Quantitative Proteomic Analysis of Exosome Protein Content Changes

Sep 29, 2014 - Fudan University, 138 YiXueYuan Road, Shanghai 200032, People's ... Affiliated to Shanghai Jiaotong University School of Medicine, 786 ...
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Quantitative Proteomic Analysis of Exosome Protein Content Changes Induced by Hepatitis B Virus in Huh‑7 Cells Using SILAC Labeling and LC−MS/MS Xue Zhao,†,‡ Yanxin Wu,† Jinlin Duan,†,§ Yanchun Ma,∥ Zhongliang Shen,† Lili Wei,† Xiaoxian Cui,† Junqi Zhang,† Youhua Xie,*,† and Jing Liu*,† †

Key Laboratory of Medical Molecular Virology (MOE/MOH) and Institutes of Biomedical Sciences, Shanghai Medical College, Fudan University, 138 YiXueYuan Road, Shanghai 200032, People’s Republic of China ‡ Microbiology Laboratory, Shanghai Municipal Center for Disease Control and Prevention, No. 1380 West Zhongshan Road, Shanghai 200336, People’s Republic of China § Department of Pathology, Tongren Hospital Affiliated to Shanghai Jiaotong University School of Medicine, 786 YuYuan Road, Shanghai 200336, People’s Republic of China ∥ Lab Center, Putuo District Center Hospital, Shanghai University of Traditional Chinese Medicine, 164 Lanxi Road, Shanghai 200062, People’s Republic of China S Supporting Information *

ABSTRACT: Hepatitis B virus (HBV) infection could cause hepatitis, liver cirrhosis, and hepatocellular carcinoma. HBV-mediated pathogenesis is only partially understood, but X protein (HBx) reportedly possesses oncogenic potential. Exosomes are small membrane vesicles with diverse functions released by various cells including hepatocytes, and HBV harnesses cellular exosome biogenesis and export machineries for virion morphogenesis and secretion. Therefore, HBV infection might cause changes in exosome contents with functional implications for both virus and host. In this work, exosome protein content changes induced by HBV and HBx were quantitatively analyzed by SILAC/LC−MS/MS. Exosomes prepared from SILAC-labeled hepatoma cell line Huh-7 transfected with HBx, wildtype, or HBx-null HBV replicon plasmids were analyzed by LC−MS/MS. Systematic analyses of MS data and confirmatory immunoblotting showed that HBx overexpression and HBV, with or without HBx, replication in Huh-7 cells indeed caused marked and specific changes in exosome protein contents. Furthermore, specific changes in protein contents were also detected in exosomes purified from HBV-infected patients’ sera compared with control sera negative for HBV markers. These results illustrate a new aspect of interactions between HBV and the host and provide the foundation for future research into roles played by exosomes in HBV infection and pathogenesis. KEYWORDS: HBV, HBx, exosome, hepatocyte, SILAC, patient serum, VCP

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INTRODUCTION Hepatitis B virus (HBV) is the type member of the Hepadnaviridae family of enveloped pararetroviruses, with human beings as the only natural hosts and hepatocytes as target cells.1,2 Infection by HBV could result in acute or chronic hepatitis, and chronic hepatitis B has a high risk of progressing onto liver cirrhosis and hepatocellular carcinoma (HCC).2,3 Despite the availability and worldwide adoption of HBV vaccines, World Health Organization estimates that HBV chronically infects an estimated 240 million people worldwide, and about 600 000 HBV-infection related deaths occur each year.4 Mature HBV virions contain a ∼3.1kb partially doublestranded DNA genome, which is converted into doublestranded covalently closed circular DNA (cccDNA) upon infection (Supplementary Figure S1A in the Supporting © XXXX American Chemical Society

Information). The cccDNA serves as the only template for viral transcription in natural infection and harbors four overlapping open reading frames (ORFs) that encode the viral structural and nonstructural proteins. These include capsid core protein (C or HBcAg) and secreted e antigen (HBeAg), three forms of transmembrane envelope proteins (large, middle, and small S or HBsAg), polymerase (P or Pol), and X protein (HBx).1 HBcAg and HBeAg are encoded by the same ORF using different start codons and so are the three forms of HBsAg. In patients, HBV-infected hepatocytes produce the infective 42 nm diameter mature virions (Dane particles) composed of the viral genome enclosed by an icosahedral capsid formed by HBcAg and an outer envelope formed by Received: March 3, 2014

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comparative quantification. Exosome content changes revealed by quantitative proteomic analyses performed on the obtained data sets were then subjected to systematic statistical and bioinfomatic analyses, and selected proteins thus identified were further analyzed using exosomes of both transfected Huh7 cells and HBV-infected patients’ sera.

three types of HBsAg embedded in cellular membrane-derived lipids.1 In addition, a large excess of noninfective filamentous and spherical subviral particles (SVPs) are also produced, which are composed of empty envelopes containing predominantly middle and small HBsAg proteins, without inner capsids or viral genomes.1,5 Unenveloped capsids with or without nucleic acid content are also secreted by cultured hepatocytes transfected with HBV replicon plasmids.6 Infection of hepatocytes by HBV in vivo does not appear to cause any visible cytopathic effects and liver damage observed in active hepatitis B is generally believed to be the result of cellmediated anti-HBV immune attacks.1,7 Interplay between HBVinfected hepatocytes and members of innate and adaptive immune systems most likely determines the clinical outcome of HBV infection and has been under investigation for decades.7,8 Mechanisms underlying progression from chronic hepatitis B toward cirrhosis and HCC are as yet poorly understood, but a multitude of published work apparently linked HBx expression in hepatocytes to the development of HCC associated with HBV infection.9,10 Results from HBx function studies also suggested that HBx might, at least in vitro, modulate a myriad of host cellular functions, some of which implicated in development of hepatitis, cirrhosis, and HCC.11,12 Exosomes are small membrane vesicles with diameters of 30−100 nm that are released from various cell types through fusion of multivesicular bodies (MVBs) with plasma membrane.13,14 Exosomes secreted by cells could be taken up by neighboring cells, degraded, or enter connecting body fluids and travel within the body.15−17 Proteomic studies of a wide variety of exosomes over recent years have demonstrated that in addition to a common set of membrane and cytosolic proteins that are most likely involved in exosome biogenesis exosomes also contain origin-specific content.18 Exosomemediated transport of lipids, proteins, as well as coding and noncoding RNA apparently participate in crucial cellular and intercellular processes like antigen presentation,19 innate immunity responses,20 as well as tumor and pathogen surveillance.14 Consequently, exosomes have become important targets for pathogenesis studies, disease biomarker discovery,21,22 and vaccine development.23 Recent evidence has shown that exosomes are also released by hepatocytes,24 and the content might reflect pathological changes in the cells.25−27 Furthermore, a series of studies on HBV virion morphogenesis have demonstrated a significant involvement of MVB vesicles and related cellular proteins in this process.28−31 It is plausible that HBV antigen expression, genome replication, as well as virion assembly and secretion could affect, directly or indirectly, the exosome biogenesis and export process, resulting in changes in exosome content released by infected hepatocytes. Given the involvement of exosomes in multiple infection and immunity-related processes as previously mentioned, such changes in exosome contents caused by HBV could be implicated in pathogenesis of HBVrelated diseases and might also be diagnostically useful. In this study, we employed stable isotope labeling with amino acids in cell culture (SILAC)-assisted quantitative proteomics32−34 to study exosome content changes induced in hepatoma cell line Huh-7 by HBx overexpression and HBV replication. Exosomes were purified from conditioned media of differently SILAC-labeled Huh-7 cells transfected with HBx expression plasmid, wild-type HBV replicon plasmid, or HBxnull mutant HBV replicon plasmid, respectively, and subjected to mass spectrometry analysis for protein identification and

