A Region of the Epstein-Barr Virus (EBV) mRNA Export Factor EB2 Containing an Arginine-rich Motif Mediates Direct Binding to RNA

Abstract

The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because ∼10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVΔEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle. The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because ∼10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVΔEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle. Several herpes viruses contain a gene whose product has the characteristics of an mRNA export factor, e.g. herpes simplex virus type 1 (HSV-1) 1The abbreviations used are: HSV-1, herpes simplex virus type 1; EBV, Epstein-Barr virus; RNP, ribonucleoprotein; GST, glutathione S-transferase; REMSA, RNA electrophoretic mobility shift assay; ARM, arginine-rich RNA-binding motif; nt, nucleotide; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline; RT, reverse transcription; NLS, nuclear localization signal; HIV-1, human immunodeficiency virus, type 1.1The abbreviations used are: HSV-1, herpes simplex virus type 1; EBV, Epstein-Barr virus; RNP, ribonucleoprotein; GST, glutathione S-transferase; REMSA, RNA electrophoretic mobility shift assay; ARM, arginine-rich RNA-binding motif; nt, nucleotide; CAT, chloramphenicol acetyltransferase; PBS, phosphate-buffered saline; RT, reverse transcription; NLS, nuclear localization signal; HIV-1, human immunodeficiency virus, type 1. protein ICP27, Epstein-Barr virus (EBV) protein EB2, human herpes virus type 8 (HHV8) protein ORF57, and herpes virus saimiri (HVS) protein ORF57 (for references, see Ref. 1Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (215) Google Scholar). At least for HSV-1 and EBV, the respective deletion of the ICP27 (2McCarthy A.M. McMahan L. Schaffer P.A. J. Virol. 1989; 63: 18-27Crossref PubMed Google Scholar) and EB2 (3Gruffat H. Batisse J. Pich D. Neuhierl B. Manet E. Hammerschmidt W. Sergeant A. J. Virol. 2002; 76: 9635-9644Crossref PubMed Scopus (72) Google Scholar) genes abolished both the cytoplasmic accumulation of specific viral mRNAs and the production of infectious viral particles, demonstrating that ICP27 and EB2 are essential viral factors whose function cannot be transcomplemented by cellular factors. The specific function(s) of these viral factors in mRNA export is, however, not yet understood. Most of the early and late HSV-1 and EBV mRNAs are transcribed from intronless genes. However, transcription, splicing, and mRNA export are linked in eukaryotic cells (4Luo M. Reed R. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14937-14942Crossref PubMed Scopus (298) Google Scholar, 5Zhou Z. Luo M. Strässer K. Katahira J. Hurt E. Reed R. Nature. 2000; 407: 401-405Crossref PubMed Scopus (397) Google Scholar, 6Luo M.L. Zhou Z. Magni K. Christoforides C. Rappsilber J. Mann M. Reed R. Nature. 2001; 413: 644-647Crossref PubMed Scopus (300) Google Scholar, 7Strässer K. Hurt E. Nature. 2001; 413: 648-652Crossref PubMed Scopus (222) Google Scholar, 8Lei E.P. Krebber H. Silver P.A. Genes Dev. 2001; 15: 1771-1782Crossref PubMed Scopus (183) Google Scholar, 9Strässer K. Masuda S. Mason P. Pfannstiel J. Poppizzi M. Rodriguez-Navarro S. Rondon A.G. Aguilera A. Struhl K. Reed R. Hurt E. Nature. 2002; 417: 304-308Crossref PubMed Scopus (632) Google Scholar, 10Hieronymus H. Silver P.A. Nat. Genet. 