at 4°C for 3 h. The lysates were then incubated with protein A/G plus agarose (Santa Cruz Biotechnology) overnight at 4°C. The agarose samples were washed three times with lysis buffer prior to analysis by Western blotting with the indicated antibodies. Cell Proliferation Assay For assessing the effect of HBx and PLSCR1 on growth rate, cells were cultured into 96-well plates in triplicate for 24 h. Then, HBx-TAP-HepG2 and TAP-HepG2 cells were transiently transfected with or without Myc-PLSCR1. After 96 h incubation, cell proliferation was analyzed using a Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan). The experiment was performed on three independent replicates in triplicate. Study Subjects The 79 healthy controls (HC), 15 chronic HBV carriers (CHB, without fibrosis) and 125 HBV-HCC cases were recruited. The fresh blood samples stored at -80

until use. CHB carriers

were defined as individuals who were positive for both HBV surface antigen (HBsAg) and immunoglobulin G antibody to HBV core antigen for at least 6 months. Diagnosis with HCC was based on (i) positive findings on cytological or pathological examination and/or (ii) positive images on angiogram, ultra-sonography, computed tomography and/or magnetic resonance imaging, combined with an α-fetoprotein concentration of ≥400 ng/ml. We confirmed that none of the individuals with HCC had other cancers through an initial screening. All controls had, by self-report, no history of HCC or other cancers.


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ELISA Assay The ELISA assay was performed in accordance with the manufacturer’s instructions (Rapidbio, West Hills, CA). Statistical Analysis Statistical evaluation was conducted using Student’s t-test.



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RESULTS AND DISCUSSION Identifying Host Proteins that Interact with HBx To identify host partners that directly bind to HBx, we performed a stringent yeast two-hybrid screening of a human liver cDNA library. Only those interactions that activated all three reporter genes were considered. Fifty-five positive clones were obtained. Non-specific interactions were eliminated by yeast re-transformation assay. The identities of HBx binding partners were determined by DNA sequencing. Thirty-eight ISTs were obtained, representing 9 unique interactions (Table 1). HBx is reported to affect various host cell functions, such as apoptosis, cell cycle progression, DNA damage repair, signal transduction and transcription. Interestingly, most of the novel HBx-interacting proteins identified in this study were reported to participate in the regulation of several signaling pathways (Figure 1A). Most of these pathways were known drivers of cancer cell signaling, such as PI3K, STAT, MAPK and NOTCH.18 Moreover, eight partners of HBx were reported to play crucial roles during oncogenesis (Table 1).19-23 Six interacting partners of HBx were related to viral infections. 14-16, 24-28 Network Analysis of HBx−host Interactions To further analyze the protein-protein interaction network of HBx, we constructed a large network by integrating the known interactions of HBx and the Y2H results from this study (Figure 1B and Supporting Information, Table S1). Most of the HBx partners formed a large network. We found that cancer driver signaling pathways were enriched in the network, including but not limited to the p53 pathway, DNA damage pathway and HIV infection pathway. One protein identified in the HBx network, PLSCR1, controls the activity of EGF signaling


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and is induced by interferon (IFN), suggesting that it might be an antiviral protein. PLSCR1 is known to inhibit the replication of hepatitis B virus.16 However, the molecular mechanism by which the inhibition occurs and its relationship with HCC is unknown. Validation of Interactions by β -galactosidase and Co-IP Assays To further confirm the interactions by Y2H screening, we performed quantitative assays for β-galactosidase (β-gal) activity in liquid cultures. All of the newly identified interactions were validated. We reasoned that interactions detected in two different binding assays were unlikely to be false-positives from the Y2H screening. Among the binding partners, the interaction between PLSCR1 and HBx had the highest ß-galactosidase activity (Figure 2A). Four positive interactions were further tested by co-immunoprecipitation (Co-IP) (Figure 2B). The bait and prey were transiently transfected into HEK293 cells as Flag-bait and Myc-prey fusions, respectively. All of these HBx interactions (PLSCR1, Notch 3, GAA and NKD2) were confirmed in mammalian cells (Figure 2B). PLSCR1 Promotes Ubiquitination and Degradation of HBx and Impairs HBx-mediated Cell Proliferation Among the obtained potential HBx-interacting clones, the most frequently selected sequences encoded PLSCR1. PLSCR1 was primarily identified as a multi-palmitoylated, calcium-binding endofacial membrane protein mediating nonselective and bidirectional transport of phospholipids between membrane leaflets.32 Structural and functional analyses revealed that PLSCR1 had several functionally important domains, including multiple PXXP and PPXY domains, a DNA binding domain, a cysteine palmitoylation motif, a nonclassical nuclear localization signal (NLS), a Ca2+ binding EF-hand-like domain, and a single transmembrane domain, indicating its potential as a