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MATERIALS AND METHODS

Chemicals, Plasmids, and Antibodies

The components of common and labeling cell culture media were purchased from Invitrogen or Sigma. L-Arginine:HCI (U-13C6, 99%), L-arginine:HCl (U-13C6, 99%; 15N4, 99%), Llysine-2HCl (4,4,5,5-D4, 96−98%), and L-lysine-2HCl (U13C6, 98%; 15N2, 98%) were purchased from Cambridge Isotope Laboratories (Andover, MA). Sequencing-grade trypsin was purchased from Promega (Madison, WI). For overexpression of HBx, HBx-encoding sequences (GenBank Accession AAD16255.1) were amplified by conventional PCR and cloned downstream of CMV promoter with an N-terminal HA tag in pcDNA3 (Invitrogen) to create pCMVHA-HBx. The empty vector pCMV was used as negative control. Plasmids carrying terminally redundant 1.1× copy wild type (pCMV-HBV1.135) and its derivative HBx-null mutant [pCMV-HBV1.1(X−)] HBV genomes under the control of CMV promoter were kindly provided by Professor Yongxiang Wang of Fudan University (Supplementary Figure 1B in the Supporting Information). Terminal redundancy of HBV genomic sequences contained in these plasmids is required for productive HBV replication upon transfection due to the circular nature of viral genome (Supplementary Figure 1A in the Supporting Information). In pCMV-HBV1.1(X−), amino acid 8 of HBx ORF was mutated to stop codon through sitedirected mutagenesis without causing amino acid changes in the overlapping polymerase ORF.36 EGFP-expressing plasmid pEGFP (Clonetech) was used in all transfections as transfection efficiency control. Antibodies against ALIX/PDCP6IP (A2215), AZGP1 (A5365), CANX (A0803), FASN (A6273), TSG101 (A2216), and VCP (A2795) were purchased from ABclonal; antibody against CTNNB1 (BD610154) was obtained from BD Biosciences; peroxidase-labeled β-actin antibody was purchased from Sigma (A3854); HSP90B1/Grp94 antibody was obtained from CST (2104); EGFP antibody was from Abmart (M20004); mouse monoclonal anti-HBx antibody was custom produced by Abmart and has been previously described;37 and rabbit anti-HBcAg antibody was purchased from DAKO (P0586). Cell Culture and SILAC Labeling

Huh-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin G/streptomycin sulfate, and 10 mM HEPES at 37 °C and 5% CO2. SILAC labeling was performed by culturing cells in labeling media containing medium (R6K4) or heavy (R10K8) amino acids for more than seven passages, as previously described.38 Labeling efficiency was confirmed by analyzing β-actin-derived peptides after trypsin digestion of samples from labeled cells using MALDITOF 4700 MALDI TOF/TOF proteomics analyzer (Applied Biosystems) as reported previously.38 B

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Transfection and Exosome Preparation

Fudan University on a hybrid quadrupole Orbitrap (QExactive) mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) coupled to a Nano Aquity UPLC system (Waters Corporation, Milford, MA). Tryptic peptides were redissolved in 10 μL of 0.1% FA solution and chromatographically separated on an analytical column (Acclaim PepMap C18, 75 μm × 15 cm). Each sample was loaded in solvent A (98% H2O/2% ACN/0.1% FA), followed by gradient elution of 2−40% solvent B (5% H2O/95% ACN/0.1% FA) over 120 min with a flow rate of 300 nL/min. The electrospray voltage of 2.2 kV versus the inlet of the mass spectrometer was used. The 15 most abundant precursor ions detected in the full MS survey scan (m/z range of 350−1200, R = 70 000) were isolated with a 3 m/z mass window for further high-energy collisional dissociation (HCD) MS/MS analysis with a resolution of 17 500. Spectra were acquired under automatic gain control (AGC) for survey spectra (AGC: 106) and MS/ MS spectra (AGC: 105). In all cases, one microscan was recorded using dynamic exclusion of 30 s. For MS/MS, precursor ions were activated using 27% normalized collision energy and an activation time of 30 ms.

Exosome-depleted serum was prepared by ultracentrifuging normal FBS at 100 000g for 16 h, followed by filtering the supernatant through 0.22 μm syringe filter (Millipore) and used for all transfection cultures. Prior to transfection, 106 unlabeled or labeled Huh-7 cells were seeded into 10 cm diameter culture dishes. After overnight growth, cells were transfected using Turbofect (Thermo Scientific) according to manufacturer’s instructions. 24 h later, cells were changed into fresh media. Fresh media were exchanged 48 h later, and culture media collected at 48 and 96 h post transfection were pooled together and stored at 4 °C. Two independent replications were performed for each transfection, and the collected media supernatants were processed in parallel in subsequent steps. Exosomes were purified using a differential centrifugation protocol as previously described.39,40 Briefly, culture media were sequentially centrifuged at 300g for 10 min and 2000g for 20 min to eliminate cell and cell debris contamination. The cleared supernatants were further centrifuged at 10 000g for 30 min at 4 °C, and exosomes were pelleted from the supernatants by ultracentrifugation at 100 000g using an SW32 rotor (Beckman) for 60 min at 4 °C. The exosome pellets were resuspended in PBS and ultracentrifuged again at 100 000g using an SW41 rotor (Beckman) for 60 min at 4 °C. Purified exosomes were resuspended in PBS and used immediately or stored at −80 °C.

Protein Identification and Quantification

Protein identification and quantification were performed using MaxQuant43 version 1.4.1.2 and UniProtKB Homo sapiens reference proteome database containing 88 479 canonical and isoform sequences (39 748 UniProtKB/Swiss-Prot and 48 731 UniProtKB/TrEMBL entries, retrieved Jan 2014) through MaxQuant’s built-in Andromeda search engine. HBV protein sequences were manually translated from corresponding nucleic acid sequences and used for HBV peptide identification in relevant samples. Default parameters in MaxQuant were used wherever applicable. Briefly, variable modifications included protein N-terminus acetylation, and oxidized methionine and fixed modification included carbamidomethylated cysteine. Full trypsin specificity was selected as digestion mode and max missed cleavages were set to 2. Peptides with lengths of a minimum of 7 amino acids were considered, with both the peptide and protein FDR set to 1%. Precursor mass tolerance was set to 20 ppm for the first search and 4.5 ppm for the main search. Product ions were searched with a mass tolerance of 20 ppm. Protein identification required a minimum of two peptides with at least one razor or unique peptide. Relative ratio quantification was performed using quantities of unique and razor peptides and required a minimum of two peptides. Protein groups marked as contaminant, reverse, or “identified by site only” in MaxQuant results were discarded. Only protein groups identified in both biological replicates using at least one of the two methods of sample preparation (see above) were collected for subsequent analyses. The mass spectrometry proteomics data, including raw MS data, MaxQuant search parameter files, and result files, have been deposited to the ProteomeXchange Consortium44 via the PRIDE partner repository with the data set identifier PXD001339.

Immunoblotting

Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes at 100 V for 80 min using Trans-blot Turbo system (Bio-Rad). The membranes were blocked with PBST containing 5% nonfat milk and incubated with the specified primary antibodies overnight, followed by incubation with secondary antibody conjugated with horseradish peroxidase. ECL substrate (Millipore) was added to the membranes, and images were captured using ChemiDoc XRS+ (Bio-Rad). Analysis of HBV Antigen and DNA

HBsAg and HBeAg were quantified using the commercial AxSYM HBsAg and HBeAg immunoassays (Abbott). Extraction of intracellular capsid-associated HBV DNA and analysis by Southern hybridization were performed as previously described.41 MS Sample Preparation

Purified exosomes were lysed in RIPA buffer containing 1% Triton X-100 and protease inhibitors (Beyotime) and total protein concentration was determined using Bradford assay (Bio-Rad). Equal amounts of heavy (R10K8) and light (unlabeled) or heavy, medium (R6K4), and light samples were then pooled together. Two methods were employed for protein digestion and peptide generation. In Method I, pooled samples were separated by 12% SDS-PAGE followed by silver staining. SDS-PAGE gel was then cut into slices, and in-gel trypsin digestion was performed as previously described.42 In Method II, electrophoresis separation step was omitted and pooled samples were boiled in 1× SDS-PAGE loading buffer and directly embedded in PAGE gel by mixing with 12% PAGE solution prior to gelling. Gels were then sliced, washed with double-distilled water, and subjected to in-gel trypsin digestion as previously described.

