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Repression by RAZ of Epstein-Barr virus bZIP transcription factor EB1 is dimerization independent Carine S e g o u f f i n , H e n r i G r u f f a t a n d Alain Sergeant Unite de Virologie Hurnaine, ENS-INSERM, U412, Ecole Normale Sup4rieure de Lyon, 46 Allee d'Italie, 69364 Lyon Cedex 07, France The hallmark of Epstein-Barr virus (EBV) infection is the establishment of a viral genome transcription pattern called latency. The EBV BZLF 1 gene product EB1 (also known as ZEBRA or Zta) is a transcription factor which is essential for the switch from latency to the lytic cycle. It has been proposed that latency is maintained (~ by the inhibition of EB1 translation via antisense hybridization of EBNA1 and EB1 hnRNAs, or (iO by the inactivation of the EB1 activating function via the direct interaction of EB1 with RelA, the retinoic acid receptor and p53, or via the titration of EBI in RAZ:EBI inactive heterodimers that are unable to bind to DNA. RAZ, a fusion protein which contains the EB1 C-terminal dimeri- Introduction Epstein-Barr virus (EBV) is a human herpesvirus that persists latently for the lifetime of the infected host. EBV is also associated with human malignancies such as Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and B and T cell lymphomas in immunocompromised individuals. EBVinfected B lymphocytes from the peripheral blood or in vitroinfected B lymphocytes can proliferate continuously when cultured (immortalization). In such cells, only a minority of the viral genes are expressed, a state defined as type III latency (Liebowitz & Kieff, 1993). Data are accumulating on the mechanisms by which the productive cycle is reactivated in such latently infected B cells, as well as the mechanisms by which reactivation can be suppressed. Various chemical agents, including the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) (zur Hausen et al., 1978), can provoke the switch from a latent to a productive viral cycle in vitro. This switch is due to the expression of two EBV-encoded transcription factors: the BZLFl-encoded factor EBI (also called Z, Zta or ZEBRA) (Chevallier-Greco et al., 1986; Countryman & Miller, 1985; Countryman et al., 1989; Authorfor correspondence:Alain Sergeant. Fax + 33 72 72 86 86. e-mail [email protected] 0001-3756 © 1996 SGM zation and DNA-binding domains fused to the Nterminal 86 amino acids of the EBV BRLF1 gene product R, has been described as a trans-dominant negative regulator of EBl-activated transcription. We demonstrate here that although RAZ efficiently represses EBl-mediated transcriptional activation, the amount of RAZ protein expressed is incompatible with repression through the titration of EB1 in inactive EB1 : RAZ heterodimers. Furthermore, we also demonstrate that RAZ efficiently represses transcription activated by an EB1 mutant carrying the GCN4 homodimerization domain (EBI~"4), despite the inability of RAZ and EBlgCn4to form stable heterodimers. Takada et al., 1986) and the BRLFl-encoded factor, called R (or Rta) (Hardwick et al., 1988) (Fig. la). Indeed, when EB1 and R expression vectors are transfected into latently infected B cells, both factors are able to activate all the EBV early genes (Buisson et al., 1989; Chevallier-Greco et al., 1986; Countryman & Miller, 1985; Flemington & Speck, 1990b; Rooney et al., 1989), probably by binding as homodimers to specific DNAbinding sites located in early EBV promoters (Chang et al., 1990; Farrell et al., 1989; Flemington & Speck, 1990a; Gruffat et al., 1990; Kouzarides et al., 1991). Moreover, only EB1 induces a productive cycle, since EB1, but not R, transactivates DNA replication from OriLyt, the origin of replication active during the lyric cycle (Cho & Tran, 1993; Fixman et al., 1995 ; Rooney et al., 1989, Schepers et al., 1993). Suppression of reactivation has been proposed to occur via antisense hybridization of EBNA1 and EB1 hnRNAs (Prang et al., 1995). It has also been proposed to occur through repression of EBl-mediated transcriptional activation, via direct interaction of EB1 with the NF-;B p65 subunit (Gutsch et al., 1994), the retinoic acid receptor (Sista et al., 1993, 1995) or p53 (Zhang et al., 1994). A trans-dominant repressor of EB1 called RAZ has also been described (Furnari et al., I994; Kelleher et al., 1995). RAZ is translated in vitro from a transcript characterized originally as a cDNA called Z8 (Manet et al., 1989), initiated at promoter PR and generated by facultative Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 16:45:23 52! iiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiii!i iiiii iiii iiii ii i i iii i iiiiiiii (a) PROTEIN TRANSLATED R (EBI?) relative amounts of RAZ and EB1 proteins expressed are incompatible with a repression through the titration of EB1 in inactive RAZ:EB1 heterodimers. Furthermore, RAZ efficiently repressed transcription activated by an EB1 mutant carrying the GCN4 homodimerization domain (EBlgen4), despite the inability of RAZ and EB1 gen4 to form stable heterodimers. cDNAs AUG ,m~ AUG Z13 ~ ~ z t s AUG R (EBI?) RAZ BZLF1 I IH l BRLFt i:il Pz V////////////////,."] ] Methods PR • Plasmids Expressionvectors. The cytomegalovirus (CMV)-based expression vec- AUG EOl (b) N Activation 1 8s D8 Oi 170 195 C 221 1 . ~' /¢ DNA binding (DB) Dlmericatlon EB1 245 RAZ Prollne rich Acidic rich C I Niiiiiiiilllll]lllllllllllllllllm 35o Actlv|tlon sos R (01) Fig, 1. Schematic representation of the EBV genome region containing the BZLF1 and BRLF1 genes. (e) Structure of full-length cDNAs corresponding to the mRNAs originating from the BomHI Z/R region, Intron sequences are indicated by thin lines. The cDNAs are listed on the right and the proteins translated from the mRNAs are listed on the left. (b) Functional domains of the EB1, R and RAZ proteins with activation, DNA binding (DB) and dimerization (Di) domains indicated, splicing (Fig. la). RAZ is a fusion protein containing the EB1 C-terminal dimerization and DNA-binding domains fused to the N-terminal 86 amino acids of R (Fig. I b). In vitro-translated RAZ does not bind stably to D N A and has been shown to recruit EB1 into RAZ:EB1 heterodimers that are unable to bind D N A (Furnari et aI., 1994). Accordingly, in co-transfection assays done in HeLa cells, RAZ expressed from plasmid pCMV-RAZ represses transcription activated by EB1 expressed from the plasmid pCMV-Z (Furnari et al., 1994). Additionally, in co-transfection assays done in B cells latently infected with EBV, RAZ expressed from plasmid pCMV-RAZ impairs the induction of EA-D (early antigen diffuse) by EB1 and R translated from bicistronic mRNAs initiated at the CMV promoter in plasmid pEBV-R/Z (Furnari et al., 1994). However, the RAZ protein has never been visualized in the repression assays, nor in EBV-infected cells. More importantly, the relative amounts of EBI and RAZ proteins were not evaluated in the repression assays, and repression through heterodimerization was not assayed directly. We have therefore evaluated the relative amounts of RAZ and EBI proteins expressed in our repression assays and have investigated whether RAZ:EB1 heterodimerization is essential for RAZ-repressed EB1transcriptional activation. Our results clearly demonstrate that although RAZ efficiently represses EBI-mediated transcriptional activation, the 53C tors for EB1 (pCMV-EB1), EB1go"4 (pCMV-EB1~en4) and RAZ (pCMVZS) contain the EB1, EB1gcn4and Z8 cDNAs cloned into the EcoRI site of the pRc-CMV vector (Invitrogen). Plasmid pRc-CMV contains the cytomegalovirus immediate-early (CMV) promoter, the SV40 early polyadenylation signal and the T7 promoter. The SV40-based expression vectors for EBI (pSV-Z41), for RAZ (pSV-Z8) and for the EB1 deletion mutant called Z59-140 (pSV-Z59-140) have been described elsewhere (Manet et al., I989; Mikaelian ef al., 1993a). The EB1 and Z8 cDNAs have been described elsewhere (Manet et al., 1989). The EB1g°n4 cDNA was excised from plasmid pAAC-EB1Cen4(Giot et al., 1991; Mikaelian et al., 1993a). The FLAG-RAZ expression plasmid pCMV-FLAG-Z8 was generated by PCR. The 5' primer contained sequences coding both for the FLAG peptide (IBI Flag system, Kodak) and for part of the N terminus of RAZ: 5' GCCCCGGATCCCACCATGGACTACAAGGA CGACGATGACAAGCATATGAGGCCTAAAAAGGATGGC3'. The 3' primer, 5' GCAAGCTTCGGTAGTGCTGCAGCAG 3', was complementary to the Z8 cDNA sequences at 622 bp from the ATG. The PCR amplified product was digested by BamHI and XmaI, and cloned into plasmid pRc-CMV to generate pCMV-FLAG-ZS. Reporter genes. The pZ-CAT reporter gene (see