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• W2004363451 abstract "The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes. The double-stranded (ds) RNA-binding protein RAX was discovered as a stress-induced cellular activator of the dsRNA-dependent protein kinase (PKR), a key regulator of protein synthesis in response to viral infection and cellular stress. We now report a novel function of RAX, independent of PKR, to enhance SV40 promoter (origin)/enhancer-dependent gene expression. Several mammalian cell lines including COS-7, CV-1, and HeLa cells were tested. Results reveal that the SV40 large T antigen is required for RAX-mediated, synergistic enhancement of gene expression. RAX augments SV40 regulatory element-dependent DNA replication and transcription. The mechanism requires the SV40 enhancer, a viral transcriptional element that is necessary for efficient SV40 DNA replication in vivo. Mutational analysis reveals that the dsRNA-binding domains of RAX are required for the gene expression enhancing function. Thus, in addition to stimulating PKR activity, RAX can positively regulate both SV40 large T antigen-dependent DNA replication and transcription in a mechanism that may alter the interaction of the cellular factor(s) with the SV40 enhancer via the dsRNA-binding domains of RAX. This novel function of RAX may have implications for regulation of mammalian DNA replication and transcription because of the many similarities between the viral and cellular processes. SV40 is a small DNA virus that has served as a powerful model system for dissecting fundamental biological processes including, DNA replication, transcription, and neoplastic transformation (1Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (456) Google Scholar, 2Herendeen D. Kelly T.J. Blow J.J. Eukaryotic DNA Replication. IRL Press, Oxford1996: 29-65Google Scholar, 3Simmons D.T. Adv. Virus Res. 2000; 55: 75-134Crossref PubMed Google Scholar, 4Damnia B. Mital R. Alwine J.C. Mol. Cell Biol. 1998; 18: 1331-1338Crossref PubMed Scopus (23) Google Scholar). The SV40 genome consists of a 5.2-kb circular duplex DNA molecule with a 300-bp regulatory region that contains SV40 promoter (including the viral origin of DNA replication) and enhancer elements (2Herendeen D. Kelly T.J. Blow J.J. Eukaryotic DNA Replication. IRL Press, Oxford1996: 29-65Google Scholar, 5Deb S. DeLucia A.L. Baur C.P. Koff A. Tegtmeyer P. Mol. Cell. Biol. 1986; 6: 1663-1670Crossref PubMed Scopus (113) Google Scholar). SV40 DNA replication and transcription occurs in the nucleus of the host cell where the SV40 genome is complexed with histones to form a nucleosomal structure (i.e. minichromosome) that is indistinguishable from cellular chromatin, indicating the similarities between viral and cellular DNA replication and transcription (2Herendeen D. Kelly T.J. Blow J.J. Eukaryotic DNA Replication. IRL Press, Oxford1996: 29-65Google Scholar, 6DePamphilis M.L. Bradley M.K. Salzman N.P. The Papovaviridae. Vol. 1. Plenum Publishing Corp., New York1986: 99-246Crossref Google Scholar, 7Alexiadis V Varga-Weisz P.D. Bonte E. Becker P.B. Gruss C. EMBO J. 1998; 17: 3428-3438Crossref PubMed Scopus (67) Google Scholar). The SV40 large T antigen (T-Ag) 1The abbreviations used are: T-Ag, SV40 large T antigen; 2-AP, 2-aminopurine; eIF2α, the α subunit of eukaryotic initiation factor-2; HA, hemagglutinin; PKR, double-stranded RNA-dependent protein kinase; RAX, double-stranded RNA-dependent protein kinase-associated protein X; ds, double stranded; CMV, cytomegalovirus; RSV, Rous sarcoma virus; RBM, RNA-binding motif.1The abbreviations used are: T-Ag, SV40 large T antigen; 2-AP, 2-aminopurine; eIF2α, the α subunit of eukaryotic initiation factor-2; HA, hemagglutinin; PKR, double-stranded RNA-dependent protein kinase; RAX, double-stranded RNA-dependent protein kinase-associated protein X; ds, double stranded; CMV, cytomegalovirus; RSV, Rous sarcoma