Regulation and Autoregulation of the Promoter for the Latency-associated Nuclear Antigen of Kaposi’s Sarcoma-associated Herpesvirus*

Kaposi’s sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 has been established as the etiological agent of Kaposi’s sarcoma and certain AIDS-as-sociated lymphomas. KSHV establishes latent infection in these tumors, invariably expressing high levels of the viral latency-associated nuclear antigen (LANA) protein. LANA is necessary and sufficient to maintain the KSHV episome. It also modulates viral and cellular transcription and has been implicated directly in oncogene-sis because of its ability to bind to the p53 and pRb tumor suppressor proteins. Previously, we identified the LANA promoter (LANAp) and showed that it was positively regulated by LANA itself. Here, we present a detailed mutational analysis and define cis -acting elements and trans -acting factors for the core LANAp. We found that a downstream promoter element, TATA box, and GC box/Sp1 site at (cid:1) 29 are all individually required for activity. This architecture places LANAp into the small and unusual group of eukaryotic promoters that contain both the downstream promoter element and TATA element but lack a defined initiation site. Further-more, we demonstrate that LANA regulates its own promoter via its C-terminal domain and does bind to a defined site within the core promoter. With

Like other herpesviruses, KSHV can enter two different life cycles: lytic replication or latency. During lytic replication, all viral proteins are expressed and viral particles are released from the host cell, eventually resulting in the destruction of the host cell. During latent infection, only a few viral genes are expressed, and these are required for the establishment and maintenance of latency (13)(14)(15)(16)(17)(18). In KS, KSHV persists latently in the majority of tumor cells, with fewer than 5% of cells undergoing spontaneous lytic replication (19 -22). All stages of KS as well as multicentric Castleman's disease and PEL, express high levels of the latent proteins, in particular the latency-associated nuclear antigen (LANA) (19,(23)(24)(25).
The LANA (open reading frame (ORF) 73, LNA) protein is the predominant target of anti-KSHV antibodies in infected individuals. Since KSHV does not encode an ORF with homology to either EBV EBNA-1 or EBNA-2, it has been surmised that KSHV LANA has subsumed the functions of the various EBNA proteins. Although LANA shows no homology at the sequence level, its phenotype and structural features are reminiscent of the EBV EBNA-1 protein, such as an extended central region of repeats. Like EBNA-1, LANA is localized to the nucleus and associated with chromatin. Two nuclear localization signals are located one each in the C-terminal region and N-terminal region (26,27). The C terminus of LANA is required for dimerization (26) and DNA binding. LANA specifically binds to two 17-bp motifs in the KSHV terminal repeats (TR). This interaction is necessary and sufficient for latent DNA replication (28 -30). Chromatin binding is mediated primarily by the N terminus of the protein and tethers the KSHV episome to cellular chromosomes, thereby ensuring proper segregation during host cell division (31)(32)(33)(34)(35)(36)(37). In addition to its role in viral episome maintenance, LANA modulates host cell behavior to create a suitable environment for latent KSHV persistence. LANA can cooperate with activated ras in fibroblast transformation assays (38) and activates the human telomerase promoter (39). Biochemical studies show that LANA binds to p53, Rb (38,40), and a growing number of cellular transcription factors: Ring3, mSin3, ATF/CREB2, and CREB-binding protein (41)(42)(43)(44).
