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• W2021574737 abstract "Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity. Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity. Bovine viral diarrhea virus (BVDV) 2The abbreviations used are: BVDV, bovine viral diarrhea virus; HCV, hepatitis C virus; CP, cytopathogenic; NCP, noncytopathogenic; S1P, sphingosine 1-phosphate; SphK1, sphingosine kinase 1; m.o.i., multiplicity of infection; SKI, sphingosine kinase inhibitor; DMEM, Dulbecco's modified Eagle's medium; p.i., postinfection; siRNA, small interfering RNA; mAb, monoclonal antibody; pAb, polyclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FCS, fetal calf serum; RT, reverse transcriptase; MDBK, Madin-Darby bovine kidney; HEK, human embryonic kidney; PBS, phosphate-buffered saline. 2The abbreviations used are: BVDV, bovine viral diarrhea virus; HCV, hepatitis C virus; CP, cytopathogenic; NCP, noncytopathogenic; S1P, sphingosine 1-phosphate; SphK1, sphingosine kinase 1; m.o.i., multiplicity of infection; SKI, sphingosine kinase inhibitor; DMEM, Dulbecco's modified Eagle's medium; p.i., postinfection; siRNA, small interfering RNA; mAb, monoclonal antibody; pAb, polyclonal antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; FCS, fetal calf serum; RT, reverse transcriptase; MDBK, Madin-Darby bovine kidney; HEK, human embryonic kidney; PBS, phosphate-buffered saline. is an enveloped, positive-sense single-stranded RNA virus classified in the genus Pestivirus of the family Flaviviridae. BVDV establishes persistent infections in cattle populations worldwide. Because BVDV shares virological and molecular properties with the Flaviviridae family member hepatitis C virus (HCV), which chronically infects an estimated 200 million patients worldwide (1Shepard C.W. Finelli L. Alter M.J. Lancet Infect. Dis. 2005; 5: 558-567Abstract Full Text Full Text PDF PubMed Scopus (2226) Google Scholar), BVDV is regarded as a surrogate model for HCV (2Buckwold V.E. Beer B.E. Donis R.O. Antiviral Res. 2003; 60: 1-15Crossref PubMed Scopus (120) Google Scholar). Both HCV and BVDV encode a single large precursor polyprotein that is processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins.BVDV NS3 exhibits serine protease and helicase/ATPase activities that require its cofactor NS4A (3Xu J. Mendez E. Caron P.R. Lin C. Murcko M.A. Collett M.S. Rice C.M. J. Virol. 1997; 71: 5312-5322Crossref PubMed Google Scholar). NS3/4A protease is essential for generating mature NS proteins that are required for viral replication. HCV NS3/4A is well characterized and has been shown to suppress type-I interferons by cleaving the cellular interferon mediators IPS-1 and TRIF (4Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (912) Google Scholar, 5Li X.D. Sun L. Seth R.B. Pineda G. Chen Z.J. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 17717-17722Crossref PubMed Scopus (644) Google Scholar). However, neither interferon suppression nor cellular targets have been identified for the BVDV NS3/4A protease (6Chen Z. Benureau Y. Rijnbrand R. Yi J. Wang T. Warter L. Lanford R.E. Weinman S.A. Lemon S.M. Martin A. Li K. J. Virol. 2007; 81: 964-976Crossref PubMed Scopus (112) Google Scholar).Lytic and persistent BVDV infections depend on the virus biotype. Cytopathogenic (CP) BVDV causes cytopathic effects via apoptosis, whereas noncytopathogenic (NCP) BVDV does not induce obvious changes in cell morphology and viability. These features are distinguished by NS2-3 processing differences; free NS3 produced by NS2-3 cleavage is generated continuously following CP BVDV infections, whereas NS3 is detected only until ∼9 h postinfection (p.i.) for NCP BVDV due to down-regulation of NS2-3 cleavage by this biotype (7Lackner T. Muller A. Pankraz A. Becher P. Thiel H.J. Gorbalenya A.E. Tautz N. J. Virol. 