Gene Ontology (GO) and KEGG Pathway Annotations and Statistical Analysis

Protein groups identified by MaxQuant were imported to Perseus 45 version 1.4.1.3 for Gene Ontology Cellular Component (GOCC), Molecular Function (GOMF) and Biological Process (GOBP) as well as KEGG annotation followed by statistical analysis. Normalized protein group quantity relative ratios were first logarithmized to base 2, and significantly changed protein groups were identified using

LC−MS/MS

LC−MS/MS experiments were performed at the core facility of Institutes of Biomedical Sciences, Shanghai Medical College, C

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Significance A43 calculation in Perseus with default parameters (threshold value 0.05). Annotations with significantly changed ratio distribution compared with all quantifiable protein groups in each comparison were then identified using 1D annotation enrichment45 analysis in Perseus using default parameters (threshold value 0.02).

then, respectively, transfected with empty vector pCMV, HBx expression plasmid pCMV-HA-HBx, and replicon plasmids harboring terminally redundant wild type (pCMV-HBV1.1) or HBx-null mutant HBV [pCMV-HBV1.1(X−)] genomes, as depicted in Figure 1. Exosomes were then prepared from culture supernatants of transfected cells and subjected to total protein quantification. To study the effects of HBx overexpression, exosomes from vector transfected unlabeled “light” cells and pCMV-HA-HBx transfected R10K8-labeled “heavy” cells were combined at 1:1 ratio (Set1). To study the effects of expression and replication of wildtype and HBx-null HBV, we combined exosomes from vector transfected unlabeled “light” cells, pCMV-HBV1.1 transfected R10K8-labeled “heavy” cells, and pCMV-HBV1.1(X−) transfected R6K4-labeled “medium” cells at 1:1:1 ratio (Set2). The combined exosome samples were then processed using two slightly different methods: in Method I, samples were subjected to SDS-PAGE first and gel slices were prepared after silver staining; in Method II, samples were embedded into a small volume of SDS-PAGE separation gel during preparation and the gels were directly used in subsequent steps without electrophoresis. Gels were then processed through the conventional in-gel trypsin digestion/ LC−MS/MS pipeline.

Human Sera Collection, Exosome Preparation, and Immunoblotting Analysis

HBV-negative and HBV-positive human sera were collected from health check and outpatient departments of Putuo District Center Hospital, Shanghai. For analysis of multiple exosome proteins, sera from 4 to 6 patients or health check recipients were pooled together, respectively, to achieve a final volume of 2−5 mL. For analysis of VCP in exosomes, 0.4 to 1 mL serum from individual subjects was used without pooling. Exosomes were prepared from pooled or individual sera using the same protocol as that used for transfection supernatant previously described. After total protein quantification, exosome proteins were analyzed by immunoblotting.

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RESULTS AND DISCUSSION

General Experiment Design

To probe possible changes in exosome protein contents induced by HBx or HBV, we designed SILAC/MS experiments, as summarized in Figure 1. Currently, efficient HBV infection

Validation of Transfection and Exosome Purification

Successful transfection of Huh-7 cells was first verified by examining the expression of EGFP from cotransfected pEGFP control plasmid in fluorescent microscopy (data not shown) and Western blot (Figure 2A). Expression of HBx in cells

Figure 2. Validation of transfection of Huh-7 cells. (A) Western blot analysis of protein expression from transfected plasmids. Huh-7 cells transfected with indicated plasmids were analyzed for the expression of HBx and HBcAg, respectively. EGFP expressed from cotransfected pEGFP control plasmid as well as endogenous beta-actin were analyzed in parallel. *, due to low level expression of HBx by pCMVHBV1.1, loading quantities were increased 10-fold to confirm expression. (B) Southern blot analysis of HBV replication intermediates in Huh-7 cells transfected with HBV genome plasmids. Intracelluar capsid-associated nucleic acids were extracted and detected using HBV-specific probes. Equal loading was based on densitometry quantification of cotransfected EGFP. RC (relaxed circular), DS (double-stranded), and SS (single-stranded) represent different forms of replication intermediates.

Figure 1. Schematic summary of experimental and data analysis process of the study.

of human hepatocytes in vitro is limited to primary hepatocytes and the human hepatoma cell line HepaRG,46 both of which are difficult to label using SILAC because neither retain HBV infection sensitivity after long-term culture and continuous passaging. Instead, we transfected human hepatoma cell line Huh-7 with HBV replicon plasmids to initiate HBV expression and replication as a simulation of HBV infection. Huh-7 cells were first labeled using “heavy” (R10K8) or “medium” (R6K4) weight isotope amino acids, and efficient labeling (>90%) was confirmed by analyzing β-actin-derived tryptic peptides in MS (Supplementary Figure 2 in the Supporting Information). Unlabeled and labeled cells were

transfected with pCMV-HA-HBx was then confirmed in Western blot using anti-HBx antibody (Figure 2A). Western blot using anti-HBc antibody showed that HBV core proteins were expressed in cells transfected with pCMV-HBV1.1 and pCMV-HBV1.1(X−), as expected (Figure 2A). Immunoassay of HBsAg and HBeAg also indicated that pCMV-HBV1.1 and pCMV-HBV1.1(X−) transfections both resulted in expression and secretion of HBsAg and HBeAg into culture media (see below and Figure 3A), whereas Southern blot analysis of D

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Figure 3. Validation of exosome preparation protocol. (A) Removal of secreted HBV antigens and particles by the exosome preparation protocol. Samples from different steps of the preparation process using media supernatants of Huh-7 cells transfected with plasmids as indicated were analyzed by quantitative immunoassay for HBsAg (top) and HBeAg (bottom) and results were normalized to percentages of starting material. (B) Western blot analysis of exosome (top) and nonexosome (bottom) markers in exosomes prepared from Huh-7 cells transfected with indicated plasmids. Cell lysates were analyzed in parallel as antibody reactivity controls.

Qualitative Analyses of Protein Content of Exosomes Produced by HBx- and HBV-Transfected Huh-7 Cells

intracellular capsid-associated DNA identified replication intermediate species characteristic of HBV (Figure 2B). Expression of HBx from pCMV-HBV1.1 was too low and only detectable when loading quantity was increased by 10-fold. Despite such a low expression level, more intracellular capsid protein and HBV replication intermediate DNA were detected in pCMV-HBV1.1-transfected cells compared with pCMVHBV1.1(X−)-transfected cells (Figure 2A,B), indicating a positive contribution by HBx toward HBV replication, which concurred with previous reports.36 These data demonstrated that transfection of Huh-7 cells with pCMV-HA-HBx led to the expected overexpression of HBx, while transfection with pCMV-HBV1.1 and pCMV-HBV1.1(X−) resulted in different levels of HBV antigen expression and genome replication. Exosomes were then prepared from culture supernatants of transfected cells following a differential centrifugation protocol.47 Because HBV expression and replication are accompanied by secretion of HBeAg, empty HBsAg-derived SVPs, and mature virions, exosome preparation from pCMV-HBV1.1 and pCMV-HBV1.1(X−) transfection media was monitored for the removal of such HBV-derived nonexosome contents by quantitative immunoassay of HBsAg and HBeAg. As shown in Figure 3A, compared with cleared culture media input, over 90% HBsAg and over 98% HBeAg were eventually removed at the end of the differential centrifugation process. Successful purification of exosomes and absence of proteins from possible contaminating cellular membranous structures was also confirmed in Western blot using antibodies to corresponding protein markers (Figure 3B). The exosome marker Tsg101 was detected in all exosome preparations, whereas commonly used cellular protein markers including Grp94 (endoplasmic reticulum), calnexin (endoplasmic reticulum), and VDAC1 (mitochondria) were not markedly present (Figure 3B). Taken together, these data indicated that the exosome preparation process successfully purified exosomes from Huh-7 culture supernatants while simultaneously removing a substantial majority of HBV-derived viral and subviral particles as well as soluble viral antigens from pCMV-HBV1.1 and pCMVHBV1.1(X−) transfection media.