virus; RBM, RNA-binding motif. is a multifunctional viral protein with DNA helicase activity and transcriptional activity and plays an essential role in SV40 DNA replication and transcription (1Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (456) Google Scholar, 2Herendeen D. Kelly T.J. Blow J.J. Eukaryotic DNA Replication. IRL Press, Oxford1996: 29-65Google Scholar, 3Simmons D.T. Adv. Virus Res. 2000; 55: 75-134Crossref PubMed Google Scholar, 4Damnia B. Mital R. Alwine J.C. Mol. Cell Biol. 1998; 18: 1331-1338Crossref PubMed Scopus (23) Google Scholar). The T-Ag has been found to interact with a number of cellular proteins that regulate DNA replication and transcription (e.g. DNA polymerase α, human TFIIB-related factor, pRb, and p53) and to modify cellular regulatory processes that may efficiently promote viral gene expression and replication in permissive cells (1Fanning E. Knippers R. Annu. Rev. Biochem. 1992; 61: 55-85Crossref PubMed Scopus (456) Google Scholar, 2Herendeen D. Kelly T.J. Blow J.J. Eukaryotic DNA Replication. IRL Press, Oxford1996: 29-65Google Scholar, 3Simmons D.T. Adv. Virus Res. 2000; 55: 75-134Crossref PubMed Google Scholar, 4Damnia B. Mital R. Alwine J.C. Mol. Cell Biol. 1998; 18: 1331-1338Crossref PubMed Scopus (23) Google Scholar). However, the mechanisms by which SV40 T-Ag cooperates with the cellular proteins in vivo to facilitate viral DNA replication and transcription have not been fully elucidated. The interferon-inducible double-stranded (ds) RNA-dependent protein kinase (PKR) is a major regulator of host antiviral defense and normal cellular protein synthesis (8Hovanessian A.G. J. Interferon Res. 1989; 9: 641-647Crossref PubMed Scopus (162) Google Scholar, 9Clemens M.J. Elia A. J. Interferon Cytokine Res. 1997; 17: 503-524Crossref PubMed Scopus (517) Google Scholar, 10Meurs E. Chong K. Galabru J. Thomas N.S. Kerr I.M. Williams B.R. Hovanessian A.G. Cell. 1990; 62: 379-390Abstract Full Text PDF PubMed Scopus (814) Google Scholar, 11Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar). For example, viral infection leads to PKR activation with phosphorylation of its physiologic substrate, the α subunit of eukaryotic initiation factor-2 (eIF2α), that leads to inhibition of translation of mRNA (12Hershey J.W.B. Annu. Rev. Biochem. 1991; 60: 717-755Crossref PubMed Scopus (840) Google Scholar). PKR also shuts down protein synthesis in uninfected cells following cellular stress responses that lead to inhibition of cell growth and apoptosis, including interleukin-3 growth factor withdrawal, serum deprivation, and treatment of cells with tumor necrosis factor-α and lipopolysaccharide (11Ito T. Jagus R. May W.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7455-7459Crossref PubMed Scopus (78) Google Scholar, 13Yeung M.C. Liu J. Lau A.S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12451-12455Crossref PubMed Scopus (160) Google Scholar, 14Der S.D. Yang Y.L. Weissamn C. Williams B.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3279-3283Crossref PubMed Scopus (362) Google Scholar). Recently, RAX and its human homologue, PACT, were independently discovered as the first cellular activators of PKR (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (370) Google Scholar). RAX/PACT is also a dsRNA-binding protein that directly binds and activates PKR following cellular stresses (such as interleukin-3 withdrawal, sodium arsenite, hydrogen peroxide, serum deprivation, or ceramide treatment) that lead to cell death (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (370) Google Scholar, 17Patel C.V. Handy I. Goldsmith T. Patel R.C. J. Biol. Chem. 2000; 275: 37993-37998Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Ruvolo P.P. Gao F. Blalock W.L. Deng X. May W.S. J. Biol. Chem. 2001; 276: 11754-11758Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). In addition, it is now clear that PKR is a key regulator of protein synthesis but may also regulate other fundamental cellular processes including cell proliferation, apoptosis, differentiation, and signal transduction (19Judware R. Petryshyn R. Mol. Cell. Biol. 1991; 11: 3259-3267Crossref PubMed Google Scholar, 20Koromilas A.E. Roy S. Barber G.N. Katze M.G. Sonenberg N. Science. 