2003; 33: 155-161Crossref PubMed Scopus (171) Google Scholar). For example, one model for mRNA export suggests that intron removal in metazoans is associated with the deposition, 20–24 nucleotides upstream of the exon-exon junction, of a multiprotein complex (EJC) including REF/Aly, Y14, RNPS1, SRm160, and magoh (11Le Hir H. Moore M. Maquat L.E. Genes Dev. 2000; 14: 1098-1108PubMed Google Scholar, 12Le Hir H. Izaurralde E. Maquat L.E. Moore M.J. EMBO J. 2000; 19: 6860-6869Crossref PubMed Scopus (697) Google Scholar, 13Kim V.N. Yong J. Kataoka N. Abel L. Diem M.D. Dreyfuss G. EMBO J. 2001; 20: 2062-2068Crossref PubMed Scopus (165) Google Scholar, 14Kataoka N. Diem M.D. Kim V.N. Yong J. Dreyfuss G. EMBO J. 2001; 20: 6424-6433Crossref PubMed Scopus (168) Google Scholar). In such a complex, REF/Aly recruits TAP/NXF1 to cellular mRNPs (15Grüter P. Tabernero C. von Kobbe C. Schmitt C. Saavedra C. Bachi A. Wilm M. Felber B.K. Izaurralde E. Mol. Cell. 1998; 1: 649-659Abstract Full Text Full Text PDF PubMed Scopus (470) Google Scholar, 16Strässer K. Hurt E. EMBO J. 2000; 19: 410-420Crossref PubMed Google Scholar, 17Stutz F. Bachi A. Doerks T. Braun I.C. Seraphin B. Wilm M. Bork P. Izaurralde E. RNA. 2000; 6: 638-650Crossref PubMed Scopus (305) Google Scholar, 18Le Hir H. Gatfield D. Izaurralde E. Moore M. EMBO J. 2001; 20: 4987-4997Crossref PubMed Scopus (593) Google Scholar). TAP/NXF1 interaction with nuclear pore components then targets mRNPs to the nuclear pore complex and promotes their translocation into the cytoplasm (19Strässer K. Bassler J. Hurt E. J. Cell Biol. 2000; 150: 695-706Crossref PubMed Scopus (183) Google Scholar). However, mRNAs are still detected in the cytoplasm of Drosophila cells depleted of all EJC proteins, suggesting that additional adaptor protein(s) may mediate the interaction between TAP and cellular mRNAs (20Gatfield D. Izaurralde E. J. Cell Biol. 2002; 159: 579-588Crossref PubMed Scopus (170) Google Scholar). Cellular mRNAs generated from intronless genes are likely to be exported out of the nucleus through nonspecific interactions with mRNA-bound adaptors like REF/Aly (21Rodriguez J.P. Rode M. Gatfield D. Blencowe B.J. Carmo-Fonseca M. Izaurralde E. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1030-1035Crossref PubMed Scopus (216) Google Scholar) or through sequence-specific interactions with SRp20, 9G8 (22Huang Y. Steitz J.A. Mol. Cell. 2001; 7: 899-905Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), or U2AF (23Zolotukhin A.S. Tan W. Bear J. Smulevitch S. Felber B.K. J. Biol. Chem. 2002; 277: 3935-3942Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It must be emphasized that SRp20 and 9G8, like REF, interact directly with TAP/NXF1 (24Huang Y. Gattoni R. Stevenin J. Steitz J.A. Mol. Cell. 2003; 11: 837-843Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Interestingly, it has been demonstrated that ICP27 recruits the cellular mRNA export factors REF/Aly and TAP/NXF1 to viral mRNAs generated from viral intronless genes (25Koffa M.D. Clements J.B. Izaurralde E. Wadd S. Wilson S.A. Mattaj I.W. Kuersten S. EMBO J. 2001; 20: 5769-5778Crossref PubMed Scopus (138) Google Scholar), thus allowing viral mRNAs to access the cellular mRNA nuclear export pathway. ICP27 also interacts with SRp20 (26Sciabica K.S. Dai Q.J. Sandri-Goldin R.M. EMBO J. 2003; 22: 1608-1619Crossref PubMed Scopus (134) Google Scholar), making it likely that ICP27-mediated mRNA export occurs by recruitment of TAP by REF and/or SRp20. The above results strongly suggest that several adapters may recruit TAP for efficient and perhaps cooperative nuclear export of mRNAs generated from both intron-containing and intronless genes (24Huang Y. Gattoni R. Stevenin J. Steitz J.A. Mol. Cell. 2003; 11: 837-843Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). The EBV EB2 protein also exports RNAs generated from both intronless and intron-containing genes (27Buisson M. Hans F. Kusters I. Duran N. Sergeant A. J. Virol. 1999; 73: 4090-4100Crossref PubMed Google Scholar, 28Semmes O.J. Chen L. Sarisky R. Gao Z. Zhong L. Hayward S.D. J. Virol. 1998; 72: 9526-9534Crossref PubMed Google Scholar, 29Farjot G. Buisson M. Duc Dodon M. Gazzolo L. Sergeant A. Mikaelian I. J. Virol. 