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• W2275029764 abstract "The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. The non-structural protein 5A (NS5A) is a hepatitis C virus (HCV) protein indispensable for the viral life cycle. Many prior papers have pinpointed several serine residues in the low complexity sequence I region of NS5A responsible for NS5A phosphorylation; however, the functions of specific phosphorylation sites remained obscure. Using phosphoproteomics, we identified three phosphorylation sites (serines 222, 235, and 238) in the NS5A low complexity sequence I region. Reporter virus and replicon assays using phosphorylation-ablated alanine mutants of these sites showed that Ser-235 dominated over Ser-222 and Ser-238 in HCV replication. Immunoblotting using an Ser-235 phosphorylation-specific antibody showed a time-dependent increase in Ser-235 phosphorylation that correlated with the viral replication activity. Ser-235 phosphorylated NS5A co-localized with double-stranded RNA, consistent with its role in HCV replication. Mechanistically, Ser-235 phosphorylation probably promotes the replication complex formation via increasing NS5A interaction with the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein. Casein kinase Iα (CKIα) directly phosphorylated Ser-235 in vitro. Inhibition of CKIα reduced Ser-235 phosphorylation and the HCV RNA levels in the infected cells. We concluded that NS5A Ser-235 phosphorylated by CKIα probably promotes HCV replication via increasing NS5A interaction with the 33-kDa vesicle-associated membrane protein-associated protein. Chronic HCV 2The abbreviations used are: HCV, hepatitis C virus; CKI and CKII, casein kinase I and II, respectively. infection affects 130–170 million people worldwide (1Scheel T.K. Rice C.M. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.Nat. Med. 2013; 19: 837-849Crossref PubMed Scopus (430) Google Scholar). The infection is often asymptomatic until development of severe liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma, making chronic HCV infection the most common cause of liver transplant (2Thomas D.L. Global control of hepatitis C: where challenge meets opportunity.Nat. Med. 2013; 19: 850-858Crossref PubMed Scopus (230) Google Scholar). HCV is an enveloped virus with a positive, single-stranded RNA genome encoding three structural (core, E1, and E2) and seven non-structural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (1Scheel T.K. Rice C.M. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.Nat. Med. 2013; 19: 837-849Crossref PubMed Scopus (430) Google Scholar). The structural proteins together with the host membranes make up the viral particles, whereas the non-structural proteins are required for a complete life cycle. Already, there are several approved highly efficient HCV antivirals targeting non-structural proteins, including NS3/4A protease inhibitors (boceprevir, telaprevir, and simeprevir) and an NS5B RNA-dependent RNA polymerase inhibitor (sofosbuvir) (3Pawlotsky J.M. New hepatitis C therapies: the toolbox, strategies, and challenges.Gastroenterology. 2014; 146: 1176-1192Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar). However, their high costs prohibit their accessibility to most patients (4Hill A. Cooke G. Medicine: hepatitis C can be cured globally, but at what cost?.Science. 2014; 345: 141-142Crossref PubMed Scopus (53) Google Scholar). New competitive alternatives are desirable. NS5A is a multitasking protein required for the HCV life cycle and thus a good antiviral target (5Reghellin V. Donnici L. Fenu S. Berno V. Calabrese V. Pagani M. Abrignani S. Peri F. De Francesco R. Neddermann P. NS5A inhibitors impair NS5A-PI4KIIIα complex formation and cause a decrease of PI4P and cholesterol levels in HCV-associated membranes.Antimicrob. Agents Chemother. 