INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) also known as human herpesvirus 8 (HHV-8) is associated with malignancies of both endothelial and lymphoid cells in humans. KSHV has been well established as the etiologic agent responsible for Kaposi's sarcoma (KS), an endothelial neoplasm frequent in homosexual men with AIDS and highly prevalent in sub-Saharan Africa (reviewed in reference 25). KSHV is also linked to two other AIDS-related malignancies, primary effusion lymphoma and multicentric Castleman's disease (5, 26). The presence of viral DNA in CD19⁺ B cells and other mononuclear cells of the peripheral blood of KS/AIDS patients (1, 2, 31), even prior to full-blown KS, suggests that infection of the lymphoid compartment is antecedent to the development of the endothelial disease.

KSHV infection, similar to infection by other herpesviruses, displays two life cycle modes, latency and lytic replication. Latency is established by the virus both in endothelial and B cells and is detectable in such cell types both in vitro and in infected hosts (1, 3, 7, 19, 22, 27, 31). KSHV latency-associated genes are expressed in most spindle cells of KS tumors and are thought to contribute to their survival and proliferation (25). However, many viral genes (e.g., vGCR and vMIPs I and II) encoding homologs of cellular signaling proteins which have been suspected of roles in the histogenesis of KS are expressed strictly as lytic cycle products (13, 20, 23, 24, 28). This suggests that the KSHV lytic cycle may also contribute to KS lesion formation. Additional support for this notion comes from studies showing that treatment of high-risk patients with the antiviral agents ganciclovir, which blocks lytic KSHV replication, reduces KS risk (18). Of course, lytic reactivation also likely contributes to KS progression by allowing spread of the virus from the lymphoid reservoir to the endothelial sites of KS tumor formation. A complete understanding of the molecular events which control and direct viral reactivation from latency is thus critical to completely understanding KSHV pathogenesis.

We and others have previously shown that ectopic overexpression of KSHV open reading frame ORF 50 (ORF50) induces lytic reactivation of the virus in B-cell models of latency (8, 15, 16, 29). Furthermore, we identified a potent carboxy-terminal activation domain in the ORF50 protein which shares homology with its counterparts in Epstein-Barr virus (EBV) and herpesvirus saimiri; deletion of this domain from the cognate KSHV protein abrogates its ability to reactivate the virus in BCBL-1 cells (15). We also identified an amino-terminal oligomerization domain which allowed us to design a dominant-negative mutant of ORF50 that strongly suppresses transcriptional activation by the wild-type (wt) protein and inhibits both spontaneous and chemically induced lytic reactivation. Thus, transcriptional activation by ORF50 is absolutely required for lytic reactivation of KSHV induced by all known reactivating signals (15).

We initiated our investigation into ORF50's transactivation mechanism by utilizing transient cotransfections of ORF50 expression vectors together with reporter plasmids in which various KSHV promoters drive expression of firefly luciferase. These studies demonstrated that ORF50 transactivates the promoters of the ORF57, K-bZIP, nut-1 (PAN), thymidine kinase (TK), kaposin, and DNA binding protein genes in CV-1, BJAB (human B-lymphoblast), and SLK (human endothelial) cells (15, 16). However, ORF50's inability to transactivate the DNA polymerase, assembly protein (AP), and glycoprotein B promoters suggested that ORF50 displays selectivity in transactivation and is not simply a promiscuous activator.

In this report, we mapped the DNA sequence required for ORF50's activation of the ORF57 promoter and show that this element is homologous to an element in the K-bZIP promoter. The element can be directly bound by recombinant ORF50 protein; the binding site consists of a 12-bp partially palindromic sequence, 5'-AACAATAATGTT-3', together with several 3' flanking nucleotides. Transfer of this element to heterologous cellular and viral TATA boxes which alone are not activated by ORF50 confers dramatic ORF50 responsiveness, and this activation is independent of orientation of the element. Mutations in the element which inhibit ORF50 binding to either promoter also inhibit ORF50-dependent activation in vivo, indicating the importance of DNA binding for activation.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Plasmids. All plasmids were propagated as described elsewhere (15, 16). pcDNA3-gORF50, described elsewhere (16), is a KSHV genomic clone which expresses the full-length ORF50 protein.

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Deletion analysis of the ORF57 promoter reveals the minimal sequences necessary for transactivation by ORF50 (50RE). This schematic depicts the features of the ORF57 promoter lying between the transcriptional start site (genomic position 82003 [12]) and 602 bp upstream of it [here named -602(WT)]. Each deletion of the promoter is depicted by the grey bars and named 1 to 5K (described in Materials and Methods); the endpoint of each deletion is also described by its distance in base pairs from the start site, listed by a negative number next to the name of the deletion. Each was cotransfected into CV-1 cells with increasing amounts of pcDNA3-FLg50 or empty pcDNA3, using Superfect, as described in Materials and Methods, and fold transactivation was calculated. The maximal point of transactivation in each titration curve is listed (as well as the standard deviation [SD], in parentheses) for each deletion. The black bar denotes promoter positions -106 to -54, referred to as 50RE. The abbreviations above the wt promoter indicate consensus binding sites for cellular transcription factors as predicted by TRANSFAC (32). Abbreviations: SREBP-1, sterol regulatory element binding protein 1; CDP, CAAT displacement protein; SRY, sex-determining region Y protein; GATA, GATA family factors; OCT, octamer proteins; CREB, cyclic AMP-responsive binding element protein; AP-1, activating protein 1; SOX, SRY-like high-mobility-group box-containing protein family; AML-1a, acute myeloid leukemia 1a protein; TATA, TATA box; ISRE, interferon-stimulated response element.