We and others have identified a cluster of three latently expressed proteins (v-FLIP, v-cyclin, and LANA) and have shown that these proteins were expressed by differentially spliced, multicistronic mRNAs. The latent messages all were regulated by the same cis-regulatory region, which is called the LANA promoter (LANAp) (19,45,46). The cis-minimal regulatory region of LANA is sufficient for the high level LANAp activity in cell lines of different origin: B cell-derived, endothelium-derived, and epithelium-derived. We and others previously identified DNA fragments upstream of the LANA ORF, which exhibited promoter activity in reporter-based assays (14,19,45). Fig. 1A summarizes the location of all LANA promoter mutants reported previously or generated for this study and summarizes their promoter activity in HEK293 cells. Similar results were observed in SLK (a KS-derived endothelial cell line) and B cell tumor lines (BJAB and BCBL-1). A set of mutants (pGL3-1 to pGL3-5) were constructed by Sarid et al. (14) and kindly provided to us by the authors. All constructs exhibited basal activity independently of the presence of other KSHV proteins. However, our studies also showed that LANAp was regulated positively by LANA and negatively by the p53 tumor suppressor protein (47). Thus, so far these two are the only known trans-acting factors that regulate LANAp. An analysis of the methylation status in the LANA promoter revealed the complete absence of methylation in LANAp in PEL and KS infection (48). This phenotype is consistent with the constitutive transcriptional activity of LANAp and sets LANAp apart from other KSHV promoters that are rapidly methylated and silenced upon infection (48). In transgenic mice, LANAp was active in CD19ϩ B cells but not in CD3ϩ T cells, demonstrating that the cis-acting elements in LANAp are sufficient for appropriate lineage specificity (49) and that LANAp, in the absence of other viral genes, is sufficient to drive latent gene expression.
Here, we present a detailed mutational analysis of LANAp to define cis-acting elements and trans-regulatory factors. We demonstrate that LANA promoter activity depends on a downstream promoter element (DPE) and TATA box as well as members of the Sp1/Krueppel family of transcription factors. LANAp is the first viral promoter that is a member of the class of eukaryotic promoters that contain both a DPE element and a TATA element but lack a defined initiation site.
For the construction of the target plasmid for transposon mutagen-esis, the NotI restriction site in pDD83 was destroyed by digestion with NotI, Klenow-mediated fill in and blunt religation. This yielded pDD365. pDD365 gives results identical to pDD83, confirming that removal of the NotI site and vector sequences distal to the LANAp insert had no effect on LANAp-dependent reporter gene transcription. Afterward, transposon mutagenesis was performed on pDD365 using the EZ::TN In-Frame Linker Insertion Kit (Epicenter Technologies). This method relies on the ability of a modified 1000-bp Tn5 transposon element-coupled to transposase to insert itself randomly into target DNA in vitro (81,84). Transposon events are identified by kanamycin resistance. DNAs from positive clones were subsequently digested with NotI to release the Kan R marker and religated to regenerate a NotI site and a 57-bp insertion in the clone. 400 mutants were randomly selected for plasmid purification and analysis. Since the size of the LANAp fragment is 552 bp compared with ϳ2500 bp (pGL3 basic vector minus origin and minus Amp R region), we expected one in five clones to contain an insertion in the LANAp region. Unfortunately, of 400 Tn5 mutants, we isolated only 12 mutants with an insertion in the 552-bp KSHV LANAp sequence. This is less than the expected ratio and suggests integration bias on the part of the Tn5 in vitro transposon reaction. Transposon insertions in plasmids pDD700 -pDD711 were confirmed by sequencing. In order to screen the TN5 mutants, we eliminated a NotI site in the vector backbone, yielding pDD365. PstI linkers (New England BioLabs, Beverly, MA) were ligated into the SmaI and PvuII sites of pDD83 to yield pDD461 and pDD413, respectively. A map of all reporter constructs is shown in Fig. 1. For the PCR-directed site mutagenesis, pDD83 served as the wild-type template. Mutants containing two base pair substitutions in the putative transcription factor binding sites of LANAp were generated using the GeneTailor TM site-directed mutagenesis system (Invitrogen). The two base pair substitutions in pDD780, pDD782, pDD783, and pDD784 are shown in Fig. 2A and were confirmed by sequencing. To eliminate the possibility that inadvertent second-site mutations in the luciferase ORF might be responsible for the loss of luciferase activity, we recloned the LANAp region containing 2-bp substitutions that result in loss of function into pGL3 basic. The "recloned" plasmids showed similar promoter activity compared with the parent plasmids, demonstrating that loss of function was due to the internal mutation (data not shown). Tissue Culture and Transfection-HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (0.05 g/ml), and streptomycin (5 units/ml) (all from Invitrogen) at 37°C under 5% CO 2. At day 1, cells were seeded at 5 ϫ 10 4 cells/ml/well (6-well plate) to reach 50% confluence after 24 h. At day 2, 4000 ng of total DNA together with 1000 ng pDD173 (pCDNA3.1-hislacZ; Invitrogen) was suspended in 150 l of Dulbecco's modified Eagle's medium (no serum, no antibiotics) and 7.5 l of Superfect (Qiagen, Valencia, CA). The transfection mixture was incubated for 30 min at room temperature, and the total volume was adjusted to 1.5 ml with complete medium. Cells were washed once with Dulbecco's modified Eagle's medium, and 0.5 ml of transfection mixture was added per triplicate well. Cells were incubated overnight at 37°C under 5% CO 2 , and the medium was exchanged with complete medium. All transfections were repeated at least three times each in triplicate.
