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• W2010808237 abstract "The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using 15N-, 13C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome. The human proteome contains myriad intrinsically disordered proteins. Within intrinsically disordered proteins, polyproline-II motifs are often located near sites of phosphorylation. We have used an unconventional experimental paradigm to discover that phosphorylation by protein kinase A (PKA) occurs in the intrinsically disordered domain of hepatitis C virus non-structural protein 5A (NS5A) on Thr-2332 near one of its polyproline-II motifs. Phosphorylation shifts the conformational ensemble of the NS5A intrinsically disordered domain to a state that permits detection of the polyproline motif by using 15N-, 13C-based multidimensional NMR spectroscopy. PKA-dependent proline resonances were lost in the presence of the Src homology 3 domain of c-Src, consistent with formation of a complex. Changing Thr-2332 to alanine in hepatitis C virus genotype 1b reduced the steady-state level of RNA by 10-fold; this change was lethal for genotype 2a. The lethal phenotype could be rescued by changing Thr-2332 to glutamic acid, a phosphomimetic substitution. Immunofluorescence and transmission electron microscopy showed that the inability to produce Thr(P)-2332-NS5A caused loss of integrity of the virus-induced membranous web/replication organelle. An even more extreme phenotype was observed in the presence of small molecule inhibitors of PKA. We conclude that the PKA-phosphorylated form of NS5A exhibits unique structure and function relative to the unphosphorylated protein. We suggest that post-translational modification of viral proteins containing intrinsic disorder may be a general mechanism to expand the viral proteome without a corresponding expansion of the genome. Hepatitis C virus (HCV) 2The abbreviations used are: HCVhepatitis C virusSIS2204I mutationNS3, NS5A, and NS5Bhepatitis C virus non-structural protein 3, 5A, and 5B, respectivelyIDDintrinsically disordered domainPPIIpoly-proline-IIRT-qPCRreal-time quantitative PCRCHXcycloheximideK2525 μg/ml kanamycinC2020 μg/ml chloramphenicolNi-NTAnickel-nitrilotriacetic acidSH3Src homology 3HSQCheteronuclear single quantum correlationPKAccatalytic subunit of PKA. establishes chronic infections in humans that can persist for decades before causing any clinical manifestations. The ability of a positive-strand RNA virus with such limited coding capacity to so effectively evade host defenses is extraordinary. A unique feature of HCV, relative to most acute RNA viruses of similar genetic size and encoded functions, is nonstructural protein 5A (NS5A). NS5A is a two-domain protein. The amino-terminal domain can form at least two structurally distinct homodimers (1.Love R.A. Brodsky O. Hickey M.J. Wells P.A. Cronin C.N. Crystal structure of a novel dimeric form of NS5A domain I protein from hepatitis C virus.J. Virol. 2009; 83: 4395-4403Crossref PubMed Scopus (206) Google Scholar, 2.Tellinghuisen T.L. Marcotrigiano J. Rice C.M. Structure of the zinc-binding domain of an essential component of the hepatitis C virus replicase.Nature. 2005; 435: 374-379Crossref PubMed Scopus (399) Google Scholar). One of these dimers binds to RNA, with a preference for GU-rich RNA (3.Huang L. Hwang J. Sharma S.D. Hargittai M.R. Chen Y. Arnold J.J. Raney K.D. Cameron C.E. Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein.J. Biol. Chem. 2005; 280: 36417-36428Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 4.Hwang J. Huang L. Cordek D.G. Vaughan R. Reynolds S.L. Kihara G. Raney K.D. Kao C.C. Cameron C.E. Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins.J. Virol. 2010; 84: 12480-12491Crossref PubMed Scopus (67) Google Scholar). The carboxyl-terminal domain is an intrinsically disordered domain (IDD) (5.Hanoulle X. Verdegem D. Badillo A. Wieruszeski J.M. Penin F. Lippens G. Domain 3 of non-structural protein 5A from hepatitis C virus is natively unfolded.Biochem. Biophys. Res. Commun. 