Fig. 2a) has been described elsewhere (Urier et al., 1989). Plasmid pCMV-CAT was made by inserting the CAT gene into the HindIII site of plasmid pCEP4 (Invitrogen). • Transfections. Plasmids were prepared by the alkaline lysis method and purified through two sequential caesium chloride gradients. The DNAs were in the same topological state as assayed by agarose gel electrophoresis. HeLa cells were grown in DMEM (Gibco) supplemented with 10 % (v/v) fetal calf serum and were seeded at I x I06 cells per 100 mm Petri dish 8 h prior to transfection. Transfections were performed by the calcium phosphate precipitation method. Cells were mixed with the appropriate DNAs : typically 15 ,g of DNA was used which included the expressing vector and plasmids carrying the reporter genes. Transfected cells were washed and collected 48 h after transfection. • CAT assays. These were performed essentially as described previously (Gorman el' al., 1982). Sonication of the cells, however, was replaced by lysis in a buffer containing 0"25 M-Tris-HC1pH 8 and 0"05% SDS. Acetylation of chloramphenicol was quantified by thin-layer chromatography followed by scintillation counting. • Immunoblots. Cells were washed with cold PBS and incubated with 100 ,l of lysis buffer as already described (Mikaelian et al., 1993 a). Equal amounts of protein were loaded and separated on a 10 % polyacrylamideSDS gel, and transferred to a nitrocellulose membrane (ScbIeicher and Schuell) by electroblotting. The membrane was then incubated with the primary antibody. The monoclonal antibody mAbZ125 detects both EB1 and EB1g°n4 proteins (see Figs 2a and 6a). The monoclonal antibody mAbZ130 detects EB1, Z59-140 and RAZ (see Fig. 3a). The monoclonal antibody mAbM2 (IBIFlag system, Kodak) detecfs FLAG-RAZ (see Figs 2 a and 3 a). The rabbit polyclonal antibody to bacterially synthesized EB1 leucine zipper, AbLZebl, detects EB1 and RAZ and does not detect the Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 16:45:23 EB1gcn4 protein (see Fig. 3a, AbLZebl). The membranes were then incubated with horseradish peroxidase-conjugatedgoat anti-mouse (for mAbZ125 and mAbM2 antibodies) or goat anti-rabbit (for AbLZebl) immunoglobulins (Amersham).The proteins were visualizedwith an ECL kit (Amersham) as specifiedby the supplier. When aSS-labelledproteins were to be visualized by ECL, a sheet of transparent plastic paper was placed between the X-ray film and the membrane in order to selectively absorb radioactiveemission from asS. • Synthesis of in vitro-translated proteins and EMSA. EBI, EB1g°n4, RAZ and Z59-93 (EB1 deleted from aa 59 to 93; Giot el a]., I991) proteins were synthesizedalone or in combinationin a reticulocyte [ysate-coupled transcription-translation system (Promega). 14C-or a~Slabelled proteins were visualizedby SDS-PAGE and autoradiographyof the dried gel. Electrophoreticmobility shift assays (EMSA) were performed by incubating 4 x I04 c.p.m, of 5' a2p-labelleddouble-stranded DNA probes with 2,1 of in vitro-translated proteins for 30 rain at room temperature in 20 mM-HEPES (pH 7"9), 100 mM-KC1, 1 mM-MgCI2, 0"5 mM-DTT,10% glycerol and 1 ~g of poly(dI-dC) in a final volume of 20,1. After incubation, the mixture was loaded onto a 4"5% (w/v) polyacrylamide gel (29 to I cross-linked), 0"2 x TBE, and run at room temperature at 10 V/cm for 3 h. The protein-DNA complexes were visualized by autoradiography. Results Efficient repression by RAZ of EBl-mediated transcriptional activation cannot be explained by the formation of RAZ: EB1 heterodimers RAZ and EB1 cannot be separated in size by SDS-PAGE (not shown). Thus, in order to specifically differentiate the RAZ protein from the EB1 protein in transient repression assays, the coding sequence for a peptide (FLAG) recognized by monoclonal antibody mAbM2 was inserted 5' to the Z8 cDNA in plasmid pCMV-Z8 to generate plasmid pCMVFLAG-Z8 (Fig. 2a). The FLAG-RAZ protein should be recognized specifically by antibody mAbM2, whereas the EBI protein should be recognized specifically by monoclonal antibody mAbZ125 directed against an epitope located between amino acids 59 and 86 of EB1, but which is absent in the RAZ protein (Fig. 2a). This was found to be the case (Fig. 2c). We then evaluated the relative amounts of EB1 and RAZ protein expressed