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• W2045163760 abstract "Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain. Regulation of viral genome expression is the result of complex cooperation between viral proteins and host cell factors. We report here the characterization of a novel cellular factor sharing homology with the specific cysteine-rich C-terminal domain of the basic helix-loop-helix repressor protein I-mfa. The synthesis of this new factor, called HIC for Human I-mfa domain-Containing protein, is controlled at the translational level by two different codons, an ATG and an upstream non-ATG translational initiator, allowing the production of two protein isoforms, p32 and p40, respectively. We show that the HIC protein isoforms present different subcellular localizations, p32 being mainly distributed throughout the cytoplasm, whereas p40 is targeted to the nucleolus. Moreover, in trying to understand the function of HIC, we have found that both isoforms stimulate in T-cells the expression of a luciferase reporter gene driven by the human T-cell leukemia virus type I-long terminal repeat in the presence of the viral transactivator Tax. We demonstrate by mutagenesis that the I-mfa-like domain of HIC is involved in this regulation. Finally, we also show that HIC is able to down-regulate the luciferase expression from the human immunodeficiency virus type 1-long terminal repeat induced by the viral transactivator Tat. From these results, we propose that HIC and I-mfa represent two members of a new family of proteins regulating gene expression and characterized by a particular cysteine-rich C-terminal domain. human T-cell leukemia virus type I human immunodeficiency virus type 1 base pair long terminal repeat CRE-binding proteins CREB-binding protein basic helix-loop-helix polyacrylamide gel electrophoresis green fluorescent protein Human T-cell leukemia virus type I (HTLV-I)1 and human immunodeficiency virus type 1 (HIV-1) are both human retroviruses that infect in vivo CD4+ T lymphocytes. However, these infections lead to two extremely different diseases. Infection with HTLV-I results in dysregulation of T-cell proliferation, sometimes causing the development of adult T-cell leukemia. By contrast, infection with HIV-1 causes the loss of T-cells, resulting in systemic immunosuppression and development of AIDS. Characterization of molecular mechanisms involved in the interactions between viral and cellular components contributes to understanding why HTLV-I and HIV-1 have different effects on the infected T-cell growth. For instance, the transcriptional regulators used by T-cells to control cell function are used differently by HTLV-I and HIV-1 to regulate expression of their genome. Both retroviruses utilize the cellular RNA polymerase II to transcribe their genome (1.Chun R.F. Jeang K.-T. J. Biol. Chem. 1996; 271: 27888-27894Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 2.Mavankal G. Ignatius Ou S.H. Oliver H. Sigman D. Gaynor R.B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2089-2094Crossref PubMed Scopus (62) Google Scholar, 3.Okamoto H. Sheline C.T. Corden J.L. Jones K.A. Peterlin B.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11575-11579Crossref PubMed Scopus (89) Google Scholar, 4.Parada C.A. Roeder R.G. Nature. 1996; 384: 375-378Crossref PubMed Scopus (237) Google Scholar) but also code for their own regulatory proteins that regulate the transcription of viral and cellular genes. Thus, HIV-1 Tat protein is able to activate transcription from the viral long terminal repeat (LTR) promoter (5.Arya S.K. Guo C. Josephs S.F. Wong-Staal F. Science. 1985; 229: 69-73Crossref PubMed Scopus (591) Google Scholar, 6.Sodroski J.G. Patarca R. Rosen C.A. Wong-Staal F. Haseltine W.A. Science. 