2009; 381: 634-638Crossref PubMed Scopus (72) Google Scholar, 6.Liang Y. Kang C.B. Yoon H.S. Molecular and structural characterization of the domain 2 of hepatitis C virus non-structural protein 5A.Mol. Cells. 2006; 22: 13-20PubMed Google Scholar) that contains numerous putative sites of phosphorylation and has been reported to bind dozens of cellular proteins (7.Cordek D.G. Bechtel J.T. Maynard A.T. Kazmierski W.M. Cameron C.E. Targeting the NS5A protein of HCV: an emerging option.Drugs Future. 2011; 36: 691-711Crossref PubMed Scopus (43) Google Scholar). The ability of NS5A to antagonize and/or to hijack numerous cellular pathways may be a key determinant of HCV persistence. hepatitis C virus S2204I mutation hepatitis C virus non-structural protein 3, 5A, and 5B, respectively intrinsically disordered domain poly-proline-II real-time quantitative PCR cycloheximide 25 μg/ml kanamycin 20 μg/ml chloramphenicol nickel-nitrilotriacetic acid Src homology 3 heteronuclear single quantum correlation catalytic subunit of PKA. Specific combinations of post-translational modifications, including phosphorylation, in the IDD of p53 confer the ability to interface with myriad cellular pathways (8.Uversky V.N. Oldfield C.J. Dunker A.K. Intrinsically disordered proteins in human diseases: introducing the D2 concept.Annu. Rev. Biophys. 2008; 37: 215-246Crossref PubMed Scopus (1058) Google Scholar). Conformational sampling of an IDD leads to multiple, unique structures capable of unique interactions (8.Uversky V.N. Oldfield C.J. Dunker A.K. Intrinsically disordered proteins in human diseases: introducing the D2 concept.Annu. Rev. Biophys. 2008; 37: 215-246Crossref PubMed Scopus (1058) Google Scholar). Although empirical examples are quite limited, the thought is that the addition of a post-translational modification to a specific location will restrict conformations sampled, thus channeling the IDD toward a single structure or subset of structures. Therefore, the interaction of NS5A with so many proteins may rely on a similar mechanism, phosphorylation-dependent acquisition of structure. Several observations suggest an important role for NS5A phosphorylation in HCV multiplication and/or pathogenesis. First, persistent replication of the prototypical genotype 1b subgenomic replicon RNA in Huh-7 cells and sublines thereof requires an adaptive mutation that changes Ser-2204 of NS5A to Ile (9.Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1275) Google Scholar). The S2204I substitution prevents formation of the hyperphosphorylated/p58 form of NS5A (9.Blight K.J. Kolykhalov A.A. Rice C.M. Efficient initiation of HCV RNA replication in cell culture.Science. 2000; 290: 1972-1974Crossref PubMed Scopus (1275) Google Scholar). It is becoming increasingly clear that full-length genomes encoding adaptive mutations fail to cause disease in chimpanzees (10.Bukh J. Pietschmann T. Lohmann V. Krieger N. Faulk K. Engle R.E. Govindarajan S. Shapiro M. St Claire M. Bartenschlager R. Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 14416-14421Crossref PubMed Scopus (217) Google Scholar, 11.Yi M. Hu F. Joyce M. Saxena V. Welsch C. Chavez D. Guerra B. Yamane D. Veselenak R. Pyles R. Walker C.M. Tyrrell L. Bourne N. Lanford R.E. Lemon S.M. Evolution of a cell culture-derived genotype 1a hepatitis C virus (H77S.2) during persistent infection with chronic hepatitis in a chimpanzee.J. Virol. 2014; 88: 3678-3694Crossref PubMed Scopus (24) Google Scholar), consistent with NS5A phosphorylation contributing to pathogenesis. Inhibitors of host cell kinases have been shown to impact HCV replication, presumably due to inhibition of NS5A phosphorylation (12.Quintavalle M. Sambucini S. Di Pietro C. De Francesco R. Neddermann P. The α isoform of protein kinase CKI is responsible for hepatitis C virus NS5A hyperphosphorylation.J. Virol. 