Exosomes purified from culture supernatants were subjected to total protein quantification and combined in equal amounts, as illustrated in Figure 1 into two sets of exosome samples: Set1 and Set2. Samples from two independent transfection replications were processed using both Method I and Method II as previously described and analyzed by LC−MS/MS following in-gel trypsin digestion. Raw data generated by MS were then passed to MaxQuant for protein identification and quantification. For maximum stringency, only protein groups identified in both replications using at least one of Methods I and II were considered in following analyses. Overall, MaxQuant identified 213 cellular protein groups encoded by 266 genes in Set1 and 265 protein groups encoded by 323 genes in Set2 (Supplementary Table S1 in the Supporting Information). Although HBx was detected by Western blot in pCMV-HAHBx and pCMV-HBV1.1 transfected Huh-7 cell lysates (Figure 2A), HBx-derived peptides were not identified in Set1 or Set2. Peptides derived from HBeAg/HBcAg were identified in Set2, consistent with data presented in Figure 3A. No HBsAg- or HBV polymerase-derived peptide was identified in Set2. Of the 213 cellular protein groups identified in Set1 and the 265 cellular protein groups identified in Set2, 144 were commonly identified in both samples (Figure 4A). When the encoding genes of Set1 and Set2 proteins as well as the ExoCarta48 database of previously published human exosome protein genes were compared, about 84% of Set1 and 82% of Set2 genes were found in the ExoCarta database and 165 genes were commonly found in Set1, Set2, and ExoCarta (Figure 4B and Supplementary Table S1 in the Supporting Information). These commonly identified genes encode proteins frequently identified in exosome preparations prepared from various sources, including those involved in membrane transport and fusion (small GTPase Rab proteins, annexins) and MVB biogenesis (Alix/PDCD6IP, clathrins), heat-shock proteins (HSP90s, HSP70s), and 14−3−3 proteins, as well as ubiquitins, cross-membrane transporters, and cytoskeleton proteins (tubulins) (Supplementary Table S1 in the Supporting Information). Multiple genes encoding ribosome proteins (RPLs, RPSs), translation elongation factors (EEFs), and histones are also prominently featured in the commonly E

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was identified in Set2. Calnexin (CANX) was identified in Set2, and Grp94 (HSP90B1) was identified in both Set1 and Set2, despite the fact that immunoblotting failed to detect significant amounts of these proteins in exosome preparations (Figure 3B). Differences in sensitivity and specificity between immunoblotting and MS methodologies might explain some of these inconsistencies. In addition, both CANX and HSP90B1 are included in ExoCarta database, indicating that their products have been identified by MS in other exosome preparations. Relative Quantification and Statistical Analyses of Exosome Protein Content Changes Using MaxQuant and Perseus

Figure 4. Summary of qualitative cellular exosome protein identification. (A) Overlap between protein groups identified by MaxQuant in Set1 and Set2. (B) Overlap between genes encoding proteins identified in Set1, Set2, and genes included in ExoCarta exosome protein gene database.

In addition to qualitative protein identification, SILAC/MS also offers the possibility of analyzing relative abundance of proteins or protein groups between the differently labeled samples, providing quantitative measurements of the protein content changes induced by the applied treatments. In this work, MaxQuant was used to calculate the normalized ratios between quantities of the same protein group in exosomes prepared from culture media of Huh-7 cells transfected with different plasmids. Such ratios provide rough estimates regarding whether and how much the relative abundance of corresponding protein groups is changed as a result of different

identified genes. Seventy-five genes that were identified in Set1 or Set2 but were not present in current ExoCarta database might represent exosome proteins specific to Huh-7 cells. The exosome marker Tsg101, which could be detected by immunoblotting in exosomes prepared using the same procedure (Figure 3B), was not identified by MS in Set1 or Set2, and among other frequently used exosome protein markers, such as tetraspannins (CD63, CD81, etc.), only CD63

Figure 5. Quantitative exosome protein changes induced by HBx and HBV in Huh-7 cells. Normalized ratios of protein group intensities between exosomes prepared from Huh-7 cells transfected with HBx expression plasmid pCMV-HA-HBx and control vector (A), pCMV-HBV1.1 and control vector (B), pCMV-HBV1.1(X−) and control vector (C), and pCMV-HBV1.1 and pCMV-HBV1.1(X−) (D) are logarithmized to base 2 and distribution of the results plotted. GO annotation terms enriching in upregulated or downregulated member protein groups were identified by 1D annotation enrichment analysis in Perseus and overlaid in red (upregulated) or green (downregulated). Full list of annotation terms identified by Perseus 1D annotation enrichment is available in Supplementary Table S2 in the Supporting Information. F

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Table 1. Protein Groups Significantly Changed in Exosomal Abundance in Response to HBx and HBV Transfection in Huh-7 Cells Log2 (normalized ratio) gene symbol

gene name

HBx

HBV vs HBV(X−)

HBV

HBV(X−)

A2M CCT3 APOC3 STAM; STAM2 DCD CALML5 LYZ LTF S100A8 FABP5 CSTA ARHGEF17; RAB43 SBSN SERPINA1 APOA1 AZGP1 CASP14 FLNA; FLNC JUP HBB; HBD DSP S100A9 KRT18 COL5A2 HSPD1 MYCBP2 GPC3 CSE1L SDC1 ATP5A1 PROM1 HSP90B1 FASN SLC35B2 DYNC1H1 NPM1 APOB DIP2B COL14A1 HSPA5 ACLY VCP HNRNPA1 PIGR VIM SYNE2 DSC1

alpha-2-macroglobulin T-complex protein 1 subunit gamma apolipoprotein C-III signal transducing adapter molecule 1; signal transducing adapter molecule 2 dermcidin calmodulin-like protein 5 lysozyme C lactotransferrin protein S100-A8 fatty acid-binding protein, epidermal cystatin-A Rho guanine nucleotide exchange factor 17; Ras-related protein Rab-43 suprabasin alpha-1-antitrypsin apolipoprotein A-I zinc-alpha-2-glycoprotein caspase-14 filamin-A; filamin-C junction plakoglobin hemoglobin subunit beta; hemoglobin subunit delta desmoplakin protein S100-A9 keratin, type I cytoskeletal 18 collagen alpha-2(V) chain 60 kDa heat shock protein, mitochondrial probable E3 ubiquitin-protein ligase MYCBP2 glypican-3 exportin-2 syndecan-1 ATP synthase subunit alpha, mitochondrial prominin-1 endoplasmin fatty acid synthase adenosine 3-phospho 5-phosphosulfate transporter 1 cytoplasmic dynein 1 heavy chain 1 nucleophosmin apolipoprotein B disco-interacting protein 2 homologue B collagen alpha-1(XIV) chain 78 kDa glucose-regulated protein ATP-citrate synthase transitional endoplasmic reticulum ATPase heterogeneous nuclear ribonucleoprotein A1 polymeric immunoglobulin receptor vimentin nesprin-2 desmocollin-1

−4.68 −2.09 −4.11 N.I. −4.47 −4.09 −4.50 −4.59 N.I. −3.49 −4.52 N.I. −2.49 N.I. −3.98 −3.65 −3.95 N.I. −3.80 −4.25 −3.57 −4.11 −1.30 −1.21 −1.26 N.I. 1.51 N.I. 1.53 N.I. 1.64 1.91 0.61 0.76 1.20 1.13 1.77 1.23 1.93 0.66 N.I. 2.09 N.I. −5.03 −4.83 −3.87 N.I.

−2.41 −1.44 −0.32 −0.74 −0.45 −0.18 −1.31 −0.81 −1.82 −0.04 −0.55 −0.82 −0.80 −0.32 0.42 −0.95 −0.87 −1.89 −1.09 −1.01 −0.59 −1.22 1.09 1.79 1.34 −3.57 −0.82 0.91 −0.74 1.14 −0.04 0.21 −0.57 −0.57 −0.30 −0.43 −0.58 −0.15 1.92 −0.07 −0.35 0.84 0.71 N.I. N.I. N.I. N.Q.

−6.68 −5.78 −5.62 −5.26 −4.87 −4.29 −4.27 −4.14 −4.12 −4.07 −4.04 −3.82 −3.50 −3.46 −3.43 −3.40 −3.37 −3.33 −3.25 −3.01 −2.94 −2.40 −1.02 −0.99 0.15 0.18 0.30 0.45 0.74 0.80 0.97 1.01 1.11 1.11 1.11 1.11 1.14 1.19 1.29 1.37 1.40 1.45 1.51 N.I. N.I. N.I. N.Q.