2014; 58: 7128-7140Crossref PubMed Scopus (28) Google Scholar). It is a phosphoprotein that appears as two bands at 56 and 58 kDa on immunoblots, respectively, referred to as hypophosphorylated (p56) and hyperphosphorylated (p58) NS5A (6Huang Y. Staschke K. De Francesco R. Tan S.L. Phosphorylation of hepatitis C virus NS5A nonstructural protein: a new paradigm for phosphorylation-dependent viral RNA replication?.Virology. 2007; 364: 1-9Crossref PubMed Scopus (131) Google Scholar). NS5A interacts with many viral and host proteins and participates in various aspects of the viral life cycle (7Ross-Thriepland D. Harris M. Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!.J. Gen. Virol. 2015; 96: 727-738Crossref PubMed Scopus (104) Google Scholar). For example, NS5A was reported to interact with the hVAP-A protein that takes part in the replication protein complex formation (8Evans M.J. Rice C.M. Goff S.P. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13038-13043Crossref PubMed Scopus (275) Google Scholar, 9Tu H. Gao L. Shi S.T. Taylor D.R. Yang T. Mircheff A.K. Wen Y. Gorbalenya A.E. Hwang S.B. Lai M.M. Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein.Virology. 1999; 263: 30-41Crossref PubMed Scopus (208) Google Scholar10Gao L. Aizaki H. He J.W. Lai M.M. Interactions between viral nonstructural proteins and host protein hVAP-33 mediate the formation of hepatitis C virus RNA replication complex on lipid raft.J. Virol. 2004; 78: 3480-3488Crossref PubMed Scopus (274) Google Scholar). NS5A mutations that disrupted the interaction with hVAP-A strongly reduced HCV RNA replication (8Evans M.J. Rice C.M. Goff S.P. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13038-13043Crossref PubMed Scopus (275) Google Scholar). A subset of the genotype 1 HCV with mutations that confer replication fitness shows enhanced NS5A interaction with hVAP-A and suppressed NS5A hyperphosphorylation (8Evans M.J. Rice C.M. Goff S.P. Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 13038-13043Crossref PubMed Scopus (275) Google Scholar). Thus, NS5A hyperphosphorylation was concluded to reduce genotype 1 HCV replication via reducing NS5A interaction with hVAP-A. A lot of effort has been devoted to identifying NS5A phosphorylation sites: by mutating potential sites followed by immunoblotting (11Appel N. Pietschmann T. Bartenschlager R. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain.J. Virol. 2005; 79: 3187-3194Crossref PubMed Scopus (191) Google Scholar, 12Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1276) Google Scholar13Fridell R.A. Valera L. Qiu D. Kirk M.J. Wang C. Gao M. Intragenic complementation of hepatitis C virus NS5A RNA replication-defective alleles.J. Virol. 2013; 87: 2320-2329Crossref PubMed Scopus (32) Google Scholar), by overexpressing NS5A in non-liver cells followed by Edman degradation or mass spectrometry (14Katze M.G. Kwieciszewski B. Goodlett D.R. Blakely C.M. Neddermann P. Tan S.L. Aebersold R. Ser(2194) is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A.Virology. 2000; 278: 501-513Crossref PubMed Scopus (61) Google Scholar, 15Reed K.E. Rice C.M. Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321.J. Biol. Chem. 1999; 274: 28011-28018Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), by transfecting the HCV replicon into liver cells followed by mass spectrometry (16Lemay K.L. Treadaway J. Angulo I. Tellinghuisen T.L. A hepatitis C virus NS5A phosphorylation site that regulates RNA replication.J. Virol. 2013; 87: 1255-1260Crossref PubMed Scopus (34) Google Scholar, 17Nordle Gilliver A. Griffin S. Harris M. Identification of a novel phosphorylation site in hepatitis C virus NS5A.J. Gen. Virol. 2010; 91: 2428-2432Crossref PubMed Scopus (10) Google Scholar18Ross-Thriepland D. Harris M. Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation.J. Virol. 