View larger version (31K): [in this window] [in a new window]   FIG. 2.   Enhancer-like function of 50RE and conservation with the K-bZIP promoter. (A) 50RE confers ORF50 responsiveness on a heterologous TATA box. 50RE was fused to the hsp70 TATA box (30) in forward and reverse orientations to generate the two reporter plasmids depicted. Each was cotransfected into CV-1 cells with various amounts of ORF50 expression vector using Fugene-6, and fold activation was calculated as for Fig. 1. Maximal activation of each reporter is given at the right, followed by standard deviations (S.D.) in parentheses. (B) The TATA-proximal promoters of the ORF57 and K-bZIP genes share three conserved sequences. The -106/-5 sequence of the ORF57 promoter is given at the top, the sequence of the TATA-proximal promoter of K-bZIP is shown at the bottom, and the consensus between them is in the center. The grey boxes denote the conserved functional blocks shared by the two promoters. The convergent arrows denote the 12-bp palindromic sequence shared by the promoters, and the open box denotes the extended, palindromic flanking sequences found only in p57. The numbers above and below the sequences refer to the genomic positions of the first nucleotide depicted in each promoter sequence. Kpn I refers to the KpnI restriction endonuclease site which demarcates the right-hand side of 50RE at nt -54 from the ORF57 start site.

Cell lines and transfections. CV-1 cells were propagated and maintained as previously described (15). Transfections for analysis of the initial ORF57 promoter deletion series were performed using Superfect reagent (Qiagen) as previously described (12). All other transfections were performed using Fugene-6 (Boehringer Mannheim). Briefly, 10⁵ CV-1 cells were plated in 2 ml of medium in each well of a six-well plate and then allowed to adhere overnight under standard growth conditions. Plasmid DNA for each transfection was aliquoted into 0.1 ml of minimal Dulbecco modified Eagle medium, using 0.5 µg of pcDNA3.1lacZ, 0.5 µg of the appropriate reporter vector (as described in the figure legends and text), and 0.5 to 2 µg of pcDNA-g50 (as described in the figure legends and text). In all cases, pcDNA3 was used as a filler plasmid to bring the total DNA amount to 3 µg for each transfection; all transfections likewise were performed in triplicate. Next, 9 µl of Fugene-6 reagent was added to each DNA-medium mixture, mixed by vigorous flicking, and allowed to incubate at room temperature for 30 min. Cells were refed with 2 ml of fresh growth medium, and the DNA-medium-Fugene-6 mixture was added dropwise to each well. Transfections were harvested at 40 to 48 h posttransfection and analyzed for luciferase and -galactosidase.

Luciferase and -galactosidase assays. Performed as described previously (15). -Galactosidase assays were performed as an internal control for each transfection to normalize for transfection efficiency and variability in the cell extract harvest.

Construction and propagation of recombinant baculovirus. Briefly, using a Bac-N-Blue transfection kit (Invitrogen) according to the manufacturer's recommendations, Sf9 cells (UCSF Cell Culture Facility) were cotransfected with Bac-N-Blue viral DNA and pBlueBacHis-50, using Insectin-Plus liposomes (Invitrogen). Recombinant virus was harvested by collection of the cell medium at 5 days posttransfection and purified by subsequent plaque assay using baculovirus agarose (Invitrogen) containing 5-bromo-4-chloro-3-indolyl--D-galactosidase (X-Gal) to identify recombinants. Plaques containing recombinant virus were transferred to fresh Sf9 monolayers in 12-well plates to generate P1 viral stocks. These viral stocks were subsequently screened for ORF50 expression by infection of fresh Sf9 monolayers in six-well plates, which were harvested in 10s buffer (4) at 48 h postinfection (hpi) and analyzed by Western blotting using our previously described anti-ORF50 rabbit serum (16). The best-expressing viral clone (named Bac-50) was then amplified into a high-titer, P2 stock by infection of a 500-ml suspension culture of Sf9 cells, which was harvested when approximately 95% of the cells had lysed. The supernatant-containing virus was subsequently separated from cell debris by centrifugation, and virus was titered by plaque assay on Sf9 cells.