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multifunction protein.33-35 Moreover, PLSCR1 has a known role as a scramblase, which catalyzes a rapid transbilayer movement of phospholipids between membrane leaflets. Increasing evidence suggests that PLSCR1 plays a role in cell signaling, maturation and apoptosis and the growth of cancer cells.10-13 Recently, PLSCR1 was reported to be a substrate of cellular protein kinases and to potentiate the antiviral activity of interferon.14-16 Furthermore, results of several reported studies revealed that PLSCR1 overexpression was associated with the differentiation of human myeloid leukemia cells into granulocytes, and the suppression of ovarian carcinoma cell growth.11-12 We first analyzed the HBx expression levels in the presence of PLSCR1. HBx was significantly reduced when co-expressed with PLSCR1 in a dose-dependent manner (Figure 3A, left panel). However, proteosome inhibitor MG132 treatment counteracted PLSCR1-mediated downregulation of the HBx protein (Figure 3A, right panel), implying that the HBx decrease is likely due to post-transcriptional regulation. Because ubiquitination is the initial step in proteasome mediated degradation, we next examined the level of HBx ubiquitination with or without co-expression of PLSCR1. Co-transfection of the PLSCR1 expression vectors resulted in increasing amounts of ubiquitinated-HBx levels after MG132 treatment (Figure 3B), suggesting that PLSCR1-mediated HBx degradation involves induction of ubiquitination. In general, the stability of the HBx protein is regulated in vivo and maintained at a very low intracellular level. HBx can be actively ubiquitinated and undergo proteolysis through the ubiquitin-proteasome pathway.36 It has been discovered that the 26S proteasome complex, heat shock protein 40 (Hsp40), HBV core proteins, tumor suppressor p53, transcriptional factor Id-1, MDM2, the X-linked tumor suppressor TSPX and Siah-1 contribute to destabilization of the HBx


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protein in a proteasome-dependent manner.36-40 We found that PLSCR1 mediates the ubiquitination and degradation of HBx. However, PLSCR1 belongs to neither the HECT nor Ring finger type E3 ligase families, and additional studies are required to further define the mechanism of PLSCR1-mediated HBx regulation. HBx acts as a transcriptional transactivator or interacts with transcriptional transactivator(s) to transactivate target genes, and transactivates some genes associated with cell proliferation, such as IL-8, TNFα, transforming growth factor (TGF)-b1, and early growth response factor (EGRF).41 We next examined whether PLSCR1-dependent degradation of HBx interfered with HBx-mediated cell proliferation in HepG2 cells stably transfected with TAP-HBx. PLSCR1 overexpression reduced HBx-TAP-HepG2 cell proliferation, whereas PLSCR1 had no significant effect on the proliferation of the control TAP-HepG2 cell lines (Figure 3C). Dong et al. reported that PLSCR1 enhanced the IFN response and suppressed the replication of vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) in vitro, partly through increased expression of a select subset of potential antiviral genes.15 The antiviral action of PLSCR1 against HBV occurs, at least partly, by activation of the Jak/Stat pathway. Our results indicated PLSCR1 might mediate the antiviral and anti-carcinogenesis activity against HBV through regulating HBx stability and inhibiting HBx-mediated cell proliferation. PLSCR1 physically interacts with HBx To understand the mechanism underlying PLSCR1 degradation of HBx, we further examined the physical interaction between PLSCR1 and HBx. Reciprocal co-IP and GST pull-down experiments revealed an association between PLSCR1 and HBx (Figure 4A and 4B). Next, we observed the sub-cellular localization of PLSCR1 and HBx. PLSCR1 is distributed between the



