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• W1973729131 abstract "Dengue virus, a member of the family Flaviviridae, poses a serious public health threat worldwide. Dengue virus is a positive-sense RNA virus that harbors a genome of ∼10.7 kb. Replication of dengue virus is mediated coordinately by cis-acting genomic sequences, viral proteins, and host cell factors. We have isolated and identified several host cell factors from baby hamster kidney cell extracts that bind with high specificity and high affinity to sequences within the untranslated regions of the dengue virus genome. Among the factors identified, Y box-binding protein-1 (YB-1) and the heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1, hnRNP A2/B1, and hnRNP Q, bind to the dengue virus 3′-untranslated region. Further analysis indicated that YB-1 binds to the dengue virus 3′ stem loop, a conserved structural feature located at the 3′ terminus of the 3′-untranslated region of many flaviviruses. Analysis of the impact of YB-1 on replication of dengue virus in YB-1+/+ and YB-1–/– mouse embryo fibroblasts indicated that host YB-1 mediates an antiviral effect. Further studies demonstrated that this antiviral impact is due, at least in part, to a repressive role of YB-1 on dengue virus translation via a mechanism that requires viral genomic sequences. These results suggest a novel role for YB-1 as an antiviral host cell factor. Dengue virus, a member of the family Flaviviridae, poses a serious public health threat worldwide. Dengue virus is a positive-sense RNA virus that harbors a genome of ∼10.7 kb. Replication of dengue virus is mediated coordinately by cis-acting genomic sequences, viral proteins, and host cell factors. We have isolated and identified several host cell factors from baby hamster kidney cell extracts that bind with high specificity and high affinity to sequences within the untranslated regions of the dengue virus genome. Among the factors identified, Y box-binding protein-1 (YB-1) and the heterogeneous nuclear ribonucleoproteins (hnRNPs), hnRNP A1, hnRNP A2/B1, and hnRNP Q, bind to the dengue virus 3′-untranslated region. Further analysis indicated that YB-1 binds to the dengue virus 3′ stem loop, a conserved structural feature located at the 3′ terminus of the 3′-untranslated region of many flaviviruses. Analysis of the impact of YB-1 on replication of dengue virus in YB-1+/+ and YB-1–/– mouse embryo fibroblasts indicated that host YB-1 mediates an antiviral effect. Further studies demonstrated that this antiviral impact is due, at least in part, to a repressive role of YB-1 on dengue virus translation via a mechanism that requires viral genomic sequences. These results suggest a novel role for YB-1 as an antiviral host cell factor. Dengue virus (DENV) 2The abbreviations used are: DENV, dengue virus; hnRNP, heterogeneous nuclear ribonucleoprotein; UTR, untranslated region; HIV-1, human immunodeficiency virus, type 1; GMP-PNP, guanosine-5′-[(β,γ)-imido]triphosphate; RT, reverse transcription; MEF, mouse embryo fibroblast; EMSA, electrophoretic mobility shift assay; m.o.i., multiplicity of infection; FCS, fetal calf serum; BHK, baby hamster kidney; MALDI, matrix-assisted laser desorption ionization; MS/MS, tandem mass spectrometry; 3′SL, 3′ stem loop.2The abbreviations used are: DENV, dengue virus; hnRNP, heterogeneous nuclear ribonucleoprotein; UTR, untranslated region; HIV-1, human immunodeficiency virus, type 1; GMP-PNP, guanosine-5′-[(β,γ)-imido]triphosphate; RT, reverse transcription; MEF, mouse embryo fibroblast; EMSA, electrophoretic mobility shift assay; m.o.i., multiplicity of infection; FCS, fetal calf serum; BHK, baby hamster kidney; MALDI, matrix-assisted laser desorption ionization; MS/MS, tandem mass spectrometry; 3′SL, 3′ stem loop. is the etiologic agent of dengue fever, currently the most prevalent arthropod-borne viral disease of humans. DENV is related to other medically important flaviviruses, including West Nile, yellow fever, and Japanese encephalitis viruses, and is transmitted to humans by the mosquitoes Aedes aegypti and Aedes albopictus. DENV contains a positive-sense RNA genome of 10.7 kb that is translated in the cytoplasm of infected cells as a single polyprotein and subsequently cleaved to yield three structural proteins (capsid, envelope, and pre-membrane) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) (1Hahn C.S. Hahn Y.S. Rice C.M. Lee E. Dalgarno L. Strauss E.G. Strauss J.H. J. Mol. Biol. 1987; 198: 33-41Crossref PubMed Scopus (292) Google Scholar). Following a requisite first round of viral translation in the cytoplasm, the subsequent steps of genome replication, second round translation, and assembly are coordinated to generate infectious DENV virions. Four serotypes of DENV (DENV1–4) co-circulate in most endemic countries, increasing the incidence of the more severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome. To date, only palliative treatments and supportive therapies are available for tens of millions of patients who acquire the disease annually. The identification of host cell factors that play a role in DENV infection remains an essential and largely unexplored area in the characterization of the mechanism of viral replication and the development of effective antiviral therapies. The DENV genome contains a 7mG cap and is flanked by 5′- and 3′-untranslated regions (UTRs) of ∼96 and 451 nucleotides, respectively. The 3′-UTR of DENV differs from nearly all host cell mRNA 3′-UTRs in that it lacks a poly(A) tail. The DENV 5′- and 3′-UTRs contain sequences and secondary structures that are highly conserved among flaviviruses and are important for the regulation of translation and replication (1Hahn C.S. Hahn Y.S. Rice C.M. Lee E. Dalgarno L. Strauss E.G. Strauss J.H. J. Mol. Biol. 1987; 198: 33-41Crossref PubMed Scopus (292) Google Scholar, 2Alvarez D.E. De Lella Ezcurra A.L. Fucito S. Gamarnik A.V. Virology. 2005; 339: 200-212Crossref PubMed Scopus (231) Google Scholar, 3Alvarez D.E. Lodeiro M.F. Luduena S.J. Pietrasanta L.I. Gamarnik A.V. J. Virol. 2005; 79: 6631-6643Crossref PubMed Scopus (280) Google Scholar, 4Chiu W.W. Kinney R.M. Dreher T.W. J. Virol. 2005; 79: 8303-8315Crossref PubMed Scopus (101) Google Scholar, 5Filomatori C.V. Lodeiro M.F. Alvarez D.E. Samsa M.M. Pietrasanta L. Gamarnik A.V. Genes Dev. 2006; 20: 2238-2249Crossref PubMed Scopus (288) Google Scholar, 6Holden K.L. Harris E. Virology. 2004; 329: 119-133Crossref PubMed Scopus (102) Google Scholar, 7Holden K.L. Stein D.A. Pierson T.C. Ahmed A.A. Clyde K. Iversen P.L. Harris E. Virology. 2006; 344: 439-452Crossref PubMed Scopus (119) Google Scholar, 8Kinney R.M. Huang C.Y. Rose B.C. Kroeker A.D. Dreher T.W. Iversen P.L. Stein D.A. J. Virol. 2005; 79: 5116-5128Crossref PubMed Scopus (106) Google Scholar, 9Olsthoorn R.C. Bol J.F. RNA (N. Y.). 2001; 7: 1370-1377PubMed Google Scholar, 10Tilgner M. Deas T.S. Shi P.Y. Virology. 2005; 331: 375-386Crossref PubMed Scopus (80) Google Scholar, 11You S. Falgout B. Markoff L. Padmanabhan R. J. Biol. Chem. 2001; 276: 15581-15591Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 12Yu L. Markoff L. J. Virol. 2005; 79: 2309-2324Crossref PubMed Scopus (74) Google Scholar). Viral end-to-end interactions have been demonstrated directly (3Alvarez D.E. Lodeiro M.F. Luduena S.J. Pietrasanta L.I. Gamarnik A.V. J. Virol. 2005; 79: 6631-6643Crossref PubMed Scopus (280) Google Scholar) and shown to be involved in DENV RNA synthesis (5Filomatori C.V. Lodeiro M.F. Alvarez D.E. Samsa M.M. Pietrasanta L. Gamarnik A.V. Genes Dev. 2006; 20: 2238-2249Crossref PubMed Scopus (288) Google Scholar). 