Overexpression and purification of ORF50 from Bac-50-infected Sf9 cells. A 500-ml spinner culture of exponentially dividing Sf9 cells at a density of 2 × 10⁶ cells/ml was infected with Bac-50 at a multiplicity of infection of 10. At 48 hpi, cells were collected by centrifugation, washed twice in 1× phosphate-buffered saline, and lysed in 100 ml of 1× urea HIS binding buffer (500 mM NaCl, 20 mM Tris [pH 8.0], 5 mM imidazole, 6 M urea, 25 mM phenylalanine, 25 mM isoleucine) supplemented with the Protease Inhibitor Cocktail Set III (Calbiochem). The resultant extract was then passed through an 18-gauge needle four times to ensure complete cell lysis and to shear the genomic DNA. Cell debris was removed by centrifugation in a GSA rotor (Sorvall) at 11,000 rpm for 30 min. The supernatant was decanted and then filtered using a 0.45-µm-pore-size filter (Nalgene) to remove any remaining particular matter.

Overexpression and purification of ORF50 from Escherichia coli. BL21(DE3) competent E. coli cells (100 µl; Life Technologies) were transformed with 3 µg of pRSET 0.8 or pET28b-N50, transferred to 1 liter of Luria broth containing 50 µg of ampicillin per ml, and grown overnight at 37°C with shaking. After about 12 h of growth, when cells reached an optical density of 0.6 to 0.8, recombinant protein expression was induced using 1 mM isopropylthio--D-galactosidase (IPTG), and cells were maintained at 37°C with shaking for 6 h. Cells were then pelleted and resuspended in 1× urea HIS lysis buffer as above and allowed to lyse overnight with nutation at 4°C. Next, NP-40 was added to 0.1%, and cells were sonicated using a large tip at a power setting of 8 for 30 s four times to complete cell lysis and to shear chromosomal DNA. The extract was clarified as above and loaded onto a 5-ml chelating column prepared as above. After washing as above, protein was eluted stepwise in six 2.5-ml fractions using 500 mM imidizole in 1× urea HIS binding buffer. Fractions were analyzed as above, and ORF50-containing fractions were pooled and dialyzed against DNA binding buffer as above. Using Coomassie blue staining, we estimated that His₆-N50 and -C50 generated in this manner comprised approximately 20% of the resulting total protein.

SDS-PAGE/Western blotting. Procedures were as previously described (15), using gels containing 10% polyacrylamide. Anti-ORF50 rabbit serum was used as previously described (15). Anti-His₆ antibody (Clontech) was used at a dilution of 1:1,000.

EMSA. Probes for EMSA (top strands are diagrammed in Fig. 4A, 5A, 6, and 8A) were assembled by annealing complementary as described above (see "Plasmids") and resuspended in dH₂O at a concentration of 100 ng/µl. Each EMSA probe was designed to contain an overhanging BglII site at each end to facilitate either cloning or labeling by Klenow DNA polymerase. One hundred nanograms of probe was labeled in a reaction containing 1× REact 2 buffer (Life Technologies), 0.017 mM each dATP, dGTP, and dTTP, 0.5 U of Klenow DNA polymerase (Life Technologies), and 20 µCi of [-³²P]dCTP (6,000 Ci/mmol; Amersham/Pharmacia). After a 30-min incubation at 25°C, the reaction was chased by the addition of 0.017 mM dCTP. Unincorporated nucleotides were removed by purification using QuickSpin TE G-25 columns (Boehringer Mannheim), followed by sequential extraction with phenol-CHCl₃ and CHCl₃. The probe was then precipitated along with 200 ng of yeast tRNA (Life Technologies) as carrier, pelleted, and resuspended at a specific activity of 10⁵ cpm/µl (usually 100 to 200 µl, quantitated by scintillation counting).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Deletion analysis of the ORF57 promoter reveals the minimal sequences necessary for transactivation by ORF50 50RE. We have previously mapped the transcripts for the KSHV ORF57 gene and showed that its promoter is responsive to ORF50 expression (12, 15). To map the elements of the ORF57 promoter that confer responsiveness to ORF50 activation, we first cloned 600 bp of the ORF57 promoter 5' to a luciferase reporter and confirmed that this fragment contained all necessary sequences. Cotransfection of this reporter with an expression vector for ORF50 revealed a 300-fold enhancement of luciferase expression in the presence of ORF50 (Fig. 1). Next, we constructed the series of deletion mutants shown in Fig. 1 and assayed each vector in the presence or absence of ORF50. As shown in Fig. 1, deletions to nucleotide (nt) -218 (relative to the start site of ORF57 mRNA) only trivially influenced ORF50 upregulation, and further deletion to nt -106 resulted in only a threefold additional loss of inducibility. However, further deletion to nt -54 completely ablated ORF50 induction (Fig. 1, -5K reporter) without affecting the basal level of luciferase expression (not shown). These data demonstrate that (i) ORF50 cannot activate transcription through the ORF57 TATA box alone and (ii) the 52 bp unique to 5, lying between nt -106 and -54, contain an element(s) required for ORF50 transactivation of the ORF57 promoter. We refer to this 52-bp DNA sequence hereafter as the ORF50 response element (50RE).