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plasma membrane and the nucleus, and confocal analysis revealed that overexpressed PLSCR1 and HBx colocalized in the nucleus and cytoplasm, suggesting a physical interaction occurs between HBx and PLSCR1 (Figure 4C). PLSCR1 contains a number of functional domains, including a WW- binding motif, a cysteine-rich palmitoylation site, an atypical nuclear localization signal (NLS), a Ca2+ binding domain, and a predicted type 2β transmembrane helix (Fig. 4D).33-35 To determine which, if any, of these motifs was necessary for the interaction with HBx, a series of PLSCR1 mutants were used in co-IP assays. To create the 5C→5A PLSCR1 mutant, the palmitoylation signal CCCPCC was altered to AAAPAA, thereby abrogating PLSCR1 trafficking to the plasma membrane and resulting in relocalization of virtually all of the expressed protein to the nucleus. The PLSCR1 5C→5A/K3A mutant contains the palmitoylation site mutation in addition to a K258A mutation within the nuclear localization signal, which abrogates nuclear trafficking of the protein. Deletion analysis indicated that the N-terminus (1-163 aa) and the palmitoylation sites (158-240 aa) of PLSCR1 interacted with HBx (Figure 4D). The C-terminus mutation (240-381 aa) had no significant effect on the interaction between PLSCR1 and HBx. Although the ∆N-terminus mutant (158-381 aa) also interacted with HBx, the affinity decreased dramatically. These results indicate that multiple domains play important roles in the interaction between PLSCR1 and HBx. Although PLSCR1 and HBx colocalize in the cytoplasm and nucleus, co-IP assays confirmed that the PLSCR1 5C→5A/K3A mutation nearly abolished its binding to HBx, indicating that HBx mainly interacted with the nuclear PLSCR1. PLSCR1 regulates various cellular processes through interactions with other proteins. PLSCR1 can itself act as a transcription factor. 42 It has been reported that PLSCR1 interacts with


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CD4 at the cell surface of T lymphocytes, and inhibition of this interaction leads to the inhibition of HIV infection.43 PLSCR1 interacts with ANG in the cell nucleus and regulates rRNA transcription.44 Interaction of PLSCR1 with HIV-1 Tat results in the repression of Tat-dependent transcription.26 Because our results suggest that the interaction between PLSCR1 and HBx occurs in the nucleus, we next investigated whether the regulation of the HBx protein by PLSCR1 required direct interaction between PLSCR1 and HBx. To test this hypothesis, we examined the effects of the regulation of wild type PLSCR1, as well as the palmitoylation mutants with or without the NLS mutants, on HBx protein expression. The PLSCR1 5C→5A/K3A mutant had no significant effect on HBx protein levels (Figure 4E). Consistent with this, we observed that this mutation did not increase the ubiquitination of HBx (Figure 4F), suggesting that PLSCR1 regulates HBx in a manner that depends on their physical interaction. Nuclear trafficking of PLSCR1 has been observed only in circumstances where its cellular expression was induced by IFN, or other cytokines or growth factors that transcriptionally activate the gene. Under conditions of transcriptional induction, PLSCR1 has been shown to traffic to both the plasma membrane and the nucleus in a manner that appears to be regulated through palmitoylation.10, 34 Once in the nucleus, PLSCR1, an acidic polypeptide, is found tightly bound to the DNA33, 34 and transcription regulators, such as human immunodeficiency virus type-1 (HIV-1) Tat and angiogenin44. Here, our work suggested that in addition to acting as a transcriptional regulator by selectively altering gene or rRNA transcription, PLSCR1 also binds to HBx and promotes its degradation in the nucleus. IFN-α Increases the Protein Level of PLSCR1 and Promotes the Ubiquitination and Degradation of HBx