5′-to-3′-UTR interactions are presumed to be involved in the regulation of viral translation as well (13Edgil D. Harris E. Virus Res. 2006; 119: 43-51Crossref PubMed Scopus (49) Google Scholar). The ∼100 nucleotides at the 3′ terminus of the 3′-UTR of all flaviviruses form a thermodynamically stable and conserved structural element, the 3′ stem loop (3′SL), that has been demonstrated to play an important role in viral replication. Deletion of the 3′SL significantly reduces translation of viral reporter RNAs (6Holden K.L. Harris E. Virology. 2004; 329: 119-133Crossref PubMed Scopus (102) Google Scholar), whereas mutagenesis of the 3′SL has been demonstrated to impair DENV translation and replication (7Holden K.L. Stein D.A. Pierson T.C. Ahmed A.A. Clyde K. Iversen P.L. Harris E. Virology. 2006; 344: 439-452Crossref PubMed Scopus (119) Google Scholar, 12Yu L. Markoff L. J. Virol. 2005; 79: 2309-2324Crossref PubMed Scopus (74) Google Scholar). The loop region of the 3′SL, referred to as the pentanucleotide loop, contains sequences that are necessary for replication of flaviviruses, including West Nile and dengue viruses (10Tilgner M. Deas T.S. Shi P.Y. Virology. 2005; 331: 375-386Crossref PubMed Scopus (80) Google Scholar). Phosphorodiamidate oligomers targeted to this region interfered with both translation and replication of DENV reporter replicons (7Holden K.L. Stein D.A. Pierson T.C. Ahmed A.A. Clyde K. Iversen P.L. Harris E. Virology. 2006; 344: 439-452Crossref PubMed Scopus (119) Google Scholar, 8Kinney R.M. Huang C.Y. Rose B.C. Kroeker A.D. Dreher T.W. Iversen P.L. Stein D.A. J. Virol. 2005; 79: 5116-5128Crossref PubMed Scopus (106) Google Scholar). Despite the delineation of many important cis-elements in the DENV genome, our understanding of the molecular basis of their action remains unclear. In particular, very little is currently understood about the role of host factors in maintenance and modulation of DENV genomic structure and function. Although host-derived proteins, including La autoantigen (La) (14De Nova-Ocampo M. Villegas-Sepulveda N. del Angel R.M. Virology. 2002; 295: 337-347Crossref PubMed Scopus (157) Google Scholar, 15Garcia-Montalvo B.M. Medina F. del Angel R.M. Virus Res. 2004; 102: 141-150Crossref PubMed Scopus (70) Google Scholar, 16Yocupicio-Monroy M. Padmanabhan R. Medina F. del Angel R.M. Virology. 2007; 357: 29-40Crossref PubMed Scopus (55) Google Scholar, 17Yocupicio-Monroy R.M. Medina F. Reyes-del Valle J. del Angel R.M. J. Virol. 2003; 77: 3067-3076Crossref PubMed Scopus (49) Google Scholar), eukaryotic elongation factor 1A (eEF1A) (14De Nova-Ocampo M. Villegas-Sepulveda N. del Angel R.M. Virology. 2002; 295: 337-347Crossref PubMed Scopus (157) Google Scholar, 18Blackwell J.L. Brinton M.A. J. Virol. 1997; 71: 6433-6444Crossref PubMed Google Scholar), and the polypyrimidine tract-binding protein (14De Nova-Ocampo M. Villegas-Sepulveda N. del Angel R.M. Virology. 2002; 295: 337-347Crossref PubMed Scopus (157) Google Scholar), have been shown to interact with the 3′-UTRs of DENV and West Nile virus genomes, their precise roles in viral replication are not clear. For example, investigators have demonstrated that La can bind to both the 5′- and 3′-UTRs of DENV (15Garcia-Montalvo B.M. Medina F. del Angel R.M. Virus Res. 2004; 102: 141-150Crossref PubMed Scopus (70) Google Scholar). Although subsequent experiments indicated that La undergoes changes in cellular localization during DENV infection and that La can repress viral RNA synthesis in vitro (16Yocupicio-Monroy M. Padmanabhan R. Medina F. del Angel R.M. Virology. 2007; 357: 29-40Crossref PubMed Scopus (55) Google Scholar), using a dominant negative La protein, we were unable to demonstrate any impact of La on DENV propagation in cultured cells. 