1992; 257: 1685-1689Crossref PubMed Scopus (495) Google Scholar, 21Jagus R. Joshi B. Barber G.N. Int. J. Biochem. Cell Biol. 1999; 31: 123-138Crossref PubMed Scopus (176) Google Scholar, 22Williams, B. R. (2001) Science's STKE http://stke.sciencemag.org/cgi/content/full/sigtrans;2001/89/re2.Google Scholar). Findings reported here indicate that RAX has a novel, unexpected function to augment SV40 T-Ag-dependent DNA replication and transcription that is dependent on its dsRNA-binding domains but independent of PKR. Cell Culture and Transfection—COS-7, CV-1, and HeLa cells were obtained from American Type Culture Collection and were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. All transient transfection or co-transfection in these cells was performed using LipofectAMINE (Invitrogen). Luciferase Reporter Constructs and Expression Constructs—pGL3-luciferase reporter vectors (including pGL3-promoter vector and pGL3-basic vector) and pRL-Renilla luciferase reporter vectors (including pRL-SV40 vector and pRL-CMV vector) were obtained from Promega. pGL3-promoter vector contains the SV40 early promoter element, whereas pGL3-basic vector lacks promoter or enhancer elements. pRL-SV40 vector and pRL-CMV vector contain the SV40 early promoter/enhancer elements and the human cytomegalovirus (CMV) early promoter/enhancer elements, respectively. pGL3-SV40, pGL3-CMV, and pGL3-RSV luciferase reporter vectors were also created by subcloning the promoter/enhancers of SV40, CMV, and Rous sarcoma virus (RSV) into the pGL3-basic vector, respectively. The SV40 promoter/enhancer sequence in the pGL3-SV40 and pRL-SV40 vectors contains the SV40 core origin of DNA replication and auxiliary elements including the 21-bp repeats and the 72-bp enhancer elements (23Pauly M. Treger M. Westhof E. Chambon P. Nucleic Acids Res. 1992; 20: 975-982Crossref PubMed Scopus (14) Google Scholar). pSV40 E-TAL luciferase reporter vector was created by subcloning the SV40 enhancer sequence from pRL-SV40 vector into pTAL-luciferase reporter vector (Clontech) that contains a TATA-like promoter (PTAL) region from the Herpes simplex virus-thymidine kinase promoter. The hemagglutin (HA) epitope-tagged RAX cDNA (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar) was subcloned into expression vectors pcDNA3 (Invitrogen) and pGC1, respectively. The pGC1 vector was derived from the pGL3-CMV vector by deleting the luciferase reporter gene cDNA. Expression plasmids of FLAG-tagged RAX and its mutants (such as K84A, K177A, ΔII and ΔI/II) were also created using expression vectors pIRESneo (Clontech) and pcDEF3 (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 24Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (151) Google Scholar), respectively. Site-directed mutagenesis experiments were performed using the Transformer Kit (Clontech). In K84A and K177A mutants the lysine codon was mutated to an alanine codon. ΔII and ΔI/II mutants were created by deleting the cDNA fragments coding amino acid residues 125–194 or residues 63–219 using the restriction enzymes followed by blunt-end ligation. An expression construct of SV40 large T antigen was created by subcloning the T-Ag cDNA fragment of SV40 genome/pRB322 into the pIRESneo vector. A FLAG-tagged eIF2α cDNA was also subcloned into the pcDNA3 vector. The expression vectors pcDNA3 and pcDEF3 contain the SV40 promoter/enhancer elements that function as the neomycin promoter, whereas pGC1 and pIRESneo do not contain such SV40 regulatory elements. Co-expression of RAX and eIF2α—Co-transfection of 0.5 μg of FLAG-RAX/pIRESneo (empty vector as a control) with 0.5 μg of FLAG-eIF2α/pcDNA3 into COS-7 cells was performed in 6-well plates. After 48 h cells were lysed in buffer A (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1% Triton X-100, and protease inhibitor mixture (Sigma)). 