2004; 78: 10765-10775Crossref PubMed Scopus (112) Google Scholar). The CP biotype is characterized by dramatic up-regulation of viral RNA synthesis that could be correlated with the induction of cytopathic effect (7Lackner T. Muller A. Pankraz A. Becher P. Thiel H.J. Gorbalenya A.E. Tautz N. J. Virol. 2004; 78: 10765-10775Crossref PubMed Scopus (112) Google Scholar, 8Vassilev V.B. Donis R.O. Virus Res. 2000; 69: 95-107Crossref PubMed Scopus (67) Google Scholar, 9Yamane D. Kato K. Tohya Y. Akashi H. J. Gen. Virol. 2006; 87: 2961-2970Crossref PubMed Scopus (32) Google Scholar). Because free NS3, but not NS2-3, can form an active viral replicase complex with other NS proteins, increased viral RNA synthesis promoted through the release of free NS3 has been suggested to be a determinant of the characteristic lytic phenotype of CP BVDV infections (10Lackner T. Muller A. Konig M. Thiel H.J. Tautz N. J. Virol. 2005; 79: 9746-9755Crossref PubMed Scopus (49) Google Scholar). However, little is known about the regulation of cellular signaling by BVDV NS2-3, NS3, and NS3/4A, which is crucial for the control of both viral replication and biotype.Recent studies on the mechanisms of viral replication revealed that HCV RNA synthesis occurs on a lipid raft membrane structure where the active viral replicase complex is found (11Aizaki H. Lee K.J. Sung V.M. Ishiko H. Lai M.M. Virology. 2004; 324: 450-461Crossref PubMed Scopus (225) Google Scholar, 12Gao L. Aizaki H. He J.W. Lai M.M. J. Virol. 2004; 78: 3480-3488Crossref PubMed Scopus (274) Google Scholar). The significance of the lipid raft as a scaffold for viral replication is further demonstrated by the identification of a novel HCV replication inhibitor, NA255, which prevents the biosynthesis of sphingolipids, the major components of lipid rafts (13Sakamoto H. Okamoto K. Aoki M. Kato H. Katsume A. Ohta A. Tsukuda T. Shimma N. Aoki Y. Arisawa M. Kohara M. Sudoh M. Nat. Chem. Biol. 2005; 1: 333-337Crossref PubMed Scopus (153) Google Scholar). Administration of NA255 results in disruption of the HCV replicase complexes from the lipid rafts. This report proposes that the interaction between HCV NS5B and sphingomyelin on lipid rafts plays a crucial role for HCV RNA replication. Cellular sphingolipid metabolism is regulated by a large number of converting enzymes that maintain a homeostasis (14Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1733) Google Scholar) but viral mechanisms that affect the sphingolipid metabolism to facilitate viral replication have yet to be identified.In a search for potential host proteins that interact with BVDV NS3, we identified sphingosine kinase 1 (SphK1) as a binding partner of NS3 using the yeast two-hybrid system. SphK1 is a lipid kinase that catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), a bioactive sphingolipid implicated in diverse cellular functions, including proliferation, survival, tumorigenesis, development, inflammation, and immunity (14Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1733) Google Scholar, 15Shida D. Takabe K. Kapitonov D. Milstien S. Spiegel S. Curr. Drug Targets. 2008; 9: 662-673Crossref PubMed Scopus (270) Google Scholar). Here, we analyze the biological significance of the SphK1 interaction with NS3, NS2-3, and NS3/4A. Using purified recombinant SphK1 and NS3, SphK activity was inhibited by NS3 in a dose-dependent manner, independently of its serine protease activity. The inhibition appears to be specific for BVDV NS3 because HCV NS3 had no effect on SphK activity. Using specific chemical inhibitors, small interfering RNA (siRNA), and a catalytically inactive mutant of SphK1, we investigated the significance of SphK inhibition in the viral replication. The present study is the first report demonstrating that SphK1 is targeted by a virus to inhibit its catalytic activity, and this mechanism may contribute to the efficient