Luciferase Activity-Firefly luciferase activity was measured 48 h after transfection using the Dual-Luciferase reporter assay system (Promega) in a Turner TD20/20 luminometer according to the manufacturer's instructions. ␤-Galactosidase activity was determined using o-nitrophenyl-␤-D-galactopyranoside as described (47) or the Galacto-Light Plus kit (Tropix, Bedford, MA) according to the manufacturer's recommendations.
Electrophoretic Mobility Shift Assay-Nuclear extracts were prepared from BCBL-1 cells and diluted with dilution buffer at 1:1 ratio (12 mM HEPES (pH 7.5), 50 mM KCl, 6 mM MgCl 2 , 5 M ZnSO 4 , 2 mM dithiothreitol, 0.1% Nonidet P-40, and 50% glycerol). Annealed doublestranded oligonucleotides (50 ng) were end-labeled with [␥-32 P]ATP using T4 polynucleotide kinase. The binding reaction was performed at room temperature for 30 min in a mixture containing 2 l of the diluted nuclear extract (ϳ10 g), 1 g of poly(dI-dC), 2 l of 1 M NaCl, 3 l of 5ϫ binding buffer (5 mM MgCl 2 , 2.5 mM EDTA, 2.5 mM dithiothreitol, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), and 20% glycerol), and 2 l of labeled oligonucleotide. For the competition experiments, unlabeled double-stranded oligonucleotides were added in up to a 100-fold molar excess in the initial reaction mixture. For supershift analysis, after the incubation for 10 min at room temperature, 3 l of a polyclonal rabbit antibody against LANA (a gift from Jae U. Jung, Harvard University) or a polyclonal goat anti-Sp1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the binding reaction mixture. DNA-protein complexes were analyzed by electrophoresis through 4.7% polyacrylamide gel at 200 V at 4°C for 2 h.
Western Blot Analysis-HEK293 cells were transfected with 10 g of total DNA using 20 l of Superfect (Qiagen, Valencia, CA) per 10-cm dish. 48 h after the transfection, whole cell extracts were prepared in 300 l of sample buffer (80 mM Tris, pH 6.8, 100 mM dithiothreitol, 2% SDS, 10% sucrose, and 0.004% bromphenol blue) with boiling for 5 min. Lysates were electrophoretically separated on Bio-Rad 4 -15% gradient- Ն10 or 0, relative promoter activity is higher than 10-fold, or less than 10-fold compared with the basal promoter activity of control vector (pGL3 basic). B, diagram of the reporter plasmids used in transient transfection assay to investigate the contribution of sequences downstream of ϩ1 transcriptional initiation site to LANAp activity. MCS, multiple cloning site. C, promoter activity of mutations in LANAp, which affect sequences downstream of the start site. HEK293 cells were transfected with the indicated plasmids. Data represent the mean relative luciferase activity after 48 h of triplicate experiments normalized by cotransfected ␤-galactosidase activity. S.D. is indicated by error bars. D, analysis of transposon-generated insertion mutants in LANAp. HEK293 cells were transfected with the indicated plasmids representing insertion mutants in LANAp. Each insertion site is indicated relative to the transcriptional initiation site (127,880 as ϩ1). Bars represent the mean relative luciferase activity in triplicate experiments. Transfection efficiency was normalized by co-transfected ␤-galactosidase activity. S.D. is indicated by error bars. ready gels. Proteins were transferred to Hybond™-P membranes (Amersham Biosciences) in transfer buffer (25 mM Tris-HCl, pH 8.3, 12 mM glycine, and 10% methanol), and the membranes were blocked with 5% milk overnight at 4°C. The membranes were washed three times with phosphate-buffered saline (0.05% Tween 20) for 10 min each time. The membranes were incubated with a polyclonal rabbit antibody (1:5000 dilution) against LANA (a gift from Jae U. Jung) in Blotto HD (28 mM Tris-HCl, 22 mM Tris base, 1.4 mM CaCl 2 , 80 mM NaCl, 2% Nonidet P-40, and 0.2% SDS) containing 0.2% bovine serum albumin for 1 h and then washed three times with Blotto HD (0.2% bovine serum albumin). The blots were incubated with horseradish peroxidase-conjugated secondary antibody against rabbit IgG (1:5000 dilution; Santa Cruz Biotechnology) in Blotto HD (0.2% bovine serum albumin) for 45 min and subsequently washed three times with Blotto HD. After final washing with phosphate-buffered saline for 10 min, the blots were developed with the Western blotting detection system (Amersham Biosciences).