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IFN-α has been used for the treatment of HBV infection for two decades.45 IFN-α belongs to the IFN-α/β system, which mediates antiviral, antiproliferative, immune, and other cellular effects. Because PLSCR1 is an interferon (IFN)-inducible gene, and IFN-α leads to a dramatic increase in PLSCR1 protein levels, we examined whether IFN-α affected the stability and ubiquitination of HBx. As expected, IFN-α treatment induced a gradual decrease in HBx protein levels (Figure 5A). During this process, PLSCR1 levels dramatically increased (Figure 5A). We next examined the role of PLSCR1 in the regulation of the effects of IFN-α on HBx using Huh7 cells stably transfected with either a PLSCR1 shRNA cassette targeting PLSCR1 or an empty vector as a control. After treatment with IFN-α for 24 h, HBx and PLSCR1 levels dramatically decreased and increased, respectively, in control Huh7 cells (Figure 5B). IFN-α increased PLSCR1 expression and had no effect on HBx expression in Huh7 cells where PLSCR1 expression was stably knocked down. These results suggest that IFN-α decreased HBx expression through modulating the stability of PLSCR1 (Figure 5B). Following pretreatment with MG132, the decrease of HBx induced by IFN-α was partially abolished, indicating that IFN-α promoted HBx degradation, in part, through proteasome-mediated degradation (Figure 5C). Consistent with this, IFN-α enhanced HBx ubiquitination when PLSCR1 was upregulated (Figure 5D). It has been reported that IFN-α suppresses viral gene expression, preventing the formation of viral RNA-containing core particles, and reducing the accumulation of viral replicative intermediates. 46 IFN-α inhibits HBV replication through a variety of mechanisms. It engages the IFN-α receptor complex to activate the Jak/Stat pathway, and induces the transcription of IFN-stimulated genes (ISGs) that activate antiviral defense pathways in infected and uninfected cells.47 However, the precise antiviral mechanisms of IFN-α and the biological functions of many


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ISGs have not been fully elucidated. Using different cell lines, it has been shown that the antiviral state induced by IFN-α is complex and can be established by induction of a wide pattern of genes and activation of a variety of cellular functional proteins. Multiple proteins interact with each other to build up the IFN-α antiviral networks and determine the level of HBV replication and transcription. Taken together, IFN-α induces the transcriptional induction and nuclear trafficking of PLSCR1, which in turn promotes the ubiquitination and degradation of HBx. The Expression of PLSCR1 is Increased in the Plasma of Chronic HBV Carriers (CHB) and HBV-HCC Patients The expression levels of IFN-α were higher in the acute and chronic HBV-infected patients compared to healthy controls (HC). PLSCR1 is one of IFN inducible targets, suggesting that the expression level of PLSCR1 might be elevated following HBV infection. To prove this hypothesis, the level of PLSCR1 protein in 94 plasma samples from 15 CHB patients and 79 HC were tested using ELISA assays. The background characteristics of the CHB patients and HC are listed in in supporting information in Tables S2 and S3. The results show that individual plasma PLSCR1 levels vary across the different groups (Figure 6A), with PLSCR1 levels substantially increased in some of the plasma of CHB patients compared with HC. To further explore the correlation between PLSCR1 protein levels in plasma and the development of HCC in chronic HBV carriers, we tested PLSCR1 levels in plasma samples from 125 HBV-HCC patients (Supporting Information, Table S4). Even though the expression level of PLSCR1 in many HCC patients was not changed. Compared with CHB, plasma protein levels of PLSCR1 in HBV-HCC patients decreased significantly (Figure 6B). This result implies that CHB



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patients with low plasma levels of PLSCR1 might have a high risk of developing HCC. PLSCR1 exhibits differential expression under various inflammatory conditions and neoplastic diseases. PLSCR1 expression correlates with acute phase response, acute myelogenous leukemia, systemic lupus erythematosus, colorectal cancer, and ovarian carcinoma.10-13,

48

PLSCR1 plays an important role in ovarian cancer development and chemoresistance12. PLSCR1 is both a novel diagnostic biomarker and an important prognostic factor for colon cancer, and the plasma level of PLSCR1 is substantially increased in early stage colon cancer.49 Our work suggested that following HBV infection, PLSCR1 levels increased in plasma, promoting the ubiquitination and degradation of HBx and participating in the host defense against HBV infection. In the CHB patients, downregulation of PLSCR1 implies that the patients have a high risk of developing HCC.