replication and pathogenesis of BVDV.EXPERIMENTAL PROCEDURESReagents and Antibodies—d-erythro-Sphingosine and sphingosine kinase inhibitor (SKI) (16French K.J. Schrecengost R.S. Lee B.D. Zhuang Y. Smith S.N. Eberly J.L. Yun J.K. Smith C.D. Cancer Res. 2003; 63: 5962-5969PubMed Google Scholar) were purchased from Calbiochem (La Jolla, CA). S1P was obtained from Cayman Chemical (Ann Arbor, MI). Anti-FLAG M2 monoclonal antibody (mAb; IgG1), anti-Myc mAb (IgG1), and isotype control IgG1 mAb were from Sigma. Anti-BVDV NS3 (IgG2a) and anti-GAPDH mAbs were from TropBio (Townsville, Australia) and Ambion (Austin, TX), respectively. Rabbit polyclonal antibodies (pAbs) against calnexin and SphK1 were from Stressgen (Victoria, Canada) and Exalpha (Maynard, MA), respectively. Mouse pAb against junctional adhesion molecule 1 was described previously (17Makino A. Shimojima M. Miyazawa T. Kato K. Tohya Y. Akashi H. J. Virol. 2006; 80: 4482-4490Crossref PubMed Scopus (124) Google Scholar). Goat anti-mouse IgG, IgG1, IgG2a, or anti-rabbit IgG antibodies conjugated with Alexa 488, Alexa 488, Alexa 594, or Alexa 568, respectively, were from Invitrogen. Rabbit pAb against HCV NS3 was kindly provided by Dr. Michinori Kohara (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan). Protein concentrations in samples were determined with the Protein Quantification Kit-Rapid (Dojindo, Rockville, MD) using bovine serum albumin as a standard. Molybdenum blue spray was from Sigma. Recombinant human SphK1 (hSphK1) was purchased from BPS Bioscience (San Diego, CA).Cells and Viruses—MDBK, LB9.K, and human embryonic kidney HEK293 cell lines were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5, 10, and 10% fetal calf serum (FCS), respectively, at 37 °C in a humidified 5% CO2 atmosphere. MDBK and LB9.K cells were confirmed to be free of BVDV by reverse transcriptase-polymerase chain reaction (RT-PCR). BVDV strains KS86-1cp, KS86-1ncp, and Nose have been described previously (18Nagai M. Sakoda Y. Mori M. Hayashi M. Kida H. Akashi H. J. Gen. Virol. 2003; 84: 447-452Crossref PubMed Scopus (46) Google Scholar). Unless otherwise indicated, MDBK and LB9.K cells were infected with BVDV using a multiplicity of infection (m.o.i.) of 5 for 1 h, washed twice with FCS-free DMEM, and incubated in DMEM containing 5 or 10% FCS, respectively. End point viral titration was performed with four replicates on MDBK cells and the 50% tissue culture infective dose (TCID50) determined as described previously (7Lackner T. Muller A. Pankraz A. Becher P. Thiel H.J. Gorbalenya A.E. Tautz N. J. Virol. 2004; 78: 10765-10775Crossref PubMed Scopus (112) Google Scholar). The intracellular synthesis of virus-specific proteins at 72 h p.i. was detected by indirect immunofluorescence analysis using anti-BVDV NS3 mAb (TropBio) and a secondary fluorescein isothiocyanate-labeled antibody as described below under “Immunofluorescence Microscopy.”RNA Extraction—Total RNA was extracted using the SV Total RNA Isolation System (Promega, Madison, WI) according to the manufacturer's protocol.Plasmids—The bovine SphK1 complementary DNA (cDNA; GenBank™ data base accession number XM_870939.1) was generated from total RNA extracted from MDBK cells by RT-PCR using primers that incorporated EcoRI and EcoRV sites at the 5′ and 3′ ends, respectively. Mammalian expression vector of N-terminal FLAG-tagged bovine SphK1, designated pFlag-SphK1, was generated by cloning the SphK1 cDNA into pFLAG-CMV2 (Sigma) using EcoRI and EcoRV sites. The fragments encoding a series of deletion mutants of SphK1 were generated by PCR-mediated site mutagenesis using pFlag-SphK1 as a template. The fragment of catalytically inactive SphK1G177D was generated by PCR-mediated mutagenesis using pFlag-SphK1 as a template with the mutagenic primer 5′-TCGTGGATCAGCCCATCATCGGACATGACCACCAG-3′ to substitute Gly177 to Asp.The