RESULTS
Sequences Downstream of the Transcription Start Site Contribute to Core Promoter Activity-The side-by-side comparison of the deletion clones from Sarid et al. (14) with those from Jeong et al. (47) allowed us to further define LANAp. Confirming previous observations, both sets of clones exhibited significant constitutive promoter activity as long as sequences up to Ϫ88 (127,968) were included. However, the deletion clones starting at ϩ11 (127,869) showed a generally lower level of activity compared with the clones starting at ϩ271 (127,609). Of the two deletion mutants that extended to Ϫ279 (128,159) and differed only by their proximal site at ϩ271 (127,609) or ϩ11 (127,869), the clone starting at ϩ271 exhibited Ն10-fold promoter activity, whereas the clone starting at ϩ11 exhibited only Ն3-fold promoter activity above vector control (Fig. 1A).
A second difference between the ϩ271 to Ϫ279 reporter construct (pDD83) and the ϩ11 to Ϫ279 reporter construct (pGL3Ϫ3) was that in the latter the KSHV promoter region was cloned into the multiple cloning site of pGL3basic (Promega), whereas pDD83 starts at the luciferase start codon and replaces the 5Ј-UTR and translation-initiation sequence originally present in pGL3 basic with KSHV sequences (Fig. 1B). To investigate the influence of this difference, we cloned the region ϩ271 to Ϫ279 into the multiple cloning sites of pGL3 basic as shown in Fig. 1B. This yielded pDD753 retained the 5Ј-UTR of pGL3basic and differed from pGL3-3 by 291 nucleotides downstream of LANA promoter start site. As shown in Fig. 1D, transfection assays in HEK293 cells with pDD465 (pGL3-3) and pDD753 showed significant differences in promoter activity, demonstrating that sequences between ϩ11 and ϩ271 contribute to LANAp activity. Similar results were obtained in the KS-derived SLK, the B cell-derived BJAB and the PEL-derived BCBL-1 cell lines (data not shown).
Transposon-mediated Insertion Mutagenesis-To identify additional cis-acting elements in the LANAp region, we subjected our reporter plasmid pDD365 (ϩ271 to Ϫ279) to in vitro transposon mutagenesis using the EZ::TN in-frame linker insertion kit (Epicenter Technologies). Fig. 1D shows the relative reporter activity of each insertion mutant in HEK293 cells. Mutants pDD700 (ϩ270), pDD701 (ϩ249), pDD702 (ϩ245), pDD703 (ϩ241), pDD704 (ϩ241), pDD705 (ϩ237), pDD707 (ϩ200), pDD709 (ϩ198), pDD710 (ϩ179), and pDD711 (Ϫ44) show decreased promoter activities compared with the wildtype control, pDD83. In contrast, mutants pDD706 and pDD708 show promoter activities higher than the wild-type control, suggesting that the transposons did not disrupt an essential site. We also investigated whether any of these insertion mutants had lost the ability to be transactivated by LANA (data not shown). None of them did, suggesting that LANA's transactivation of LANAp uses other sites in the promoter. Based on our deletion analysis, we conclude that essential LANAp elements are located between ϩ11 and ϩ271 but not distal to Ϫ88 relative to the transcription start site. The lossof-function transposon mutant at Ϫ43 prompted us to investigate the core LANA promoter in detail.