CONCLUSIONS In this study, we used HBx as bait to screen a human liver cDNA library by a yeast two-hybrid screening to characterize the binary interaction between HBx and host factors. Nine host factors were identified as novel interacting partners of HBx, one of which is PLSCR1. PLSCR1 interacts with the HBx protein in the nucleus, facilitates its degradation in cells through a proteasome and ubiquitin-dependent mechanism, and inhibits HBx-mediated cell proliferation. IFN-α promotes ubiquitination and degradation of HBx by inducing the transcriptional induction and nuclear trafficking of PLSCR1. After HBV infection, PLSCR1 levels in the plasma increased. The protein level of PLSCR1 in plasma samples of HBV-HCC patients is decreased, indicating the important role of PLSCR1 in the host defense to HBV infection and development of HCC in HBV patients.


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ASSOCIATED CONTENT Supporting Information Table S1 highlights available information on 50 known HBx interacting proteins from the literature.

Tables S2-S4 show the concentration of PLSCR1 in plasma of HC, CHB and

HBV-HCC subjects. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *JW: E-mail, [email protected]; Phone: +86-10-80705118; Fax: +86-10-80705155.

FH:

E-mail, [email protected]; Phone: +86-10-68171208; Fax: +86-10-80705155 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by grants from the National High Technology Program (2012AA020201), the Special Funds for Major State Basic Research of China (2011CB910600, 2014CBA02001), the Chinese Program of International S&T Cooperation (2014DFB30020), and the Beijing NOVA Program (2011014). REFERENCES (1) Urban S, Schulze A, Dandri M, Petersen J. The replication cycle of hepatitis B virus. J Hepatol. 2010, 52(2), 282-4. (2) Neuveut C, Wei Y, Buendia MA. Mechanisms of HBV-related hepatocarcinogenesis. J Hepatol. 2010, 52(4), 594-604. (3) Bartosch B. Hepatitis B and C viruses and hepatocellular carcinoma. Viruses. 2010, 2(8),



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FIGURE LEGENDS Figure 1. The protein-protein interaction network of HBx. (A) The HBx interactome characterized using Y2H screening. EGF, epidermal growth factor; IFN, interferon; TCR, T cell receptor; Wnt, wingless-type; TNFα, tumor necrosis factor α; PI3K, phosphatidylinositol 3'-kinase; UPR, unfolded protein response. (B) The integrated network of HBx. Green nodes, direct partners of HBx; gray nodes, indirect partners of HBx; red nodes, enriched pathways in the HBx network. Figure 2. Verification of Y2H interactions. (A) Quantitative Assays for β-galactosidase (β-gal) activity in liquid cultures was performed using o-nitrophenyl-β-D-galactopyranoside (ONPG) according to the manufacturer’s instructions (ProQuest two-hybrid system, Invitrogen, CA). Data are expressed as the mean ± SD (n = 2). Student’s t test was used. (B) Co-IP assay. Flag- or Myc-tagged plasmids were transfected into HEK293 cells. Immunoprecipitations were performed using anti-Flag antibody and protein A/G plus agarose. The lysates and immunoprecipitates were detected using the indicated antibodies. Figure 3. PLSCR1 promotes the proteasomal degradation of HBx. (A) PLSCR1-mediated HBx reduction is proteasome-dependent. HEK293 cells were co-transfected with a constant amount of Flag-HBx plasmid and increasing amounts of Myc-PLSCR1. Twenty-four hours after transfection, Western blotting was performed with antibodies as indicated. The transfected cells were treated with or without 20 µM MG132 for 6 h. (B) PLSCR1 facilitates poly-ubiquitination of HBx. Huh7 cells were transfected with plasmids encoding HA-ubiquitin, Flag-HBx and Myc-PLSCR1. The transfected cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-Flag antibody followed by