mammalian expression vectors of BVDV NS proteins were generated using SRα promoter vector, pME18S. The Myc tag sequence together with the multiple cloning site from pGBKT7 was amplified by PCR using pGBKT7-NS3 as a template and cloned between XhoI and PstI sites of pME18S vector. A DNA fragment encoding BVDV NS3, NS2-3, NS3/4A, and NS5A was generated from the BVDV Nose strain (genotype 1a; GenBank data base accession number AB078951) by SuperScript III One-Step RT-PCR System with Platinum Taq High Fidelity (Invitrogen) using primers that incorporated NdeI and PstI sites at the 5′ and 3′ ends, respectively. The fragments were then cloned into pME18S-Myc using NdeI and PstI sites to generate pME-NS3, pME-NS2-3, pME-NS3/4A, and pME-NS5A. A series of N-terminal deletion mutants of NS3 was generated by PCR-mediated site mutagenesis using pME-NS3 as a template. The fragment of serine protease-negative NS3/4AS2051A was generated by PCR-mediated mutagenesis using pME-NS3/4A as a template with the mutagenic primer 5′-AATATAGGCAGGCCCGCCCATCCCTTCAAGTT-3′ to substitute Ser2051 to Ala. Hybrid cytomegalovirus enhancer/chicken β-actin (CAG) promoter-driven pME18S vectors, termed pCAG, encoding BVDV NS proteins were constructed by a replacement of the SRα promoter with the CAG promoter fragment from the pCAGGS vector using SspI and XhoI sites of pME18S vectors. pEF-HCV NS3/4A, which contains the HCV NS3/4A cDNA of the HCV HCR6 strain (genotype 1b; GenBank data base accession number AY045702) cloned into pEF-1 vector (Invitrogen), was kindly provided by Dr. Michinori Kohara. All constructs were confirmed by sequencing with an ABI PRISM 3150 genetic analyzer (Applied Biosystems, Tokyo, Japan).Yeast Two-hybrid Screening—Potential interacting partners of NS3 were sought using the yeast two-hybrid system according to the manufacturer's manual for the MATCHMAKER Library Construction and Screening Kit (Clontech, Palo Alto, CA). The N-terminal domain of NS3 (amino acids 1889 to 2032) derived from the BVDV Nose strain was amplified by RT-PCR using primers that incorporated NdeI and EcoRI sites at the 5′ and 3′ ends, respectively, and cloned into NdeI and EcoRI sites of pGBKT7 in-frame with the Gal4 DNA-binding domain to express N-terminal Myc-tagged partial NS3, designated pGBKT7-NS3. For the construction of the MDBK cDNA library, first strand cDNA was synthesized using random primers from 0.6 μg of mRNA, which was purified from total RNA using the oligotex-dT30<Super> mRNA Purification Kit (TaKaRa, Shiga, Japan), and double-stranded cDNA amplified by 22 cycles long distance (LD) PCR as described in manufacturer's protocol. Saccharomyces cerevisiae strain AH109 was transformed with the bait plasmid pGBKT7-NS3, and selected in synthetic medium lacking tryptophan. A positive clone harboring pGBKT7-NS3 was confirmed to express N-terminal 142 amino acids of the NS3 protein with anti-Myc mAb by Western blot analysis (data not shown). The MDBK cell double-stranded cDNA library together with SmaI-linearized pGADT7-Rec (Clontech) was cotransformed in an AH109 clone harboring pGBKT7-NS3 to clone cDNA into the GAL4 AD expression vector pGADT7-Rec by homologous recombination. The transformed yeast cells were grown on agar plates of synthetic medium lacking histidine, leucine, and tryptophan containing 20 μg/ml 5-bromo-4-chloro-3-indolyl-α-O-galactopyranoside (X-α-gal). A total of 145 clones were identified from 1 × 106 colonies screened in the library. The insert DNA fragments of isolated clones were amplified by PCR using LD-Insert Screening Amplimer Sets (Clontech) according to the manufacturer's protocol, and then determined by sequencing.Transfection and Immunoprecipitation—LB9.K cells were transiently transfected using Lipofectamine 2000 (Invitrogen) as described in the manufacturer's protocol. Transfection efficiency of LB9.K cells was