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• W2887501590 abstract "Interferon lambda (IFN-λ) is a relatively unexplored, yet promising antiviral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of antiviral proteins to multiple points of the viral life cycle, as well as facilitating the identification of antiviral proteins not previously known to be elicited upon HBV infection (e.g. IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through several essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated whereas cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation toward the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 versus IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication. Interferon lambda (IFN-λ) is a relatively unexplored, yet promising antiviral agent. IFN-λ has recently been tested in clinical trials of chronic hepatitis B virus infection (CHB), with the advantage that side effects may be limited compared with IFN-α, as IFN-λ receptors are found only in epithelial cells. To date, IFN-λ's downstream signaling pathway remains largely unelucidated, particularly via proteomics methods. Here, we report that IFN-λ3 inhibits HBV replication in HepG2.2.15 cells, reducing levels of both HBV transcripts and intracellular HBV DNA. Quantitative proteomic analysis of HBV-transfected cells was performed following 24-hour IFN-λ3 treatment, with parallel IFN-α2a and PBS treatments for comparison using a dimethyl labeling method. The depth of the study allowed us to map the induction of antiviral proteins to multiple points of the viral life cycle, as well as facilitating the identification of antiviral proteins not previously known to be elicited upon HBV infection (e.g. IFITM3, XRN2, and NT5C3A). This study also shows up-regulation of many effectors involved in antigen processing/presentation indicating that this cytokine exerted immunomodulatory effects through several essential molecules for these processes. Interestingly, the 2 subunits of the immunoproteasome cap (PSME1 and PSME2) were up-regulated whereas cap components of the constitutive proteasome were down-regulated upon both IFN treatments, suggesting coordinated modulation toward the antigen processing/presentation mode. Furthermore, in addition to confirming canonical activation of interferon-stimulated gene (ISG) transcription through the JAK-STAT pathway, we reveal that IFN-λ3 restored levels of RIG-I and RIG-G, proteins known to be suppressed by HBV. Enrichment analysis demonstrated that several biological processes including RNA metabolism, translation, and ER-targeting were differentially regulated upon treatment with IFN-λ3 versus IFN-α2a. Our proteomic data suggests that IFN-λ3 regulates an array of cellular processes to control HBV replication. Chronic hepatitis B (CHB) 1The abbreviations used are:HBVhepatitis B virusCHBchronic hepatitis BHCChepatocellular carcinomapgRNApregenomic RNAIFN-α2ainterferon-alpha2aIFN-λ3interferon-lambda3ISGinterferon-stimulated geneSDCsodium deoxycholateqPCRquantitative polymerase chain reactionGOgene ontologyFDRfalse discovery rate. 1The abbreviations used are:HBVhepatitis B virusCHBchronic hepatitis BHCChepatocellular carcinomapgRNApregenomic RNAIFN-α2ainterferon-alpha2aIFN-λ3interferon-lambda3ISGinterferon-stimulated geneSDCsodium deoxycholateqPCRquantitative polymerase chain reactionGOgene ontologyFDRfalse discovery rate. is a major health problem worldwide, affecting 240 million people throughout the world with a prevalence in Africa and South-East Asia (1Schweitzer A. Horn J. Mikolajczyk R.T. Krause G. Ott J.J. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013.Lancet. 2015; 386: 1546-1555Abstract Full Text Full Text PDF PubMed Scopus (1811) Google Scholar). Chronic HBV-infected individuals mostly acquire the virus at a young age through vertical transmission or contact with the blood or other body fluids of an infected person. CHB eventually progresses to severe and high-mortality liver diseases including liver cirrhosis and hepatocellular carcinoma (HCC) resulting in 600,000 deaths annually (2Grimm D. Thimme R. Blum H.E. HBV life cycle and novel drug targets.Hepatol. Int. 2011; 5: 644-653Crossref PubMed Scopus (91) Google Scholar, 3Wright T.L. Introduction to chronic hepatitis B infection.Am. J. Gastroenterol. 