3J. Walker, S. Paranjape, and E. Harris, unpublished results.3J. Walker, S. Paranjape, and E. Harris, unpublished results. Clearly, the identification and characterization of host cell factors that are involved in the orchestration of different stages of the DENV life cycle will extend our understanding of the molecular mechanisms of DENV replication. One of the challenges to identification of host cell proteins that interact with a viral genome is to ensure that isolated proteins bind with sufficient specificity and affinity to suggest biological significance. To increase the likelihood of biochemical isolation of physiologically relevant host cell binding factors, we have utilized an in vitro biochemical system that mimics properties of DENV infection observed in vivo. Specifically, we employed RNA affinity chromatography to isolate DENV UTR-interacting host cell factors from a baby hamster kidney cell extract that recapitulates DENV translation dynamics observed in vivo. In further experiments, we investigated the role of one of the host cell factors identified, Y box-binding protein 1 (YB-1), in DENV replication. YB-1 is a cold shock domain protein that has been shown previously to be involved in diverse cellular processes, including the regulation of cap-dependent translation, transcription, and signal transduction (reviewed in Ref. 19Kohno K. Izumi H. Uchiumi T. Ashizuka M. Kuwano M. BioEssays. 2003; 25: 691-698Crossref PubMed Scopus (432) Google Scholar). YB-1 has previously been implicated in oncogenesis and has been shown to associate with several DNA viruses. Our results indicate that YB-1 elicits an antiviral effect on DENV replication that is mediated, in part, through repression of DENV viral translation. Cell Lines, Extracts, Virus Infection, and Flow Cytometry—Baby hamster kidney (BHK)-21 cells (clone 15) were maintained in minimum essential media α (Invitrogen) containing 5% fetal calf serum (FCS, HyClone, Logan, UT), 2 mm Glutamax (Invitrogen), and 100 units of penicillin/100 μg of streptomycin (P/S) (Invitrogen) at 37 °C in 5% CO2. Immortalized mouse embryo fibroblasts (MEFs) generated from YB-1+/+ and YB-1–/– mice (20Lu Z.H. Books J.T. Ley T.J. Mol. Cell. Biol. 2005; 25: 4625-4637Crossref PubMed Scopus (134) Google Scholar) were obtained from Zhi Hong Lu and Timothy Ley (Washington University, St Louis). Cells were maintained in Dulbecco's modified Eagle's media (Invitrogen) containing 10% FCS, 0.1 mm nonessential amino acids (Invitrogen), 2 mm Glutamax (Invitrogen), and P/S. C6/36 A. albopictus mosquito cells were maintained in Leibovitz's L-15 medium (Invitrogen) supplemented with 10% FCS, P/S, and 100 mm HEPES, pH 7.2. For extract preparation, BHK cells were adapted for growth in suspension by maintenance in a rich growth medium. Specifically, cells were grown in RPMI 1640 medium (Invitrogen) containing 10% FCS, 10 mm HEPES, pH 7.2, and P/S. Cells were seeded in a spinner bottle and supplemented with growth medium every 2–3 days to maintain a density of 106 cells/ml. Twelve to 24 h prior to harvesting, growth medium was added to ensure that harvested cells were in growth phase. Translation extracts were prepared as described previously (21Edgil D. Diamond M.S. Holden K.L. Paranjape S.M. Harris E. Virology. 2003; 317: 275-290Crossref PubMed Scopus (52) Google Scholar). Briefly, BHK cells were harvested, washed with PBS, resuspended in hypotonic lysis buffer (21Edgil D. Diamond M.S. Holden K.L. Paranjape S.M. Harris E. Virology. 