typically 80–90%. LB9.K cells were seeded onto 6-well tissue culture plates 24 h before transfection. Cells were then transfected with 4 μg of plasmids per well. At 24 h post-transfection, cells were washed twice with ice-cold phosphate-buffered saline (PBS) and scraped into 0.2 ml of lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 20 mm sodium fluoride, 1 mm Na3VO4) supplemented with Complete protease inhibitor mixture (Roche Diagnostics). The lysates equalized with the same amount of proteins were immunoprecipitated with 3 μg of anti-FLAG, anti-Myc, anti-BVDV NS3, or control mouse IgG1 mAbs for 2 h at 4 °C, respectively. The immune complexes were precipitated by incubation with protein G-Sepharose beads (GE Healthcare) for another 1 h. The agarose beads were washed four times with 1 ml of wash buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 0.1% Triton X-100, 20 mm sodium fluoride, 1 mm Na3VO4). The immunoprecipitates were separated by SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad), probed with antibodies, and immunocomplexes detected by enhanced chemiluminescence (ECL). Antibodies used were: horseradish peroxidase-conjugated mAbs against FLAG (1:1000 dilution; Sigma) and Myc (1:1000; Santa Cruz Biotechnology), and a rabbit pAb against NS3 (1:3000). Images were taken by LAS-4000mini image analyzer system (Fujifilm, Tokyo, Japan).Subcellular Fractionation—Cells were harvested into lysis buffer lacking Triton X-100, sonicated, and centrifuged at 1,000 × g for 10 min. Subcellular fractionation was performed by sequential centrifugation as described previously (19Maceyka M. Sankala H. Hait N.C. Le Stunff H. Liu H. Toman R. Collier C. Zhang M. Satin L.S. Merrill Jr., A.H. Milstien S. Spiegel S. J. Biol. Chem. 2005; 280: 37118-37129Abstract Full Text Full Text PDF PubMed Scopus (481) Google Scholar). In brief, postnuclear supernatants were centrifuged at 17,000 × g for 15 min to obtain the inner membrane fraction. The resulting supernatants were centrifuged at 100,000 × g for 1 h to obtain cytosolic and pelleted plasma membrane fractions. The pellet containing inner or plasma membrane was resuspended in lysis buffer (volume comparable with supernatant) and sonicated.Sphingosine Kinase Assay—Sphingosine kinase activity was determined as described previously (20Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). The labeled S1P was separated by TLC on Silica Gel G-60 (Whatman) with 1-butanol/ethanol/acetic acid/water (80:20:10:20, v/v) and visualized by autoradiography. The radioactive spots corresponding to S1P were scraped and counted in a scintillation counter.Generation of Recombinant Bovine Sphingosine Kinase 1— pFlag-SphK1 was transfected into HEK293 cells using Lipofectamine 2000 (Invitrogen) to express the Flag-SphK1, which was subsequently purified by binding to FLAG(M2)-Sepharose (Sigma), followed by elution with the FLAG peptide (0.2 mg/ml). The eluted Flag-SphK1 was concentrated using an Ultrafree-0.5 Centrifugal Filter Device (50,000 Da cutoff; Millipore, Billerica, MA) and diluted in the sphingosine kinase buffer (20 mm Tris-HCl, pH 7.4, 20% glycerol, 1 mm β-mercaptoethanol, 1 mm EDTA, 15 mm sodium fluoride, 20 mm Na3VO4, and 0.5 mm 4-deoxypyridoxine) supplemented with a Complete protease inhibitor mixture (Roche Diagnostics). This procedure was repeated five times to reduce the concentration of the FLAG peptide.Generation of Glutathione S-Transferase (GST) Fusion Proteins—NS3, NS3/4A, and NS5A sequences of BVDV Nose strain were amplified by PCR using plasmid pME-NS3/4A or pME-NS5A as a template. The PCR product of NS3, NS3/4A, or NS5A was cloned into bacterial expression vector pGEX5X-2 using SmaI and NotI sites (NS3 and NS3/4A) or EcoRI and NotI sites (NS5A). HCV NS3 and NS3/4A sequences were amplified by PCR using plasmid pEF-HCV NS3/4A as a template. The PCR products