Core Promoter Function Depends on TATA Box, DPE, and Sp1 Elements-To investigate specific transcriptional factors that would be important for core LANAp activity, we introduced targeted mutations into predicted cis-acting elements in the LANAp (pDD83). Fig. 2A shows a detailed sequence analysis and insertion mutagenesis. In general, eukaryotic promoters are considered either TATA-containing or TATA-less. In TATA-containing promoters, the TATA box is located ϳ30 nucleotides upstream of the transcription initiation site (50,51). The LANA promoter did not contain a TATA box consensus site 30 bp upstream of the latent LANA mRNA start site (indicated by ϩ1 in Fig. 2A) as previously determined (19). TATA-less promoters invariably contain a DPE located at about 30 nucleotides downstream of the transcription initiation site. The DPE consensus sequence is 5Ј-(A/G)G(A/T)(C/T)(G/A/C) (52). We identified such a sequence in the LANA promoter at position Ϫ32 (127,848) (i.e. within the required distance of the LANA start site at 127,880). Alternatively, Sarid et al. (14) identified additional, distal transcription initiator sites at Ϫ19 (127,899, indicated by a star in Fig. 2A) and Ϫ69 (127,949) relative to the start site at ϩ1. The transcription initiation site at Ϫ19 is located 34 bp downstream of a TATA consensus motif. Therefore, we investigated two hypotheses. (a) Is the DPE required for LANAp activity and/or (b) is the TATA element required for LANAp activity?
The activity of DPE-containing promoters is dramatically decreased if the exact spacing between the DPE and the transcription initiation site is altered (50). To examine the possibility that LANAp was dependent on the DPE, we introduced a 6-bp PstI linker insertion between the DPE and the transcription initiation site (pDD413). As shown in Fig. 2, B-E, this insertion mutation completely abrogated LANAp activity in every cell line tested. To determine the importance of the DPE directly, we introduced a targeted mutation (CG to AT) into the DPE element (pDD788). This point mutation also destroyed LANAp activity (Fig. 2, B-E), which demonstrates that LANAp activity was dependent on the DPE.
To examine the possibility that LANAp activity was dependent on the TATA box, we performed mutagenesis of the TATA box. Mutation (TA to GC) of the putative TATA box abolished LANAp activity (pDD784 in Fig. 2, B-E). tion (pDD461) between ϩ1 and the putative TATA box also abolished activity. A larger 57-bp transposon insertion (pDD711) between the start site and putative TATA box totally demolished LANAp activity, suggesting that the TATA box is also required for LANAp activity. These data strongly suggest that the activity of LANAp was simultaneously dependent on a TATA box as well as DPE.
Sequence analysis revealed two putative GC box/Sp1 binding sites at positions Ϫ29 and Ϫ46 in LANAp. To examine the contribution of these sites to LANAp activity, we introduced 2-bp substitution mutations into the sites. As shown in Fig. 2, B-E, the mutation introduced into the first GC box/Sp1 binding site (Sp1.B at Ϫ29 and Ϫ30) completely abrogated LANAp activity (pDD780), whereas two independent mutations in the second GC box/Sp1 binding site (Sp1.C at Ϫ46 and Ϫ47 or at Ϫ50 and Ϫ51) reduced the promoter activity by 50% (pDD782 and pDD783). These data demonstrate that the GC box/Sp1 site at Ϫ29 was essential for LANAp activity, whereas the GC box/Sp1 site at position Ϫ46 augmented promoter activity. In summary, we identified three cis-acting elements (TATA, DPE, and GC box/Sp1) within the core LANAp that were independently required for activity.