2006; 101: S1-S6Crossref PubMed Scopus (158) Google Scholar). hepatitis B virus chronic hepatitis B hepatocellular carcinoma pregenomic RNA interferon-alpha2a interferon-lambda3 interferon-stimulated gene sodium deoxycholate quantitative polymerase chain reaction gene ontology false discovery rate. hepatitis B virus chronic hepatitis B hepatocellular carcinoma pregenomic RNA interferon-alpha2a interferon-lambda3 interferon-stimulated gene sodium deoxycholate quantitative polymerase chain reaction gene ontology false discovery rate. Current therapeutic agents for CHB are interferon (IFN)-α and nucleos(t)ide analogs (NAs) (4EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.J. Hepatol. 2017; 67: 370-398Abstract Full Text Full Text PDF PubMed Scopus (2987) Google Scholar, 5Yuen M.F. Lai C.L. Treatment of chronic hepatitis B: Evolution over two decades.J. Gastroenterol. Hepatol. 2011; 26: 138-143Crossref PubMed Scopus (142) Google Scholar, 6Lau D.T.Y. Bleibel W. Current status of antiviral therapy for Hepatitis B.Therap. Adv. Gastroenterol. 2008; 1: 61-75Crossref PubMed Scopus (24) Google Scholar). IFN-α is a cytokine that possesses antiviral and immunomodulatory effects, which promotes control and eradication of viral infection. The advantages of using IFN-α for CHB treatment are the decreased incidence of viral resistance following this treatment and the finite duration of therapy with a higher rate of seroconversion of viral antigens, i.e. HBeAg and HBsAg, compared with NAs. However, the drawbacks of IFN-α treatment are its inconvenient route of administration and its adverse effects such as influenza-like symptoms, nausea, vomiting, cytopenia, and psychiatric disorders. Regarding the latter agent, NAs against CHB, the lack of a 3′-hydroxyl group in these compounds allows competitive incorporation into the viral genome resulting in termination of viral replication (7Fung J. Lai C.L. Seto W.K. Yuen M.F. Nucleoside/nucleotide analogues in the treatment of chronic hepatitis B.J. Antimicrobial Chemother. 2011; 66: 2715-2725Crossref PubMed Scopus (140) Google Scholar). Although NAs can directly suppress HBV replication and have fewer unfavorable side effects compared with IFN-α, CHB patients usually require a long-term treatment of these agents, thus increasing the chance of the emergence of viral resistance. In addition, the low rate of HBeAg and HBsAg seroconversion is another limitation of NAs. Therefore, new drugs that overcome restrictions of current anti-HBV treatments are still needed. Several lines of reasoning suggest that IFN-λ could have a superior combination of efficacy and reduced side-effects. IFN-λ or type III IFN is a cytokine in the class II cytokine family that has recently been used in clinical trials of CHB treatment (8Phillips S. Mistry S. Riva A. Cooksley H. Hadzhiolova-Lebeau T. Plavova S. Katzarov K. Simonova M. Zeuzem S. Woffendin C. Chen P.-J. Peng C.-Y. Chang T.-T. Lueth S. De Knegt R. Choi M.-S. Wedemeyer H. Dao M. Kim C.-W. Chu H.-C. Wind-Rotolo M. Williams R. Cooney E. Chokshi S. Peg-interferon lambda treatment induces robust innate and adaptive immunity in chronic Hepatitis B patients.Front. Immunol. 2017; 8: 621Crossref PubMed Scopus (42) Google Scholar, 9Chan H.L.Y. Ahn S.H. Chang T.T. Peng C.Y. Wong D. Coffin C.S. Lim S.G. Chen P.J. Janssen H.L.A. Marcellin P. Serfaty L. Zeuzem S. Cohen D. Critelli L. Xu D. Wind-Rotolo M. Cooney E. Peginterferon lambda for the treatment of HBeAg-positive chronic hepatitis B: A randomized phase 2b study (LIRA-B).J. Hepatol. 2016; 64: 1011-1019Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). IFN-λ has 4 subtypes, namely IFN-λ1, -λ2, -λ3, and –λ4. IFN-λ3 was shown to have superior antiviral potency compared with other IFN-λ subtypes (10Dellgren C. Gad H.H. Hamming O.J. Melchjorsen J. Hartmann R. Human interferon-lambda3 is a potent member of the type III interferon family.Genes Immunity. 