The 50RE confers ORF50 responsiveness on heterologous TATA boxes in an enhancer-like fashion. To determine whether the ORF57 upstream promoter sequences can confer ORF50 responsiveness on a heterologous TATA box, we next fused the 50RE sequence (-106/-54 [Fig. 1]) to the well-characterized TATA box for the cellular gene hsp70 (30), driving firefly luciferase as a reporter. Importantly, Fig. 2A shows that, similar to the ORF57 TATA box alone (5K deletion in Fig. 1), ORF50 is incapable of transactivating the hsp70 TATA box alone (Fig. 2A, line 1). However, fusion of the 50RE in either orientation relative to the TATA box allows robust ORF50 transactivation in both CV-1 cells (Fig. 2A) and BJAB cells (not shown). Furthermore, although ORF50 cannot activate transcription directed by the promoter of the late KSHV AP gene (16), the ORF57 50RE promoter sequences also confer strong ORF50-dependent activation on the AP TATA box (data not shown). This suggests that ORF50 shows little TATA box specificity. Together, Fig. 1 and 2 establish that the 50RE, between -106 and -54 of the ORF57 promoter, is both necessary and sufficient for ORF 50 transactivation.

The TATA-proximal promoters of the ORF57 and K-bZIP genes share three conserved sequences. We have previously demonstrated that among the promoters transiently transactivated by ORF50, the ORF57 and K-bZIP promoters were most strongly activated (15). Therefore, we compared the -106/-5 sequence of the ORF57 promoter (containing the 50RE and the TATA box) with the TATA-proximal K-bZIP promoter to look for similarities.

Generation of a recombinant baculovirus expressing ORF50 for use in DNA binding assays. To determine whether or not the ORF50 protein interacted directly with the ORF57 promoter, we initially attempted to detect an interaction between the 50RE (diagrammed in Fig. 4A) and crude ORF50 protein present in (i) nuclear extracts of transiently transfected Cos9 cells, (ii) rabbit reticulocyte lysate (RRL) or wheat germ lysate in which ORF50 cDNA had been transcribed and translated in vitro, or (iii) nuclear extracts of BCBL-1 cells induced with tetradecanoyl phorbol acetate. Lysates containing ORF50 from each of these sources failed to interact with ORF57 promoter sequences in a manner that corresponded with ORF50's ability to activate transcription (not shown). This suggested that detection of this interaction might require more highly purified and concentrated material.

View larger version (51K): [in this window] [in a new window]   FIG. 3.   Time course of ORF50 protein expression in Sf9 cells infected with recombinant baculovirus. A recombinant baculovirus in which ORF50 is expressed under control of the polyhedrin promoter (Bac-50) was constructed and grown to high titer as described in Materials and Methods. Sf9 cells were incubated with medium alone (Mock) or infected with Bac-50 at a multiplicity of infection of 10; whole-cell extracts were prepared at the indicated hours postinfection and analyzed by immunoblotting with anti-ORF50 antibody. Extract from Sf9 cells infected with a recombinant baculovirus expressing an irrelevant, non-KSHV protein ("WT"; a gift from David Morgan) and mock extract were harvested at 48 hpi and similarly analyzed. ORF50 was also expressed by transcription and translation in vitro in RRL and detected together with the Sf9 proteins or with a shorter exposure to film (short).

Both the palindrome and flanking sequences contribute to DNA binding and transactivation by ORF50. To analyze the interactions of baculovirus-generated ORF50 with the ORF57 promoter using EMSA, we created four 26-bp probes (named 5A to 5D) which span the 50RE and are shown in Fig. 4A. Each probe was labeled with ³²P and then incubated with binding buffer alone or increasing amounts of the partially purified baculovirus-derived protein. These DNA-protein mixtures were then separated on nondenaturing polyacrylamide gels; a typical example is shown in Fig. 4B. These data demonstrate the formation of four different complexes, designated 1, 1*, 2, and 3. Complexes 1 and 1* form only on probes 5C and 5D. Complex 2, however, forms on all of the probes, suggesting that it may contain nonspecific DNA binding proteins. In this experiment, complex 3 was observed only on probes 5A and 5B; however, complex 3 can occasionally be detected on probes 5C and 5D as well (Fig. 5B), suggesting that it, too, may be sequence nonspecific. The low intensity and variable appearance of complex 3 precluded further rigorous characterization.