were then cloned into pGEX5X-1 using EcoRI and XhoI sites. BVDV and HCV NS proteins were expressed in Escherichia coli BL21 as GST fusion proteins at the N terminus. Overnight cultures were grown with shaking at 37 °C in Luria-Bertani broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol. The culture was then diluted into fresh Luria-Bertani broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol, and grown with shaking at 37 °C to an A600 of 0.6–1.0. Expression of the GST fusion proteins was then induced with 1.2 mm isopropyl β-d-thiogalactopyranoside, and the cultures were incubated with shaking at 37 °C for a further 3 h. The bacterial cells were then harvested by centrifugation at 6,000 × g for 10 min at 4 °C, resuspended in 10 ml of GST-soluble buffer (40 mm Tris-HCl, pH 7.5, 5 mm EDTA, 0.5% Triton X-100), and lysed by sonication. The lysates were mixed well and centrifuged at 20,000 × g for 20 min at 4 °C. The resultant clarified bacterial lysate was then incubated with GSH-Sepharose 4B for 2 h at 4 °C with constant mixing. Subsequently, the GSH-Sepharose beads (with bound protein) were pelleted by centrifugation at 3,000 × g for 5 min at 4 °C and washed five times in GST-soluble buffer. These beads were then either used directly in a pull-down assay, or the GST fusion proteins were eluted by incubation with cold PBS containing 10 mm GSH for 30 min with constant mixing. This elution procedure was repeated three times. Eluted proteins were concentrated by using Ultrafree-0.5 Centrifugal Filter Device (10,000 Da cutoff; Millipore).Immunofluorescence Microscopy—LB9.K cells were seeded on an eight-well chamber slide (Nunc, Roskilde, Denmark) at 2 × 104 per well 24 h before transfection. Nontransfected cells or the cells transfected with Flag-SphK1 were inoculated with BVDV as described in the figure legends. At 18 h p.i., cells were washed twice with PBS, fixed with PBS containing 4% paraformaldehyde, permeabilized with PBS containing 0.5% Triton X-100, and blocked with PBS containing 10% bovine serum albumin for 10 min. Nontransfected cells were then incubated with anti-calnexin pAb and mouse anti-BVDV NS3 mAb for 1 h followed by incubation with Alexa 568-conjugated goat anti-rabbit IgG and Alexa 488-conjugated goat anti-mouse IgG antibodies for 1 h at room temperature. Transfected cells were double-stained with mouse anti-FLAG mAb (IgG1) and mouse anti-BVDV NS3 mAb (IgG2a) followed by Alexa 488-conjugated goat anti-mouse IgG1 and Alexa 594-conjugated goat anti-mouse IgG2a antibodies, or with mouse anti-FLAG mAb and anti-calnexin pAb followed by Alexa 488-conjugated goat anti-mouse IgG and Alexa 568-conjugated goat anti-rabbit IgG antibodies. Cells incubated with secondary antibodies were then washed three times with PBS, mounted in Dako fluorescent mounting medium (Dako Corporation, Carpinteria, CA), then sealed and observed under an LSM 510 microscope (Carl Zeiss, Tokyo, Japan).Measurement of S1P Synthesis—LB9.K cells transiently transfected with pCAG vectors encoding BVDV NS proteins or MDBK cells infected with BVDV were incubated for 4 h in phosphate-free DMEM (Invitrogen), then labeled with fresh phosphate-free DMEM containing [32P]orthophosphate (0.2 mCi/ml) and incubated for 4 h at 37 °C in a humidified 5% CO2 atmosphere. Cells were then scraped on ice into 400 μl of methanol, 1 m NaCl, 5 m NaOH (100:100:3, v/v), then 200 μl of chloroform added. Samples were vortexed thoroughly and centrifuged at 14,000 × g for 5 min. The upper aqueous phase containing S1P was transferred to a new tube, and acidified through addition of 20 μl of 1 m HCl and 400 μl of chloroform/methanol/HCl (100/200/1, v/v). Samples were vortexed thoroughly and phases separated by addition of 120 μl of chloroform and 120 μl of 2 m KCl. After centrifugation, the lower organic phase was dried under vacuum and resuspended in