2009; 10: 125-131Crossref PubMed Scopus (129) Google Scholar). IFN-λ receptor is composed of an IFNLR1 and IL10R2 dimer; hence upon ligand binding, the receptor activates both IFN and IL-10-like signaling pathways. The type I IFN-receptors also form a heterodimer (IFNAR1 and IFNAR2), however, the sequences of all these receptors do diverge, particularly at the C terminus (11Pestka S. The interferon receptors.Sem. Oncol. 1997; 24 (S9-18-S19-40)Google Scholar, 12de Weerd N.A. Samarajiwa S.A. Hertzog P.J. Type I Interferon Receptors: Biochemistry and Biological Functions.J. Biol. Chem. 2007; 282: 20053-20057Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar). IFNLR1 is expressed only in epithelial cells including hepatocytes in contrast to IFN-α receptor, which is widely expressed in many cell types throughout the body; thus IFN-λ treatment results in fewer side-effects when compared with IFN-α treatment (9Chan H.L.Y. Ahn S.H. Chang T.T. Peng C.Y. Wong D. Coffin C.S. Lim S.G. Chen P.J. Janssen H.L.A. Marcellin P. Serfaty L. Zeuzem S. Cohen D. Critelli L. Xu D. Wind-Rotolo M. Cooney E. Peginterferon lambda for the treatment of HBeAg-positive chronic hepatitis B: A randomized phase 2b study (LIRA-B).J. Hepatol. 2016; 64: 1011-1019Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 13Miller D.M. Klucher K.M. Freeman J.A. Hausman D.F. Fontana D. Williams D.E. Interferon lambda as a potential new therapeutic for hepatitis C.Ann. N.Y. Acad. Sci. 2009; 1182: 80-87Crossref PubMed Scopus (54) Google Scholar, 14Ramos E.L. Preclinical and clinical development of pegylated interferon-lambda 1 in chronic hepatitis C.J. Interferon Cytokine Res. 2010; 30: 591-595Crossref PubMed Scopus (49) Google Scholar). IFN-λ has been shown to activate the JAK-STAT pathway, inducing formation of the ISGF3 transcription complex, leading to expression of interferon-stimulated genes (ISGs) in similar fashion to type I IFN, but with a different temporal profile compared with those induced by type I IFN (15Jilg N. Lin W. Hong J. Schaefer E.A. Wolski D. Meixong J. Goto K. Brisac C. Chusri P. Fusco D.N. Chevaliez S. Luther J. Kumthip K. Urban T.J. Peng L.F. Lauer G.M. Chung R.T. Kinetic Differences in the Induction of Interferon Stimulated Genes by Interferon-α and IL28B are altered by Infection with Hepatitis C Virus.Hepatology. 2014; 59: 1250-1261Crossref PubMed Scopus (80) Google Scholar, 16Bolen C.R. Ding S. Robek M.D. Kleinstein S.H. Dynamic expression profiling of type I and type III interferon-stimulated hepatocytes reveals a stable hierarchy of gene expression.Hepatology. 2014; 59: 1262-1272Crossref PubMed Scopus (134) Google Scholar, 17Marcello T. Grakoui A. Barba Spaeth –G Machlin E.S. Kotenko S.V. Macdonald M.R. Rice C.M. Interferons α and λ inhibit Hepatitis C virus replication with distinct signal transduction and gene regulation kinetics.Gastroenterology. 2006; 131: 1887-1898Abstract Full Text Full Text PDF PubMed Scopus (479) Google Scholar). A limited number of reports regarding IL-10-like signaling pathways have been published, with evidence of STAT3/5 biological activities (18Dumoutier L. Tounsi A. Michiels T. Sommereyns C. Kotenko S.V. Renauld J.C. Role of the interleukin (IL)-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/interferon-lambda 1: similarities with type I interferon signaling.J. Biol. Chem. 2004; 279: 32269-32274Abstract Full Text Full Text PDF PubMed Scopus (281) Google Scholar, 19Donnelly R.P. Sheikh F. Kotenko S.V. Dickensheets H. The expanded family of class II cytokines that share the IL-10 receptor-2 (IL-10R2) chain.J. Leukocyte Biol. 2004; 76: 314-321Crossref PubMed Scopus (242) Google Scholar). In fact, no reports have comprehensively investigated the downstream molecular signaling effects of IFN-λ. To better understand molecular mechanisms underlying direct antiviral and immunomodulatory effects of IFN-λ3, a comprehensive catalogue of protein effectors regulated by IFN-λ3 was compiled using quantitative proteomics analysis in the well-established HBV-transfected hepatoblastoma cell line model viz. HepG2.2.15 cells (20Sells M.A. Chen M.L. Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 1005-1009Crossref PubMed Scopus (997) Google Scholar). HepG2.2.15 cells have been widely used as a model of chronic hepatitis B because they support HBV replication and virion secretion (21Witt-Kehati D. Bitton Alaluf M. Shlomai A. Advances and challenges in studying Hepatitis B virus in vitro.Viruses. 2016; 8: 21Crossref Scopus (21) Google Scholar, 22Verrier E.R. Colpitts C.C. Schuster C. Zeisel M.B. Baumert T.F. Cell culture models for the investigation of Hepatitis B and D virus infection.Viruses. 2016; 8: 261Crossref Scopus (42) Google Scholar, 23Wang J. Jiang D. Zhang H. Lv S. Rao H. Fei R. Wei L. Proteome responses to stable hepatitis B virus transfection and following interferon alpha treatment in human liver cell line HepG2.Proteomics. 2009; 9: 1672-1682Crossref PubMed Scopus (24) Google Scholar, 24Otsuka M. Aizaki H. Kato N. Suzuki T. Miyamura T. Omata M. Seki N. Differential cellular gene expression induced by hepatitis B and C viruses.Biochem. Biophys. Res. Commun. 2003; 300: 443-447Crossref PubMed Scopus (39) Google Scholar, 25Xin X.M. Li G.Q. Guan X.R. Li D. Xu W.Z. Jin Y.Y. Gu H.X. Combination therapy of siRNAs mediates greater suppression on hepatitis B virus cccDNA in HepG2.2.15 cell.Hepato-gastroenterology. 2008; 55: 2178-2183PubMed Google Scholar, 26Li G.Q. Xu W.Z. Wang J.X. Deng W.W. Li D. Gu H.X. Combination of small interfering RNA and lamivudine on inhibition of human B virus replication in HepG2.2.15 cells.World J. Gastroenterol. 2007; 13: 2324-2327Crossref PubMed Scopus (18) Google Scholar, 27Ding X.R. Yang J. Sun D.C. Lou S.K. Wang S.Q. Whole genome expression profiling of hepatitis B virus-transfected cell line reveals the potential targets of anti-HBV drugs.Pharmacogenomics J. 2008; 8: 61-70Crossref PubMed Scopus (21) Google Scholar). These new findings could improve the treatment strategy of HBV infection and expand potential applications of IFN-λ3. Mixing of dimethyl-labeled samples corresponding to IFN-λ3, IFN-α2a, and control (PBS) treatments were performed at n = 5 versus the typical n = 3 to emphasize depth. Only biological replicates were performed. The normality of all proteomics, western-blotting and qPCR data was evaluated with the Shapiro-Wilk test. In all cases, the unpaired Student's t test or one-way ANOVA was selected when the distribution of the data was normal; otherwise the Mann-Whitney U test was applied. For proteomic analysis, peak intensity log2 ratios (L/M, L/H, M/H) were compared against a value of 0 (no change, log2(1)). Significance was based on the following criterion: p value < 0.05 or cases where proteins were detected in only one condition making standard statistical analysis inapplicable, with the requirement that these proteins must be identified in at least 4 of 5 experiments. Proteins not fulfilling the above criteria as well as those appearing in fewer than 3 experiments, were excluded from downstream analysis. Regarding western-blotting and qPCR experiments, n = 3 was set. One-way ANOVA was performed for these experiments with p value < 0.05 considered significant. In the case of DAVID enrichment analysis, all proteins identified by mass spectrometry were input as background. When Fisher-based enrichment analysis was performed against an in-house database of proteomic/transcriptomic studies, resulting log (p values) were conservatively adjusted by subtracting the log value corresponding to the total of all possible study/study combinations in the database. HepG2.2.15 cells are stable HBV-transfected cells derived from a hepatoblastoma HepG2 cell line (20Sells M.A. Chen M.L. Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 1005-1009Crossref PubMed Scopus (997) Google Scholar). These cells contain a plasmid which expresses the complete genome of HBV, which integrates into host cellular DNA (20Sells M.A. Chen M.L. Acs G. Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA.Proc. Natl. Acad. Sci. U.S.A. 1987; 84: 1005-1009Crossref PubMed Scopus (997) Google Scholar, 28Wang L.Y. Li Y.G. Chen K. Li K. Qu J.L. Qin D.D. Tang H. Stable expression and integrated hepatitis B virus genome in a human hepatoma cell line.Gen. Mol. Res. 2012; 11: 1442-1448Crossref PubMed Scopus (4) Google Scholar). Because HBV within HepG2.2.15 replicates and secretes HBsAg, HBeAg and HBV DNA into the culture media, HepG2.2.15 has been widely used as a model of chronic hepatitis B (22Verrier E.R. Colpitts C.C. Schuster C. Zeisel M.B. Baumert T.F. Cell culture models for the investigation of Hepatitis B and D virus infection.Viruses. 2016; 8: 261Crossref Scopus (42) Google Scholar, 28Wang L.Y. Li Y.G. Chen K. Li K. Qu J.L. Qin D.D. Tang H. Stable expression and integrated hepatitis B virus genome in a human hepatoma cell line.Gen. Mol. Res. 2012; 11: 1442-1448Crossref PubMed Scopus (4) Google Scholar). The HepG2.2.15 cell line was kindly provided from Professor Antonio Bertoletti (Singapore Institute for Clinical Sciences, A*Star). These cells were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco, MA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco), 1% MEM Non-Essential Amino Acids (MEM-NEAA; Gibco), 1% Penicillin/Streptomycin (Gibco) and Geneticin (G418; Gibco) at a final concentration of 150 μg/ml. The cultured cells were grown in a humidified incubator at 37 °C with 5% CO2. One million HepG2.2.15 cells were seeded into 6-well plates with 1 ml media and maintained in complete DMEM for 24 h. For determining the effects of IFN-λ3 on HBV replication, these cells were left untreated or treated with 1, 10, 100 or 1000 ng/ml of IFN-λ3 (5259-IL-025, R&D Systems, MN) and incubated for another 24 h. For proteomics analysis, HepG2.2.15 cells were plated at 5 × 106 cells in T-75 flasks and grown in complete DMEM for 24 h at 37 °C. These cells were subsequently cultured in media with 100 ng/ml of IFN-λ3 or 100 ng/ml of IFN-α2a (11100–1, pbl assay science, NJ) or PBS (control) for another 24 h. For further qPCR experiments with an optimized concentration of IFN-λ3, HepG2.2.15 cells were stimulated with or without 100 ng/ml of IFN-λ3 for 0, 8, 16, and 24 h. TRIzol Reagent (Thermo, MA) was used to extract total RNA as specified in the accompanying manual. Complementary DNA (cDNA) synthesis was carried out using the Taqman Reverse transcription kit (Applied Biosystems, MA). Conditions for reverse transcription were as specified in the manual. Relative gene expression was measured with the ABI Prism 7500 sequence detection system (Applied Biosystems). All primers and probes were designed with the “primer express 3” program (Applied Biosystems), and are shown in supplemental Table S1. To monitor gene amplification, the intensity of fluorescence from the 18S housekeeping gene was monitored via Taqman probe whereas all target gene levels were monitored via SYBR green dye (Applied Biosystems). To test the specificity of SYBR green dye, melting curve analysis was conducted for all target genes. The condition of amplification for both target and housekeeping genes was 1 cycle at 95 °C for 5 min followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. Relative gene expression was calculated with the 2−ddCt method. The Student's t test and one-way ANOVA were used to compare the relative expressions of target genes in cells treated with various doses of IFN-λ3. A p value less than 0.05 was considered significant. After trypsinization and PBS-washing, cellular DNA was extracted using the QIAamp DNA Blood Mini Kit (Qiagen, MD) according to manufacturer's instructions. Quantification of HBV viral load was performed by absolute quantitative real-time PCR using the ABI Prism 7500 sequence detection system (Applied Biosystems). For ease of plasmid amplification, we used plasmids containing only the HBV gene preS1 as a surrogate marker for HBV DNA (preS1 was chosen because it is highly conserved across all HBV genotypes). First, preS1 plasmids were extracted from E. coli transformants with the GeneJET Plasmid Miniprep Kit (Fermentas, MA). The concentration of extracted plasmids was measured by spectrophotometer (Nanodrop, Thermo) and copy/μl was determined. The plasmid concentration was adjusted and diluted in a range of 107, 106, 105, 104, 103, and 102 copy/μl. These concentrations were used to construct a standard curve. Both standard and sample preS1 were amplified at the same time using conditions described above. The fluorescent intensities were specified as Ct values. For standards, Ct values at the above concentrations were used to plot a standard curve. This curve and sample Ct values were used to calculate the amount of HBV DNA in the samples. The Student's t test and one-way ANOVA were used to compare viral loads in cells treated with various levels of IFN-λ3, as well as untreated cells. A p value less than 0.05 was considered significant. HepG2.2.15 cells were seeded in 96-well plates at a density of 1 × 104 cells per well and incubated for 24 h. The culture media was removed and replaced with fresh complete media in the absence or presence of IFN-λ3 (1, 10, 100 and 1000 ng/ml). The cells were further incubated for 24 h followed by addition of 10 μl 5 mg/ml MTT (Sigma, MO) solution in each well with gentle shaking. After 4h incubation, the resulting purple formazan crystals were dissolved with dimethyl sulfoxide (DMSO, Riedel-deHaën, NJ) and subsequently measured with ELISA plate reader (Thermo) at wavelength 570 nm. The absorbance values of cells treated with each concentration of drug were compared with that of control (untreated) cells and % cell viability was calculated. This experiment was performed in triplicate. After trypsinization, the cells were lysed with 5% sodium deoxycholate (SDC) and 1X protease inhibitor (Thermo) mixture, followed by sonication. All cell debris was removed by centrifugation and supernatant protein concentrations of each sample were measured via BCA Protein Assay (Thermo). Equal amounts of protein from treated and untreated HepG2.2.15 were reduced and alkylated by dithiothreitol (DTT) treatment for 30 min at 37 °C and iodoacetamide (IA) treatment for 30 min at room temperature in the dark, respectively. These samples were further quenched with DTT at least 15 min at room temperature before incubating with trypsin at a ratio of 1:50 at 37 °C overnight. These mixtures were incubated with 0.5% trifluoroacetic acid (TFA) for 30 min and then centrifuged to remove SDC precipitate. The amount of tryptic peptide of each sample was determined with the Pierce Quantitative Fluorometric Peptide Assay (Thermo). Peptides from untreated, IFN-α2a treated, and IFN-λ3 treated HepG2.2.15 were labeled with light reagents (formaldehyde and cyanoborohydride), medium reagents (formaldehyde-d2 and cyanoborohydride), and heavy reagents (deuterated and 13C-labeled formaldehyde and cyanoborodeuteride), respectively, for an hour at room temperature. Ammonia solution and formic acid (FA) were sequentially used to stop the reaction. Labeling efficiency was tested, and we found that greater than 99% of peptides were labeled (data not shown). After combining these three samples, the mixed labeled-peptides were dried in a SpeedVac centrifuge at room temperature. Next, the pooled peptides were separated into 10 fractions to reduce complexity using the Pierce High pH Reversed-Phase Peptide Fractionation Kit (Thermo). Eluates of each fraction were dried in a SpeedVac centrifuge before LC-MS/MS analysis. The fractionated samples were resuspended in 0.1% FA (Sigma) to a final volume of 15 μl prior to MS injection. The peptides were then analyzed via an EASY-nLC1000 system (Thermo) coupled to a Q-Exactive Orbitrap Plus mass spectrometer (Thermo) equipped with a nano-electrospray ion source (Thermo). The peptides were eluted in 5–40% acetonitrile in 0.1% FA for 70 min followed by 40–95% acetonitrile in 0.1% FA for 20 min at a flow rate of 300 nl/min. The MS methods included a full MS scan at a resolution of 70,000 followed by 10 data-dependent MS2 scans at a resolution of 17,500. The normalized collision energy of HCD fragmentation was