−3.89 −4.27 −4.94 −5.44 −4.29 −3.15 −2.91 −3.10 −2.78 −4.59 −4.64 −3.32 −3.11 −3.52 −3.84 −2.54 −2.61 −2.27 −1.96 −2.17 −2.97 −1.77 −1.66 −2.73 −0.99 3.70 1.15 −0.46 1.61 −1.07 0.85 0.42 1.49 1.60 1.50 1.34 1.75 1.18 −0.60 1.05 0.89 0.60 0.99 N.I. N.I. N.I. −2.98

Normalized ratios calculated by MaxQuant between exosomes from HBx, wildtype HBV, HBx-null HBV [HBV(X−)], and vector-transfected Huh-7 cells are logarithmized to base 2 and listed. Normalized ratios between wildtype HBV and HBx-null HBV transfections are also listed alongside HBx ratios for easy comparison. Ratios calculated as significant in Perseus (Significance A, default parameters) are highlighted in bold and italicized. N.I., not identified; N.Q., not quantifiable. a

transfections. Statistical significance of these normalized ratios calculated by MaxQuant was then probed by importing them into Perseus, logrithmizing to base 2, and then analyzing using two methods in Perseus. First, Significance A analysis43 was used to identify protein groups with statistically significant higher or lower ratios compared with all quantified protein

groups. In other words, abundance of these protein groups in exosomes is most likely markedly increased or decreased between the two compared transfections. Second, 1D annotation enrichment analysis45 was performed on all GO and KEGG annotation terms associated with the quantified proteins to identify annotations enriched in high or low ratios. G

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significantly increased in exosome abundance (Table 1 and previous section). However, similar to the case with wildtype HBV transfection, 1D annotation enrichment analysis identified the two closely related GOCC annotations “intrinsic to membrane” and “integral to membrane” as displaying overall increased abundance in exosomes after HBx-null HBV transfection (Supplementary Table S2 in the Supporting Information and Figure 5C). As shown in Figure 2, although HBx expression from wildtype HBV is much lower than pCMV-HA-HBx, the removal of such low expression of HBx by introducing premature termination into HBx ORF resulted in a marked drop in HBV expression and replication level. In light of this, it could be concluded that HBV X protein expression and elevated HBV replication are required for causing the significantly increased presence of one set of proteins (FASN, SLC35B2, DYNC1H1, NPM1, APOB, DIP2B, COL14A1, HSPA5, ACLY, VCP, HNRNPA1) in Huh-7 exosomes, but they are not involved in causing the decreased presence of another set of proteins (A2M, CCT3, APOC3, STAM/ STAM2, DCD, CALML5, LYZ, LTF, S100A8, FABP5, CSTA, ARHGEF17/RAB43, SBSN, SERPINA1, APOA1, DSP) in exosomes from HBV replicon-transfected cells. The latter is most likely a result of non-X HBV protein expression and HBV genome replication.

Instead of individual protein groups, 1D annotation enrichment sheds light on what structural and functional groups of proteins tend to be increased or decreased between the two compared transfections. HBx-Induced Exosome Protein Content Changes in Huh-7

Of the 213 protein groups identified by MaxQuant in Set1, 210 were quantifiable (Supplementary Table S1 in the Supporting Information), and their logarithmized normalized ratios between exosomes from pCMV-HA-HBx transfected versus pCMV vector transfected Huh-7 cells display a nearly normal distribution surrounding zero, with 75 protein groups falling between −1 and 0 and 78 protein groups between 0 and 1 (Figure 5A). Significance A analysis in Perseus identified 18 protein groups (PIGR, VIM, A2M, LTF, CSTA, LYZ, DCD, HBB/HBD, APOC3, S100A9, CALML5, APOA1, CASP14, SYNE2, JUP, AZGP1, DSP, FABP5) that significantly decreased in exosomes upon HBx overexpression and 7 protein groups (GPC3, SDC1, PROM1, APOB, HSP90B1, COL14A1, VCP) whose abundance in exosomes significantly increased upon HBx overexpression (Table 1). However, 1D annotation enrichment analysis failed to identify any annotation term whose members displayed overall increase or decrease in exosome abundance in response to HBx overexpression. HBV-Induced Exosome Protein Content Changes in Huh-7

Of the 265 cellular protein groups identified by MaxQuant in Set2, 263 and 265 were quantifiable for wildtype HBV versus vector and HBx-null HBV versus vector ratios, respectively (Supplementary Table S1 in the Supporting Information). Logarithmized normalized ratios in these two comparisons exhibited roughly normal distribution around zero (compare Figure 5B,C), similar to HBx-induced changes (Figure 5A). Significance A analysis identified 22 protein groups (A2M, CCT3, APOC3, STAM/STAM2, DCD, CALML5, LYZ, LTF, S100A8, FABP5, CSTA, ARHGEF17/RAB43, SBSN, SERPINA1, APOA1, AZGP1, CASP14, FLNA/FLNC, JUP, HBB, DSP, S100A9) and 11 protein groups (FASN, SLC35B2, DYNC1H1, NPM1, APOB, DIP2B, COL14A1, HSPA5, ACLY, VCP, HNRNPA1) that significantly decreased and increased, respectively, in abundance in exosomes prepared from Huh-7 cells transfected with wildtype HBV replicon plasmid pCMVHBV1.1 (Table 1). Meanwhile, 1D annotation enrichment analysis identified two closely related GOCC annotations “intrinsic to membrane” and “integral to membrane”, whose members tended to be increased in exosomes upon wildtype HBV transfection (Supplementary Table S2 in the Supporting Information and Figure 5B).

Comparison between HBV- and HBV(X−)-Induced Exosome Protein Content Changes in Huh-7 in Relation to HBx-Induced Changes

Because Set2 contained differently labeled exosomes from both wildtype and HBx-null HBV transfected Huh-7 cells, quantitative analysis of exosome protein content differences between the two could be performed. When logarithmized normalized ratios of protein group quantities from wildtype versus HBx-null HBV transfections were plotted, the distribution pattern slightly skewed toward the left, with 167 of the 263 (63%) quantifiable protein groups having ratios below zero (Figure 5D). Significance A analysis in Perseus identified four protein groups (MYCBP2, A2M, FLNA/FLNB, S100A8) with significantly lower abundance in wildtype HBV transfection exosomes and eight protein groups (HNRNPA1, VCP, CSE1L, KRT18, ATP5A1, HSPD1, COL5A2, COL14A1) with significantly lower abundance in HBx-null HBV transfection exosomes (Table 1). These changes between HBx-containing HBV and HBx-null HBV displayed little overlapping with changes between HBx and vector transfected Huh-7 cells, the only exceptions being A2M, COL14A1, and VCP (Table 1). Clearly, although presence/absence of HBx was a common factor in these two set of comparisons, the amount of HBx expressed as well as presence/absence of other HBV antigens and viral genome replication (Figure 2) also heavily influenced the effects on exosome contents. When 1D annotation enrichment was analyzed, annotations GOMF “structural constituent of ribosome” and “RNA binding” as well as KEGG “Ribosome” were identified as significantly decreased in overall member abundance in exosomes from HBV-transfected Huh-7 compared with exosomes from HBV(X−)-transfected Huh-7. In consistence with this result, 21 GOBP and 2 GOCC annotations, mostly related to ribosome and transcription functions, were also identified as decreased (Figure 5D and Supplementary Table S2 in the Supporting Information).