2014; 88: 1421-1432Crossref PubMed Scopus (52) Google Scholar), and by screening kinases that interact with NS5A in the liver cells (19Masaki T. Matsunaga S. Takahashi H. Nakashima K. Kimura Y. Ito M. Matsuda M. Murayama A. Kato T. Hirano H. Endo Y. Lemon S.M. Wakita T. Sawasaki T. Suzuki T. Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.J. Virol. 2014; 88: 7541-7555Crossref PubMed Scopus (66) Google Scholar). Most of the identifications centered around eight highly conserved serine residues in the LCS I region of NS5A. Among them, phosphorylation-ablated alanine mutation at Ser-229 or Ser-235 resulted in a profound reduction in the HCV genotype 2a activity (13Fridell R.A. Valera L. Qiu D. Kirk M.J. Wang C. Gao M. Intragenic complementation of hepatitis C virus NS5A RNA replication-defective alleles.J. Virol. 2013; 87: 2320-2329Crossref PubMed Scopus (32) Google Scholar, 19Masaki T. Matsunaga S. Takahashi H. Nakashima K. Kimura Y. Ito M. Matsuda M. Murayama A. Kato T. Hirano H. Endo Y. Lemon S.M. Wakita T. Sawasaki T. Suzuki T. Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.J. Virol. 2014; 88: 7541-7555Crossref PubMed Scopus (66) Google Scholar); however, the alanine mutations at these two sites seemed to have different effects on the levels of NS5A hyperphosphorylation. Potentially, the above observations are due to the lack of phosphorylation site-specific antibodies that could distinguish the so-called hyperphosphorylated band (p58) of NS5A. Recently, an antibody against Ser-222 phosphorylation was developed (18Ross-Thriepland D. Harris M. Insights into the complexity and functionality of hepatitis C virus NS5A phosphorylation.J. Virol. 2014; 88: 1421-1432Crossref PubMed Scopus (52) Google Scholar), but the functions of Ser-222 phosphorylation remain unclear because alanine mutation at Ser-222 does not have an apparent phenotype (11Appel N. Pietschmann T. Bartenschlager R. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain.J. Virol. 2005; 79: 3187-3194Crossref PubMed Scopus (191) Google Scholar, 13Fridell R.A. Valera L. Qiu D. Kirk M.J. Wang C. Gao M. Intragenic complementation of hepatitis C virus NS5A RNA replication-defective alleles.J. Virol. 2013; 87: 2320-2329Crossref PubMed Scopus (32) Google Scholar, 19Masaki T. Matsunaga S. Takahashi H. Nakashima K. Kimura Y. Ito M. Matsuda M. Murayama A. Kato T. Hirano H. Endo Y. Lemon S.M. Wakita T. Sawasaki T. Suzuki T. Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.J. Virol. 2014; 88: 7541-7555Crossref PubMed Scopus (66) Google Scholar). Moreover, whereas alanine mutation at Ser-225, Ser-229, Ser-232, and Ser-235 reduced HCV genotype 2a activity, the same mutations enhanced genotype 1b activity (11Appel N. Pietschmann T. Bartenschlager R. Mutational analysis of hepatitis C virus nonstructural protein 5A: potential role of differential phosphorylation in RNA replication and identification of a genetically flexible domain.J. Virol. 2005; 79: 3187-3194Crossref PubMed Scopus (191) Google Scholar), adding another layer of complexity to the functions of NS5A phosphorylation (7Ross-Thriepland D. Harris M. Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on!.J. Gen. Virol. 2015; 96: 727-738Crossref PubMed Scopus (104) Google Scholar). To discover HCV phosphoproteins in the conditions that resemble viral infection, we took advantage of the cell culture-derived infectious HCV system (20Wakita T. Pietschmann T. Kato T. Date T. Miyamoto M. Zhao Z. Murthy K. Habermann A. Kräusslich H.G. Mizokami M. Bartenschlager R. Liang T.J. Production of infectious hepatitis C virus in tissue culture from a cloned viral genome.Nat. Med. 2005; 11: 791-796Crossref PubMed Scopus (2409) Google Scholar) and identified three serine phosphorylation sites (Ser-222, Ser-235, and Ser-238) in the LCS I region of NS5A in the HCV (J6/JFH-1)-infected Huh7.5.1 liver cells using LC-MS/MS-based phosphoproteomics. Subsequent study using molecular virology and a phosphorylation site-specific antibody showed that Ser-235 is a CKIα phosphorylation site of NS5A responsible for enhancing genotype 2a HCV replication, probably via enhancing interaction with hVAP-A. Human hepatocarcinoma 7.5.1 cell line (Huh7.5.1) originated in Francis V. Chisari's laboratory in the Scripps Research Institute was used in most experiments (21Zhong J. Gastaminza P. Cheng G. Kapadia S. Kato T. Burton D.R. Wieland S.F. Uprichard S.L. Wakita T. Chisari F.V. Robust hepatitis C virus infection in vitro.Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 9294-9299Crossref PubMed Scopus (1513) Google Scholar). The cells were cultured in DMEM (Invitrogen, catalogue no. 12100-046) with 10% fetal bovine serum (Biological Industries, catalogue no. 040011B) without antibiotics. A rabbit antibody specific to Ser-235-phosphorylated NS5A was custom-made by GeneTex Corp. using a synthetic peptide (SQLpSAPSLKATC, where pS indicates phosphorylated serine). Antibodies against HCV NS5A (7B5 and 2F6) and NS3 (2E3) were obtained from BioFront Technologies. The double-stranded RNA antibody (J2-1402) was from English and Scientific Consulting Kft., the casein kinase Iα antibody (sc-6477) was from Santa Cruz Biotechnology, Inc., and the β-actin antibody (A5316) was from Sigma-Aldrich. The hVAP-A antibody (15275-1-AP) was from the Proteintech Group. DTT (catalogue no. 3483-12-3) and iodoacetamide (catalogue no. 144-48-9) were obtained from Thermo. Casein kinase inhibitor (D4476, catalogue no. D1944) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (catalogue no. M2003) were obtained from Sigma-Aldrich. The lipid droplet was stained with BODIPY 493/503 (catalogue no. D-3922) from Life Technologies. The full HCV genome (J6/JFH-1, 5′C19Rluc2AUbi) construct and the subgenome replicon (JFH-1/SG-Neo) were kindly provided by Charles M. Rice (Rockefeller University). The replication defect replicon construct (pSGR-JFH-1) was a gift from Timothy Tellinghuisen (Scripps Research Institute). The cytomegalovirus (CMV) promoter-driven NS3-NS5A expression construct was made via PCR amplification of an NS3-NS5A fragment from the full HCV genome (5′C19Rluc2AUbi) construct with the following primers: forward, 5′-AAGCTTATGGCTCCCA-3′ with a HindIII site at the 5′ end; reverse, 5′-TCTAGATCAGCAGCAC-3′ containing a stop codon and an XbaI site at the 3′ end. The NS3-NS5A fragment was first inserted into the pSTBlue-1 vector (Novagen, catalogue no. 70596). The NS3-NS5A fragment was then excised from the vector via HindIII and XbaI double digestion and ligated into the expression vector pcDNA3.1 (+) (Invitrogen, catalogue no. V790-20). The CMV-driven NS5A expression vectors were made using the Gateway system (Invitrogen). Briefly, the NS5A fragment was amplified with PCR and ligated into the entry vector using the pENTR directional TOPO cloning kits (catalog no. 2400-20). After a sequencing check, the NS5A insert was moved from the entry vector to the destination vector pcDNA-DEST40 (catalog no. 12274-015) using the Gateway LR Clonase II enzyme mix (catalog no. 11791-020). After transformation using the One Shot TOP10 chemically competent cells (catalog no. C4040-52, Invitrogen), the correct vectors were verified with DNA sequencing. Single or combinatory alanine mutations at Ser-222, Ser-235, and Ser-238 were made via site-directed mutagenesis using PCR (KOD hot start polymerase, Merck-Millipore, catalogue no. 71086-4) followed by DpnI (New England Biolabs, catalogue no. R0176L) digestion and transformation into DH5α competent cells. All plasmids were verified with DNA sequencing. Casein kinase Iα small hairpin RNA (shCSNK1A1) plasmid was obtained from the RNAi core facility at the Academia Sinica (Taipei, Taiwan). The target sequence is GCCACAGTTGTGATGGTTGTT. The control non-targeting shRNA sequence is CAAATCACAGAATCGTCGTAT. The procedures were described previously (22Rinschen M.M. Yu M.J. Wang G. Boja E.S. Hoffert J.D. Pisitkun T. Knepper