set at 32%. An MS scan range of 350 to 1400 m/z was selected and precursor ions with unassigned charge states, a charge state of +1, or a charge state of greater than +8 were excluded. A dynamic exclusion of 30 s was used. The peaklist-generating software used in this study was Proteome Discoverer™ Software 2.1 (Thermo). The SEQUEST-HT search engine was employed in data processing. MS raw data files were searched against the Human Swiss-Prot Database (20,219 proteins, June 2017) and the Hepatitis B Virus Swiss-Prot Database (225 proteins, June 2017), as well as a list of common protein contaminants (www.thegpm.org/crap/). The following parameters were set for the search: (1Schweitzer A. Horn J. Mikolajczyk R.T. Krause G. Ott J.J. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013.Lancet. 2015; 386: 1546-1555Abstract Full Text Full Text PDF PubMed Scopus (1811) Google Scholar) digestion enzyme: trypsin; (2Grimm D. Thimme R. Blum H.E. HBV life cycle and novel drug targets.Hepatol. Int. 2011; 5: 644-653Crossref PubMed Scopus (91) Google Scholar) maximum allowance for missed cleavages: 2; (3Wright T.L. Introduction to chronic hepatitis B infection.Am. J. Gastroenterol. 2006; 101: S1-S6Crossref PubMed Scopus (158) Google Scholar) maximum of modifications: 4; (4EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection.J. Hepatol. 2017; 67: 370-398Abstract Full Text Full Text PDF PubMed Scopus (2987) Google Scholar) fixed modifications: carbamidomethylation of cysteine (+57.02146 Da), as well as light, medium, and heavy dimethylation of N termini and lysine (+28.031300, +32.056407, and +36.075670 Da); (5Yuen M.F. Lai C.L. Treatment of chronic hepatitis B: Evolution over two decades.J. Gastroenterol. Hepatol. 2011; 26: 138-143Crossref PubMed Scopus (142) Google Scholar) variable modifications: oxidation of methionine (+15.99491 Da). The mass tolerances for precursor and fragment ions were set to 10 ppm and 0.02 Da, respectively. Known contaminant ions were excluded. The Proteome Discoverer decoy database together with the Percolator algorithm were used to calculate the false positive discovery rate of the identified peptides based on Q-values which were set to 1%. The Precursor Ions Quantifier node in Proteome Discoverer™ Software was employed to quantify the relative MS signal intensities of dimethyl labeled-peptides. The control channels were used as denominators to generate abundance ratios of IFN-λ3/control and IFN-α2a/control. Log2 of the normalized ratio was used to calculate the mean and standard deviation of fold change across all five biological replicates. When these ratios were found in less than three experiments, the relevant proteins were excluded. Significantly differentially regulated proteins were determined by Mann-Whitney U test and unpaired t-tests with p value < 0.05 considered significant. We compiled a list of defense response to virus using a variety of resources as follows. The online resource Database for Annotation, Visualization and Integrated Discovery (DAVID, v6.8, https://david.ncifcrf.gov/) and Reactome (https://reactome.org/) were employed to classify the proteins regulated by IFN-λ3 into functional categories using all proteins identified by MS as background (for DAVID). We used terms such as “antiviral,” “antigen processing/presentation” to help extract a custom list of broad antiviral proteins. Additionally, the list contains proteins involved in the HBV life-cycle that were derived from manual literature curation. Further analysis of up- and down-regulated proteins was per" @default.
• W2887501590 created "2018-08-22" @default.
• W2887501590 creator A5016385732 @default.
• W2887501590 creator A5024511017 @default.
• W2887501590 creator A5026991644 @default.
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• W2887501590 date "2018-11-01" @default.
• W2887501590 modified "2023-10-16" @default.
• W2887501590 title "Comprehensive Proteomics Identification of IFN-λ3-regulated Antiviral Proteins in HBV-transfected Cells" @default.
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