HBV(X−)-Induced Exosome Protein Content Changes in Huh-7

Seventeen protein groups (A2M, CCT3, APOC3, STAM/ STAM2, DCD, CALML5, LYZ, LTF, S100A8, FABP5, CSTA, ARHGEF17/RAB43, SBSN, SERPINA1, APOA1, DSP, DSC1) significantly decreased in abundance in Huh-7 exosomes after transfection with HBx-null HBV replicon plasmid pCMVHBV1.1(X−) (Table 1). Interestingly, all of these protein groups except DSC1, which were not quantifiable in wildtype HBV transfection, were also identified as significantly decreased in exosomes from wildtype HBV replicon-transfected cells. Only one protein group (MYCBP2) significantly increased in abundance, which was only slightly increased in wildtype HBV transfection exosomes. This is in clear contrast with wildtype HBV replicon transfection, wherein 11 protein groups H

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Correlation between Quantitative Proteomics and Semiquantitative Immunoblotting

Huh-7 cells. The next obvious question was whether the observed changes, or at least some of them, also occur in vivo after HBV infection. To answer this question, we prepared exosomes using pooled sera from HBV-infected patients as well as HBV antigen and DNA-negative control subjects. As shown in Figure 7A, exosomes from hepatitis B patients’ sera

To evaluate the correlation between quantitative proteomics results and semiquantitative immunoblotting, we selected and detected five proteins identified and quantified in both Set1 and Set2 using commercial antibodies in exosomes prepared from transfected Huh-7 cells. Densitometry scanning was performed and ratios between different transfections were calculated and compared with ratios produced by MaxQuant. As shown in Figure 6, the correlation between MS and Western blot results

Figure 7. Serum-derived exosome protein content changes in HBVinfected patients. (A) Exosomes were prepared from pooled HBVinfected patients (HBV) or HBV antigen and DNA-negative control subjects (Control) and analyzed in immunoblotting after total protein quantification. Equal amounts of total exosome protein were loaded in each lane. (B) Exosomes were prepared from six individual HBVinfected patient’s serum and six control subjects and analyzed as in panel A.

Figure 6. Comparison of exosome protein content changes identified by MS and immunoblotting (WB) data. Antibodies against selected proteins identified and quantified in Set1 and Set2 using MS were used to detect corresponding proteins in exosome preparations from Huh-7 cells transfected with indicated plasmids loaded at equal amounts of total protein (left). Densitometry scanning was performed to calculate the folds of change in protein quantity normalized against pCMV vector control and listed against corresponding normalized ratios calculated by MaxQuant using MS data (right).

contained lower levels of ALIX, higher levels of Grp94 (HSP90B1) and VCP, and roughly similar levels of AZGP1 and FASN compared with exosomes from control subjects. Among these changes, only those of Grp94 and VCP were in agreement with changes observed in MS and immunoblotting results obtained using Huh-7 cells (Table 1 and Figure 6). These results clearly demonstrated that HBV infection is indeed associated with detectable changes in serum exosome protein content in patients. However, because Huh-7 is an immortal hepatoma cell line rather than normal hepatocytes, extrapolating in vitro observations in Huh-7 to HBV-infected patients requires extreme caution. Nevertheless, as demonstrated with Grp94 and VCP, results obtained in vitro could provide valuable hints for studying similar in vivo conditions. VCP (valosin-containing protein), also termed transitional endoplasmic reticulum ATPase or p97, belongs to the AAA (ATPase with multiple cellular activities) family and has been accredited with diverse chaperone functions involved in ubiquitin-dependent protein quality and degradation control at ER, endosome/lysosome, autophagosome, mitochondria, cytoplasm, as well as chromosome locations.49 Furthermore, association of VCP abnormality with certain types of cancer has been noticed in recent years and suggested possible involvement in tumorigenesis.50 In liver, elevated VCP expression has been found to be linked to postoperative recurrence of HCC,51 whereas the multitarget inhibitor sorafenib, certified for treating late stage HCC, has been shown to also target VCP and disrupt secretory pathways, resulting in ER stress and cell death of HCC cells.52 As previously mentioned, HBV infection is an important risk factor for HCC, and HBx has been shown to be overexpressed in at least a portion of HBV-related HCC patients. In light of this, the identification of VCP as elevated both in exosomes from HBx and HBV transfected Huh-7 cells (Table 1 and Figure 6) and serum exosomes from HBV-infected patients (Figure 7A) was interesting. Because such results with pooled sera reflect averaged differences between the two groups, we went on to examine whether there were variations between HBV-infected patients as far as VCP content in serum

varies considerably depending on the protein in question. For example, MS identified VCP as increased in Huh-7 exosomes upon transfection with HBx and HBV (with or without HBx) plasmids, which was concurred by immunoblotting and densitometry scanning. However, although MS results suggested that AZGP1 decreased in exosome abundance after transfection with HBx and HBV (with or without HBx) plasmids, in immunoblotting, the level of AZGP1 apparently increased in exosomes from such transfections (Figure 6). For the remaining three proteins tested, ALIX, CTNNB1, and FASN, obvious correlation between MS and Western results was observed only for one or two of the three transfections (Figure 6). Although conventional immunoblotting is inherently much less quantitative compared with MS, these two methods also have other significant differences that might underline some of the observed differences. For example, MS does not distinguish degraded protein fragments from fulllength proteins very well, and quantification of peptides with possible post-translational modification like glycosylation in MS is difficult, whereas in immunoblotting, these issues may or may not affect results, depending on targeted epitope(s), specificity, and sensitivity of the antibody used. Nevertheless, these observations suggested that caution should be exercised when drawing conclusions solely based on either immunoblotting or MS results. When MS and immunoblotting data are in good agreement, such as in the case of VCP, the reliability of the results could be expected to be high. Characterization of Exosome Protein Content Changes in HBV-Infected Patients

In the previous sections, exosome content changes induced by HBx and HBV were studied in vitro by transfecting cultured I

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to and taken up by neighboring tissue cells, resident or circulating immune cells, and even cells of remotely located tissues and organs suggested that virus-induced exosome content changes might be implicated in more diverse processes involving intercellular communication, such as virus spread, antivirus immune reactions, and tumor surveillance. Although HBV exclusively infects hepatocytes, HBV infection of hepatocytes leads to a series of events directly and indirectly involving other cell types, not only in the liver but also at remote sites, through antigens and virions secreted into serum by infected hepatocytes as well as cytokines and chemokines released by hepatocytes and immune cells. Consequently, the origin of the serum exosome content changes observed in HBV-infected patients (Figure 7), or in other words, to what degree infected hepatocytes and other cell types, respectively, contribute to such changes, is an interesting but complex question, which most certainly warrants further research efforts. An equally challenging and more clinically relevant question is whether and how such changes are related to hepatitis B pathogenesis, including infection resolution, chronic progression, and carcinogenesis. The current study would serve as a foundation for future studies aimed at addressing these important questions. In conclusion, data presented in this work reveal a new aspect of HBV−host interactions and offer multiple interesting candidates, at both the individual gene level and multigene annotation correlation level, for future research addressing whether and how exosomes mechanistically fit into HBV life cycle and pathogenesis. Furthermore, detection of HCC-related proteins like VCP in patient serum exosomes suggests novel applications of exosome-targeted diagnostics in clinical oncology.

exosomes was concerned. Immunoblotting using exosomes prepared from individual sera showed that serum exosome from two of six HBV-infected patients contained clearly detectable VCP, whereas VCP was near or below detection level in serum exosomes from the four remaining patients and six HBVnegative controls (Figure 7B). AZGP1 level, which was analyzed in parallel, showed much less significant variations among different samples. Expanding the analyzed samples to include a total of 10 HBV-negative controls and 30 HBVinfected patients led to similar results: VCP was not detected in the 10 control samples but was detected in 20 of the 30 (67%) HBV samples (Figure 7B and Supplementary Figure S3 in the Supporting Information). These data demonstrated that elevated VCP content in serum exosomes is indeed specific to HBV-infected patients but only occurs in a portion of patients. Studies involving much larger sample sizes are required to probe the clinical relevance of this observation and might help elucidate what host and viral factors are involved in determining VCP level in serum exosomes in HBVinfected patients. Comparison between high-exosome VCP patients and other patients regarding responses to drug treatment, as well as progression to fibrosis, cirrhosis, and HCC, is also an interesting direction worth following. In the meantime, detection of VCP in some patients’ serum and reported association of elevated VCP expression in liver with HCC recurrence51 suggest the possibility of using serum exosome VCP as a possible marker for monitoring HCC progression and recurrence.