View larger version (56K): [in this window] [in a new window]   FIG. 4.   Both the palindrome and flanking sequences contribute to DNA binding and transactivation by ORF50. (A) Sequences of four 26-bp oligonucleotides used as probes in EMSA and cloned into reporter plasmids. The sequence of the top strand of 50RE is shown at top, with the grey box depicting the 12-bp palindrome and the open box depicting the extended, flanking palindromic sequences. Each sequence below that represents the top strand of each of four oligonucleotides named 5A to 5D, which were annealed to complementary oligonucleotides as described in Materials and Methods. The resultant products were radiolabeled for use in EMSA or cloned as dimers upstream of the hsp70 TATA box in plasmid hsp-luc for use in transactivation assays (generating plasmids p57-5Ahsp-luc, p57-5Bhsp-luc, p57-5Chsp-luc, and p57-5Dhsp-luc; see Materials and Methods). The palindromic and flanking sequences which are included in each oligonucleotide are indicated by the boxes. (B) Four DNA-protein complexes are formed by incubation of Bac-50-infected Sf9 cell extracts with the EMSA probes. ORF50 was partially purified from Bac-50-infected Sf9 cells as described in Materials and Methods, and 0, 2.5, 5.0, and 10.0 µg, of extract (containing ca. 0, 50, 100, and 200 ng of His6-tagged ORF50 [see Materials and Methods]) were analyzed for interaction with probes 5A to 5D by EMSA. Each set of four binding reactions is indicated above the autoradiogram according to the EMSA probe used, and resulting complexes are indicated by the numbers to the right of the autoradiogram (see text). (C) Transactivation of the 5A to 5D sequences by ORF50. Each reporter plasmid (A) was cotransfected into CV-1 cells with increasing amounts of pcDNA3-FLg50 or empty expression vector, and fold activation was assayed as for Fig. 2A. The entire titration curve is depicted for each reporter. (D) ORF50 is a component of complexes 1 and 1*. Partially purified extract from Bac-50-infected Sf9 cells (250 µg) was depleted with Ni-NTA-agarose or mock depleted with glutathione-Sepharose (glutathione-seph.) as described in Materials and Methods. The Ni-NTA-depleted extract was further depleted by a second incubation with a fresh aliquot of beads; 5 µg of untreated extract or an equivalent volume of each depleted extract was then subjected to EMSA with probe 5D as for panel B (top). The migration of complexes 1 to 3 is indicated to the right. Alternatively, 1/10 volume of each extact was analyzed by immunoblotting for the presence of ORF50 (bottom). (E) Competition EMSAs demonstrate a hierarchy of binding preferences for ORF50 binding to the 5A to 5D sequences. Partially purified extract from Bac-50-infected Sf9 cells (6 µg) was incubated with a 10-, 20-, 40-, or 80-fold molar excess of each nonradiolabelled EMSA probe before incubation with a 1-fold molar equivalent of radiolabeled EMSA probe 5D. These preparations were subject to EMSA for panel B, and each set of four binding reactions is indicated above the autoradiogram. Included for comparison are an EMSA of a binding reaction lacking protein (No protein) and a reaction containing labeled 5D probe and ORF50 without specific competitor (No competitor); the migration of complexes 1 to 3 is indicated to the right.

View larger version (78K): [in this window] [in a new window]   FIG. 5.   ORF50 binding to sequences within the palindrome is necessary for transcriptional activation. (A) Sequences of four 26-bp oligonucleotides used as probes in EMSA and cloned into reporter plasmids. The sequence of the top strand of p57-5Dwt is shown at the top, with the grey box depicting the 12-bp palindrome and the open box depicting the extended, flanking palindromic sequences. Each sequence below that represents the top strand of each of four mutant oligonucleotides named 5Dm1 to 5Dm3, in which the 6-bp linker substitution represented by the hatched box was substituted for the wt sequence indicated in each probe. These were annealed to a complementary oligonucleotide and then radiolabeled for use in EMSAs; 5Dwt, 5Dm2, and 5Dm3 were also cloned to produce the reporter vectors p57-5Dwthsp-luc, p57-5Dm2hsp-luc, and p57-5Dm3hsp-luc (see Materials and Methods). (B) The right half of the palindrome and flanking sequences are required for binding of ORF50 to p57-5D. Aliquots of 0, 2.5, 5.0, and 10.0 µg of partially purified extract (containing ca. 0, 50, 100, and 200 ng of His6-tagged ORF50 [see Materials and Methods]) from Bac-50-infected Sf9 cells were analyzed for interaction with probes 5Dwt and 5Dm1 to 5Dm3 by EMSA as for Fig. 4B. Each set of four binding reactions is indicated above the autoradiogram according to the EMSA probe used. (C) Competition EMSAs confirm the sequence requirements for ORF50 binding to p57-5D. Six micrograms of partially purified extract from Bac-50-infected Sf9 cells was incubated with a 50-, 100-, or 200-fold molar excess of each nonradiolabeled EMSA probe before incubation with a 1-fold molar equivalent of radiolabeled EMSA probe 5Dwt. These preparations were subject to EMSA as for Fig. 4E, and each set of four binding reactions is indicated above the autoradiogram. Included for comparison are an EMSA of a binding reaction lacking protein (No protein) and a reaction containing labeled 5Dwt probe and ORF50 without specific competitor (No competitor). (D) DNA binding of the 5D sequence by ORF50 is required for transcriptional activation. Each reporter plasmid (A) was cotransfected into CV-1 cells with increasing amounts of pcDNA3-FLg50 or empty expression vector, and fold activation was assayed and depicted as in Fig. 4C.