50 μg of the lysate was loaded into a 10% SDS-PAGE gel followed by Western blot analysis using an anti-FLAG antibody (Roche Diagnostics) and the same blot was also stained by Fast Green dye. Luciferase Assays—Co-transfection of 1 μg of HA-RAX/pGC1 (empty vector as a control) with 0.1 μg of pGL3-SV40 vector, pGL3-RSV vector, or pGL3-CMV vector into COS-7 cells was performed in 6-well plates. After 48 h cells were lysed and assayed for luciferase activity using the luciferase assay system (Promega). 0.1 μg of pGL3-SV40 luciferase vector was also co-transfected into CV-1 cells with a total of 1 μg of plasmids containing empty vector, RAX alone, T-Ag alone, or both RAX and T-Ag followed by luciferase assays. The plasmid combinations were 0.5 μg of pGC1 plus 0.5 μg of pIRESneo for the “empty vector,” 0.5 μg of HA-RAX/pGC1 plus 0.5 μg of pIRESneo for the “RAX alone,” 0.5 μg of pGC1 plus 0.5 μg of T-Ag/pIRESneo for the “T-Ag alone,” and 0.5 μg of RAX/pGC1 plus 0.5 μg of T-Ag/pIRESneo for the “both RAX and T-Ag,” respectively. Similar experiments were also carried out in HeLa cells. The total protein was measured for normalizing luciferase activity readings. To rule out the possibility that the observed effect of RAX on luciferase activity might be an artifact of a particular RAX expression vector or luciferase reporter vector used, other RAX expression plasmids (including HA-RAX/pcDNA3 and Flag-RAX/pIRESneo) and other luciferase reporter vectors (including pRL-SV40 and pRL-CMV Renilla luciferase vectors as well as pGL3-control vector (Promega), which contains both SV40 promoter and enhancer elements and was alternatively used as a pGL3-SV40 vector) were also tested in the studies. Similar results were obtained and some examples are shown under “Results.” Treatment of the PKR inhibitor 2-aminopurine (2-AP) was performed (25Hu Y. Conway T.W. J. Interferon Res. 1993; 13: 323-328Crossref PubMed Scopus (106) Google Scholar). After the HA-RAX/pGC1 plasmid (empty vector as a control) was co-transfected with the pGL3-SV40 luciferase vector into COS-7 cells using the LipofectAMINE method, 2-AP was immediately added into the Dulbecco's modified Eagle's media to a final concentration of 10 mm. After 48 h cells were lysed and luciferase activity was assayed as described above. Southern Blot Analysis—Co-transfection in COS-7 and CV-1 cells was carried out as described before. For plasmid isolation, cells were harvested and lysed in the plasmid lysis buffer (0.6% SDS, 10 mm EDTA). The lysate was added with NaCl to a final concentration of ∼1 m and incubated on ice overnight. The lysate was then centrifuged for 15 min at 13 × g to pellet chromosome DNA. The plasmids in the supernatant were extracted by phenol/chloroform and then precipitated by ethanol. The purified plasmids were linearized by the restriction enzymes and then digested by DpnI (26Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6973-6977Crossref PubMed Scopus (352) Google Scholar) followed by Southern blot analysis using the luciferase cDNA insert or the FLAG-eIF2α cDNA insert as probe that was radiolabeled using the RadPrime DNA labeling system (Invitrogen). Northern Blot Analysis—Co-transfection was performed as described above. Total RNAs were isolated using the Trizol method (Invitrogen) and probed with the radiolabeled luciferase cDNA insert. [35S]Methionine Labeling and Immunoblotting—Co-transfection was performed as described above. After 48 h, proteins in COS-7 cells that transiently express HA-RAX (or empty vector as a control) were pulse-labeled with [35S]methionine for 60 min to assess protein synthesis (27Thomis D.C. Samuel C.