2006; 80: 11305-11312Crossref PubMed Scopus (67) Google Scholar, 13.Neddermann P. Quintavalle M. Di Pietro C. Clementi A. Cerretani M. Altamura S. Bartholomew L. De Francesco R. Reduction of hepatitis C virus NS5A hyperphosphorylation by selective inhibition of cellular kinases activates viral RNA replication in cell culture.J. Virol. 2004; 78: 13306-13314Crossref PubMed Scopus (123) Google Scholar). In addition, the most potent direct acting anti-HCV drug reported to date, daclatasvir, targets NS5A and alters the phosphorylation state of the protein (14.Gao M. Nettles R.E. Belema M. Snyder L.B. Nguyen V.N. Fridell R.A. Serrano-Wu M.H. Langley D.R. Sun J.H. O'Boyle 2nd, D.R. Lemm J.A. Wang C. Knipe J.O. Chien C. Colonno R.J. Grasela D.M. Meanwell N.A. Hamann L.G. Chemical genetics strategy identifies an HCV NS5A inhibitor with a potent clinical effect.Nature. 2010; 465: 96-100Crossref PubMed Scopus (838) Google Scholar, 15.Lemm J.A. O'Boyle 2nd, D. Liu M. Nower P.T. Colonno R. Deshpande M.S. Snyder L.B. Martin S.W. St Laurent D.R. Serrano-Wu M.H. Romine J.L. Meanwell N.A. Gao M. Identification of hepatitis C virus NS5A inhibitors.J. Virol. 2010; 84: 482-491Crossref PubMed Scopus (172) Google Scholar). Finally, a clear connection has been made between casein kinase II phosphorylation in the carboxyl-terminal region of the IDD and viral assembly (16.Appel 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, 17.Tellinghuisen T.L. Foss K.L. Treadaway J. Regulation of hepatitis C virion production via phosphorylation of the NS5A protein.PLoS Pathog. 2008; 4: e1000032Crossref PubMed Scopus (333) Google Scholar), although a molecular explanation for the gain of function caused by phosphorylation remains a mystery. Therefore, knowledge of the kinases responsible for NS5A phosphorylation and the corresponding sites of phosphorylation (the NS5A phosphoproteome) is essential to understand the role of individual phosphorylation states during the HCV life cycle and to test the hypothesis that inhibitors of NS5A may function by perturbing one or more phosphorylated states of NS5A. The inability to directly reveal the NS5A phosphoproteome (e.g. by using mass spectrometry to analyze NS5A isolated from HCV-infected hepatocytes) demands an alternative experimental approach. Here we define a new experimental paradigm to study phosphorylation of NS5A. We show that protein kinase A (PKA) phosphorylation occurs near the poly-proline-II (PPII) motif of NS5A, leading to changes in the conformational sampling of this SH3 binding determinant. This phosphorylation-dependent change in NS5A dynamics is important for replication because it contributes to the integrity of the replication organelle. We suggest that phosphorylation and other post-translational modifications of viral IDPs represent an important strategy for expanding the functional proteome of viruses with limited coding capacity. NS5A 2005–2419 was generated as described previously (18.Huang L. Sineva E.V. Hargittai M.R. Sharma S.D. Suthar M. Raney K.D. Cameron C.E. Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli.Protein Expr. Purif. 2004; 37: 144-153Crossref PubMed Scopus (59) Google Scholar). Oligonucleotides 1 and 2 (Table 1) were used with pET26-Ub-Δ32-NS5A-C(His) (encoding residues 2005–2419 of the polyprotein) to amplify residues 2194–2419. The NS5A deletion mutant 2005–2306 was created using oligonucleotides 3 and 4 (Table 1). NS5A containing ΔP3, T2332A, or T2332E was amplified using oligonucleotides 2 and 3 (Table 1) on pHCVbart.rep1/Ava-II-ΔP3 (3.Huang L. Hwang J. Sharma S.D. Hargittai M.R. Chen Y. Arnold J.J. Raney K.D. Cameron C.E. Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein.J. Biol. Chem. 2005; 280: 36417-36428Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), pHCVbart.rep1/Ava-II-T2332A, or pHCVbart.rep1/Ava-II-T2332E. The PCR fragments were digested and ligated into pET26Ub-CHIS vector. All plasmid constructs were verified by restriction enzyme digestion and DNA sequencing.TABLE 1Oligonucleotides used in this studyNumberNameSequence1HCV-NS5A-2194-NcoI-for5′-CGC GCC ATG GAT CCT CTG GTT CTC CCC CCT CCT TGG CC-3′2HCV-NS5A-HindIII-rev5′-GGT ACC AAG CTT CTA TTA GCA GCA GAC GAC GTC CTC ACT-3′3HCV-Δ32-NS5A-NcoI- for5′-GCG GGT ACC CCA TGG ATC CTC TGG TGG AGT CCC CTT CTT-3′4HCV-NS5A- 2307-HindIII-rev5′-GGC AAG CTT CTA TTA GTA GTC CGG GTC CTT CCA-3′5HCV-NS5A-T2332A-for5′-CCA CGG AGG AAG AGG GCG GTT GTC CTG TCA GAA-3′6HCV-NS5A-T2332A-rev5′-TTC TGA CAG GAC AAC CGC CCT CTT CCT CCG TGG-3′7HCV-NS5A-XhoI-for5′-GCG AAA TTC CCT CGA GCG ATG CCC ATA-3′8HCV-NS5B-MfeI-rev5′-GCG GGT GGT GTC AAT TGG TGT CTC-3′9HCV-NS5A-T2332E-for5′-CCA CGG AGG AAG AGG GAG GTT GTC CTG TCA GAA-3′10HCV-NS5A-T2332E-rev5′-TTC TGA CAG GAC AAC CTC CCT CTT CCT CCG TGG-3′11HCV-rep-RT-PCR-for5′-GGA AGC GGT CAG CCC AT-3′12HCV-rep-RT-PCR-rev5′-GCG TTG GCT ACC CGT GAT-3′13HCV-Northern-for5′-GGG TGC TTG CGA GTG CC-3′14HCV-Northern-rev5′-GGC CAG TAA CGT TAG GGG-3′ Open table in a new tab Oligonucleotides 5–8 (Table 1) were used to perform overlap extension PCR on pHCVbart.rep1/Ava-II to create the T2332A derivative. The overlap PCR fragment was digested and ligated into vector to generate pHCVbart.rep1/Ava-II-T2332A (T2332A). The T2332E derivative, pHCVbart.rep1/Ava-II-T2332E, was constructed with oligonucleotides 7–10 (Table 1) in the same manner as the T2332A derivative. Both Thr-2332 derivatives were also cloned in the context of the NS5A cell culture adaptive mutation S2204I (SI), to generate SI/T2332A and SI/T2332E, using pHCVbart.rep1/Ava-II-SI (3.Huang L. Hwang J. Sharma S.D. Hargittai M.R. Chen Y. Arnold J.J. Raney K.D. Cameron C.E. Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein.J. Biol. Chem. 2005; 280: 36417-36428Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), as described above. The plasmids pJC1/GLuc2A and pJC1/GLuc2A(GNN) were described previously (19.Phan T. Beran R.K. Peters C. Lorenz I.C. Lindenbach B.D. Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.J. Virol. 2009; 83: 8379-8395Crossref PubMed Scopus (112) Google Scholar). To construct the pJC1/GLuc2A(T2332A) and (T2332E) replicons, a 3,676-bp fragment from pYSGR-JFH(T2332A) and T2332E was subcloned into pJC1/GLuc2A using common SpeI and BsrGI restriction sites, generating pJC1/GLuc2A(T2332A) and (T2332E) replicons. All NS5A, NS3, and NS5B proteins (genotype 1b) used in this study were expressed and purified as described previously (4.Hwang J. Huang L. Cordek D.G. Vaughan R. Reynolds S.L. Kihara G. Raney K.D. Kao C.C. Cameron C.E. Hepatitis C virus nonstructural protein 5A: biochemical characterization of a novel structural class of RNA-binding proteins.J. Virol. 2010; 84: 12480-12491Crossref PubMed Scopus (67) Google Scholar, 18.Huang L. Sineva E.V. Hargittai M.R. Sharma S.D. Suthar M. Raney K.D. Cameron C.E. Purification and characterization of hepatitis C virus non-structural protein 5A expressed in Escherichia coli.Protein Expr. Purif. 2004; 37: 144-153Crossref PubMed Scopus (59) Google Scholar, 20.Wang Q. Arnold J.J. Uchida A. Raney K.D. Cameron C.E. Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase.Nucleic Acids Res. 2010; 38: 1312-1324Crossref PubMed Scopus (27) Google Scholar, 21.Zhong W. Ferrari E. Lesburg C.A. Maag D. Ghosh S.K. Cameron C.E. Lau J.Y. Hong Z. Template/primer requirements and single nucleotide incorporation by hepatitis C virus nonstructural protein 5B polymerase.J. Virol. 