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CONCLUSIONS Although exosome was discovered decades ago, its functions in various aspects of physiology, pathology, and immunology have only just begun to be studied and appreciated. Virus infection, even infection by nonlytic and noncytopathic viruses such as HBV, constitutes a significant disturbance to normal cell physiology, and expression of potentially oncogenic viral proteins such as HBx might eventually change the fate of the infected cell. It is therefore not surprising that, as an important cellular process, externalization of cellular proteins through exosome biogenesis and export could be affected by viral expression and replication, as demonstrated by results obtained using transfected Huh-7 cells presented in this study. The fact that HBV utilizes some host-cell MVB functions for progeny virion morphogenesis28−31 might have also contributed to some exosome content changes induced by HBV and HBV(X−) replicons observed in Huh-7 cells. To our knowledge, this is the first report addressing the direct or indirect effects of HBV on the exosome production and secretion process of infected cells. Delineating the biological functions of the exosome protein content changes identified in this work will be a major challenge. Externalization through exosome export might constitute mechanisms for regulating cellular levels or turnover rates for certain proteins, and virus-induced exosome content changes therefore might help create an intracellular environment more favorable for virus life cycle progression, intracellular virus persistence, or virus-associated malignant transformation. Intracellular levels of some of the proteins with increased or decreased exosomal abundances identified in this work (Figure 6) demonstrated only marginal variation in response to HBx or HBV replicon transfection (data not shown). More extensive and in-depth analysis are required to help identify cellular proteins affected by HBV infection in such a way. The possibility of secreted exosomes to be transported

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ASSOCIATED CONTENT

* Supporting Information S

Schematic diagrams of HBV genome and terminally redundant HBV replicon plasmid used for transfection (Supplementary Figure S1). Analysis of SILAC labeling efficiency (Supplementary Figure S2). Analysis of VCP in serum exosomes from additional HBV-negative and HBV-infected subjects (Supplementary Figure S3). Full listing of protein groups identified by MaxQuant in Set1 and Set2 and comparison of encoding genes with ExoCarta exosome gene database (Supplementary Table S1). Full listing of GO and KEGG annotations with enriched upregulated or downregulated members in exosome protein content changes identified using 1D annotation enrichment analysis in Perseus (Supplementary Table S2). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*Y.X.: E-mail: [email protected]. Fax: +86-21-54237973. Tel: +86-21-54237972. *J.L.: E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (31071143, 31170148), the National Science and Technology Major Project for Infectious Diseases (2012ZX10002-006, 2012ZX10004-503, J

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2012ZX10002-012, and 2013ZX10002-001), “973” program (2012CB519002), and Shanghai Municipal R&D Grant (GWDTR201216). We thank Prof. Yongxiang Wang of Fudan University for providing the HBV replicon plasmids and staff at the Proteomics Core Facilities of IBS, Fudan University for their technical assistance in MS analysis.

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urinary proteomics: exosomes as a source of urinary biomarkers. Nephrology 2005, 10 (3), 283−290. (23) Hao, S.; Moyana, T.; Xiang, J. Review: cancer immunotherapy by exosome-based vaccines. Cancer Biother. Radiopharm. 2007, 22 (5), 692−703. (24) Conde-Vancells, J.; Rodriguez-Suarez, E.; Embade, N.; Gil, D.; Matthiesen, R.; Valle, M.; Elortza, F.; Lu, S. C.; Mato, J. M.; FalconPerez, J. M. Characterization and comprehensive proteome profiling of exosomes secreted by hepatocytes. J. Proteome Res. 2008, 7 (12), 5157−5166. (25) Kogure, T.; Lin, W. L.; Yan, I. K.; Braconi, C.; Patel, T. Intercellular nanovesicle-mediated microRNA transfer: a mechanism of environmental modulation of hepatocellular cancer cell growth. Hepatology 2011, 54 (4), 1237−1248. (26) Bala, S.; Petrasek, J.; Mundkur, S.; Catalano, D.; Levin, I.; Ward, J.; Alao, H.; Kodys, K.; Szabo, G. Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, druginduced, and inflammatory liver diseases. Hepatology 2012, 56 (5), 1946−1957. (27) Pan, Q.; Ramakrishnaiah, V.; Henry, S.; Fouraschen, S.; de Ruiter, P. E.; Kwekkeboom, J.; Tilanus, H. W.; Janssen, H. L.; van der Laan, L. J. Hepatic cell-to-cell transmission of small silencing RNA can extend the therapeutic reach of RNA interference (RNAi). Gut 2012, 61 (9), 1330−1339. (28) Hoffmann, J.; Boehm, C.; Himmelsbach, K.; Donnerhak, C.; Roettger, H.; Weiss, T. S.; Ploen, D.; Hildt, E. Identification of alphataxilin as an essential factor for the life cycle of hepatitis B virus. J. Hepatol. 2013, 59 (5), 934−941. (29) Lambert, C.; Doring, T.; Prange, R. Hepatitis B virus maturation is sensitive to functional inhibition of ESCRT-III, Vps4, and gamma 2adaptin. J. Virol 2007, 81 (17), 9050−9060. (30) Patient, R.; Hourioux, C.; Roingeard, P. Morphogenesis of hepatitis B virus and its subviral envelope particles. Cell. Microbiol. 2009, 11 (11), 1561−1570. (31) Watanabe, T.; Sorensen, E. M.; Naito, A.; Schott, M.; Kim, S.; Ahlquist, P. Involvement of host cellular multivesicular body functions in hepatitis B virus budding. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (24), 10205−10210. (32) Zhu, H.; Pan, S.; Gu, S.; Bradbury, E. M.; Chen, X. Amino acid residue specific stable isotope labeling for quantitative proteomics. Rapid Commun. Mass Spectrom. 2002, 16 (22), 2115−2123. (33) Chen, X.; Smith, L. M.; Bradbury, E. M. Site-specific mass tagging with stable isotopes in proteins for accurate and efficient protein identification. Anal. Chem. 2000, 72 (6), 1134−1143. (34) Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 2002, 1 (5), 376−386. (35) Nassal, M.; Rieger, A. A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. J. Virol. 1996, 70 (5), 2764−2773. (36) Tang, H.; Delgermaa, L.; Huang, F.; Oishi, N.; Liu, L.; He, F.; Zhao, L.; Murakami, S. The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication. J. Virol. 2005, 79 (9), 5548−5556. (37) Wei, L.; Shen, Z.; Zhao, X.; Wu, Y.; Liu, W.; Zhang, J.; Xie, Y.; Liu, J. A broadly reactive monoclonal antibody detects multiple genotypes of hepatitis B virus X protein. Arch. Virol. 2014, 159 (10), 2731−2735. (38) Du, R.; Long, J.; Yao, J.; Dong, Y.; Yang, X.; Tang, S.; Zuo, S.; He, Y.; Chen, X., Subcellular quantitative proteomics reveals multiple pathway cross-talk that coordinates specific signaling and transcriptional regulation for the early host response to LPS. J. Proteome Res. 9, (4), 1805−1821. (39) Khatua, A. K.; Taylor, H. E.; Hildreth, J. E.; Popik, W. Exosomes packaging APOBEC3G confer human immunodeficiency virus resistance to recipient cells. J. Virol. 2009, 83 (2), 512−521.