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 10837-10841Crossref PubMed Scopus (86) Google Scholar). The cells were lysed in buffer B (10 mm HEPES, pH 7.4, 5 mm EDTA, 150 mm NaCl, 1% Triton X-100, 20 mm sodium fluoride, 20 mm sodium pyrophosphate, 20 mm β-glycerophosphate, 20 mm sodium molybdate, 20 mm microcystin-LR and protease inhibitor mixture (Sigma)) followed by SDS-PAGE, Western transfer, and autoradiography. The same blot was also immunoblotted using antibodies against phospho-eIF2α (Cell Signaling Technology), eIF2α (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), HA (12CA5, Roche Diagnostics), and SV40 T-Ag (BD Pharmingen). Poly(I)·Poly(C)-Agarose Beads Binding Assays—Expression plasmids of FLAG-tagged wild-type and mutant RAX as described earlier were transfected into COS-7 cells, respectively. After 48 h cells were lysed in buffer A. Aliquots (50 μg) of lysates were incubated with 20 μl of poly(I)·poly(C) or poly(C)-agarose beads (Amersham Biosciences) for 1.5hat4 °C. These beads were washed three times with buffer A before boiling in SDS-PAGE sample buffer. The eluted samples and 50 μg of the whole cell lysate (as input) were then loaded onto a 10% SDS-PAGE gel followed by immunoblotting using an anti-FLAG antibody. RAX Selectively Stimulates SV40 Promoter/Enhancer-dependent Gene Expression—To investigate the mechanism(s) by which RAX may regulate PKR, COS-7 cells were co-transfected with plasmids containing FLAG-tagged RAX and FLAG-tagged eIF2α. Surprisingly, Western blot analysis revealed that FLAG-RAX could significantly enhance the expression of the FLAG-eIF2α plasmid that was created in a pcDNA3 vector (Invitrogen, Fig. 1A). Fast Green staining confirmed that expression of total cellular proteins was not affected (Fig. 1B). RAX-mediated augmentation of exogenous gene expression was unexpected because RAX was initially identified and characterized as a cellular activator of PKR that inhibits protein synthesis (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (370) Google Scholar). Because viral promoter/enhancers (such as those of SV40, CMV, and RSV) are commonly used to drive exogenous gene expression, we tested whether RAX could affect the transcriptional activity of these promoter/enhancer elements. A commercial pGL3-luciferase reporter vector (Promega) containing these viral promoter/enhancer elements was constructed and co-transfected into COS-7 cells along with a plasmid containing either an HA-tagged RAX or an empty vector. Luciferase expression results indicated that HA-RAX could significantly enhance SV40 promoter/enhancer-dependent gene expression (i.e. ∼10-fold increase) but had only a minimal effect on gene expression driven by the CMV or RSV promoter/enhancers (i.e. ∼1-fold increase, Fig. 2A). Western analysis demonstrates equal levels of HA-RAX or T-Ag expression (Fig. 2B).Fig. 2Effects of RAX on luciferase reporter gene expression in COS-7 cells. 1 μg of HA-RAX/pGC1 plasmid (empty vector as a control) was co-transfected into COS-7 cells with 0.1 μg of pGL3-luciferase reporter vector containing the promoter/enhancer of SV40, RSV, or CMV (pGL3-SV40, pGL3-RSV, or pGL3-CMV). After 48 h cells were lysed and assayed for luciferase activity as described under “Materials and Methods.” A, relative luciferase activity. Luciferase expression is displayed relative to that of the vector. V, empty vector pGC1; RAX, HA-RAX/pGC1. B, Western blot analysis using antibodies of anti-HA and anti-T-Ag. Lanes 1 and 4, pGL3-SV40; lanes 2 and 5, pGL3-RSV; lanes 3 and 6, pGL3-CMV.View Large Image Figure ViewerDownload Hi-res image Download (PPT) SV40 Large T Antigen Is Required for RAX-mediated Enhancement of Luciferase Reporter Gene Expression—COS-7 cells were derived from CV-1 monkey kidney cells following transformation with a replication origin-defective SV40 mutant that encodes the wild type T-Ag (28Gluzman Y. Cell. 1981; 23: 175-182Abstract Full Text PDF PubMed Scopus (1454) Google Scholar). Abundant expression of T-Ag occurs in COS-7 but