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Western blotting with indicated antibodies. (C) PLSCR1 impairs HBx-mediated cell proliferation. The Myc-PLSCR1 was transiently transfected into HBx-TAP-HepG2 and TAP-HepG2 cells. After 96 h, cells were collected for CCK8 cell proliferation assay. Data are expressed as the mean ± SD (n = 3). Figure 4. HBx interacts with PLSCR1. (A) Confirmation of the interaction between HBx and PLSCR1 by co-IP assay. HEK293 cells were cotransfected with Myc-PLSCR1 and Flag-HBx or Flag expression vectors. Cell lysates were immunoprecipitated with anti-Myc antibody followed by Western blotting with indicated antibodies. (B) Interaction between PLSCR1 and HBx is revealed by GST pull-down assays. Input and pull-down samples were both subjected to immunoblotting with anti-GST and anti-myc antibodies. Input represents 10% of that used for pull-down. (C) HBx is colocalized with PLSCR1. HepG2 cells were transfected with pEGFP-HBx and pDsRed1-PLSCR1. Scale bar, 10 µm. (D) Determination of mutual interaction regions in PLSCR1 and HBx. A diagram for the deletion mutants of PLSCR1 is shown (top panel). Mapping of the PLSCR1 domain for HBx binding. Extracts from HEK293 cells transfected with the indicated plasmid DNA encoding deletion mutants of PLSCR1 and Flag-HBx were immunoprecipitated with Myc antibody, followed by immunoblotting with Flag or Myc antibodies (bottom panel). (E) PLSCR1 reduces HBx protein levels. HepG2 cells were co-transfected with a constant amount of Flag-HBx plasmid and with Myc-PLSCR1, a Myc-PLSCR1(5C→5A) mutant, or a Myc-PLSCR1(5C→5A, K-A) mutant. Twenty-four hours after transfection, Western blot was performed with antibodies as indicated. In the 5C→5A PLSCR1 mutant, the palmitoylation signal CCCPCC was altered to AAAPAA. The 5C→5A/K→A mutation contains the palmitoylation site mutation plus mutation K258A within



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the nuclear localization signal, which abrogates nuclear trafficking of the protein. (F) PLSCR1 facilitates poly-ubiquitination of HBx. Huh7 cells were transfected with plasmids as indicated. The transfected cells were treated with MG132 for 6 h. Cell lysates were immunoprecipitated with anti-Flag antibody followed by Western blotting with indicated antibodies. Figure 5. IFN-α enhances the expression of PLSCR1 and induces the degradation of HBx. (A) IFN-α enhances the expression of PLSCR1 and downregulates the expression of HBx. HepG2 cells was transfected and treated as indicated. PLSCR1 and HBx were analyzed by western blotting with the indicated antibody. (B) IFN-α inhibits HBx through induction of PLSCR1. Huh7 cells stably transfected with either an shRNA cassette targeting PLSCR1 or an empty vector were transfected and treated as indicated. PLSCR1 and HBx were analyzed by Western blotting with the indicated antibody. (C) MG132 partially abolishes IFN-α induced degradation of HBx. The transfected HepG2 cells were treated with IFN-α for 24 h and MG132 for 6 h as indicated. (D) IFN-α facilitates poly-ubiquitination of HBx. Huh7 cells were transfected with plasmids encoding HA-ubiquitin and Flag-HBx. The transfected cells were treated with IFN-α for 24 h and MG132 for 6 h as indicated. Cell lysates were immunoprecipitated with anti-Flag antibody followed by Western blotting with indicated antibodies. Figure 6. PLSCR1 changes in the plasma of CHB and HBV-HCC patients. (A) PLSCR1 was elevated in the plasma of CHB patients. (B) Compared with CHB, plasma protein levels of PLSCR1 in HBV-HCC patients decreased significantly. HC, healthy control; CHB, chronic HBV carriers. Student’s t test was used.