REFERENCES

(1) Seeger, C.; Mason, W. S. Hepatitis B virus biology. Microbiol Mol. Biol. Rev. 2000, 64 (1), 51−68. (2) Liang, T. J. Hepatitis B: the virus and disease. Hepatology 2009, 49 (5 Suppl), S13−S21. (3) McMahon, B. J. The natural history of chronic hepatitis B virus infection. Hepatology 2009, 49 (5 Suppl), S45−S55. (4) World Health Organization, Hepatitis B. World Health Organization Fact Sheet 204 (Revised July 2013). http://www.who. int/mediacentre/factsheets/fs204/en/ (accessed Dec 10, 2013). (5) Patient, R.; Hourioux, C.; Sizaret, P. Y.; Trassard, S.; Sureau, C.; Roingeard, P. Hepatitis B virus subviral envelope particle morphogenesis and intracellular trafficking. J. Virol 2007, 81 (8), 3842−3851. (6) Hong, R.; Bai, W.; Zhai, J.; Liu, W.; Li, X.; Zhang, J.; Cui, X.; Zhao, X.; Ye, X.; Deng, Q.; Tiollais, P.; Wen, Y.; Liu, J.; Xie, Y. Novel recombinant hepatitis B virus vectors efficiently deliver protein and RNA encoding genes into primary hepatocytes. J. Virol. 2013, 87 (12), 6615−6624. (7) Chisari, F. V.; Isogawa, M.; Wieland, S. F. Pathogenesis of hepatitis B virus infection. Pathol Biol. 2010, 58 (4), 258−266. (8) Dandri, M.; Locarnini, S. New insight in the pathobiology of hepatitis B virus infection. Gut 2012, 61 (Suppl 1), i6−i17. (9) Guerrieri, F.; Belloni, L.; Pediconi, N.; Levrero, M. Molecular mechanisms of HBV-associated hepatocarcinogenesis. Semin. Liver Dis. 2013, 33 (2), 147−156. (10) Ng, S. A.; Lee, C. Hepatitis B virus X gene and hepatocarcinogenesis. J. Gastroenterol. 2011, 46 (8), 974−990. (11) Murakami, S. Hepatitis B virus X protein: a multifunctional viral regulator. J. Gastroenterol. 2001, 36 (10), 651−660. (12) Feitelson, M. A.; Reis, H. M.; Tufan, N. L.; Sun, B.; Pan, J.; Lian, Z. Putative roles of hepatitis B x antigen in the pathogenesis of chronic liver disease. Cancer Lett. 2009, 286 (1), 69−79. (13) Denzer, K.; Kleijmeer, M. J.; Heijnen, H. F.; Stoorvogel, W.; Geuze, H. J. Exosome: from internal vesicle of the multivesicular body to intercellular signaling device. J. Cell Sci. 2000, 113 (Pt 19), 3365− 3374. (14) Schorey, J. S.; Bhatnagar, S. Exosome function: from tumor immunology to pathogen biology. Traffic 2008, 9 (6), 871−881. (15) Meckes, D. G., Jr.; Raab-Traub, N., Microvesicles and viral infection. J. Virol. 85, (24), 12844−12854. (16) Thery, C.; Ostrowski, M.; Segura, E. Membrane vesicles as conveyors of immune responses. Nat. Rev. Immunol. 2009, 9 (8), 581− 593. (17) Cocucci, E.; Racchetti, G.; Meldolesi, J. Shedding microvesicles: artefacts no more. Trends Cell Biol. 2009, 19 (2), 43−51. (18) Raimondo, F.; Morosi, L.; Chinello, C.; Magni, F.; Pitto, M. Advances in membranous vesicle and exosome proteomics improving biological understanding and biomarker discovery. Proteomics 2011, 11 (4), 709−720. (19) Bobrie, A.; Colombo, M.; Raposo, G.; Thery, C. Exosome secretion: molecular mechanisms and roles in immune responses. Traffic 2011, 12 (12), 1659−1668. (20) Li, J.; Liu, K.; Liu, Y.; Xu, Y.; Zhang, F.; Yang, H.; Liu, J.; Pan, T.; Chen, J.; Wu, M.; Zhou, X.; Yuan, Z. Exosomes mediate the cell-tocell transmission of IFN-alpha-induced antiviral activity. Nat. Immunol. 2013, 14 (8), 793−803. (21) Pisitkun, T.; Johnstone, R.; Knepper, M. A. Discovery of urinary biomarkers. Mol. Cell. Proteomics 2006, 5 (10), 1760−1771. (22) Hoorn, E. J.; Pisitkun, T.; Zietse, R.; Gross, P.; Frokiaer, J.; Wang, N. S.; Gonzales, P. A.; Star, R. A.; Knepper, M. A. Prospects for K

dx.doi.org/10.1021/pr5008703 | J. Proteome Res. XXXX, XXX, XXX−XXX

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(40) Li, J.; Liu, K.; Liu, Y.; Xu, Y.; Zhang, F.; Yang, H.; Liu, J.; Pan, T.; Chen, J.; Wu, M.; Zhou, X.; Yuan, Z. Exosomes mediate the cell-tocell transmission of IFN-alpha-induced antiviral activity. Nat. Immunol 2013, 14 (8), 793−803. (41) Qin, J.; Zhai, J.; Hong, R.; Shan, S.; Kong, Y.; Wen, Y.; Wang, Y.; Liu, J.; Xie, Y. Prospero-related homeobox protein (Prox1) inhibits hepatitis B virus replication through repressing multiple cis regulatory elements. J. Gen. Virol. 2009, 90 (Pt 5), 1246−1255. (42) Taoka, M.; Ikumi, M.; Nakayama, H.; Masaki, S.; Matsuda, R.; Nobe, Y.; Yamauchi, Y.; Takeda, J.; Takahashi, N.; Isobe, T. In-gel digestion for mass spectrometric characterization of RNA from fluorescently stained polyacrylamide gels. Anal. Chem. 2010, 82 (18), 7795−7803. (43) Cox, J.; Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 2008, 26 (12), 1367−1372. (44) Vizcaino, J. A.; Deutsch, E. W.; Wang, R.; Csordas, A.; Reisinger, F.; Rios, D.; Dianes, J. A.; Sun, Z.; Farrah, T.; Bandeira, N.; Binz, P. A.; Xenarios, I.; Eisenacher, M.; Mayer, G.; Gatto, L.; Campos, A.; Chalkley, R. J.; Kraus, H. J.; Albar, J. P.; Martinez-Bartolome, S.; Apweiler, R.; Omenn, G. S.; Martens, L.; Jones, A. R.; Hermjakob, H. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotechnol. 2014, 32 (3), 223−226. (45) Cox, J.; Mann, M. 1D and 2D annotation enrichment: a statistical method integrating quantitative proteomics with complementary high-throughput data. BMC Bioinf. 2012, 13 (Suppl 16), S12. (46) Gripon, P.; Rumin, S.; Urban, S.; Le Seyec, J.; Glaise, D.; Cannie, I.; Guyomard, C.; Lucas, J.; Trepo, C.; Guguen-Guillouzo, C. Infection of a human hepatoma cell line by hepatitis B virus. Proc. Natl. Acad. Sci. U. S. A. 2002, 99 (24), 15655−15660. (47) Liang, B.; Peng, P.; Chen, S.; Li, L.; Zhang, M.; Cao, D.; Yang, J.; Li, H.; Gui, T.; Li, X.; Shen, K. Characterization and proteomic analysis of ovarian cancer-derived exosomes. J. Proteomics 2013, 80C, 171−182. (48) Mathivanan, S.; Fahner, C. J.; Reid, G. E.; Simpson, R. J. ExoCarta 2012: database of exosomal proteins, RNA and lipids. Nucleic Acids Res. 2012, 40 (Databaseissue), D1241−D1244. (49) Meyer, H.; Bug, M.; Bremer, S. Emerging functions of the VCP/ p97 AAA-ATPase in the ubiquitin system. Nat. Cell Biol. 2012, 14 (2), 117−123. (50) Fessart, D.; Marza, E.; Taouji, S.; Delom, F.; Chevet, E. P97/ CDC-48: proteostasis control in tumor cell biology. Cancer Lett. 2013, 337 (1), 26−34. (51) Yamamoto, S.; Tomita, Y.; Nakamori, S.; Hoshida, Y.; Nagano, H.; Dono, K.; Umeshita, K.; Sakon, M.; Monden, M.; Aozasa, K. Elevated expression of valosin-containing protein (p97) in hepatocellular carcinoma is correlated with increased incidence of tumor recurrence. J. Clin. Oncol. 2003, 21 (3), 447−452. (52) Yi, P.; Higa, A.; Taouji, S.; Bexiga, M. G.; Marza, E.; Arma, D.; Castain, C.; Le Bail, B.; Simpson, J. C.; Rosenbaum, J.; Balabaud, C.; Bioulac-Sage, P.; Blanc, J. F.; Chevet, E. Sorafenib-mediated targeting of the AAA(+) ATPase p97/VCP leads to disruption of the secretory pathway, endoplasmic reticulum stress, and hepatocellular cancer cell death. Mol. Cancer Ther. 2012, 11 (12), 2610−2620.

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