M.A. Quantitative phosphoproteomic analysis reveals vasopressin V2-receptor-dependent signaling pathways in renal collecting duct cells.Proc. Natl. Acad. Sci. U.S.A. 2010; 107: 3882-3887Crossref PubMed Scopus (146) Google Scholar). Briefly, the Huh7.5.1 cells were infected with HCV (J6/JFH-1) for 72 h before being harvested in a lysis buffer containing 8 m urea, 75 mm NaCl, and 50 mm Tris (pH 8.0). The proteins were reduced with 10 mm DTT, alkylated with 55 mm iodoacetamide, and digested into peptides with trypsin. The tryptic peptides were then desalted using an OASIS HLB column (Waters, catalogue no. WAT094225). To enrich for phosphopeptides, strong cation exchange high performance liquid chromatography (polysulfoethyl A column, 200 × 4.6 mm, 5 μm, 200 Å, capacity: 4 mg of peptides, PolyLC) and immobilized metal affinity chromatography (Pierce IMAC spin column) were used. The conditions used for strong cation exchange were 100% solvent A (5 mm KH2PO4 in 25% acetonitrile, pH 2.6), 2 min; 86% solvent A, 14% solvent B (5 mm KH2PO4 and 500 mm KCl in 25% acetonitrile, pH 2.6), 15 min; 30% solvent A, 70% solvent B, 16 min; 100% solvent B, 11 min; 100% solvent A, 11 min. Phosphopeptide identification was performed on an LTQ-Orbitrap mass spectrometer. Spectra were collected in a data-dependent mode with one full precursor MS1 scan in the Orbitrap followed by six-fragment MS2 scans in the LTQ. Collision-induced dissociation was done at 35% normalized energy level. Peptide peak lists were generated with the Bioworks software package (version 3.3.1, SP1 (23Eng J.K. McCormack A.L. Yates J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5420) Google Scholar)) using the following criteria: precursor mass range between 600 and 3500 atomic mass units, precursor tolerance 1.4 atomic mass units for grouping spectra, a total ion current of >1000 (arbitrary units)/spectrum, and a minimum of 15 peaks/spectrum. Peptide identifications were done using the Sequest search algorithm against a database containing protein sequences of Homo sapiens (34,942 entries, NCBI RefSeq) and HCV JFH1 isolate (10 entries, Uniprot Q99IB8) plus common protein contaminants: porcine (P00761) and bovine (P00760) trypsin and human keratins (P35908, Q01546, P04264, P12035, P08729, and P35527). The search parameters were as follows: precursor mass tolerance 20 ppm, product mass tolerance 1.0 atomic mass unit, 2 missed cleavages, fixed cysteine carboxyiodomethylation, variable modifications: methionine oxidation plus serine, tyrosine, and threonine phosphorylation. The target-decoy strategy based on reversed protein sequences was used to set a false discovery rate of <1% for identified peptides (24Elias J.E. Gygi S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry.Nat. Methods. 2007; 4: 207-214Crossref PubMed Scopus (2827) Google Scholar). Common contaminants were excluded. Phosphorylation site-specific identification confidence was based on the Ascore algorithm (25Beausoleil S.A. Villen J. Gerber S.A. Rush J. Gygi S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization.Nat. Biotechnol. 2006; 24: 1285-1292Crossref PubMed Scopus (1205) Google Scholar). Cells were washed with ice-cold phosphate-buffered saline (PBS) and resuspended in a lysis buffer (50 mm HEPES, 150 mm NaCl, 5 mm EDTA, 0.2% Nonidet P-40, pH 7.6) containing protease inhibitor (Calbiochem, catalogue no. 539134) and phosphatase inhibitor (Calbiochem, catalogue no. 524625). After centrifugation at 10,000 × g at 4 °C, the supernatant was collected and quantified using a BCA assay kit (BIOTOOLS, catalogue no. TAAR-ZBE6). 