2003; 317: 275-290Crossref PubMed Scopus (52) Google Scholar), and lysed via passage through a 21-gauge needle. Lysates were subjected to centrifugation (10,000 × g) for 5 min. Supernatant was harvested and supplemented with glycerol to a final concentration of 10% prior to flash-freezing in liquid nitrogen. Translation extracts were thawed and supplemented with a freshly prepared mixture of ATP, GTP, creatine phosphokinase, phosphocreatine, and protease inhibitors prior to use. Infection of MEFs by DENV2 strain 16681 was performed as described previously (22Diamond M.S. Edgil D. Roberts T.G. Lu B. Harris E. J. Virol. 2000; 74: 7814-7823Crossref PubMed Scopus (212) Google Scholar). Briefly, cells were incubated with virus at an indicated m.o.i. for 2 h in a volume of medium sufficient to cover the cells. At 2 h post-infection, cells were washed four times and then incubated in growth medium. At indicated times post-infection, cell supernatants were collected and supplemented with FCS to 20%. Detached cells and cellular debris were removed by centrifugation at 1200 rpm for 3 min. Viral supernatants were stored at –80 °C until plaque assays were conducted. Plaque assays were performed on BHK cells as described previously (22Diamond M.S. Edgil D. Roberts T.G. Lu B. Harris E. J. Virol. 2000; 74: 7814-7823Crossref PubMed Scopus (212) Google Scholar). Flow cytometric analysis was performed to determine intracellular levels of DENV2 NS3 protein in infected MEF cells. Flow cytometry was conducted as described previously (22Diamond M.S. Edgil D. Roberts T.G. Lu B. Harris E. J. Virol. 2000; 74: 7814-7823Crossref PubMed Scopus (212) Google Scholar, 23Helt A.M. Harris E. J. Virol. 2005; 79: 13218-13230Crossref PubMed Scopus (39) Google Scholar). DENV NS3 protein was detected using a mouse monoclonal antibody to DENV NS3. 4P. R. Beatty and E. Harris, unpublished results. Following overnight incubation with the primary antibody at 4 °C, samples were washed with PBS and stained with a secondary Alexa 488-conjugated goat anti-mouse antibody (Invitrogen) for 1–2 h at 25 °C. NS3 expression was measured using an EPICs XL flow cytometer (Beckman-Coulter, Fullerton, CA). RNA Constructs, in Vitro Transcription, and Transfection—A region consisting of five nucleotides of NS5 and the entire 3′-UTR of DENV2 (451 nucleotides) was amplified from infectious clone pD2/IC (a gift of Richard Kinney, Centers for Disease Control and Prevention, Fort Collins, CO) using a forward primer containing an EcoRI site and a T7 promoter (5′-CCGGAATTCTAATACGACTCACTATAGGTAGAAAGCAAAAC-3′) and a reverse primer containing an XbaI restriction site (5′-GACTTCTAGCCTTGTTTCATGTTAG-3′). PCR products were cloned into pBSKS II (Stratagene, La Jolla, CA) to create a vector from which sense and antisense 3′-UTR could be transcribed in separate reactions following digestion with different restriction enzymes and gel purification. DENV2 3′SL constructs were generated from the DENV2 3′-UTR constructs using a forward primer containing an EcoRI site, a T7 consensus site, and homology to the 5′ end of the 3′SL (5′-CGGAATTCTAATACGACTCACTATAGGGAAAGACCAGAGATCCTGCTGTCTCC-3′) and a reverse primer with homology to the 3′ end of the 3′SL (5′-GGTCGACTCTAGAGAACCTGTTGATTCAAAC-3′). PCR fragments were subcloned into pTOPO (Invitrogen), excised, and cloned into pBSKS II (Stratagene). DENV2 3′-UTR RNA and firefly luciferase reporter RNAs, 5DLuc3D and 5DLuc3ΔSL (6Holden K.L. Harris E. Virology. 2004; 329: 119-133Crossref PubMed Scopus (102) Google Scholar), and DENV2 Renilla luciferase replicons, (p)DRrep and (p)DRrep-RdRPmut, 5K. Clyde, J. Barrera, and E. Harris, submitted for publication. were transcribed using a Ribomax T7 RNA polymerase kit (Promega, Madison, WI). A 7-methyl-GpppA nucleotide was incorporated at the initial adenosine residue during transcription of luciferase reporter constructs (New England Biolabs, Ipswich, MA). Unincorporated nucleotides were removed using Nuc-Away columns (Ambion, Austin, TX). RNA was concentrated, when necessary, by ethanol/ammonium acetate precipitation. DENV2 luciferase reporter constructs were transfected into YB-1–/– and YB-1+/+ MEF and BHK cells using Lipofectamine 2000 (Invitrogen) as described previously (7Holden K.L. Stein D.A. Pierson T.C. Ahmed A.A. Clyde K. Iversen P.L. Harris E. Virology. 