Autoregulation of the LANAp by LANA Protein-We previously reported that LANA transactivates its own promoter (47, 53) but had been unable to demonstrate full-length LANA binding to the LANAp ϩ271 to Ϫ279 fragment directly (29). This may have been due to technical difficulties of LANA-based electrophoretic mobility shift assay, but it left open the alternative possibility that LANA, like adenovirus E1A or EBV EBNA-2, regulated transcription via proteinprotein interactions and did not contact DNA directly (54,55). To map the domain of LANA that was required to activate LANAp, we transfected different deletion mutants of the LANA protein together with a reporter plasmid containing the LANAp (Fig. 3A). As depicted in Fig. 3B, two mutants, each containing different C-terminal deletions, both lost their ability to transactivate the LANAp. In contrast, mutants that contain central domain deletions but retain their intact Cterminal domain transactivated the LANAp similarly to wildtype LANA. These data suggest that the central domain of LANA is dispensable for the transactivation of its own promoter. We obtained essentially similar data using a second set of mutants (29) with larger deletions of the central domain (data not shown).
To confirm expression of these mutants, we performed Western blot analysis using polyclonal rabbit antiserum against LANA (Fig. 3C). All mutants were expressed upon transient transfection and migrated at the expected size. The wild type LANA in pDD105 contained shorter repeats in central domain FIG. 4. DNA binding activities for the essential GC box/Sp1 site in LANAp. Nuclear extracts were prepared from BCBL-1 cells. Gel shift assay with 32 P-labeled oligonucleotide containing a consensus Sp1 binding site (5Ј-CCCTTG-GTGGGGGCGGGGCCTAAGCTGCG-3Ј). Competition experiments were performed by the addition of increasing concentrations of unlabeled DNA oligonucleotides, as indicated above the gel, and gel shift assay with 32 P-labeled LANAp oligonucleotide containing the second putative Sp1-binding-site (5Ј-ATTGTCCCGGGCG-CCGCGTAG-3Ј). DNA-protein complexes are labeled with the letters A-G on the right. P, free probe. The first lane is probe only. with a total length of 1003 aa, whereas the other mutants were constructed from wild type LANA containing longer repeats with a total length of 1162 aa.
To confirm the functionality of the LANA mutants with regard to their known TR-specific DNA binding, we transfected the different LANA expression plasmids together with a reporter plasmid containing seven copies of the LANA binding site in the KSHV TR region downstream of a minimal SV40 promoter. Previously, Garber et al. (28) showed that purified LANA protein bound directly to the TR element and that in the context of this reporter construct, LANA binding resulted in strong transcriptional suppression of the adjacent heterologous SV40 promoter. As shown in Fig. 3D, plasmids expressing the intact C-terminal region of LANA showed the transcriptional suppression comparable with wild-type LANA, whereas the two C-terminal LANA mutants did not. This demonstrates that all LANA mutants were expressed, that all were capable of binding to the high affinity TR LANA binding site, and that the central domain was dispensable for LANA's transcriptional activation activity on LANAp.
LANA Protein Binds to an Essential GC Box/Sp1 Motif in Its Own Promoter-To investigate the binding of cellular transcription factors to the essential GC box/Sp1 binding site at Ϫ29 of the LANAp, we performed an electrophoretic mobility shift assay. Seven protein-DNA complexes (complexes a, b, c, d, e, and f) were observed in BCBL-1 cells using an oligonucleotide containing the predicted GC box/Sp1 binding site (Ϫ29) in LANAp (Fig. 4). Increasing amounts of the unlabeled probe diminished the complexes in a dose-dependent fashion. Since complexes a and b changed size upon competition, they probably represent multimeric complexes (Fig. 4, lanes 2-4). A 100fold molar excess of an unrelated sequence (NF-B) did not compete for binding (Fig. 4, lane 5). Competition experiments with increased amounts of an unlabeled Sp1 consensus oligonucleotide did not compete these complexes (Fig. 4, lanes 6 -8).
The addition of anti-LANA antiserum yielded a supershifted band (Fig. 4, lane 9), indicating that some of these complexes contained LANA. As a positive control, a Sp1 consensus oligonucleotide was incubated with BCBl-1 extract (Fig. 4, lanes  10 -19). Here, increasing amounts of the unlabeled Sp1 binding site diminished the complexes in a dose-dependent fashion (Fig. 4, lanes 15-17). Increasing amounts of unlabeled LANAp oligonucletide competed for some of the lower migrating complexes but not the main Sp1 complex (Fig. 4, lanes 11-13), consistent with the limited sequence similarity of both sites. A 100-fold molar excess of an unrelated sequence (NF-B) did not compete with any complex (Fig. 4, lane 14). The addition of anti-LANA antiserum did not change the banding pattern (Fig.  4, lane 18), but the addition of an anti-Sp1 specific antiserum did (Fig. 4, lane 19).