ORF50 binding to sequences within the palindrome is necessary for transcriptional activation. To determine the role of the palindrome and flanking sequences in DNA binding and transcriptional activation by ORF50 in the context of the optimized 5D EMSA probe sequence, we generated the three linker scanning mutations shown in Fig. 5A. The 6-bp sequence CTGCAG (a PstI restriction endonuclease site) was substituted either for half of the core palindrome (5Dm1 or 5Dm2) or for the 6 bp on the TATA-proximal side of the core palindrome (5Dm3). The wt probe and each mutant probe were then labeled and analyzed by EMSA as before. Figure 5B shows that ORF50 binds to 5Dm1 in a quantitatively similar manner as to the 5Dwt probe. However, ORF50 binds to neither 5Dm2 nor 5Dm3. These data therefore suggest that the right half of the palindrome and the 3' flanking sequences are the primary determinants for ORF50 binding to this element. In fact, these data agree well with the ORF50 binding preferences determined for probes 5A through 5D (compare probe 5C [Fig. 4A and B] with 5Dm1 [Fig. 5A and 5B]).

Fine mutational analysis of the 50RE. To confirm that the negative effects of the 5Dm1 to 5Dm3 mutations were not specific to the chosen linker sequence or to the large size of the substitutions, we generated a second series of nine linker substitution mutations across the 5D element in which 1 to 4 bp were changed in each mutation; these are shown in Fig. 6 and named 5Dm4 to 5Dm9 (for 5Dm4 and 5Dm9, we constructed one and two additional variants, respectively).

View larger version (21K): [in this window] [in a new window]   FIG. 6.   Fine mutational analysis of 50RE. A second series of nine linker substitution mutants across the 5D element was generated as EMSA probes and cloned into hsp-luc using oligonucleotides (as described in Materials and Methods). All of the mutants are named at the left; the new series of mutants, containing 1- to 4-bp changes of the wt sequence, are listed in lines 5 to 13. The specific base pairs changed in each clone of both series are listed in the center column underneath the sequence of 5Dwt. Each was tested both for DNA binding by the Bac-50-generated ORF50 and for transcriptional activation as in Fig. 4 and 5. DNA binding data were semiquantitated for each mutant by comparison to the binding of ORF50 to the 5Dwt probe, which was assigned the value of +++; similarly, activation was quantitated for each mutant by comparison to ORF50's activation of the 5Dwt reporter construct, which was assigned the value of 100% (the first four lines summarize the data from Fig. 5).

The homologous sequences within the K-bZIP promoter are required for full activation by ORF50. Thus far, we have analyzed the functional consequences of DNA binding by ORF50 in the context of the isolated, 25-bp 5D sequences from 50RE assembled as a dimer upstream of the heterologous TATA box from the cellular hsp70 gene. Such an analysis places this response element in an artificial position relative to the TATA box and eliminates the potential influences of natural flanking sequences in DNA binding by ORF50, heterologous cellular proteins, or both. Therefore, we introduced these linker substitution mutations into the context of the wt, full-length ORF57 or K-bZIP promoter. In both promoters, we mutated the entire shared palindrome by introducing m1 (to substitute the left side of the palindrome) together with the sequence TCCGGA (a BspEI restriction site) to substitute for the right side of the palindrome. The resultant mutant reporters were named m1+2.

View larger version (17K): [in this window] [in a new window]   FIG. 7.   The palindromic sequences within the K-bZIP promoter are required for full activation by ORF50. A 12-bp linker substitution mutation was introduced to replace the 12-bp palindromic sequence of the wt, full-length ORF57 (A) or K-bZIP (B) promoter (see text). Each mutant promoter was compared to its corresponding wt promoter in cotransfections of CV-1 cells as described for Fig. 5D.

DNA binding of the K-bZIP promoter by ORF50 protein correlates with transcriptional activation. To determine whether or not the ORF50 protein could bind directly to the K-bZIP promoter in a manner that correlated with transcriptional activation, we performed EMSA using oligonucleotides representing the wt K-bZIP promoter sequence and the K-bZIP m1+2 sequence (ZIPwt and ZIPm1+2, respectively [Fig. 8A]). The second lane of Fig. 8B demonstrates that a single, major complex (indicated by an arrow) forms when the labeled ZIPwt probe is incubated with partially purified ORF50 protein. (An additional weak complex of unclear origin is also observed.)

View larger version (86K): [in this window] [in a new window]   FIG. 8.   ORF50 binds the TATA-proximal K-bZIP promoter with specificity that corresponds with ORF50's ability to activate transcription. (A) Sequence of the top strand of two 28-bp oligonucleotides used as probes in EMSA. Both were annealed to their complementary oligonucleotides as described in Materials and Methods. The box depicts the palindrome shared with the ORF57 promoter, and the grey nucleotides correspond to positions which are homologous to the ORF57 promoter (as in Fig. 2B). (B) Partially purified extract from Bac-50-infected Sf9 cells (6 µg) was incubated with a 50- or 200-fold molar excess of each nonradiolabeled 5Dwt or 5Dm2 EMSA probe (Fig. 4A) or 5- or 10-fold molar excess of each nonradiolabeled ZIPwt or ZIPm1+2 EMSA probe (A) and then incubated with a 1-fold molar equivalent of radiolabeled EMSA probe ZIPwt. These preparations were subject to EMSA as for Fig. 4B, and each set of four binding reactions is indicated above the autoradiogram. (C) Partially purified ORF50 protein was incubated with a 50- or 200-fold molar excess of nonradiolabeled ZIPwt or ZIPm1+2 EMSA probe and then incubated with 1-fold molar equivalent of radiolabeled 5Dwt probe. Included for comparison are an EMSA of a binding reaction lacking protein (No protein) and a reaction containing labeled 5Dwt probe and ORF50 without specific competitor (No competitor).