20 μg of total protein were separated on a 7.5% SDS-polyacrylamide gel before being transferred to a nitrocellulose membrane (Bio-Rad, catalogue no. 162-0112). The membrane was blocked with TBS-T (150 mm NaCl, 50 mm Tris plus 0.05% Tween 20 and 0.1% bovine serum albumin (BSA), pH 7.6) and incubated with primary antibodies followed by infrared dye-conjugated secondary antibodies (LI-COR, IRDye 680 or 800). The proteins of interest were quantified using the LI-COR Odyssey scanner and software. Monolayer cells were seeded at a subconfluent density on a coverglass inside a Petri dish. Before staining, the cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 20 min. The cells were treated with a permeabilization buffer (0.3% Triton X-100 and 0.1% BSA in PBS) for 30 min, followed by a blocking buffer (1% BSA, 0.05% saponin, and 0.2% gelatin) for 10 min three times. The cells were probed with primary antibody and then a fluorescence (Alexa 488, 568, or 594)-labeled secondary antibody (Invitrogen). The cell nuclei were counterstained with DAPI (Sigma-Aldrich, catalogue no. D9542). Immunofluorescence images were taken using a Leica SP5C spectral confocal laser-scanning microscope and software. HCV constructs were linearized with XbaI (New England Biolabs, catalogue no. R0145) digestion before being transcribed into viral RNAs using the Ambion MEGAscript T7 in vitro transcription kit (Invitrogen, catalogue no. AM1334). The viral RNA was purified using phenol/chloroform extraction and isopropyl alcohol precipitation. The quality and quantity of the RNA were assessed by gel electrophoresis and a Thermo NanoDrop spectrophotometer. Electroporation was performed using the Amaxa 4D-Nucleofector system and the Amaxa SF 4D-Nucleofector X solution kit from Lonza. Briefly, the Huh7.5.1 cells were subcultured 1–2 days before electroporation so that they reached between 80 and 90% confluence on the day of electroporation. Prior to electroporation, the cells were trypsinized, washed in PBS, and suspended in the SF solution at a density of 1 × 106 cells/ml. A 100-μl aliquot of the cell suspension was mixed with 5 μg of the viral RNA and then transferred to a 100-μl cuvette for electroporation using the built-in program CM-137. The transfected cells were allowed to recover at room temperature for 10 min and then cultured in the DMEM supplemented with 10% FBS. One day before the experiment, the Huh7.5.1 cells were seeded at a density so that they reached 80% confluence on the next day for transfection. The viral RNA was mixed with DMRIE-C (Invitrogen, catalogue no. 10459-014) at a ratio of 1 μg/3 μl and used to transfect the Huh7.5.1 cells. At various time points (4, 12, 24, 48, and 72 h) after transfection, the cells were lysed and subjected to luciferase assay using the luciferase assay kit (Promega, catalogue no. E1500). Active casein kinase Iα was purchased from SignalChem (catalogue no. C64-10G-10). Biotinylated synthetic peptides mapped to amino acids 216–243 of NS5A (101 peptide, biotin-RRLARGpSPPSEASSSVSQLSAPpSLRATC; 111 peptide, biotin-RRLARGpSPPSEASSSVSQLpSAPpSLRATC) were synthesized by GeneTex Corp. The active kinase was mixed with the 101 peptide on ice. ATP solution was then added to the mixture and incubated at 30 °C for 15 min prior to detection of phosphorylation using dot blotting. The Huh7.5.1 cells were infected with HCV virus (J6/JFH-1) for 72 h at a multiplicity of infection of 0.001. The infected cells were then seeded in a 6-well plate at a density of 5 × 106 cells/well for 24 h before kinase inhibition with D4476. One μl of D4476 was dissolved in 3 μl of lipid-based transfection reagent (Mirus Bio, TransIT-LT1, catalogue no. MIR 2304) plus 6 μl of serum-free DMEM before being added to the cells. The cells were incubated with the inhibitor for 24 h before immunoblotting. For small hairpin RNA-based kinase silencing, shRNA harboring lentivirus was prepared and used to infect the cells before they were harvested for RNA and protein measurements. The cells were lysed with the TRIzol LS reagent (Ambion, catalogue no. 10296-010), and the total RNA was isolated with the direct-zol RNA Mini Prep kit (ZYMO Research, catalogue no. R2052). The RNA