in a transient repression assay. The BZLF1 gene promoter PZ carrying two EBl-binding sites and linked to the CAT gene (pZ-CAT, Fig. 2a) was co-transfected into HeLa cells together with the EBI expression vector pCMVEB1. As expected, transcription of the CAT gene from plasmid pZ-CAT was activated by EB1 (Fig. 2b, lane 2), and this activation was strongly repressed by the addition of increasing amounts of plasmid pCMV-FLAG-Z8 to the DNA used for transfection (Fig. 2 b, lanes 3 to 5). Surprisingly, the decrease in the EBI-mediated transactivation of promoter PZ (Fig. 2b, lanes 3 to 5) correlated with a decrease in the amount of EBI protein expressed from plasmid pCMV-EBI (Fig. 2c, mAbZI25, lanes 3 to 5). More importantly, although the amount of FLAG-RAZ protein increased (Fig. 2 c, mAbM2, lanes 3 to 5) when plasmid pCMV-FLAG-Z8 was transfected in increasing amounts, the amount of FLAG-RAZ protein was far less than the amount of EB1 protein, even in the transfections where the amount of EB1 was reduced (Fig. 2c, compare mAbZ125 with mAbM2, lane 5). It should be stressed that 5,1 of total cell protein extract was used to visualize the amount of EB1 protein present in the transfected cells whereas 20 I11of the same extract was used to visualize the FLAG-RAZ protein. Since using different antibodies to compare the amounts of EB1 and RAZ is not state of the art, we had to show that at the dilution used, antibodies mAbM2 and mAbZ125 detect EBI and RAZ with comparable efficiencies. To do this, EB1 and RAZ were in vitro-translated and labelled with [aSS]methionine in rabbit reticulocyte lysates (RRL), size-separated by SDS-PAGE, transferred to nitrocellulose, and visualized by autoradiography. As shown on the autoradiogram in Fig. 3 (b), equivalent amounts of RAZ (lanes I to 3) and EBI (lanes 6 to 7) proteins were present on the membrane (EB1 contains three methionines and RAZ four). As shown on the immunoblot in Fig. 3 (b), the antibodies mAbM2 (dil.: 1/500 of the original solution) and mAbZI25 (dil.: I / I 0 0 of the original solution) detected the in vitro-translated RAZ (lanes 1 to 3) and EB1 proteins (lanes 6 to 8) with comparable efficiencies. By using the two antibodies at the dilutions indicated above, we observed that when 150 ng of pCMV-EB1 was co-transfected in HeLa cells with 300 ng of pCMV-FLAG-Z8, more EBI protein (Fig. 3 b, lane 5) than RAZ protein (Fig. 3 b, lane 4) was detected. It must be noted that under the conditions of EB1 and RAZ transfection described above, EB1 activation was strongly repressed by RAZ (Fig. 2 b, lane 5). We also evaluated the relative levels of RAZ and EB1 proteins in HeLa cells by using as an internal reference a shorter EB1 mutant called Z59-I40, and the antibodies mAbZ130 and AbLZ ebl directed against epitopes present in EB1, RAZ and Z59-140 (Fig. 3 a). Firstly, and as shown in Fig. 3 (c), comparable amounts of RAZ (autoradiogram, lanes 1 to 3) and EBI (autoradiogram, lanes 6 to 8) proteins translated in vitro and labelled with [14C]leucine were separated by SDS-PAGE and transferred to nitrocellulose. The antibodies mAbZ130 (immunoblot mAbZI30) and AbLZ e61 (immunoblot AbLZ ebl) efficiently detected the in vitro-translated RAZ (lanes 1 to 3) and EB1 (lanes 6 to 8) proteins. When HeLa cells were cotransfected either with i ~g of pSV~EB1 and I ~g of pSV-Z59140, or with 1 lag of pSV-FLAG-Z8 and I ~tg of pSV-Z59-140, both mAbZI30 and AbLZ ebl antibodies clearly detected Z59140 expressed at comparable levels in both co-transfections. However, much less RAZ protein than EB1 protein was detected in transfected cells suggesting that RAZ was either less stable than EB1 and/or that RAZ was less efficiently translated than EB1. Indeed, equal amounts of cytoplasmic RAZ and EB1 poly(A) + RNAs were found in HeLa cells transfected as described above (not shown), confirming that the difference in the amounts of RAZ and EB1 proteins expressed in HeLa cells was also not transcriptional. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 16:45:23 53 iiiiiiiiiiiiiiiiiiiiiiiiiii iii iii iiii !i!iiiiiiiii!i (a) (a) -22 pZ-CAT TATAm -72 P-1 -86 ZREZ1 -225 mAbZ125 s,X EB1 mAbZ125 A N EB1 1 / 140 170 195 221 Z59-140 ¥ ss X I 40 t't~o~''' 195 221 245 1 140 1to lss ~r 2~ s'6 14o ,;'~,Sy ~., 245 -"->-4 " FLAG FLAG-RAZ 111111 y 245 8'6 FLAG rm FLAG-RAZ C 86 mAbZ130 ZREZ2 mAbM2 AbLzebl mAbM2 (b) (b) Exlracls (MI) FIAG-RAZ EB1 [2,5% O~ lanes 10.0% in vitro translated HeLa cells extract ~ + 1 in vitro translated + + 1"-5~'5l ~ + + ÷ + + + + 2 3 4 7 8 5 6 Autoradiogram ~' ~' 75,% ~ ~ Immunoblot < - - mAbM2-- --mAbZ12$-- 5.o~ 2,5% (c) 0,0% lanes pCMV-EB1 (ng) pCMV-FLAG-Z8 (rig) I I I I I 1 / 2 3 4 5 ~ 1 5 0 / 0 75 150 300 Extracts (,ul) FLAG-RAZ EB1 Z59-140 lanes in vitro translated I ' - ' ~ + + + 1 2 3 HeLa cells in vitro extract translated 1"3'-0-""'3'~ I " ~ + + + + + + + 4 5 6 7 8 Autoradiogram (c) RAZ -,~ 1 2 3 4 5 Extract (HI) Z59-140-~ mAbZ130 mAbZ125 5 mAbM2 20 mmunoblot RAZ Z59-140 AbLZ e b l Fig. 2 Fig. 3 Fig. 2. RAZ repression of EBI-mediated transactivation cannot be explained by the formation of RAZ; EBI heterodimers. (a) Schematic structure of the pZCAT reporter gene construct. Regions in the EBI and FLAG-RAZ proteins specifically immunodetected by antibodies mAbZ125 and mAbM2 are indicated. (b) The reporter plasmid pZCAT was cotransfected in HeLa cells alone (lane I ) or cotransfected with pCMV-EBI (lane 2) or cotransfected with pCNV-EBI and increasing amounts of pCMV-FLAG-Z8 (lanes 3 to 5), as indicated. The amount of CNV promoter was kept constant by co-transfecting, where necessary, with plasmid pCMV-O. CAT activity is expressed as percentage chloramphenicol acetylation. (c) Visualization of the EBI (mAbZ125) and FLAG-RAZ (mAbM2) proteins expressed in HeLa cells transfected as described in (b), lanes I to 5. The volume of total cell extract analysed is indicated on the right. Fig. 3. The difference in the amount of EBI and RAZ proteins detected in HeLa cells is not due to a difference in the relative detection efficiencies of the antibodies used. (o) Schematic representation of the proteins used in the immunoblotting experiments and of the epitopes recognized by mAb7125, mAbZ130, mAbM2 and AbLZebl. (b) The FLAG-RAZ (lanes I to 3) and EBI (lanes 6 to 8) proteins, translated in vitro and labelled with [3SS]methionine (autoradiogram), were detected by antibodies mAbN2 and mAbZ125 with similar efficiencies (immunoblot). Visualization by mAbM2 and mAbZ125 of the FLAGRAZ (lane 4) and EBI (lane 5) proteins expressed in HeLa cells cotransfected with 150 ng of pCMV-EBI and 300 ng of pCMV-FLAG-Z8 (immunoblot). The volume of extracts analysed and which protein they contained are indicated. (c) The FLAGRAZ (lanes I to 3) and EBI (lanes 6 to 8) proteins, translated in vitro and labelled with [14C]leucine (autoradiogram), were efficiently detected by antibodies mAbZ130 and AbLZebl (immunoblot). Visualization by mAbZ130 and AbLZebl antibodies of the FLAG-RAZ (lane 4) and EBI (lane 5) proteins expressed in HeLa cells cotransfected with I llg of pSV-Z8 and I pg of pSV-Z59-140, or cotransfected with I l~g of pSV-Z41 and I pg of pSV-Z59-140 (immunoblot). The volume of extracts analysed and which protein they contained are indicated. i3; Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 16:45:23 (a) ~,0,0 % - 70,0% - 60,0% - 50,0% - 40.0% - 30.0% - ,< 20,0% - ] 0.0% - 0 " 0 ¢'¢~ l I t I I 2 I 3 4 5 6 / 0.07 0,15 0,3 / / / I / 6 7 lanes pCMV-CAT (~g) I 7 0.25 pCMV-FLAG.Z8 (l~g) pCMV-EB1 (pg) 0.6 / 0.6 0.15 0.15 (b) 1 2 m AbZ l 2 5 mAbM2 3 4 5 ~;~!:i!i:,;: Extract (~1) 5 20 Fig. 4. RAZ repression of EB1 -mediated transactivation of the CMV promoter cannot be explained by the formation of RAZ: EB1 heterodimers. (a) HeLa cells were transfected with pCMV-CAT alone (lane 1 ) or with pCMV-CAT and increasing amounts of pCIvlV-FLAG-Z8 (lanes 2 to 5) or with pCMV-EB1 (lane 6) or with both pCMV-EB1 and pCMV-FLAG-Z8 (lane 7), as indicated. CAT activity was expressed as percentage chloramphenicol acetylation. (b) Visualization of the EB1 (mAbZ125) and FLAG-RAZ (mAblVl2) proteins expressed in HeLa cells transfected as indicated in (o), lanes 1 to 7. The volume of total cell extract analysed is indicated. The results presented above definitely exclude the possibility that the difference in the amount of EB1 and RAZ proteins detected in HeLa cells was due to a difference in the relative detection efficiencies of the antibodies used. They also exclude the possibility of simple promoter interference since the amounts of Z59-140 protein expressed were comparable