2006; 344: 439-452Crossref PubMed Scopus (119) Google Scholar). MEF and BHK cells transfected with Renilla luciferase replicons were harvested at various times post-transfection as indicated. MEF cells transfected with 5DLuc3D and 5DLuc3ΔSL firefly luciferase reporter constructs were harvested at 4–7 h post-transfection. Translation was monitored by luciferase production as described previously (6Holden K.L. Harris E. Virology. 2004; 329: 119-133Crossref PubMed Scopus (102) Google Scholar, 7Holden K.L. Stein D.A. Pierson T.C. Ahmed A.A. Clyde K. Iversen P.L. Harris E. Virology. 2006; 344: 439-452Crossref PubMed Scopus (119) Google Scholar). Briefly, cells were washed with PBS and lysed with Cell Culture Lysis Reagent (Promega) for assessment of firefly luciferase levels or with Renilla Luciferase Assay Lysis Buffer (Promega) for measurement of Renilla luciferase. Luciferase production was monitored using Luciferase Assay Reagent Substrate or Renilla Luciferase Assay Substrate (Promega) and a TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA). Assessment of transfection efficiency was determined by quantitative RT-PCR analysis as described elsewhere (7Holden K.L. Stein D.A. Pierson T.C. Ahmed A.A. Clyde K. Iversen P.L. Harris E. Virology. 2006; 344: 439-452Crossref PubMed Scopus (119) Google Scholar).5 Specifically, RNA was harvested from cells at 2 h post-transfection using the RNeasy system (Qiagen, Valencia, CA) or mini RNA Isolation II kits (Zymoresearch, Orange County, CA) and then quantitated using the Lux system (Invitrogen) for RNA reporter constructs or the Taqman system (Applied Biosystems, Foster City, CA) for DENV replicon constructs. Relative transfection efficiencies of YB-1–/– and YB-1+/+ cells were determined and used to normalize luciferase activity. Affinity Chromatography—To prepare affinity resin, 5 μm of DENV reporter RNA was oxidized by incubation in 0.1 m sodium periodate, 0.1 m NaOAc, pH 5.0, for 1 h at 25°C. Following oxidation, RNA was precipitated by addition of 1 ml of ice-cold ethanol and isolated by centrifugation at 10,000 × g for 15 min. Precipitated RNA was washed with 70% ethanol, dried briefly in a speedvac, and resuspended in 0.1 m NaOAc, pH 5.0. Coupling of RNA to the resin was accomplished by incubating oxidized RNA with 100 μl of hydrazide-agarose resin (Sigma) (equilibrated in 0.1 m NaOAc, pH 5.0) on an end-over-end rotator at 4 °C for 12–18 h. Uncoupled RNA was removed by washing three times each with 2.0 m NaCl, 0.1 m NaOAc, and translation buffer (21Edgil D. Diamond M.S. Holden K.L. Paranjape S.M. Harris E. Virology. 2003; 317: 275-290Crossref PubMed Scopus (52) Google Scholar). Equilibrated resin was incubated with BHK translation extract at 30 °C for 30–60 min. Reactions were conducted in the presence of translation inhibitors and/or RNA competitors as indicated. Translation competence of the extract was monitored by luciferase production using Luciferase Assay Reagent (Promega) as above. Following binding, protein-RNA complexes were washed extensively in buffer containing 100 mm KCl and then eluted with a step gradient of KCl (0.1–1 m). Eluted protein fractions were concentrated with 20% trichloroacetic acid containing deoxycholate salt and resolved by SDS-PAGE. Proteins were stained with Coomassie Colloidal Blue (Invitrogen). Protein bands of interest were excised and submitted for MS/MS mass spectrometry as described below. Mass Spectrometry—Gel bands of interest were excised and digested with trypsin (Promega) (25Bienvenut W.V. Deon C. Pasquarello C. Campbell J.M. Sanchez J.C. Vestal M.L. Hochstrasser D.F. Proteomics. 