concentrations were determined by the NanoDrop spectrophotometer. For absolute quantification, 10 ng of RNA were subjected to one-step quantitative analysis using the One-Step qRT-PCR kit (KAPA Biosystems, catalogue no. KK4660) and the StepOnePlus Real-Time PCR system (Applied Biosystem). For relative quantification, 500 ng of RNA were subjected to reverse transcription using SuperScript III reverse transcriptase (Invitrogen, catalogue no. 18080-044) and 2 pm HCV gene-specific primer (5′-CACTCGCAAGCACCCTATCA-3′). qPCR was done using HCV-specific primers: sense, 5′-TCTGCGGAACCGGTGAGTA-3′; antisense, 5′-TCAGGCAGTACCACAAGGC-3′. Abundance of the 18S rRNA was used as an internal control. The primers were 5′-AAACGGCTACCACATCCAAG-3′ (sense) and 5′-CCTCCAATGGATCCTCGTTA-3′ (antisense). To identify phosphorylation sites of the HCV proteins, we analyzed the phosphoproteome of HCV (genotype 2a, J6/JFH-1)-infected Huh7.5.1 cells. 1,087 proteins with 1,773 phosphorylation sites were identified with high confidence (Ascore > 20; i.e. FDR < 0.01) (25Beausoleil S.A. Villen J. Gerber S.A. Rush J. Gygi S.P. A probability-based approach for high-throughput protein phosphorylation analysis and site localization.Nat. Biotechnol. 2006; 24: 1285-1292Crossref PubMed Scopus (1205) Google Scholar). Fig. 1 classifies the phosphorylation sites identified. Serine phosphorylation sites were the largest group, which was dominated by proline-directed sites, followed by acidophilic sites and then basophilic sites. The Phosphoproteome Database of HCV-infected Human Hepatocellular Carcinoma 7.5.1 Cells was established. NS5A was the only HCV phosphoprotein identified with three high confidence serine phosphorylation sites (Table 1, Ser-222, Ser-235, and Ser-238) in the LCS I region and conserved across major HCV strains (Fig. 2A). All three sites were identified individually in singly phosphorylated peptides (Table 1). Ser-235 and Ser-238 were identified in a doubly phosphorylated peptide. Three additional phosphorylation sites identified with lower confidence were Ser-225 (Ascore = 16.8), Ser-229 (8.6), and Ser-232 (5.1). Raw mass spectrometry data were deposited in the ProteomeXchange Consortium with the identifier PXD000988.TABLE 1Phosphopeptides identified for the HCV non-structural protein NS5APeptide sequenceMH+ChargeXcorrΔCnIonsAscoreSer-222Ser-235Ser-238GSPPSEASSSVSQLSAPSLR2,023.934.960.0941/11224.75GSPPSEASSSVSQLSAPSLR2,023.924.980.1732/5644.73GSPPSEASSSVSQLSAPSLR2,023.922.990.3122/5627.99GSPPSEASSSVSQLSAPSLR2,103.933.810.2645/14839.6345.69 Open table in a new tab To investigate roles of Ser-222, Ser-235, and Ser-238 phosphorylation sites, we made single, double, and triple mutations of the three sites to phosphorylation-ablated alanine in a full-length HCV reporter construct (Rluc-J6/JFH-1). Fig. 2B summarizes the reporter activity at various time points after the in vitro transcripts of the viral constructs were transfected into the Huh7.5.1 cells. As seen, the reporter activities of the serine-to-alanine mutants, either at Ser-222 (S222A) or Ser-238 (S238A), did not differ from that of the wild type. In contrast, the reporter activity of the S235A mutant was significantly suppressed, suggesting a crucial role of Ser-235 phosphorylation in the HCV activity. In fact, as long as Ser-235 was mutated in double or triple alanine mutation, the reporter activity remained significantly lower than that of the wild type virus (Fig. 2C), consistent with a predominant role of Ser-235 phosphorylation over Ser-222 and Ser-238 phosphorylation in the HCV" @default.
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• W2275029764 date "2016-02-01" @default.
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• W2275029764 title "Phosphoproteomics Identified an NS5A Phosphorylation Site Involved in Hepatitis C Virus Replication" @default.
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