in each co-transfection. These results demonstrate that the amount of FLAG-RAZ protein expressed is incompatible with a repression of EBl-mediated transcriptional activation through the titration of EB1 in inactive ILAZ:EB1 heterodimers. Moreover, these results also suggest that the CMV promoter driving the expression of mRNAs from which EB1 and FLAGRAZ were translated was activated by EB1 and repressed by FLAG-RAZ. RAZ repression of E B l - m e d i a t e d transactivation of t h e C M V p r o m o t e r c a n n o t be e x p l a i n e d by t h e f o r m a t i o n of RAZ: EB 1 h e t e r o d i m e r s When HeLa cells were transfected with a plasmid carrying the CMV promoter linked to the CAT gene (pCMV-CAT), the CAT gene was detectably transcribed (Fig. 4a, lane 1). CAT gene transcription was unchanged when plasmid pCMVFLAG-Z8 was added in increasing amounts to plasmid pCMVCAT in the transfections (Fig. 4a, lanes 2 to 5), and the FLAGRAZ protein was clearly detected by monoclonal antibody mAbM2 (Fig. 4 b, mAbM2, lanes 3 to 5). However, the activity of the CMV-CAT reporter gene was strongly increased by EBI expressed from plasmid pCMV-EBI co-transfected into HeLa cells with plasmid pCMV-CAT (Fig. 4a, lane 6), and EB1 protein was detected by the monoclonal antibody mAbZI25 (Fig. 4 b, lane 6). Moreover, EBI-activated CAT transcription was strongly repressed when plasmid pCMV-FLAG-Z8 was co-transfected with plasmids pCMV-CAT and pCMV-EB1 (Fig. 4a, lane 7). Again, the decrease in EBl-activated transcription was associated with a decrease in the amount of EB1 protein translated from mRNAs transcribed from plasmid pCMV-EB1 (Fig. 4 b, mAbZ125, compare lane 6 with lane 7). It is noteworthy that although equal amounts of plasmid pCMVFLAG-Z8 were added to the cells in the transfections depicted in lanes 5 and 7, the amount of FLAG-RAZ protein detected was only slightly higher in the presence of EB1 (Fig. 4b, mAbM2, lane 7) than in the absence of EB1 (Fig. 4 b, mAbM2, lane 5). This indicates that RAZ represses the EBl-mediated expression of both EB1 and FLAG-RAZ from plasmids pCMVEB1 and pCMV-FLAG-Z8 respectively. But when the amount of EB1 protein is compared to the amount of FLAG-RAZ protein expressed, this is again not compatible with a repressive effect of RAZ through the titration of EB1 into inactive RAZ:EB1 heterodimers. These results clearly demonstrate that transcription initiated at the CMV promoter is responsive to EB1, and that this EBI-mediated transcriptional activation is repressed by FLAG-RAZ by a mechanism which cannot be explained by invoking the formation of RAZ:EB1 heterodimers. The f o r m a t i o n of R A Z : E B 1 h e t e r o d i m e r s is not r e q u i r e d for RAZ to repress E B l - a c t i v a t e d transcription If the repressing effect of RAZ described above cannot be explained by the titration of EB1 into inactive RAZ:EB1 heterodimers, then repression should be seen if EBI and RAZ cannot form heterodimers. To directly evaluate this possibility, we constructed a hybrid protein, EB1een4, in which the homodimerization domain of EB1 is replaced by the homodimerization domain of the yeast transcription factor GCN4 (Fig. 5a). In order to demonstrate that the GCN4 and EBI dimerization domains do not stably heterodimerize, we performed a mobility shift assay using a 32P-labelled doublestranded oligonucleotide containing an EBl-binding site (Fig. 5a). EBI gen4 (Fig. 5b, lanes I to 3) or EB1 (Fig. 5b, lanes 5 to 7) were co-translated with increasing amounts of the EB1 deletion mutant called Z59-93 (Fig. 5 a). The formation of the heterodimers EB1gen4 : Z5 9-93 or EBI : Z59-93 and their binding to DNA was evaluated by incubating the in vitro-translated proteins with the 32P-labelled DNA probe described in Fig. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 16:45:23 ;3: (a) (a) ~la Ipc~bes S ' ) ' - .... ~ 3' ° S E81 ' 170 195 221 170 195 221 245 EB1 gcn4 S9 EB1 Y ebl e b ! OB DI 93 ..... 140 mAbZ125 I 245 ZSg*g3 RAZ I D ~ ¢n4 EBlg en4 I AbLZ (b) (b) EB1 g©n4 + + + 4- EB1 4- + = Z59-93 lanes i KDa M 46 1 2 3 4 5 6 7 .