chloroform, and resolved by TLC as described above. The radioactive spots corresponding to S1P were scraped from the plates and counted in a scintillation counter.RNA Interference—Duplex siRNAs were purchased from Invitrogen. The siRNA sequence targeting SphK1 was 5′-GCAGUGGCCGCUUCUUUGAACUAUU-3′ (sense) and 5′-AAUAGUUCAAAGAAGCGGCCACUGC-3′ (antisense), corresponding to 634–658 relative to the first nucleotide of the start codon. The sequence used for scrambled control siRNA was 5′-GCAGGCCCGUUUCUUAGCAUUGAUU-3′ (sense) and 5′-AAUCAAUGCUAAGAAACGGGCCUGC-3′ (antisense). LB9.K cells were transfected with 20 nm siRNA using siLentfect (Bio-Rad) according to the manufacturer's protocol.Quantitative Real-time RT-PCR—cDNA synthesis was performed with the PrimeScript RT Reagent Kit (TaKaRa) according to the manufacturer's protocol. GAPDH mRNA and viral RNA were quantified using Power SYBR Green PCR Master Mix (Applied Biosystems) as previously described (21Yamane D. Kato K. Tohya Y. Akashi H. Vet. Microbiol. 2008; 129: 69-79Crossref PubMed Scopus (28) Google Scholar).Apoptosis Assay—The DEVDase activity assay was performed as described previously (22Yamane D. Nagai M. Ogawa Y. Tohya Y. Akashi H. Microbes. Infect. 2005; 7: 1482-1491Crossref PubMed Scopus (30) Google Scholar) using Ac-DEVD-AMC as a substrate.RESULTSIdentification of SphK1 as a Binding Partner of BVDV NS3 and NS2-3— To identify potential cellular binding partners of BVDV NS3, we conducted a yeast two-hybrid screen using the N-terminal 142 amino acids of NS3 as bait, and isolated a cDNA clone encoding partial SphK1 from 1 of 145 total positive colonies. The cDNA sequence encoded 164 C-terminal amino acids of SphK1 (Fig. 1A). Employing the cDNA sequence for bovine SphK1 from the GenBank data base, specific primers were designed and used to clone a bovine SphK1 by RT-PCR cloning using total RNA isolated from MDBK cells. Subsequently, a catalytically inactive form of bovine SphK1 was constructed by substituting aspartic acid (D) for glycine (G) at position 177 in the ATP-binding site of the diacylglycerol catalytic domain, according to the previous study (23Pitson S.M. Moretti P.A. Zebol J.R. Xia P. Gamble J.R. Vadas M.A. D'Andrea R.J. Wattenberg B.W. J. Biol. Chem. 2000; 275: 33945-33950Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). This SphK1G177D was used for further functional studies. To investigate whether the cloned bovine SphK1 encodes a bona fide SphK, LB9.K cells were transiently transfected with expression vectors containing FLAG-tagged SphK1. Similar to the previous study (24Olivera A. Kohama T. Edsall L. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (460) Google Scholar), SphK activity in cell lysates from LB9.K cells transiently transfected with SphK1 was increased ∼300-fold (Fig. 1, B and C). By comparison, expression of catalytically inactive SphK1G177D produced no detectable increase in SphK activity. Western blot analysis using anti-FLAG antibody revealed a specific protein band with an apparent molecular mass consistent with the predicted size (∼55 kDa) of FLAG-tagged SphK1, which was absent in vector-transfected cells (Fig. 1C).To confirm the interaction between NS3 and SphK1, we cotransfected Flag-SphK1 and Myc-NS3 in LB9.K cells, immunoprecipitated NS3 using an anti-Myc mAb, and determined whether SphK1 was coprecipitated with NS3 by Western blotting. We also cotransfected an empty vector or NS5A as a negative control and uncleaved NS2-3. Both NS3 and NS2-3, but neither NS5A nor vector control, coprecipitated with SphK1 (Fig. 1D). We attempted to express Myc-tagged NS2 in the same way as above to demonstrate that NS2 does not mediate the binding of NS2-3 to SphK1, but failed to express detectable levels of NS2 protein, most likely due to its instability in LB9.K c" @default.
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