1985; 229: 74-75Crossref PubMed Scopus (355) Google Scholar, 7.Cullen B. Cell. 1986; 46: 973-982Abstract Full Text PDF PubMed Scopus (431) Google Scholar) by interacting with the transactivation responsive element located at the 5′ end of viral mRNAs (8.Berkhout B. Silverman R.H. Jeang K.T. Cell. 1989; 59: 273-282Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 9.Dingwall C. Ernberg I. Gait M.J. Green S.M. Heaphy S. Karn J. Lowe A.D. Singh M. Skinner M.A. EMBO J. 1990; 9: 4145-4153Crossref PubMed Scopus (340) Google Scholar, 10.Weeks K.M. Ampe C. Schultz S.C. Steitz T.A. Crothers D.M. Science. 1990; 249: 1281-1285Crossref PubMed Scopus (380) Google Scholar) and then with general transcription factors (for reviews see Refs. 11.Jones K.A. Genes Dev. 1997; 11: 2593-2599Crossref PubMed Scopus (196) Google Scholar and 12.Cullen B.R. Cell. 1998; 93: 685-692Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). By contrast, HTLV-I Tax protein is unable to bind specifically to nucleic acids (13.Jeang K.T. Boros I. Brady J. Radanovich M. Khoury G. J. Virol. 1988; 62: 4499-4509Crossref PubMed Google Scholar, 14.Beimling P. Moelling K. Oncogene. 1989; 4: 511-516PubMed Google Scholar, 15.Giam C.-Z. Xu Y.-L. J. Biol. Chem. 1989; 264: 15236-15241Abstract Full Text PDF PubMed Google Scholar). To stimulate transcription from LTR promoter, Tax is recruited by interaction with the activating transcription factors/CRE-binding proteins (ATF/CREB) (16.Suzuki T. Fujisawa J.I. Toita M. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 610-614Crossref PubMed Scopus (246) Google Scholar, 17.Yin M.-J. Paulssen E.J. Seeler J.-S. Gaynor R.B. J. Virol. 1995; 69: 3420-3432Crossref PubMed Google Scholar, 18.Bantignies F. Rousset R. Desbois C. Jalinot P. Mol. Cell. Biol. 1996; 16: 2174-2182Crossref PubMed Scopus (53) Google Scholar, 19.Reddy T.R. Tang H. Li X. Wong-Staal F. Oncogene. 1997; 14: 2785-2792Crossref PubMed Scopus (63) Google Scholar, 20.Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar) that bind to the three imperfect 21-bp cAMP-responsive elements located in the U3 of the LTR (21.Fujisawa J.I. Seiki M. Kiyokawa T. Yoshida M. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2277-2281Crossref PubMed Scopus (195) Google Scholar, 22.Paskalis H. Felber B.K. Pavlakis G.N. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6558-6562Crossref PubMed Scopus (105) Google Scholar, 23.Shimotohno K. Takano M. Teruuchi T. Miwa M. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8112-8116Crossref PubMed Scopus (130) Google Scholar, 24.Brady J. Jeang K.T. Duvall J. Khoury G. J. Virol. 1987; 61: 2175-2181Crossref PubMed Google Scholar, 25.Rosen C.A. Park R. Sodroski J.G. Haseltine W.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4919-4923Crossref PubMed Scopus (48) Google Scholar). Then, Tax could stimulate transcription by recruiting the coactivator CREB-binding protein (CBP) (26.Kwok R.P.S. Laurance M.E. Lundblad J.R. Goldman P.S. Shih H.-M. Connor L.M. Marriott S.J. Goodman R.H. Nature. 1996; 370: 223-226Crossref Scopus (1282) Google Scholar, 27.Giebler H.A. Loring J.E. Van Orden K. Colgin M.A. Garrus J.E. Escudero K.W. Brauweiler A. Nyborg J.K. Mol. Cell. Biol. 1997; 17: 5156-5164Crossref PubMed Scopus (164) Google Scholar, 28.Bex F. Yin M.J. Burny A. Gaynor R.B. Mol. Cell. Biol. 1998; 18: 2392-2405Crossref PubMed Scopus (114) Google Scholar) and by interacting with basal transcription factors (29.Caron C. Rousset R. Béraud C. Moncollin V. Egly J.-M. Jalinot P. EMBO J. 1993; 12: 4269-4278Crossref PubMed Scopus (101) Google Scholar, 30.Clemens K.E. Piras G. Radonovich M.F. Choi K.S. Duvall J.F. DeJong J. Roeder R. Brady J.N. Mol. Cell. Biol. 1996; 16: 4656-4664Crossref PubMed Scopus (47) Google Scholar, 31.Caron C. Mengus G. Dubrowskaya V. Roisin A. Davidson I. Jalinot P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3662-3667Crossref PubMed Scopus (34) Google Scholar). Basic helix-loop-helix (bHLH) proteins regulate cell determination and differentiation during embryogenesis (for reviews see Refs. 32.Jan Y.N. Jan L.Y. Cell. 1993; 75: 827-830Abstract Full Text PDF PubMed Scopus (392) Google Scholar and 33.Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar). For example, the cell fate of skeletal muscle precursors is regulated by the MyoD subfamily of bHLH factors includingMyoD, Myf5, myogenin, and MRF4 (33.Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (931) Google Scholar,34.Rudnicki M.A. Jaenisch R. BioEssays. 1995; 17: 203-209Crossref PubMed Scopus (375) Google Scholar). bHLH proteins are also involved in lineage commitment and differentiation as Mash2 and Hand1, which play a role in trophoblast development (35.Guillemot F. Nagy A. Auerbach A. Rossant J. Joyner A.L. Nature. 1994; 371: 333-336Crossref PubMed Scopus (525) Google Scholar, 36.Firulli A.B. McFadden D.G. Lin Q. Srivastava D. Olson E.N. Nat. Genet. 1998; 18: 266-270Crossref PubMed Scopus (303) Google Scholar, 37.Riley P. Anson-Cartwright L. Cross J.C. Nat. Genet. 1998; 18: 271-275Crossref PubMed Scopus (431) Google Scholar). The activity of bHLH factors is regulated to achieve the coordinated expression of genes during development. Recently, a novel myogenic repressor has been identified called I-mfa for Inhibitor of MyoD family (38.Chen C.-M.A. Kraut N. Groudine M. Weintraub H. Cell. 1996; 86: 731-741Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar, 39.Kraut N. Snider L. Chen C.-M.A. Tapscott S.J. Groudine M. EMBO J. 1998; 17: 6276-6288Crossref PubMed Scopus (99) Google Scholar). I-mfa is generated with two additional proteins, I-mfb and I-mfc, by alternative splicing (38.Chen C.-M.A. Kraut N. Groudine M. Weintraub H. Cell. 1996; 86: 731-741Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The three I-mf proteins share a common N-terminal region, but each has a different C terminus, the I-mfa-specific C terminus being characterized by a high content of cysteines. I-mfa is distributed mainly throughout the cytoplasm, although it is also detectable in the nucleus (38.Chen C.-M.A. Kraut N. Groudine M. Weintraub H. Cell. 1996; 86: 731-741Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). I-mfa inhibits myogenic bHLH proteins by retaining them in the cytoplasm and by interfering with their DNA binding activity in the nucleus (38.Chen C.-M.A. Kraut N. Groudine M. Weintraub H. Cell. 1996; 86: 731-741Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). I-mfa is also able to inhibit the transactivation activity of Mash2 and plays an important role in trophoblast and chondrogenic differentiation (39.Kraut N. Snider L. Chen C.-M.A. Tapscott S.J. Groudine M. EMBO J. 1998; 17: 6276-6288Crossref PubMed Scopus (99) Google Scholar). On the other hand, the functions of I-mfb and I-mfc still remain unclear. In this study, we report the isolation and characterization of a human cDNA clone encoding a novel protein, which exists in two isoforms differing by the presence or absence of a basic amino acid-rich N-terminal domain and by their localization in the cell. Both isoforms contain a common C terminus sharing homology with the specific C-terminal domain of I-mfa. We show that this new factor, called HIC for human I-mfa domain-containing protein, stimulates the expression of a luciferase reporter gene driven by the HTLV-I LTR in the presence of Tax. By using mutagenesis, we demonstrate that the I-mfa-like domain of HIC is required to stimulate luciferase expression. Finally, we also show that HIC is able to down-regulate expression from HIV-1 LTR in the presence of Tat. From these results, we propose that HIC and I-mfa represent a new family of proteins regulating gene expression and are characterized by the presence of a specific cysteine-rich C-terminal domain. Our results also suggest that HIC could differently regulate expression of HTLV-I and HIV-1 genomes. HIC cDNA clones were first isolated from a MT-2 cDNA library by the yeast two-hybrid approach. MT-2 cDNA fused to the GAL4 activation domain of the pGAD10 vector (20.Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar) was screened by using the cytoplasmic tail of CD4 as a bait fused to the LEXA DNA binding domain of the pBTM116 vector. The two-hybrid screen was performed as already described (20.Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar) with the Saccharomyces cerevisiae L40 reporter strain. The multiple tissue Northern blot and the human spleen cDNA library cloned into λgt11 were purchased from CLONTECH. RNA hybridization and library screening were performed as described by the manufacturer. The HIC cDNA encompassing the nucleotides from position 1148 to 1639 was labeled with [α-32P]dCTP using the method of random priming and was used as a probe. All the different HIC cDNAs cloned into pSPORT 1 (Life Technologies, Inc.) were transcribed and translated in the presence of [35S]methionine and [35S]cysteine by using the TNT T7 coupled transcription-translation reticulocyte lysate system of Promega. Translation products were analyzed by SDS-PAGE and autoradiography. pHIC-1 corresponds to anSalI-SpeI fragment that contains the first 1532 bp of the HIC cDNA subcloned into pSPORT 1. The 5′-deleted plasmids, pHIC-2, -3, -4, and -5, were either constructed by restriction endonuclease digestion and religation of pHIC-1 or generated by PCR amplification on pHIC-1 and subcloned into pSPORT 1. Templates where CTG or GTG were mutated were also generated by PCR amplification; CTG at position 246 was transformed into ATG (pHIC-I-atg) or CGG (pHIC-I-cgg), GTG at position 264 into ATG (pHIC-II-atg) or CGG (pHIC-II-cgg), and CTG at position 321 into ATG (pHIC-III-atg). To express HIC p32 and p40 with a GFP ormyc tag, the complete coding sequences of both proteins were subcloned into the vectors pEGFP-N1 (CLONTECH), pEGFP-C1 (CLONTECH), and pcDNA3.1(−)/Myc-His (Invitrogen). We also cloned an NheI-KpnI fragment of HIC cDNA (from position 95 to 1328) into pEGFP-N1 to analyze the production of HIC protein isoforms in vivo from the wild type leader. COS7 cells were transfected using the calcium phosphate-mediated transfection method with 20 μg of expression vector. Cells were cultivated on the glass slides and then analyzed by fluorescence 24 h after transfection. p32 and p40 tagged with themyc epitope were detected by using the anti-mycmonoclonal antibody purchased from Sigma and goat anti-mouse immunoglobin G antibody coupled to fluorescein isothiocyanate. Analysis of the green, red, and yellow fluorescence was performed with a Bio-Rad MRC 1024 confocal microscope. The coding sequences of p32 and p40 were cloned into a eukaryotic expression vector, pcDNA3.1/His (Invitrogen). The plasmid pcDNA3.1/His-HICΔ, which contains the entire coding sequence of p40 except for the I-mfa-like domain, was constructed by digesting p40 cDNA cloned into pcDNA3.1/His with EcoRV and XhoI. The resulting digest was treated with Klenow and religated as blunt ends. This approach resulted in the deletion of the last 101 amino acids. The Tat and Tax expression vectors, pBg312HIV-1Lai-Tat and pSG-Tax, respectively, have been described previously (40.Hirsch I. Spire B. Tsunetsugu-Yokota Y. Neuveut C. Sire J. Chermann J.C. Virology. 1990; 177: 759-763Crossref PubMed Scopus (24) Google Scholar, 41.Rousset R. Desbois C. Bantignies F. Jalinot P. Nature. 1996; 381: 328-331Crossref PubMed Scopus (126) Google Scholar). CEM cells were transiently cotransfected according to the procedure published previously (42.Lemasson I. Thébault S. Sardet C. Devaux C. Mesnard J.M. J. Biol. Chem. 1998; 273: 23598-23604Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). 5 μg of pACβ1 (β-galactosidase-containing reference plasmid) was included in each transfection for controlling the transfection efficiency. The total amount of DNA in each transfection was the same, the balance being made up with empty pcDNA3.1/His. Cell extracts equalized for protein content were used for luciferase and β-galactosidase assays. For the assays with the GAL4-binding site promoter-reporter plasmid, HIC (amino acids 120 to 355), Tax, and Tat were fused in frame with the DNA-binding domain of GAL4 (cloned into pBIND vector, Promega). Cotransfection assays were performed in CEM cells in the presence of the luciferase reporter plasmid pG5luc containing five GAL4-binding sites upstream of a minimal TATA box. Protein extracts were electrophoresed onto SDS-10% polyacrylamide