< EB1 gcn4 I~ 3O (c) lanes L 1 2 3 4 5 6 ~ 8 7 plasmids (lag) pZ-CAT gcn4 gcn4 EB1 : EB1 Z59-93:Z59-93 ~ _ _ . EB1 :EB1 I~ 11 EB1:Z59-93 Z59-93:Z59-93 9 lo 11 s pCMV-EB1 pCMV-EB1 g©n4 / pCMV-Z8 / o . l s ~ O.15 / (c) 0.07 o.ls 7 0.3 o.s 8 9 I 10 0.07 O.lS 0.3 0.6 11 mAbZ12S AbLZ ebl Fig. 5 Fig. 6 Fig. 5. The GCN4 and EB1 leucine-zippers do not stably heterodimerize in vitro. (o) DNA sequence of the double-stranded oligonucleotide used to evaluate specific binding of EB1, EB1 gcn4,Z59-93 and their dimeric combinations (the EB1 -binding site is underlined), and schematic representation of the proteins used in the electrophoretic mobility shift assay (EMSA) experiments. (b) [~4C]Leucine-labelled EB1, EB1 gcn4and Z59-93 proteins used in the EMSAs; lane M, molecular mass markers. (c) EMSAs were performed with the proteins described in (b), lanes 1 to 7, or with the reticulocyte lysate (lane L). The protein-DNA complexes are indicated on the left and right of (c). Fig. 6. Repression of EBl-mediated activation by RAZ is dimerization-independent. (o) Schematic representation of the proteins and epitopes recognized by mAbZ125 and AbI_Zebl. (b) The reporter plasmid pZCAT was cotransfected in HeLa cells with the expression plasmids pCMV-EB1, pCMV-EBlgCn4 and pCMV-Z8 as indicated. CAT activity is expressed as percentage chloramphenicol acetylation. (c) Visualization of the EBlgCn4 (mAbZ125) and RAZ (AbI_Zebl) proteins expressed in HeLa cells transfected as in (b) lanes 7 to 11. 4 (a). As expected, the EB1 : EB1/DNA complexes (Fig. 5 c, lane 5) formed as efficiently as the EBlgen4:EBlgen4/DNA complexes (Fig. 5c, lane I) and could be separated from the Z5993 :Z59-93/DNA complexes (Fig. 5 c, lane 4). However, while EB1 : Z59-93 :DNA complexes formed efficiently (Fig. 5 c, lanes 6 and 7), no EBIgen4:Z59-93/DNA complexes formed (Fig. 5 c, lanes 2 and 3). From these results we conclude that EB1 and EBI gen4 dimers bind the DNA probe in vitro with the same apparent efficiency and that RAZ cannot form heterodimers with EBI gen4.We then considered whether RAZ would repress 53 z EBIgen4-activated transcription. As expected, CAT transcription from plasmid pZ-CAT in HeLa cells was very inefficient (Fig. 6 b, lane 1), but was strongly increased, to the same extent, by EBI (Fig. 6b, lane 2) and by EBI gen4 (Fig. 6b, lane 7). Moreover, strong repression of both EBl-activated transcription (Fig. 6b, lanes 3 to 6) and EBlgen4-activated transcription (Fig. 6b, lanes 8 to 11) was observed when plasmid pCMV-Z8 was added in increasing amounts to the DNA used for transfection. Therefore, despite the inability of EB1gen4 and RAZ to form stable heterodimers, RAZ repressed Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Sat, 12 Aug 2017 16:45:23 EBlgen4-activated transcription. Again, the amount of EBI gen4 protein expressed from pJasmid pCMV-EB1 gen4 decreased (Fig. 6c, mAbZ125, lanes 8 to 11) as increasing amounts of RAZ proteins were expressed (Fig. 6c, AbLZ ebl, lanes 8 to 11). Discussion Transient expression assays have been used to show that the EBV protein RAZ is a trans-dominant negative regulator of EBl-activated transcription, and can be a potential regulator of the switch from EBV latency to the lytic cycle (Fumari et al., 1994). Furthermore, as RAZ does not bind stably to DNA in vitro, Furnari et al. suggested that RAZ represses EBl-activated transcription by titrating the EB1 protein in RAZ:EB1 heterodimers which are unable to bind to DNA. However, the effect of RAZ on EBl-mediated transcriptional activation can be considered to be repression by heterodimerization only if the amount of RAZ protein is equal in each transfection to the amount of EB1 protein. We show here that this is clearly not the case in transient expression assays where the CMV-based expression vectors used are similar to those used in the repression assays published by Fumari eta]. Indeed, we show that in transient assays where repression by RAZ of EBl-activated transcription is clearly seen, the amount of RAZ protein expressed is much lower than that of EBI, and therefore is not compatible with the titration of EB1 in inactive EBI:RAZ heterodimers. The difference in the amounts of RAZ and EB1 proteins detected is not due to the use of different antibodies (Fig. 3c). The difference in the amount of RAZ and EB1 proteins detected is also not transcriptional, since RAZ and EB1 RNAs were transcribed with similar efficiency from the SV40 promoter (not shown). However, although both RAZ and EB1 were located in the nuclei, much less RAZ protein was found in transfected cells suggesting that RAZ was either less stable than EB1 and/or that RAZ was less efficiently translated than EB1. Upon cotransfection of pCMV-FLAG-Z8 and pCMV-EBI in the EBVpositive Raji B cell line, no FLAG-RAZ protein could be detected whereas large amounts of EB1 protein were present in transfected cells (not shown), suggesting that the RAZ protein is even less stable in B cells than in HeLa (epithelial) cells. These last observations cast serious doubts on the validity of the model in which RAZ would be a negative regulator of the switch from latency to the lyric cycle. It must be emphasized at this point that the Z8 cDNA was very poorly represented (one copy) in the cDNA libraries originally screened, and could simply represent a product of aberrant splicing (Manet et al., 1989). We also observed in the repression assays that the amount of EB1 protein expressed from plasmid pCMV-EB1 decreased as the amount of FLAG-RAZ protein expressed from plasmid pCMV-FLAG-Z8 increased. Since the total amount of CMV promoter sequences was equal in each transfection, this observation cannot be explained by what is called 'promoter interference'. However, this observation can be explained by our finding that EB1 strongly activates transcription initiated at the CMV promoter. Thus, when plasmids pCMV-EB1 and pCMV-FLAG-Z8 are co-transfected, EB1 activates EB1 and FLAG-RAZ expression from plasmids pCMV-EBI and pCMVFLAG-Z8 respectively whereas FLAG-RAZ represses EB1activated expression of EB1 and FLAG-RAZ, leading to a net decrease in EB1 production. In these experiments, the amount of FLAG-RAZ protein produced increased, probably because the increase in the amounts of plasmid pCMV-FLAG-Z8 transfected partially compensated for the repressive effect of RAZ. Nevertheless, it is clear that in the repression assays described here, RAZ represses EBl-mediated transcriptional activation of both the PZ promoter and the CMV promoter, probably by mechanisms independent of the formation of inactive RAZ:EB1 heterodimers. We directly confirmed this hypothesis by demonstrating that RAZ similarly efficiently repressed EB1- and EBlgen4-mediated transcriptional activation, although RAZ and EB1gena could not form stable heterodimers. A probable mechanism to explain how RAZ represses EB1and EBlgen4-activated transcription is that RAZ titrates a cellular factor essential for EB1 function which is present in limiting amounts in the cell. Indeed, RAZ did not detectably repress transcription initiated at the CMV promoter unless EB1 was expressed, demonstrating that RAZ interferes specifically with the function of EB1, rather than with a factor required for the activity of the CMV promoter. The domain of RAZ involved in repression is likely to be the basic domain, and/or the R protein moiety present at the N terminus of RAZ, since the repressive effect of RAZ was also seen on EB1gen4activated transcription. It is noteworthy that the EB1 basic domain has been shown to interact with cellular factors important for EB1 transcriptional activation (Mikaelian el al., I993 b), and to titrate a cellular factor required for R-activated transcription (Giot et al., 1991). Moreover, in some efficient transfections where pCMV-EB1 was transfected in increasing amounts together with fixed amounts of the reporter gene pZCAT, EBl-activated CAT transcription decreased as the amount of EBI protein increased (not shown). This last result strongly suggests that a cellular factor required for EB1activated transcription is present in limiting amount in the cells. In conclusion, it must be stressed that although RAZ represses EBl-activated transcription in transient expression assays, such a situation has not been yet documented in EBV infected cells, and it remains to be seen if and when RAZ is efficiently expressed upon activation of the EBV switch from latency to the lyric cycle. We thank Conrad B. Bluinkfor helpfuldiscussionsand commentson the manuscript.Thisworkwas supportedby INSERM,by the Association pour la recherche sur le cancer (contract no. 6810 to A.S) (contract no. 2049 to H. G), and by FNCLCC.C.S is a recipientof an MRT fellowship. 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