The amino terminus of ORF50 generated in E. coli binds to the ORF57 response element. To independently confirm ORF50's ability to bind the DNA palindrome, we attempted to express full-length ORF50 polypeptide as a His₆ fusion in E. coli. Unfortunately, this protein was highly insoluble and also unstable, and it yielded little full-length material after purification. Alternatively, we generated His₆ fusions of ORF50 truncated at both amino and carboxy termini (amino acids [aa] 1 to 272 [N50] and aa 525 to 691 [C50]). These are displayed schematically in Fig. 9A. Each was expressed to very high levels and purified to near homogeneity as described in Materials and Methods (Fig. 9A, immunoblots).

View larger version (56K): [in this window] [in a new window]   FIG. 9.   The amino terminus of ORF50 generated in E. coli binds to the response element. (A) Expression of N- and C-terminal truncations of ORF50 in E. coli. The top displays a schematic of full-length ORF50 (FL50). Below are schematics of the truncations of ORF50 expressed as fusions to the His6 epitope tag: aa 1 to 272 (N50) and aa 525 to 691 (C50 [16]). The bottom shows Western blot analysis of the proteins expressed and purified from E. coli transfected with pET28b-N50 (N50) and pRSET 0.8 (C50 [16]), detected using anti-His6 (Clontech) and anti-ORF50 (16) antibodies, respectively. Positions of migration of protein molecular weight standards (Life Technologies) are shown to the left and right. Abbreviations: NLS, putative nuclear localization signal; Basic, highly basic domain (described in the text); LZ, leucine repeats; ST, serine/threonine-rich region; AD, activation domain (15). (B) N50 but not C50 binds stably to the 5D sequence. N50 and C50 were expressed and purified from E. coli as described in Materials and Methods and then diluted to equal final concentrations; 0, 0.5, 1.0, and 2.0 µg of each protein (containing ca. 0, 0.1, 0.2, and 0.4 µg of truncated ORF50 polypeptide; see Materials and Methods) was incubated with radiolabeled 5Dwt probe and analyzed by EMSA as described in Materials and Methods. Each set of four binding reactions is indicated above the autoradiogram according to the polypeptide used. (C) N50 binds to the ORF57 5D but not 5B sequence. EMSA was performed as for panel B using the indicated radiolabeled probes and 0, 0.5, 1.0, and 2.0 µg of N50 polypeptide. (D) Unlabeled competitors confirm N50's binding specificity. One microgram of N50 polypeptide was incubated with a 50-, 100- or 200-fold molar excess of nonradiolabeled 5B or 5Dwt EMSA probe and then incubated with a 1-fold molar equivalent of radiolabeled 5Dwt probe. Included for comparison are an EMSA of a binding reaction lacking protein (B, No Protein) and a reaction containing labeled 5Dwt probe and ORF50 without specific competitor (D, No Protein).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this investigation, we have used deletion and linker scanning mutations to identify and partially characterize a binding site for the KSHV lytic switch protein ORF50. Our deletion analyses of the ORF57 promoter enabled us to reduce the ORF50-responsive region to a 25-bp sequence (5Dwt) which conferred strong activation by ORF50 on a heterologous TATA box (Fig. 4). ORF50 polypeptide produced recombinantly in both baculovirus-infected Sf9 cells (Fig. 3) and E. coli (Fig. 9) bound avidly to this element in vitro. The response element contains a 12-bp palindrome (5'-AACAATAATG-3') which is shared between the viral ORF57 and K-bZIP promoters (Fig. 2B), two DE promoters that are potently transactivated by ORF50 (15). Substitution of the 12-bp palindrome with a 12-bp linker scanning mutation severely inhibited ORF50's ability to transactivate either cognate promoter (Fig. 7) and abrogated its ability to bind to the K-bZIP proximal promoter (Fig. 8). However, smaller mutations within the palindrome from ORF57's promoter indicate that its two halves do not make equal contributions to its activitiesthe left-hand side of the sequence is substantially more tolerant of mutations than the right-hand side. This suggests that it is the primary sequence within this region rather than its palindromic nature that is critical for DNA binding and activation. Together, these data conclusively demonstrate that DNA binding of this region by ORF50 is required for activation of both the ORF57 and K-bZIP promoters. Based on this evidence, we propose that the 25-bp element defined by fragment 5D be named 50RE₅₇ (for ORF50 response element from the ORF57 promoter).