gel (SDS-10% PAGE) and blotted to polyvinylidene difluoride membranes (Millipore). The blot was then incubated overnight at 4 °C with a blocking solution (phosphate-buffered saline containing 5% milk) prior to addition of antiserum. After 2 h at 20 °C, the blot was washed three times with 0.5% phosphate-buffered saline/Tween 20 and incubated for 2 h with goat anti-mouse or anti-rabbit immunoglobulin-peroxidase conjugate (Immunotech, Marseille, France). After three washes, the membrane was incubated with enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech). The membrane was then exposed for 0.5 to 5 min to hyperfilms-ECL (Amersham Pharmacia Biotech). The anti-Xpress serum was purchased from Invitrogen and recognizes the tag found in the Xpress leader peptide in the vector pcDNA3.1/His. The anti-HIC serum was obtained by immunizing rabbits with purified His6-tagged HIC polypeptide corresponding to the first 163 amino acids of p32. HIC cDNA was cloned into the bacterial expression vector pQE-30 (Qiagen). The N-terminal His6-tagged protein was purified as described by the manufacturer. Immunoprecipitation assay was carried out as described previously (43.Coudronnière N. Corbeil J. Robert-Hebmann V. Mesnard J.-M. Devaux C. Eur. J. Immunol. 1998; 28: 1445-1457Crossref PubMed Scopus (12) Google Scholar). Two cDNA clones coding, respectively, for the last 60 and 153 C-terminal amino acids of a novel protein were isolated from an HTLV-I-infected T-cell line cDNA library (20.Gachon F. Péléraux A. Thébault S. Dick J. Lemasson I. Devaux C. Mesnard J.M. J. Virol. 1998; 72: 8332-8337Crossref PubMed Google Scholar). As the predicted polypeptide presented homologies with the specific C-terminal domain of I-mfa, a cellular factor known to be a bHLH repressor, we decided to characterize further this novel protein. At first, the tissue distribution of the mRNA coding for this novel HIC protein was analyzed. All the tested lymphoid organs (spleen, thymus, and peripheral blood leukocytes) expressed an mRNA of about 4.4 kb (Fig. 1). This mRNA is not specific to lymphoid tissues since it is expressed in prostate, uterus, and small intestine. Finally, it is almost absent in testis and colon. From these observations, we decided to screen a human spleen cDNA library cloned into λgt11 to characterize the complete sequence of HIC cDNA. By this approach, we were able to isolate a 4,152-bp full-length HIC cDNA that was completely sequenced (GenBankTM number AF054589). The full-length HIC cDNA contains an open reading frame encoding a polypeptide of 246 amino acids if the ATG at position 591 is the initiation codon (Fig. 2). We examined the proteins synthesized in a cell-free system with a cDNA containing the first 1532 nucleotides of HIC sequence (the stop codon TAA is at position 1329; see Fig. 2). SDS-PAGE of the translational products effectively revealed a protein of 32 kDa, p32, but also an unexpected product of 40 kDa, p40 (Fig. 3 a, lane pHIC-1). To determine the origin of both polypeptides, a series of 5′ truncation mutants of pHIC-1 was generated. These experiments (Fig.3) demonstrated that p32 translation initiation was located between position 393 and 617 (compare the lanes pHIC-3 andpHIC-5) suggesting that ATG at position 591 could be an initiation codon. To confirm this hypothesis, the entire region 5′ to this ATG was deleted. SDS-PAGE of the translation products synthesized from this template revealed one major protein of 32 kDa (lane pHIC-4) indicating that the first ATG effectively is the initiation codon involved in p32 synthesis.Figure 3Mapping of the translation initiation sites of the HIC cDNA clone with a cell-free system. a, all mRNAs were translated in rabbit reticulocyte lysates with either an HIC cDNA clone containing the first 1532 bp or PCR-generated DNAs as templates for transcription. [35S]Methionine and [35S]cysteine were used in translation reactions to label proteins. Translation products were analyzed by SDS-PAGE and autoradiography. 