not CV-1 cells (Figs. 2B and 3B). To test whether T-Ag expression was required for the RAX-mediated enhancement of luciferase gene expression, CV-1 cells were also co-transfected with a pGL3 SV40-luciferase vector and the plasmids containing HA-tagged RAX and/or T-Ag. Results indicated that while expression of HA-RAX alone had essentially no effect on luciferase gene expression, co-expression with the T-Ag can synergistically enhance the activity by over 45-fold (Fig. 3A). Because both HA-RAX and T-Ag were expressed at similar levels in CV-1 cells when transfected individually or together, variable expression levels could not account for HA-RAX-mediated enhancement of luciferase expression (Fig. 3B). Furthermore, in addition to monkey COS-7 and CV-1 cells that are naturally SV40 permissive (28Gluzman Y. Cell. 1981; 23: 175-182Abstract Full Text PDF PubMed Scopus (1454) Google Scholar), RAX was also demonstrated to synergize with the T-Ag to stimulate SV40 promoter/enhancer-dependent luciferase gene expression in human HeLa cells (Fig. 4A). These findings clearly demonstrate that T-Ag expression is necessary for the synergistic enhancing effect of RAX on luciferase gene expression.Fig. 4A, RAX-mediated enhancement of luciferase reporter gene expression in HeLa cells. pGL3-SV40 luciferase reporter vector was co-transfected into HeLa cells with plasmids containing RAX and/or T-Ag followed by luciferase assays similarly as described in the legend to Fig. 3. B, effects of RAX on Renilla luciferase reporter gene expression in COS-7 cells. pRL-SV40 and pRL-CMV Renilla luciferase reporter vectors were used to repeat luciferase assays as described in the legend to Fig. 2.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To rule out a nonspecific effect from the pGL3-Firefly luciferase reporter gene vector that might account for any differential reporter gene expression observed, a second luciferase reporter gene from another species was also tested. The pRL Renilla luciferase reporter vector (Promega) that contains either SV40 or CMV promoter/enhancer elements was expressed in COS-7 cells (Fig. 4B). Results confirm that HA-RAX exerts a selective and specific effect that was not dependent on the luciferase gene because expression of the two different luciferase enzymes were both synergistically enhanced. These data rule out a nonspecific, species-specific enhancement of gene expression. Therefore, it is concluded that RAX-mediated enhancement of luciferase gene expression is specific for the SV40 promoter/enhancer elements and independent of the plasmid or reporter genes employed. RAX-mediated Enhancement of Gene Expression Occurs at the Level of Both DNA Replication and mRNA Expression—The wild-type SV40 origin of DNA replication is contained in both the pGL3- and pRL-luciferase reporter vectors. Plasmids containing this origin can replicate in cells expressing the T-Ag, such as COS-7 cells (28Gluzman Y. Cell. 1981; 23: 175-182Abstract Full Text PDF PubMed Scopus (1454) Google Scholar). Therefore, we tested whether HA-RAX could enhance SV40 origin-dependent plasmid DNA replication. To distinguish a newly replicated pGL3-luciferase plasmid in mammalian cells from the plasmid that was transfected into the cells, DpnI endonuclease was used because it would only cleave the transfected plasmid containing DpnI sites that were methylated during plasmid preparation in growing bacteria (26Li J.J. Kelly T.J. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 6973-6977Crossref PubMed Scopus (352) Google Scholar). Southern blot analysis using a radiolabeled luciferase cDNA probe revealed that pGL3-plasmid DNA replication occurred following transfection into mammalian cells and was significantly enhanced by HA-RAX (Fig. 5A). These findings indicated that RAX can stimulate SV40 promoter (origin)/enhancer-dependent DNA replication. This novel property of RAX was verified when it was shown that HA-RAX could also increase the replication of an unrelated FLAG-eIF2α/pcDNA3 plasmid in COS-7 cells (Fig. 5B). The pcDNA3 vector contains both CMV promoter/enhancer elements (for expressing the gene of interest) and the SV40 promoter (origin)/enhancer elements to allow replication of the FLAG-eIF2α/pcDNA3 in COS-7 cells. These findings help to explain how RAX can enhance expression of FLAG-eIF2α/pcDNA3 (Fig. 1A). Because RAX only slightly or moderately increased CMV promoter/enhancer activity (Figs. 2A and 4B) while significantly enhancing the FLAG-eIF2α expression, the mechanism may result primarily from RAX enhancement of SV40 promoter (origin)/enhancer dependent plasmid replication. Furthermore, because both pGL3-luciferase and the FLAG-eIF2α/pcDNA3 plasmids are different plasmid constructs, these results strongly suggest that RAX-mediated augmentation of DNA replication is not an artifact of a particular plasmid construct but rather is specific for the SV40 promoter (origin)/enhancer elements. To test whether the T-Ag was required for RAX-mediated enhancement of DNA replication, plasmid replication studies were carried out in CV-1 cells that lack T-Ag expression. Results indicated that expression of the T-Ag is required for RAX-mediated enhancement of SV40 promoter (origin)/enhancer dependent DNA replication (Fig. 5C). Northern analysis confirmed that HA-RAX synergistically increased SV40 promoter/enhancer-dependent luciferase mRNA expression in COS-7 and CV-1 cells (Fig. 6, A and B). Results in CV-1 cells indicated that although the basal level of luciferase mRNA expression was undetectable, probably because of the small amount of luciferase vector used in the cotransfection, HA-RAX synergized with the co-expressed T-Ag to stimulate mRNA expression (Fig. 6B, comparing lanes 1 and 2 with lanes 3 and 4). Because (luciferase) gene transcription primarily occurs downstream of plasmid replication, HA-RAX-enhancement of mRNA expression resulted, at least in part, from enhanced plasmid replication. As shown in Figs. 5 and 6, the magnitude of HA-RAX-mediated enhancement of mRNA expression appeared to be greater than that of plasmid DNA synthesis, especially in CV-1 cells, indicating that RAX likely stimulated SV40 regulatory element-dependent DNA transcription as well. Because the T-Ag can function in both SV40 DNA synthesis and transcriptional activation that are tightly coupled processes, RAX may cooperatively stimulate these two processes. DsRNA-binding Domains Are Required to Enhance Luciferase Gene Expression—RAX/PACT contains three putative dsRNA-binding motifs that are required for PKR activation (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (370) Google Scholar, 29Peters G.A. Hartmann R. Qin J. Sen G.C. Mol. Cell. Biol. 2001; 21: 1908-1920Crossref PubMed Scopus (123) Google Scholar). The first two dsRNA-binding motifs (dsRBM) display high homology with the first dsRBM of PKR (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (370) Google Scholar, 30Green S.R. Mathews M.B. Genes Dev. 1992; 6: 2478-2490Crossref PubMed Scopus (217) Google Scholar). Lys-84 and Lys-177 are conserved lysine residues located within the first and second dsRBMs that are necessary for the dsRNA binding of RAX (15Ito T. Yang M. May W.S. J. Biol. Chem. 1999; 274: 15427-15432Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Patel R.C. Sen G.C. EMBO J. 1998; 17: 4379-4390Crossref PubMed Scopus (370) Google Sc" @default.
• W2004363451 created "2016-06-24" @default.
• W2004363451 creator A5002006406 @default.
• W2004363451 creator A5031853527 @default.
• W2004363451 creator A5050126506 @default.
• W2004363451 date "2003-10-01" @default.
• W2004363451 modified "2023-10-05" @default.
• W2004363451 title "A Novel Role for RAX, the Cellular Activator of PKR, in Synergistically Stimulating SV40 Large T Antigen-dependent Gene Expression" @default.
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