2002; 2: 868-876Crossref PubMed Scopus (135) Google Scholar). The resulting digests were purified with C18 μZipTips (Millipore) and subjected to mass spectrometric analysis on an Applied Biosystems 4700 Proteomics Analyzer, which is a MALDI-time-of-flight tandem mass spectrometer (26Jiminez, A., Huang, L., Qiu, Y., and Burlingame, A. L. (1998) in Current Protocols in Protein Science (Coligan, J., Dunn, B. M., Ploegh, H. L., Speicher, D. W., and Wingfield, P. T., eds) pp. 16.14.11–16.14.15, Wiley Interscience, New YorkGoogle Scholar). The purified sample was mixed 1:1 with α-cyano-4-hydroxycinnamic acid matrix (10 g/liter), and 1.2 μl of the mixture was spotted onto the MALDI target plate. A reflector mode mass spectrum of the digest was first obtained, after which several individual peptide peaks were manually selected for MS/MS analysis. The peptide sequence for each was manually deduced from its MS/MS spectrum. Viral RNA Immunoprecipitation—BHK cells were infected with DENV2 strain 16681 at an m.o.i. of 1. Specifically, BHK cells at 80% confluency were incubated with DENV2 16681 in RPMI medium with 2% FCS and P/S. Following incubation for 2 h, cells were washed four times to remove unincorporated virus and then incubated in RPMI growth media. At the indicated times post-infection, cells were harvested and cross-linked with 1% formaldehyde. Cells were disrupted by bead-beating in lysis buffer (50 mm HEPES/KOH, pH 7.5, 140 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride, 1 mm leupeptin, 1 mm aprotinin, and 20 units/ml Superasin (Ambion)), and resultant cell lysates were immunoprecipitated with anti-YB-1 polyclonal rabbit antibody (gift of Valentina Evdokimova, University of British Columbia, Vancouver, Canada). Co-immunoprecipitated RNA was isolated using an RNeasy mini kit (Qiagen). One-step RT-PCR (Qiagen) was conducted using forward (5′-TTCCACACAATGTGGCACGTCAC-3′) and reverse (5′-GGAGATCCTGACGTTCCRGG-3′) primers homologous to DENV NS3. RT-PCR products were resolved via electrophoresis on 1% agarose and visualized with ethidium bromide. Footprinting and Electrophoretic Mobility Shift Analysis—RNA encoding the 3′SL of DENV was transcribed in vitro with T7 RNA polymerase. Following purification using NucAway columns (Ambion) to remove unincorporated nucleotides, RNA probes were labeled with [γ-32P]ATP. Probes were resolved on a 5% denaturing acrylamide gel, excised, and eluted. Gel-purified probe was extracted with phenol/chloroform/isoamyl alcohol, ethanol-precipitated, and resuspended in double distilled H2O. Probe concentration was determined by measurement of absorbance at 260 nm, and specific activity was determined by scintillation counting. Recombinant His-YB-1 was expressed in Escherichia coli using pHISYB-1 (gift of Valentina Evdokimova) following the procedure of Evdokimova et al. (27Evdokimova V. Ruzanov P. Imataka H. Raught B. Svitkin Y. Ovchinnikov L.P. Sonenberg N. EMBO J. 2001; 20: 5491-5502Crossref PubMed Scopus (227) Google Scholar). For footprinting reactions, 10 pm of probe was incubated with purified His-YB-1 protein (at molar ratios indicated) for 15 min at room temperature. Subsequently, RNase T2 (Sigma), RNase T1 (Ambion), or RNase VI (Ambion) was added at the concentrations indicated, and reactions were incubated for an additional 10 min at room temperature before being stopped by the addition of phenol/chloroform/isoamyl alcohol. Following phenol/chloroform extraction, reactions were ethanol-precipitated in the presence of 3 μg of glycogen (Ambion). Samples were resuspended in formaldehyde loading buffer, heated to 95 °C for 3 min, and immediately cooled on ice. One-third of each sample was resolved on a 10% denaturing polyacrylamide gel. For electrophoretic mobility shift assays, 3′SL RNA probes were synthesized via in vitro transcription with T7 RNA polymerase, and [α-32P]CTP was incorporated during transcription. 