35S-Labeled p40 and p32 are designated byarrows. Molecular size markers in kilodaltons are shown on the right. The exact start of the different HIC constructs are shown in b below the autoradiographs.b, nucleic acid sequence of the 5′ end of the HIC cDNA clone. The stop codon TAG at position 171 and the putative initiation codons are indicated in bold. The exact position of the 5′ end of the different HIC cDNA clones is indicated by anarrow. pHIC-1 and the 5′ deleted plasmids, pHIC-2, -3, -4, and -5, encode the wild type nucleotide sequence of HIC cDNA. For the other deleted constructs, the 5′ end was modified as follows: CTG at position 246 was transformed into ATG (pHIC-I-atg) or CGG (pHIC-I-cgg), GTG at position 264 into ATG (pHIC-II-atg) or CGG (pHIC-II-cgg), and CTG at position 321 into ATG (pHIC-III-atg).View Large Image Figure ViewerDownload Hi-res image Download (PPT) On the other hand, from the template pHIC-3, p40 was no longer synthesized, whereas it was still produced from pHIC-2 (Fig.3 a). This observation suggests that the HIC cDNA clone contains another initiation site upstream of the first ATG, located between positions 174 and 393. There are many examples of proteins where codons other than ATG are initiation codons (44.Kozak M. J. Biol. Chem. 1991; 266: 19867-19870Abstract Full Text PDF PubMed Google Scholar, 45.Boeck R. Kolakofsky D. EMBO J. 1994; 13: 3608-3617Crossref PubMed Scopus (81) Google Scholar, 46.Drabkin H.J. Rajbhandary U.L. Mol. Cell. Biol. 1998; 18: 5140-5147Crossref PubMed Scopus (75) Google Scholar). In human cells, TTG and CTG have been found as initiation codons. To determine whether one or both CTG located upstream of the ATG (Fig.3 b) could be translational initiator, templates starting at positions 246 or 321, where CTG was transformed into ATG (respectively, pHIC-I-atg and pHIC-III-atg), were constructed. Only the ATG which starts at position 246 generated a 40-kDa major product that could correspond to p40 (Fig. 3 a, lane pHIC-I-atg). However, when the CTG at position 246 was mutated into a non-initiation codon (CGG), p40 was still synthesized (Fig. 3 a, lane pHIC-I-cgg). Based on these results, it appears that both CTGs are not initiation codons and that the initiation codon must be contained within the nucleotide region between position 246 and 321. In this region, only the GTG located at position 264 belongs to the non-ATG codons known to be able to initiate translation in mammalian cells. For this reason, this GTG was mutated into ATG or CGG corresponding, respectively, to the plasmids pHIC-II-atg and pHIC-II-cgg (Fig. 3 b). From pHIC-II-atg a 40-kDa protein was produced, whereas the synthesis of this product was abolished with pHIC-II-cgg (Fig. 3 a). This result clearly demonstrates that the GTG at position 264 is initiator in our cell-free system. Taken together, our data clearly demonstrate that HIC mRNA codes for two protein isoforms, p32 and p40, synthesized from two different initiation codons as follows: a standard ATG initiator for p32, and a GTG located upstream of the ATG for p40. p32 and p40 only differ by the presence of a N-terminal sequence containing two basic subdomains (Fig.4 a). Moreover, their common C-terminal region shares 77% identical amino acids with the specific C-terminal domain of I-mfa (Fig. 4 b). Next we investigated the subcellular localization of both HIC protein isoforms in vivo. COS7 cells were transfected with vectors expressing p32 or p40 tagged with green fluorescent protein (GFP), fused either to their N-terminal end or their C-terminal end. As shown in Fig.5, the position of the GFP tag apparently has no influence on the localization of the HIC proteins. p32 is distributed primarily throughout the cytoplasm, although weak staining is detectable in the nucleus (Fig. 5, a and c). Two different cytoplasmic patterns of p32 localization are observed, a diffuse (Fig. 5, a and c) and a bright punctate staining (Fig. 5, b and d). p40 also exhibits a granular distribution in the cytoplasm, but in addition, it shows a staining pattern in the nucleus, localized around