LANAp probe (Fig. 5A, lanes 5). This demonstrates that LANA protein can bind to an essential GC box/Sp1 binding site in its own promoter but not a consensus Sp1 site and that the predicted GC box/Sp1 in the LANAp does not bind purified human Sp1. Whether this site can bind other Sp1/Krueppel family members is currently under investigation.
To test the relative affinities of the three related sites LANAp (5Ј-ATTGTCCCGGGCGCCGCGTAG), TR (5Ј-CCCCA-TGCCCGGGCGGGAGG), and consensus Sp1 (5Ј-CCCTTGGT-GGGGGCGGGGCCTAAGCTGCG) directly, we performed cross-competition experiments in BCBl-1 extracts. Equal amounts of LANAp, NF-B, TR, and Sp1 probe were used to compete a labeled LANAp probe (Fig. 6A) or a TR probe (Fig.  6B) or a Sp1 probe (Fig. 6C). The LANAp site did not compete efficiently with the TR site (Fig. 6B), whereas the TR site competed efficiently with itself and with the LANAp site (Fig.  6A), suggesting that the TR site had a higher affinity for DNA binding activities in BCBL-1 sites than the LANAp. An unrelated probe (NF-B) did not compete with any of the three probes. An Sp1 consensus oligonucleotide did not compete with either the LANAp or TR sites (Fig. 6C), reemphasizing that in BCBL-1 cells under the binding conditions used here these sites were different.
KSHV ORF 50/Rta Inhibits LANAp-As an immediate early lytic gene product, KSHV ORF 50/Rta plays a critical role in the induction of the viral lytic promoters (56 -61). The ORF 50/Rta also transactivated the promoter of vGPCR/ORF 74, which partially overlaps with the LANA 5Ј-UTR (47,62). This close proximity suggested the hypothesis that ORF 50 was involved in regulating the LANAp. Since LANA (and LANA-2) are the only KSHV mRNAs that are not induced in response to TPA or ORF 50/Rta (15), 2 we hypothesized that perhaps ORF 50/Rta inhibited the LANAp directly. To test this hypothesis, we transfected an ORF 50/Rta expression vector or an empty expression vector together with reporter plasmids containing different deletion mutants of the LANAp into HEK293 cells. As shown in Fig. 7A, ORF 50/Rta expression reduced LANAp activity to as little as 12% of vector control. Repression by ORF 50/Rta did not map to any particular cis-regulatory region of the promoter, suggesting that ORF 50/Rta squelched the LANA promoter by competing for cellular factors. Fig. 7B confirmed ORF 50/Rta protein expression and functionality by testing its transactivation ability on the ORF 74/vGPCRp, as previously defined (47). DISCUSSION The results of side-by-side transient transfection studies comparing the deletion clones from Sarid et al. (14) with those from Jeong et al. (47) demonstrated that important regulatory elements in LANAp are present downstream of the mRNA start site (Fig. 1A). The comparison of isogenic reporter plasmids differing only by the presence or absence of 260 bp within the LANA 5Ј-UTR revealed significant differences in promoter activity. This implied that sequences between ϩ11 and ϩ271 contribute to LANAp activity (Fig. 1D). We found at least one essential promoter element, the DPE, to be located in this region.