3′SL probe (10,000 cpm per reaction) was incubated with buffer or 5- or 10-fold molar excess of YB-1 at 25 °C for 15 min. Samples were resolved by electrophoresis on a 5%, 0.5 × TBE nondenaturing acrylamide minigel. Gels were dried and visualized using a PhosphorImager (GE Healthcare). Isolation and Identification of Host Cell Proteins That Bind to the DENV 3′-UTR—To isolate and identify mammalian host factors that are involved in various stages of DENV replication, we employed an RNA affinity chromatography strategy that involved the use of a mammalian cell extract that was competent for translation. BHK cells support high levels of DENV replication and, consequently, are widely employed to study DENV infection. This prompted us to develop a methodology for large scale production of BHK extracts. The BHK extract faithfully recapitulated DENV 3′-UTR-dependent translation observed in cells (6Holden K.L. Harris E. Virology. 2004; 329: 119-133Crossref PubMed Scopus (102) Google Scholar). For example, translation of DENV reporter RNAs (described in Fig. 1A) harboring the full-length 3′-UTR (5DLuc3D) was significantly more efficient than translation of constructs containing a deletion of the 3′SL (5DLuc3ΔSL) or lacking the DENV 3′-UTR (Fig. 1B and data not shown), similar to results obtained in cultured cells (6Holden K.L. Harris E. Virology. 2004; 329: 119-133Crossref PubMed Scopus (102) Google Scholar). Moreover, translation in BHK cell extracts was effectively competed with excess sense (Fig. 1B) but not antisense DENV 3′-UTR RNA (Fig. 1C). At high concentrations (100-fold molar excess) of antisense RNA, nonspecific titration of translation factors is likely responsible for the slight decrease in translation that is observed (Fig. 1C). BHK extracts were distinct from rabbit reticulocyte lysates; translation of 5DLuc3D reporter constructs could not be efficiently competed by excess 3′-UTR sense competitor when reactions were performed in rabbit reticulocyte lysate extracts (data not shown). We reasoned that RNA affinity chromatography employing this functional BHK extract should therefore enable us to identify factors present in BHK cells that are potentially important for mediating DENV translation by the 3′-UTR. To isolate proteins that associate with the UTRs of the DENV genome, 5DLuc3D RNA reporter constructs (Fig. 1A) were transcribed in vitro with T7 RNA polymerase, oxidized with sodium periodate, and coupled to hydrazide-agarose resin. Neither oxidation of RNA nor immobilization of reporter constructs prevented translational competency of reporter RNAs (data not shown). After extensive washing to remove uncoupled RNA, immobilized reporter constructs were incubated at 30 °C in a BHK S-10 translation extract. Importantly, the BHK S-10 lysate was not nuclease-treated, thereby ensuring the presence of endogenous competitor RNAs and more closely mimicking physiologic conditions. In these experiments, BHK extracts were treated with the nonhydrolyzable GTP analogue, GMP-PNP, which prevents translation by inhibiting the for" @default.
• W1973729131 created "2016-06-24" @default.
• W1973729131 creator A5027442064 @default.
• W1973729131 creator A5045237456 @default.
• W1973729131 date "2007-10-01" @default.
• W1973729131 modified "2023-09-28" @default.
• W1973729131 title "Y Box-binding Protein-1 Binds to the Dengue Virus 3′-Untranslated Region and Mediates Antiviral Effects" @default.
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