and in the nucleoli (Fig. 5, e and f). To be certain that the GFP tag did not influence their localization, p32 and p40 were fused to a smaller tag, containing either themyc epitope fused to their C-terminal end (Fig.6) or the X-press leader peptide fused to their N-terminal end (data not shown). In COS7 cells transfected with these constructs, p32 and p40 give the same staining pattern as that observed with the GFP tag (compare Fig. 5 and 6). In conclusion, p32 is predominantly cytoplasmic, whereas p40 is both cytoplasmic and nuclear. Moreover, p32 is distributed in both a diffuse and a punctate staining pattern throughout the cytoplasm. We do not know why two different patterns are observed with p32. However, preliminary data 2S. Thébault, unpublished results. suggest that the I-mfa-like domain of HIC could be involved in the formation of these cytoplasmic granular structures. To raise polyclonal antibodies against HIC, rabbits were immunized with highly purified HIC polypeptide corresponding to the first 163 amino acids of p32. We studied the expression of HIC protein isoforms in vivo with this antiserum by using two additional approaches. First, we analyzed by Western blotting (Fig. 7 a) protein extracts of 293T cells transfected with the eukaryotic expression vector pcDNA3.1(−)/Myc-His containing the HIC coding sequence under the control of the wild type leader (from nucleotide 95 to 1300, see Fig. 2). Second, we performed immunoprecipitation with anti-HIC from extracts of HTLV-I infected T-cells (Fig. 7 b). In both cases, a single protein was detected with anti-HIC but not with preimmune serum (Fig. 7), the size of this protein being consistent with the size of recombinant p32 when expressed in eukaryotic cell lines (data not shown). These results demonstrate that p32 is the major protein isoform produced from HIC mRNA in vivo. p40 was not detected by both approaches suggesting that p40 is weakly expressed in vivo, probably because GTG is a poor translation initiator. To check the existence of p40 in vivo, the HIC coding sequence under the control of the wild type leader (from nucleotide 95 to 1328, see Fig. 2) was cloned into the vector pEGFP-N1, in frame with the GFP nucleotide sequence. If both HIC isoforms are expressed in vivo, the products synthesized from our construct should correspond to p40- and p32-GFP fusion proteins, easily detectable by fluorescence microscopy. Indeed, after transfection of COS7 cells, different patterns of staining were observed with a confocal microscope. At first, a diffuse cytoplasmic staining (Fig. 8 a) was detected confirming that p32 is expressed in vivo from HIC cDNA. We also found a granular staining in the cytoplasm (Fig. 8,a–c), a pattern common to p32-GFP and p40-GFP (see Fig. 5,d and f). Finally, staining around and in the nucleoli (Fig. 8 c), similar to the pattern described for p40-GFP (Fig. 5 f), was observed. In conclusion, taken together, our analyses demonstrate that both p32 and p40 can be synthesized in vivo. Our results suggest that HIC protein belongs with I-mfa to the same family of proteins that is characterized by the presence of a specific cysteine-rich C-terminal domain. Therefore, by analogy with I-mfa, we postulated that HIC should be involved in gene expression regulation. Moreover, we first isolated the HIC cDNA clone from a cDNA library of MT-2, a T-cell line persistently infected by HTLV-I and producing high quantity of viral particles (47.Miyoshi I. Taguchi H. Kubonishi I. Yoshimoto S. Ohtsuki Y. Shiraishi Y. Akagi T. Gann. 1982; 28: 219-228Google Scholar). For all these reasons and in order to try to understand the function of HIC, w" @default.
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• W2045163760 date "2000-02-01" @default.
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• W2045163760 title "Molecular Cloning of a Novel Human I-mfa Domain-containing Protein That Differently Regulates Human T-cell Leukemia Virus Type I and HIV-1 Expression" @default.
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