Extensive linker scanning mutations of 50RE₅₇ revealed that additional sequences flanking the 12-bp palindrome were also required for both DNA binding and activation by ORF50 (Fig. 5 and 6). Although these flanking sequences extend the palindrome in the ORF57 but not the K-bZIP promoter, both promoters contain shared GT/CA and GG/CC doublets in the left (TATA-distal) and right (TATA-proximal) flanking sequences (Fig. 2B). In fact, a single G-to-T mutation in the GG/CC doublet completely abrogates both DNA binding and activation by ORF50 (5Dm9 in Fig. 6). In general, the linker scanning mutations of the sequences flanking the palindrome revealed that, like the palindrome itself, only the right-side (TATA-proximal) flanking sequences were strictly required for DNA binding and activation by ORF50; every mutation in this part of the element which eliminated ORF50 binding also abrogated activation. Mutations in flanking sequences to the left of the palindrome had only modest effects on DNA binding and small (and variable) impacts on transactivation in vivo. Further biochemical analyses (i.e., DNA footprinting and interference of chemical alterations of specific nucleotides) should reveal the specific contacts between ORF50 and 50RE₅₇ and will likely be required to explain the specific sequence requirements for ORF50 binding of the element in both promoters.

Further biochemical experiments should also likely aid the derivation of a consensus binding site for ORF50, especially since sequence analysis of the entire KSHV genome (23) did not reveal a similar 12-bp palindrome in any other inter or intragenic regions of the viral nucleic acid. Direct comparison of 50RE₅₇ to the promoters for three other KSHV genes which ORF50 transactivates (encoding nut-1, kaposin, and DNA binding protein [16]) also did not reveal significant homology, even within response elements which we have recently mapped in those promoters (50RE_nut-1, and 50RE_kap [J. Chang, D. M. Lukac, and D. Ganem, unpublished data]). This suggests significant heterogeneity between 50REs within DE promoters, indicating that other, unrelated sequences may also bind ORF50 protein. We have recently observed binding of ORF50 to an element required for transactivation of the KSHV TK promoter; this element lacks the 12-bp palindrome of 50RE₅₇ but shares homology to its 3' flanking sequences (Chang et al., unpublished). Interestingly, 50RE_TK is inverted relative to its TATA box and to 50RE₅₇, in agreement with Fig. 2B, which demonstrates orientation independence of ORF50-mediated activation.

Alternatively, as has been well documented for the ORF50 homolog in EBV (9, 10, 14, 17, 21) and other viral transcriptional activators (6), KSHV ORF50 may activate promoters not only directly by binding DNA but also indirectly by (i) piggybacking on cellular DNA binding proteins or (ii) altering their activity and/or abundance. This indirect mode of activation by ORF50 may in fact also be operative on the ORF57 promoter, since we saw small effects of TATA-distal deletions on ORF50 activation (Fig. 1, deletions of nt -602 to -504 and -218 to -106). The fact that the K-bZIP promoter bearing a lesion in the 12-bp palindrome retained residual ORF50 inducibility is also compatible with the presence of unrelated response elements that directly or indirectly contribute to ORF50 responses. We are currently analyzing the interactions of ORF50 with cellular factors to address these effects.

The remarkable structural similarity between the TATA-proximal promoters of both ORF57 and K-bZIP included conservation of the relative placement of not only the 12-bp palindrome and TATA-containing elements but also the potential binding site for the cellular protein Aml-1a. Although our studies have not demonstrated a role for the latter site in activation by ORF50, the conservation of this element in the ORF57 and K-bZIP promoters may reflect an as yet unidentified regulatory significance for this lymphoid-specific transcription factor in viral replication.

Generation of amino and carboxy-terminal truncations of ORF50 in E. coli allowed us to confirm that ORF50 bound directly to 50RE₅₇ and, more importantly, demonstrated that ORF50's N terminus (aa 1 to 272), but not its C terminus (aa 525 to 691), contains its primary DNA binding activity (Fig. 9). This is not surprising, since the N terminus contains an 80-aa sequence which is 18% basic. Furthermore, this extends the structure-function similarity of KSHV ORF50 with EBV Rta, which share N-terminal DNA binding and oligomerization domains and C-terminal activation domains (11, 15-17). It will be interesting to determine whether or not KSHV ORF50 binds DNA as an oligomer and if its DNA binding and oligomerization domains can be separated genetically.

Although both the baculovirus- and E. coli-generated ORF50 polypeptides could bind to 50RE₅₇, they showed subtle differences. As demonstrated in Fig. 4E, the 5A probe (Fig. 4A) was able to weakly compete with 5D for binding to the baculovirus-generated ORF50. However, although this weak interaction of ORF50 with 5A was not evident under conditions in which 5A was labeled (Fig. 4B), we have found that the N-terminal ORF50 polypeptide from E. coli is able to interact detectably with labeled 5A under similar conditions (data not shown). Among other possibilities, this discrepancy between ORF50 from the two sources might be explained by structural differences between the full-length and truncated proteins or by posttranslational modifications. For example, the serine/threonine-rich region of ORF50, a likely target of the known extensive phosphorylations of the full-length polypeptide (15), is absent in the N50 fragment expressed in E. coli (Fig. 9). If indeed phosphorylation and/or other modifications of ORF50 affect its DNA binding or transactivation activities, this would raise the intriguing possibility that ORF50's functions in vivo may be subject to multiple levels of regulation.