2000; 74: 6068-6076Crossref PubMed Scopus (61) Google Scholar) and binds REF/TAP-containing mRNPs in vivo (30Hiriart E. Farjot G. Gruffat H. Nguyen M.V. Sergeant A.P. Manet E. J. Biol. Chem. 2003; 278: 335-342Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). EB2 and its HSV-1 functional homologue, ICP27, are likely to be factors involved in the specific export of intronless viral mRNAs, probably via direct interaction. ICP27-mediated, intronless viral mRNA export is thought to occur through direct RNA-binding via an "RGG box" located within the N terminus of the protein (1Sandri-Goldin R.M. Genes Dev. 1998; 12: 868-879Crossref PubMed Scopus (215) Google Scholar, 31Mears W.E. Rice S.A. J. Virol. 1996; 70: 7445-7453Crossref PubMed Google Scholar). However, three C-terminal domains that display homology to the KH RNA interaction motifs of hnRNP K could also be involved in RNA-binding affinity and specificity (32Soliman T.M. Silverstein S.J. J. Virol. 2000; 74: 2814-2825Crossref PubMed Scopus (56) Google Scholar). EB2 expressed as a GST fusion protein has also been shown to interact directly with RNA in vitro (27Buisson M. Hans F. Kusters I. Duran N. Sergeant A. J. Virol. 1999; 73: 4090-4100Crossref PubMed Google Scholar, 28Semmes O.J. Chen L. Sarisky R. Gao Z. Zhong L. Hayward S.D. J. Virol. 1998; 72: 9526-9534Crossref PubMed Google Scholar, 33Ruvolo V. Wang E. Boyle S. Swaminathan S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8852-8857Crossref PubMed Scopus (61) Google Scholar, 34Ruvolo V. Gupta A.K. Swaminathan S. J. Virol. 2001; 75: 6033-6041Crossref PubMed Scopus (42) Google Scholar). However, Northwestern blot experiments labeled a discrete band of 55 kDa but not the full-length 90-kDa GST-EB2 protein, indicating that RNA interaction was restricted to GST-EB2 degradation products in which the EB2 RXP repeats were unmasked and C-terminal (34Ruvolo V. Gupta A.K. Swaminathan S. J. Virol. 2001; 75: 6033-6041Crossref PubMed Scopus (42) Google Scholar). This finding confirmed our previously published results showing that a GST fusion protein in which the RXP repeats are C-terminal bound RNA in vitro (27Buisson M. Hans F. Kusters I. Duran N. Sergeant A. J. Virol. 1999; 73: 4090-4100Crossref PubMed Google Scholar), and this is also where the RXP repeats are located in the HSV-1 US11 RNA-binding factor (35Roller R.J. Roizman B. J. Virol. 1990; 64: 3463-3470Crossref PubMed Google Scholar). However, deletion of the RXP domain did not affect EB2-mediated mRNA export, which demonstrates that the RXP repeat is not an RNA-binding motif essential to this function (27Buisson M. Hans F. Kusters I. Duran N. Sergeant A. J. Virol. 1999; 73: 4090-4100Crossref PubMed Google Scholar). It is therefore not known whether EB2 binds RNA and, if so, by which motif(s). In this study we have used EB2 and EB2 mutants affinity-purified from human cells as well as GST-EB2 fusion peptides purified from Escherichia coli to locate, by Northwestern analysis and RNA electrophoretic mobility shift assay (REMSA), an RNA-binding motif present in a 33-amino acid domain of EB2 (peptide Da). This motif is not the RXP repeat but is rich in arginines and has structural features of the arginine-rich RNA-binding motif (ARM) also found in several RNA-binding proteins (36Lazinski D. Grzadzielska E. Das A. Cell. 1989; 59: 207-218Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 37Tan R. Frankel A.D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5282-5286Crossref PubMed Scopus (176) Google Scholar). The motif has an arginine-rich core and is predicted to fold into an α-helix, and, as a synthetic peptide, it binds RNA in the μm range in vitro. At least 10 GST-Da molecules bind onto a 180-nt-long, highly structured RNA