2000; 74: 9134-9143Crossref PubMed Scopus (105) Google Scholar). PKA- or CK2-mediated phosphorylation of HCV non-structural proteins was performed in 50 mm HEPES, pH 7.5, 0.5 mm tris(2-carboxyethyl)phosphine, 20 mm MgCl2, 100 mm NaCl, 125 μm ATP, 0.5 μCi/μl [γ-32P]ATP (MP Biomedicals), and 1 μm NS3, NS5A, NS5A derivative, or NS5B. For reactions that did not require the use of radiolabeled ATP, the [γ-32P]ATP was omitted. Reactions performed in the presence of PKA inhibitors or NS5A inhibitor BMS-790052 were performed by incubating 5 nm PKA with inhibitor at 37 °C for 30 min, after which point NS5A was added to 0.5 μm, and phosphorylation proceeded for the specified time. The phosphorylation reaction was quenched with an equal volume of 2× SDS-PAGE sample buffer. The samples were resolved by 8% SDS-PAGE. For reactions containing radiolabeled ATP, the gels were analyzed with the Typhoon phosphorimager (GE Healthcare) and quantified with ImageQuant software. The amount of phosphorylation was normalized to the amount of radioactivity present in each lane. Gels of reactions not using radiolabeled ATP were analyzed by Western blot. PKA-phosphorylated NS5A IDD used for NMR was prepared as described above but with 100 μm isotopically labeled IDD, 250 μm ATP, and 10 μm PKA. Reactions proceeded for 60 min. Phosphorylation reactions were then desalted using a Zeba column pre-equilibrated with NMR buffer. Analysis of the PKA-phosphorylated residues on NS5A was performed by mass spectrometry with 8 μg of NS5A phosphorylated by PKA. A sample of the quenched reaction was resolved by 8% SDS-PAGE and stained with Pro-Q Diamond phosphoprotein gel stain (Invitrogen) to verify that NS5A had been phosphorylated by PKA. The remaining reaction was resolved by 8% SDS-PAGE, and the NS5A band was visualized by Coomassie staining. Peptides obtained from in-gel trypsin digestion of the Coomassie-stained band corresponding to in vitro phosphorylated NS5A were analyzed by LC/MS/MS. Tandem MS spectra obtained by collision-induced dissociation were acquired using an LC/MS/MS system that consisted of a Surveyor HPLC pump, a Surveyor Micro AS autosampler, and an LTQ linear ion trap mass spectrometer (ThermoFinnigan). The acquired spectra were processed using Xcalibur version 2.0. The raw tandem MS spectra were also converted to Mascot generic files (.mgf). Detection and mapping of the phosphorylation site were achieved by database searching of tandem mass spectra of the proteolytic peptides against a current Swiss-Prot protein database using the Bioworks version 3.2 and Mascot version 2.2 (Matrix Science) search engines. The database searches were performed with cysteine carbamidomethylation as a fixed modification. The variable modifications were methionine oxidation (+16 Da) and phosphorylation (+80 Da) of Ser, Thr, and Tyr residues. Up to two missed cleavages were allowed for trypsin digestion. Peptide mass tolerance and fragment mass tolerance were set to 3 and 2 Da, respectively. Tandem MS spectra that are matched to phosphorylated peptides were manually evaluated at the raw data level with the consideration of overall data quality, signal/noise ratio of matched peaks, and the presence of dominant peaks that did not match to any theoretical m/z value. HCV sequence alignments were performed using the Los Alamos HCV Sequence Database (22.Kuiken C. Yusim K. Boykin L. Richardson R. The Los Alamos hepatitis C sequence database.Bioinformatics. 2005; 21: 379-384Crossref PubMed Scopus (315) Google Scholar). Genotype references of the NS5A protein sequence were compared to determine the conservation of the PKA phosphorylation site, residue Thr-2332. Phosphorylation of NS5A in the presence of specified PKA inhibitors was performed in 50 mm HEPES, pH 7.5, 0.5 mm tris(2-carboxyethyl)phosphine, 20 mm MgCl2, 100 mm NaCl, 50 μm ATP, 0.5 μCi/μl [γ-32P]ATP, and 0.005 μm PKA. Reactions were incubated with 0–50 μm PKA inhibitor H-7 dihydrochloride, H-89 dihydrochloride, KT-5720, or the myristoylated peptide fragment 14–22 (Sigma), each in a final DMSO concentration of 2.5% in the reaction, at 37 °C for 30 min. After 30 min, NS5A was added to 0.5 μm, and phosphorylation proceeded for 30 min. The phosphorylation reaction was quenched with 2× SDS-PAGE sample buffer. The samples were resolved by 8% SDS-PAGE. Data were fit to a hyperbolic equation to calculate the respective IC50 for each