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Table 1. List of HBx interactions by Y2H screening. Gene ID

Official Symbol

Official Full Name

Y2H Hits

5359

PLSCR1

phospholipid scramblase 1

17

2896

GRN

granulin

4

10252

SPRY1

sprouty homolog 1

4

85409

NKD2

naked cuticle homolog 2

4

84447

SYVN1

synovial apoptosis inhibitor 1

4

Relationship to Cancer

Relationship to Virus

AML; colorectal cancer (CRC), ovarian and metastatic liver cancer

ovarian cancer, AML, breast cancer prostate cancer; Ewing sarcoma; rhabdomyosarcoma tumors astrocytic gliomas; soft tissue sarcomas

Pathways

Inhibits HBV replication; mediates initial attachment of HCV onto hepatoma cells; represses the Tat-dependent transactivation of the EGF, IFN HIV-1 long terminal repeat and reduces the nuclear translocation of Tat; mediates antiviral activity against VSV and EMCV. inhibits Tat transactivation TNFα EGF, IFN， HIV-1 Vif upregulates the expression of SPRY1 TCR Wnt

Refs

12-15, 27

19, 27 20, 28 21

UPR

4854

NOTCH3

Notch homolog 3

1

HCC; ovarian cancer; breast cancer; glomus tumors; neuroblastoma; ALL; non-small-cell lung cancer

10319

LAMC3

laminin, gamma 3

1

Small cell lung cancer

29950

SERTAD1

SERTA domain containing 1

1

breast cancer; carcinoma

2548

GAA

glucosidase, alpha; acid

1

squamous

HIV-1 Tat upregulates the expression of LAMC3 cell

22

PI3K-Akt

23, 29 24,25, 30

interacts with HPV11 and HPV16 E6 proteins involved in the initial stage of infection by HIV-1


NOTCH

NOTCH

31


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Graphical Abstract


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Page 29 of 34 A

1 2 3 4 5 6 7 8 9 10 11 12 HBx 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27


B Pathway PLSCR1 SPRY1

EGF, IFN EGF, IFN, TCR

NOTCH3

NOTCH

GAA

NOTCH

NKD2 GRN

Wnt TNFα

LAMC3

PI3K-Akt

SYVN1

UPR

SERTAD1

TNFalpha

TGF_beta_Receptor

p53 pathway

p53-Dependent G1 S DNA ... p53-Dependent G1 DNA Damage ... Stabilization of p53

Regulation of nuclear SMAD2 ... Glucocorticoid receptor regulatory network

TP53

EFEMP1

CFLAR

FGB

TM4SF4

AIP

NCOA6

LAMC3

YWHAQ

COPS5 PIN1 HSPA4

E4F1

DDIT3

DDB1

MDM2

CEBPB

SIAH1

PSMA7

Metabolism of mRNA AKT1

POLR2E

CREB1

HIF-1-alpha transcription factor network HIV Infection Disease

FETUB

RXRA

GRN UGT1A9FBXW4

SPRY1

Regulation of mRNA Stability ... Validated targets of C-MYC ...

RPL11

ACS Paragon Plus Environment ATP6AP1

Regulation of retinoblastoma protein

HPN

TBP PSMC1

Gene Expression

SMAD4

MKRN1 DDB2 PLSCR1 MDM4MAPK14CCNA2 COPS3 NKD2 MAPK8 MAPK9 UBE2D1 MIF GNB5 PRKCD EP300 SMAD3 RFWD2 MPDU1 PSMD14CDKN2A ATF3 SYVN1 PSMD7 JUN YY1 GAA FBXO11 VDAC3 ATM CDK2KAT2B PPP2CA NOTCH3 XPO1 SKP2 GTF2B HSPD1 SERTAD1 PSMC3 CREBBP CREM HIF1A

Apoptosis

COPS4

PSMB8

G1 S DNA Damage ...


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Figure 2 128x105mm (300 x 300 DPI)


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Figure 3 158x111mm (300 x 300 DPI)



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167x255mm (300 x 300 DPI)


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162x109mm (300 x 300 DPI)



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144x60mm (300 x 300 DPI)


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Interactome Map Reveals Phospholipid Scramblase 1 as a Novel

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