probe in vitro, suggesting that the EB2 ARM is not sequence specific. A deletion in the ARM disrupts the capacity of EB2 to bind RNA in vitro and in vivo and export mRNA, but without affecting its nucleocytoplasmic shuttling. Using a transcomplementation assay, we also show here that EB2-RNA binding is essential for the production of EBV-infectious particles. Plasmids—The eukaryotic expression plasmids are cytomegalovirus immediate early (CMV IE) promoter-based vectors (pAAC derived from pBluescript KSII+ (Stratagene) (27Buisson M. Hans F. Kusters I. Duran N. Sergeant A. J. Virol. 1999; 73: 4090-4100Crossref PubMed Google Scholar). When F precedes the name of the protein, the corresponding protein has been tagged at the N terminus with the FLAG epitope, which is detected by the monoclonal antibody M2 (Sigma). pAAC.F.EB2 (29Farjot G. Buisson M. Duc Dodon M. Gazzolo L. Sergeant A. Mikaelian I. J. Virol. 2000; 74: 6068-6076Crossref PubMed Scopus (61) Google Scholar) expresses the wild-type EB2 protein initiated at the BSLF2 AUG. pAAC.F.NLS.EB2.Cter expresses a nuclear EB2 protein with the N-terminal 184 amino acids deleted (30Hiriart E. Farjot G. Gruffat H. Nguyen M.V. Sergeant A.P. Manet E. J. Biol. Chem. 2003; 278: 335-342Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). pAAC.F.EB2.ΔD1 expresses EB2 with amino acids 189–211 deleted (Fig. 5). Several partially overlapping EB2 cDNA fragments were cloned in pGex4T-2 digested by BamHI and XhoI to generate GST-B to H (Fig. 1). The EB2 cDNA fragments B, Ba, Bb, C, D, Da, Db, E, F, G, and H were generated by PCR using the following DNA primers: fragment B, primers 5′-CGGGATCCGATGAAGATCCAACT-3′ and 5′-CCGCTCGAGACTTTCATCGGTGCA-3′; fragment Ba, 5′-CGGGATCCTCATCCTCAGAGGAGG-3′ and 5′-CGGAATTCGTCCTGTAGGTCCCAC-3′; fragment Bb, 5′-CGGGATCCCCTACCTCGGCATC-3′ and 5′-CGGAATTCGGCCTCTGGTGTAAGAGG-3′; fragment C, primers 5′-CGGGATCCTCTTACACCAGAGGC-3′ and 5′-CCGCTCGAGGCGTTCTTGCCTCGC-3′; fragment D, primers 5′-CGGGATCCCCCCGGTCAGAATCT-3′ and 5′-CGGCTCGAGCTGCCAGGCTCCAAT-3′; fragment Da, 5′-CGGGATCCAGGAGCACAAGGAAGCA-3′ and 5′-CGGAATTCTGTAATCTTGGAGACAGGC-3′; fragment Db, 5′-CGGGATCCATGAGTCTGGTTAAGC-3′ and 5′-CGGAATTCGTCTTGGATGGGCTC-3′; fragment E, primers 5′-CGGGATCCGACCCGTTCCTACAG-3′ and 5′-CCGCTCGAGGTAGGTGATCTCCTG-3′; fragment F, primers 5′-CGGGATCCCTCTGCACCCTGGTG-3′ and 5′-CCGCTCGAGCTTGTTTTGACGGGC-3′; fragment G, primers 5′-CGGGATTCCGACTACAACTTTGTG-3′ and 5′-CCGCTCGAGCTCTGTCAAAAGGGA-3′; and fragment H, primers 5′-CGGGATCCTTCCTGGGCCACTAC-3′ and 5′-CCGCTCGAGTTGATTTAATCCAGG-3′. GST and GST fusion proteins were expressed in a BL21 codon plus bacteria and purified with glutathione-agarose beads using standard procedures. The concentration of the purified proteins was determined using the Bio-Rad protein assay. The reporter plasmid pDM128 has been described elsewhere (38Hope T.J. Bond B.L. McDonald D. Klein N.P. Parslow T.G. J. Virol. 1991; 65: 6001-6007Crossref PubMed Google Scholar) and was a generous gift from Dr. B. R. Cullen.Fig. 1Schematic representation of the EB2 protein and derived polypeptides is shown. The boundaries of the EB2 deletion mutant Cter are also shown. The 80-amino acid-long EB2 peptides B to H and the peptides Ba, Bb, Da, and Db, used in this study as GST fusion proteins, are delineated by black rectangles. Numbers on each end of the rectangles indicate the boundaries of the polypeptides on the EB2 amino acid sequence. NES, nuclear export signal. The two independent EB2 NLSs are indicated by thick vertical bars. RXP represents arginine-X-proline repeats.