inhibitor. Rabbit polyclonal anti-NS5A, anti-NS3, and anti-NS5B were generated using purified protein immunogens by Covance, Inc. for our use. Rabbit polyclonal phospho-specific anti-Thr(P)-2332-NS5A was generated using the synthetic peptide PPRRKRpTVVLSESC conjugated to keyhole limpet hemocyanin by Covance Research Products, Inc. for our use. Mouse monoclonal anti-NS5A was purchased from Advanced Immunochemical Services, Inc. Rabbit polyclonal anti-NS4B was a gift from Dr. Kouacou Konan (Albany Medical College). Rabbit polyclonal anti-giantin and mouse monoclonal anti-β-actin were purchased from Abcam. Mouse monoclonal anti-PKAc was purchased from BD Transduction Laboratories. Goat monoclonal anti-calnexin was purchased from Origene. Alexa Fluor 488 goat anti-mouse, Alexa Fluor 488 goat anti-rabbit, Alexa Fluor 594 goat anti-rabbit, and Alexa Fluor 594 donkey anti-mouse IgG were purchased from Invitrogen. The goat anti-rabbit HRP, goat anti-rabbit AP, goat anti-mouse AP, bovine anti-goat HRP, and bovine anti-goat AP were purchased from Santa Cruz Biotechnology, Inc. Huh-7.5 cells were maintained in DMEM, as described previously (3.Huang L. Hwang J. Sharma S.D. Hargittai M.R. Chen Y. Arnold J.J. Raney K.D. Cameron C.E. Hepatitis C virus nonstructural protein 5A (NS5A) is an RNA-binding protein.J. Biol. Chem. 2005; 280: 36417-36428Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Stable cell lines were maintained in supplemented DMEM containing 0.5 mg/ml G418. Where specified, cells were treated with DMSO; PKA inhibitor H-7, H-89, or KT-5720 or the myristoylated peptide fragment 14–22 (all from Sigma); or NS5A inhibitor Daclatasvir (BMS-790052) for the designated times prior to harvesting. In vitro transcribed subgenomic RNA was transfected into Huh-7.5 cells using the TransMessenger transfection system (Qiagen). Briefly, 1.6 × 106 cells were mixed with 2 μg of RNA per the manufacturer's protocol. For colony formation assays, 0.5 × 106 cells were plated in 100-mm dishes. Cells were selected under DMEM containing 0.5 mg/ml G418 for 3 weeks, exchanging G418-containing media every 3 days. Huh-7.5 cells were maintained in DMEM supplemented with 10% fetal calf serum (HyClone, Logan, UT) and 1 mm nonessential amino acids (Invitrogen). Cells were seeded at 0.2 × 106 cells/ml/well in 12-well plates and transfected with HCV RNA transcripts by using the TransIT-mRNA transfection kit (Mirus Bio, Madison, WI). To measure the relative replication of JC1/GLuc2A reporter viruses, cell culture medium was collected at various time points post-transfection, clarified by centrifugation (16,000 × g for 5 min), and mixed with ¼ volume of Renilla 5× lysis buffer (Promega, Madison, WI) to kill infectious HCV. GLuc activity was measured, as described previously (19.Phan T. Beran R.K. Peters C. Lorenz I.C. Lindenbach B.D. Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.J. Virol. 2009; 83: 8379-8395Crossref PubMed Scopus (112) Google Scholar), on a Berthold Centro LB 960 luminescent plate reader with 20 μl of sample injected with 50 μl of Gaussia luciferase assay reagent (New England Biolabs), integrated over 10 s. To measure relative infectivity, cell culture media containing infectious virus were collected at various times post-transfection, clarified by centrifugation, and stored at −80 °C. The virus supernatants were used to infect naive Huh-7.5 cells seeded at 0.2 × 106 cells/ml/well in 12-well plates. After 6 h of virus adsorption, cells were washed three times with Dulbecco's PBS and incubated with complete medium for an additional 72 h. Cell culture media were collected, clarified, and assayed for GLuc activity as described above. Total RNA was extracted from subgenomic replicon transfected Huh-7.5 cells using the RNeasy Plus RNA extraction system (Qiagen). The RT-qPCR was performed at the Nucleic Acid Facility at Penn State. Oligonucleotides 11 and 12 were used for reverse transcription. The Taqman primer 