Generally eukaryotic promoters are composed of DNA sequences that direct accurate transcriptional initiation by the RNA polymerase II complex (63,64). One or all of these sequence motifs can be recognized in most eukaryotic promoters: the TATA box, the initiator element (INR), and the DPE. In TATA-dependent promoters, both the TATA box, typically located about 30 bp upstream of the transcriptional initiation site and bound by the TATA-binding protein subunit of TFIID, and the INR are used to accurately position the basal transcription machinery, leading to a single defined mRNA initiation site (50,65). In TATA-less promoters, the DPE in conjunction with the INR is used for the same purpose (50,65). The DPE is typically located 28 -32 bp downstream of the transcriptional  Fig. 1) containing LANAp region between Ϫ20 and Ϫ279. B, luciferase activity in HEK293 cells after cotransfection with an ORF 50 expression vector or an empty expression vector together with a reporter plasmid containing vGPCR promoter (pDD163). vGPCR, viral G protein-coupled receptors. Bars represent the mean relative luciferase activity after 48 h of triplicate experiments. The -fold promoter activity with the expression of ORF 50 relative to the promoter activity without ORF 50 expression is indicated (A and B). initiation site, and the precise spacing between DPE and the INR is critical for the cooperative binding of holo-TFIID to a TATA-less core promoter (50,63). Consistent with this notion, it has been shown that TAF II 60 and TAF II 40 of TFIID interact with the DPE to stabilize the transcription initiation complex (50). So far, all known DPE-containing promoters are TATAless, and all TATA-containing promoters are DPE-less, although most experimental data are derived from Drosophila promoters. The INRs encompassing the transcription initiation site can be found in TATA-dependent and TATA-independent promoters, and TFIID has been found to contact the INR through its TAF II 150 and TAF II 250 subunits (66).
The LANAp is different in its core architecture. LANAp does not contain a recognizable INR, which is consistent with the multiple start sites that we and others previously described (19,46). We found that by mutational analysis, both DPE and TATA box elements were independently required for LANAp function (Fig. 2). This arrangement is rarely found in eukaryotic promoters and is in contrast to the latent EBV EBNA Qp promoter (67,68), which is TATA-independent. By sequence analysis, only 14% of eukaryotic promoters contain both a DPE and TATA box, whereas ϳ31% contain neither and 55% contain one or the other (65). Understanding LANAp, in addition to understanding KSHV latency, will yield general insights into the mechanism and significance of this unique group of promoters for which no other experimental model currently exists.
We propose the arrangement shown in Fig. 8 to accommodate the results of our mutational analyses. Point mutants in the TATA, the DPE, or GC box/Sp1 sites independently abolished promoter activity. In the case of LANAp, the TATA box and DPE alone or in conjunction is not sufficient to stabilize the basal transcription machinery because of the aberrant spacing. In the LANAp, the distance between the DPE and TATA box is ϳ90 bp (as opposed to the 30-bp distance between TATA and INR or DPE and INR that has been demonstrated for other promoters). We surmise that this would prevent appropriate interactions between these cis-acting elements and their cognate DNA binding subunits in TFIID and that a third DNA contact, in our case through a GC box/Sp1 site, is required to ensure proper spacing and transcription initiation. Since we have been unable to demonstrate direct binding of Sp1 (Fig. 5) and Sp3 (data not shown) to LANAp, we surmise that other Sp1/Krueppel family members supply this function and that in BCBL-1 cells, LANA itself is part of this complex (Fig. 4).
We found that LANA transactivated LANAp through aa 751-1161, which contains the putative leucine zipper and DNA binding region. By contrast, the central region containing the repeats was not required for LANAp transactivation (Fig. 4). The requirements for autoactivation of LANAp differ from the requirements for activation of the EBV LMP-1 promoter, since Groves et al. (69) showed that a LANA mutant containing amino acids 267-971 but lacking the C-terminal region was able to activate this promoter (69). At this point, we do not know which domains within the C-terminal DNA binding domain are required for LANAp transactivation. We cannot exclude the possibility that more than one domain of LANA may be needed for transactivation, since, for instance, Lim et al. (70) previously demonstrated that the N-terminal 90 amino acids were required for the C-terminal region-mediated DNA binding and dimerization function of LANA (70). In addition, amino acids 1129 -1143 were shown to be required for the transactivation of the EBV Cp by LANA (71). Clearly, there are multiple mechanisms through which LANA can regulate target promoters, and these involve multiple regions of the LANA protein.
Since KSHV latent persistence is dependent on LANA protein and LANA protein positively regulates its own expression, studies of this feedback circuitry such as presented here may identify novel targets for antiviral drugs, with the potential to clear latent infection.