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Transfections and CAT Assays—Plasmids used for transfection were prepared by the alkaline lysis method and purified through two CsCl gradients. HeLa cells or 293T cells were grown at 37 °C in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum and were seeded at 8 × 105 cells per 100-mm-diameter Petri dish 10 h prior to transfection. Transfections were performed by the calcium precipitate method as described previously (29Farjot G. Buisson M. Duc Dodon M. Gazzolo L. Sergeant A. Mikaelian I. J. Virol. 2000; 74: 6068-6076Crossref PubMed Scopus (61) Google Scholar). To evaluate CAT protein expression, we used the Roche Applied Science CAT enzyme-linked immunosorbent assay kit. 48 h after transfection, cells were collected in phosphate buffered saline (PBS). Half of the cells were used for CAT assays according to the manufacturer's instructions. The other half was used to monitor protein expression by Western blots using the anti-FLAG M2 antibody. Immunoprecipitation of EB2-bound RNAs—To cross-link proteins to RNA, UV irradiation of transfected cells was made at 254 nm for 12 s in a Stratalinker 1800 (Stratagene). Total cell extracts were obtained after incubation of the transfected cells (106) in 300 μl of TNE buffer (10 mm Tris, pH8, 100 mm NaCl, and 1 mm EDTA) containing 1 mm Pefabloc (Uptima) and 500 units of RNasin (Promega) and several passages of the cells through a syringe needle (gauge 26). After centrifugation for 10 min at 12,000 × g, supernatants were incubated for 2 h at 4 °C with 7 μl of anti-EB2 antibody raised against peptide B (Fig. 1) and for 2 additional hours with 30 μl of protein A/G-agarose beads pre-equilibrated in TNE buffer. Beads were successively washed three times in high salt extraction buffer (PBS containing 0.5 m NaCl, 0.5% Nonidet P-40, 1 mm Pefabloc, and 500 units of RNasin), twice in PBS containing 0.5% Nonidet P-40, and once in PBS. They were finally treated for 1 h with proteinase K (500 μg/ml) in 1× PBS in the presence of 0.5% SDS. RNA recovered by phenol/chloroform extraction and ethanol precipitation was analyzed by RT-PCR essentially as described previously (29Farjot G. Buisson M. Duc Dodon M. Gazzolo L. Sergeant A. Mikaelian I. J. Virol. 2000; 74: 6068-6076Crossref PubMed Scopus (61) Google Scholar). Briefly, primers located in the pDM128 CAT coding sequence were used both for cDNA synthesis and PCR amplification (Fig. 5A). Heterokaryons Assays—24 h post transfection, 2 × 105 HeLa cells were seeded on glass coverslips with an equal number of NIH3T3 cells in 35-mm dishes and left to grow overnight at 37 °C. Cells were then treated for 2 h with 100 μg/ml cycloheximide to inhibit protein synthesis. Subsequently, cells were washed in PBS, and heterokaryon formation was performed by incubating the coverslips for 2 min in 50% polyethylene glycol (PEG) 3000–3700 (Sigma) in PBS. Following cell coverslips were washed in PBS and to medium containing 100 μg/ml After h at 37 cells were with and was performed essentially as described previously (29Farjot G. Buisson M. Duc Dodon M. Gazzolo L. Sergeant A. Mikaelian I. J. Virol. 2000; 74: 6068-6076Crossref PubMed Scopus (61) Google Scholar), that EB2 antibody or monoclonal anti-FLAG or by Dr. G. were and (Sigma) was at μg/ml the antibody of EB2 and EB2 from extracts were prepared from × HeLa or 293T cells 48 h post transfection essentially as described previously R.M. 11: PubMed Scopus Google Scholar) and overnight against buffer mm 10% 2 mm 100 mm 1 mm Pefabloc and 500 units of RNasin extracts in supplemented with Nonidet were incubated on a with anti-FLAG M2 (Sigma) overnight at 4 °C. Beads were then washed with (500 mm containing Nonidet of the proteins with buffer containing Nonidet and 0.5 FLAG proteins were analyzed on by Western and Northwestern blots were performed as described previously with monoclonal anti-FLAG M2 antibody (Sigma) and anti-EB2 antibody (29Farjot G. Buisson M. Duc Dodon M. Gazzolo L. Sergeant A. Mikaelian I. J. Virol. 