5′-CGC CGC CAA GCT CTT CAG CAA-3′ was used on an Applied Biosystems 7300 system. In vitro transcribed RNA was used to quantify the copy number in cells. Recombinant proteins or cell extracts from transiently transfected cells or stable cell lines were separated on 8% SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Membranes were probed with the appropriate antibodies. Proteins were detected by ECL (Millipore) or ECF (GE Healthcare) Western blot detection reagents. ECF-detected blots were visualized by TyphoonImager (GE Healthcare) and quantified using NS5A or PKA-phosphorylated NS5A standards via ImageQuant software. For Northern blotting, 0.5 and 2.0 μg of total RNA from stable cell lines was separated on a 0.6% agarose gel of 0.8 m formaldehyde in 20 mm MOPS buffer containing 5 mm sodium acetate and 1 mm EDTA and transferred to a nylon membrane in 150 mm sodium chloride, 15 mm sodium citrate, pH 7.0. Additionally, 0.5, 1.0, 5.0, and 10.0 ng of in vitro transcribed subgenomic replicon RNA was used as a positive control. The RNA was UV-cross-linked to the membrane and incubated with radiolabeled probes for 12 h. The probes were generated by PCR using oligonucleotides 13 and 14 (Table 1) with pHCVbart.rep1/Ava-II and labeled with [α-32P]dATP (MP Biomedicals). RNA was visualized by the Typhoon imager (GE Healthcare) and quantified using ImageQuant software. Huh-7.5 cells stably replicating the SI, SI/T2332A, and SI/T2332E subgenomic replicons were incubated in the presence of 100 μg/ml CHX for 48 h and harvested every 12 h for analysis via Northern and Western blotting and immunofluorescence. Cells were seeded on coverslips in 6-well plates. After the designated time and any specified treatment, the cells were washed with PBS and fixed for 15 min in 4% formaldehyde in PBS. Cells were washed with PBS, permeabilized for 5 min in 0.05% Triton X-100 in PBS, and washed with PBS. The cells were blocked with 3% BSA in PBS for 15 min and double-stained by incubation for 1 h each with primary antibody A followed by primary antibody B. Cells were incubated for 1 h each with Alexa-488- or Alexa-594-conjugated secondary antibodies (Invitrogen). After each antibody incubation, PBS was used to wash cells. The coverslips were mounted on glass slides in Vectashield with DAPI (Vectashield Laboratories, Inc., Burlingame, CA) and sealed with nail polish. Samples were analyzed by fluorescence microscopy (Zeiss Axiovert 200 M) with a ×63 lens, and digital images were taken with an Axiocam MRm CCD camera. Optical sections were deconvolved using Axiovision software. Slides prepared for immunofluorescence were also used for confocal microscopy. Confocal analysis was performed on an Olympus Fluoview 1000 microscope with an Olympus IX70 inverted microscope with fluorescence burner and four single-line lasers with individual shutters that are software-controlled for sequential acquisition: violet (405 nm, 10 milliwatts), blue argon (488 nm, 10 milliwatts), green HeNe (543 nm, 10 milliwatts), and red HeNe (633 nm, 10 milliwatts). Images were taken using the PlanApo ×60/1.4 oil objective and the two-dimensional X-Y scanning mode. Data were analyzed with the Olympus Fluoview version 3.0a software. Quantification was performed using Pearson's coefficient of colocalization between the green (NS5A) and red (NS4B) channels, and the statistical significance was calculated using a two-tailed paired Student's t test. Colocalization of NS5A and NS4B was calculated using 20 cells per cell line from three independent experiments. For ultrastructural analysis, cells were trypsinized, resuspended in complete DMEM, and processed as follows. Single cell suspensions were transferre" @default.
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• W2010808237 title "Expanding the Proteome of an RNA Virus by Phosphorylation of an Intrinsically Disordered Viral Protein" @default.
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