2000; 74: 6068-6076Crossref PubMed Scopus (61) Google Scholar). Northwestern blots were performed essentially as described previously L. P. Google Scholar), that the RNA was incubated with the interactions were performed with GST and GST fusion proteins purified from E. The 33-amino acid peptide was from which partially with the peptide, was from RNAs were used as and prepared as A sequence corresponding to the of the human gene was using primers and a RNA at the of the DNA fragment. A sequence corresponding to the of the of the human gene was using primers and A sequence corresponding to the intron of the human gene was using primers and RNA were transcribed from these DNA fragments with RNA and using standard in vitro were performed with proteins and RNA as indicated in the to for min at 4 °C in binding buffer (10 mm Tris, 100 mm 2 mm and were analyzed by in buffer. To binding the of the bound RNA was determined by the of the RNA on to a and with an of and bound RNA was made using EB2 RNA-binding motif to GST binds to several sites on RNA in vitro. of affinity-purified GST-Da and GST were incubated with a RNA fragment of the human of affinity-purified GST-Da were also incubated with a RNA fragment of the human in The were by on a in Large Image Figure ViewerDownload Hi-res image Download EB2 motif binds RNA in vitro as a synthetic peptides and and B, the of the RNA was determined with C, of peptide or peptide were incubated in vitro with a RNA fragment of the human the The were by on a in Large Image Figure ViewerDownload Hi-res image Download in the EB2 RNA-binding motif EB2-mediated mRNA export but not nucleocytoplasmic shuttling. HeLa cells were transfected with plasmid pDM128 and the expression vectors as indicated at the left of the CAT mRNA export was as the of CAT protein detected by enzyme-linked immunosorbent assay, with a of 100 to the of CAT protein expressed in the presence of the wild-type B, Western blot analysis of and proteins expressed in transfected HeLa was made using the anti-FLAG antibody C, HeLa cells were transfected with vectors or After 48 transfected HeLa cells were with NIH3T3 cells to and were incubated for 2 h in the presence of cycloheximide as described Cells were then with anti-FLAG or generous gift from Dr. G. and as a antibody and then with which HeLa and to be in the Numbers on the of the indicate the number of with cell protein number of with HeLa indicate the Large Image Figure ViewerDownload Hi-res image Download (PPT) EBV was into a called T. Pich D. R. Hammerschmidt W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar, Pich D. T. C. Hammerschmidt W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: PubMed Scopus Google Scholar). the F factor of DNA the chloramphenicol and the EBV with the gene for the protein the of the cytomegalovirus immediate early plus the gene as a in eukaryotic this plasmid was derived an EBV mutant deleted of the EB2 gene (3Gruffat H. Batisse J. Pich D. Neuhierl B. Manet E. Hammerschmidt W. Sergeant A. J. Virol. 2002; 76: 9635-9644Crossref PubMed Scopus (72) Google Scholar). This plasmid was transfected into 293T and a called was (3Gruffat H. Batisse J. Pich D. Neuhierl B. Manet E. Hammerschmidt W. Sergeant A. J. Virol. 2002; 76: 9635-9644Crossref PubMed Scopus (72) Google Scholar). The cells were transfected with an or an or with both the and vectors as described previously (3Gruffat H. Batisse J. Pich D. Neuhierl B. Manet E. Hammerschmidt W. Sergeant A. J. Virol. 2002; 76: 9635-9644Crossref PubMed Scopus (72) Google Scholar). the medium was collected and through a of cells with the medium was out as described previously R. M. M. A. E. Hammerschmidt W. EMBO J. 2000; 19: PubMed Scopus Google Scholar, A. M. C. J. Pich D. M. Hammerschmidt W. J. Virol. 2000; 74: