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Journal of Virology, July 2005, p. 8493-8505, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8493-8505.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Activation of the Kaposi's Sarcoma-Associated Herpesvirus Major Latency Locus by the Lytic Switch Protein RTA (ORF50)

Satoko Matsumura, Yuriko Fujita, Evan Gomez, Naoko Tanese, and Angus C. Wilson*

Department of Microbiology and NYU Cancer Institute, New York University School of Medicine, New York, New York 10016

Received 23 November 2004/ Accepted 2 March 2005


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ABSTRACT
 
Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma cells but can be induced to enter full lytic replication by exposure to a variety of chemical inducing agents or by expression of the KSHV-encoded replication and transcription activator (RTA) protein. During latency, only a few viral genes are expressed, and these include the three genes of the so-called latency transcript (LT) cluster: v-FLIP (open reading frame 71 [ORF71]), v-cyclin (ORF72), and latency-associated nuclear antigen (ORF73). During latency, all three open reading frames are transcribed from a common promoter as part of a multicistronic mRNA. Subsequent alternative mRNA splicing and internal ribosome entry allows for the expression of each protein. Here, we show that transcription of LT cassette mRNA can be induced by RTA through the activation of a second promoter (LTi) immediately downstream of the constitutively active promoter (LTc). We identified a minimal cis-regulatory region, which overlaps with the promoter for the bicistronic K14/v-GPCR delayed early gene that is transcribed in the opposite direction. In addition to a TATA box at –30 relative to the LTi mRNA start sites, we identified three separate RTA response elements that are also utilized by the K14/v-GPCR promoter. Interestingly, LTi is unresponsive to sodium butyrate, a potent inducer of lytic replication. This suggests there is a previously unrecognized class of RTA-responsive promoters that respond to direct, but not indirect, induction of RTA. These studies highlight the fact that induction method can influence the precise program of viral gene expression during early events in reactivation and also suggest a mechanism by which RTA contributes to establishment of latency during de novo infections.


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INTRODUCTION
 
Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) is a gamma-2 herpesvirus associated with three human malignancies: Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (reviewed in references 10, 23, 44, and 68). Like all herpesviruses, KSHV exploits contrasting lytic and latent modes of replication. During latency, the viral genomes are maintained in the nucleus as minichromosomes, and only a very limited number of viral genes are expressed. Several latency-associated proteins have growth-promoting functions likely to contribute to host cell immortalization and malignancy (13, 14, 46, 49, 67, 73). In response to poorly understood environmental cues, latent viral genomes may switch from latency into lytic or productive replication, a process known as reactivation. The lytic cycle involves a complex program of gene expression leading to massive amplification of viral DNA, production of infectious viral particles, and ultimately death of the host cell. In advanced KS lesions or PEL, the majority of abnormal cells harbor latent KSHV with a much smaller fraction of cells actively engaged in lytic replication. It is clear, however, that both modes of replication are essential for long-term persistence of the virus and that proteins expressed by both programs contribute to the pathogenesis of KSHV-associated disease (5, 21, 23).

In PEL lines, lytic reactivation can be induced by treatment of the cells with a variety of chemical compounds, most notably phorbol esters such as 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and deacetylase inhibitors such as sodium butyrate (42, 43, 51, 56, 76). TPA is thought to act by promoting the expression of viral open reading frame 50 (ORF50) through activation of the cellular transcription factor AP1, an end point of the protein kinase C signaling pathway (35, 70). In a similar vein, deacetylase inhibitors may overcome repressive chromatin structures on the ORF50 promoter (76). ORF50 encodes the regulator of transcriptional activation (RTA), and expression of this virus-encoded transcription factor alone is sufficient to induce reactivation (17, 37, 38, 63). RTA directly transactivates a battery of KSHV genes associated with lytic replication or cytokine production, as well as its own promoter (4, 7, 11, 47, 55, 60, 62). In addition to driving the program of lytic gene expression, RTA is required for the initiation of lytic DNA replication (2, 72). Promoters that respond to RTA do so through direct DNA binding or indirect association with cellular transcription factors, including CCAAT/enhancer-binding protein alpha (C/EBP{alpha}), CSL (CBF1/RBP-J{kappa}), and Oct-1 (reviewed in reference 74).

Mechanisms underlying the establishment and maintenance of KSHV latency remain elusive. There is evidence that latency is established relatively inefficiently following de novo infection but that once in place, the latent program is remarkably stable (15, 18). Three viral proteins are consistently expressed in latently infected cells: an antiapoptotic FLICE inhibitor (v-FLIP), a viral D-type cyclin (v-cyclin), and the multifunctional latency-associated nuclear antigens (LANA, LANA1, and LNA-1). These are encoded by ORF71, ORF72, and ORF73, respectively, and together the three genes form a multicistronic transcriptional unit, known as the latency transcript (LT) cluster or cassette (8, 9, 57, 65). During latency, the LT cluster is transcribed from a constitutively active promoter, giving rise to ~5.8- or ~5.4-kb mRNAs containing ORF71, -72, and -73 and a ~1.7-kb transcript that is generated by mRNA splicing and contains only ORF71 and -72 (see Fig. 2A for additional details). The ~5.8 and ~5.4-kb transcripts differ by the presence or absence of a short (337-nucleotide) intron in the 5' untranslated leader between ORF73 and the promoter. Last, an additional but infrequent splice can give rise to a 1.1-kb mRNA that contains only ORF71. Of low abundance compared to the ~1.7-kb mRNA, this transcript can be detected in RNA from PEL lines (BCBL-1 and BC3) and enriched by treatment with TPA (19). It is likely that LANA is principal translation product of the tricistronic mRNAs, whereas v-cyclin and v-FLIP are predominantly synthesized from the bicistronic transcript, making use of an efficient internal ribosome entry site within ORF72 (19, 34). The LT promoter (LANAp or LTc) is constitutively active in many tissue culture cells but shows tissue-restricted activity in transgenic mice where it is presented in the context of chromatin. Tissue-specific differences in promoter activity may contribute to the cell type tropism of KSHV latency (26). In addition, LANAp is stimulated by the LANA protein, creating a positive feedback loop that may help maintain latency in rapidly dividing cells (25, 27, 50).



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  		FIG. 2. LT transcripts are induced by RTA expression. (A) Arrangement of known transcripts encoding the three genes of the LT cluster and neighboring open reading frames. In latently infected PEL cells, transcription from a constitutively active promoter (LTc) gives rise to a ~5.8-kb precursor RNA that undergoes alternative splicing to produce ~5.3-kb tricistronic (ORF71, -72, and -73) and ~1.7-kb dicistronic (ORF71 and -72) transcripts (9, 56, 65). A less abundant 1.1-kb monocistronic (ORF71) transcript (not shown) has also been detected in PEL lines (19). Open reading frame K12 (T0.7) encoding the kaposins is actively transcribed during latency from both proximal and upstream promoters and is induced further during lytic replication (31, 54). The bicistronic mRNA encoding K14 (v-Ox) and v-GPCR (ORF74) is essentially silent during latency but strongly induced by RTA (28, 65). (B) Autoradiograms from Northern blot analyses of poly(A)+-selected RNA isolated from TRExBCBL1-Rta cells either mock treated (lane 1) or Dox treated (lane 2) for 36 h. The probes used (indicated by solid bars in panel A) correspond to the coding region of ORF71 (top) and K14 (middle). As a loading control, the blot was reprobed with a GAPDH-specific probe (bottom). Positions of size markers (in kilobases) are shown on the left. Filled arrowheads indicate the major LT mRNAs at 5.3 and 1.7 kb and major K14/v-GPCR mRNA at 2.5 kb. An induced transcript at ~1.4 kb is indicated by an open arrowhead.

Here, we show that LT mRNAs can be induced in latently infected BCBL1 PEL cells by forced expression of RTA. Inducible transcripts initiate from a novel promoter (termed LTi) located ~270 bp downstream of the constitutive promoter. Activation of LTi requires RTA and is independent of other viral proteins. Mapping of functional elements within LTi showed that there is extensive overlap with an inducible promoter directing expression of a bicistronic mRNA transcribed in the opposite direction that encodes the signaling proteins K14 (v-Ox) and ORF74 (v-GPCR). Both promoters make use of three RTA-responsive elements, two of which correspond to binding sites for the transcription factor CSL (CBF1/RBP-J{kappa}), an endpoint of the Notch-signaling pathway. Lastly, we show that in latently infected cells, LTi cannot be induced by sodium butyrate, a commonly used inducer of KSHV reactivation. In fact, butyrate suppressed activation mediated by forced expression of RTA and shows that choice of induction cue (direct versus indirect induction of RTA) can significantly affect the profile of gene expression. It also provides evidence that the ability of RTA to activate specific promoters can be modulated by environmental conditions and this has implications for understanding KSHV pathogenesis.


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MATERIALS AND METHODS
 
Plasmids. To construct the luciferase reporter pLT1-luc, a 1.77-kb fragment (GenBank accession no. U75698; nucleotides 127607 and 129376) spanning the divergent LT and K14/v-GPCR promoters was amplified by high-fidelity PCR (Expand High Fidelity PCR system; Roche) from a bacteriophage lambda subclone of KSHV genomic DNA and subcloned between the HindIII and XhoI sites of a modified version of the promoterless vector pGL3basic-luc. Truncations pLT3-luc and pLT4-luc were generated by limited exonuclease III digestion from the introduced XhoI site. Truncations pLT5-luc, pLT6-luc, pLT7-luc, pLT8-luc, and pLT9-luc were generated by PCR amplification using specific oligonucleotide primers. A luciferase reporter carrying the ORF50/RTA promoter was constructed by PCR amplification of KSHV genomic DNA (nucleotides 68593 to 71593). Luciferase reporter pPAN (–102/+15) luc (pSEW-PP1) was a kind gift of Gary Hayward (Johns Hopkins University School of Medicine).

A cytomegalovirus enhancer-driven expression plasmid encoding LANA (pCGT-LANANC) has been described previously (59). This derivative omits the central repetitive region that is present in the full-length protein, resulting in increased protein expression and more effective transactivation (75). To construct pCGFlag-RTA, a 3.03-kb KSHV genomic fragment (nucleotides 71599 to 74629) encoding full-length RTA was generated by high-fidelity PCR amplification (Expand High Fidelity PCR System; Roche) from BC3 cell DNA using specific oligonucleotide primers that added unique XbaI and BglII restriction sites and subcloned into pCGFlag. Site-directed mutagenesis was performed using the QuikChange Mutagenesis protocol (Stratagene) or by PCR amplification, and ligation was carried out via an added restriction site. Mutations (substitutions are shown in lowercase letters, and deleted bases are shown in square brackets) were as follows: m1, 5'-GGGAGTAGCGatatCCCACTTGTT-3' (EcoRV); m2, 5'-CTTGTTTCGGaattCCC[G]TAAGGC-3' (EcoRI); m3, 5'-CTTATCTTTGgccaGCT[A]TAAGAT-3' (MscI); m4, 5'-ATAAG[A]TGTGaattcAATAGTAATA –3' (EcoRI); m5, 5'-ATACCAGGTGgaattcATTTGTGTTA-3' (EcoRI); m6, 5'-TGTACATGATgaaTcTAAGGTGTGT-3' (EcoRI); m7, 5'-TATTGGCCGTactaGTTTCTCACG-3' (SpeI); m8, 5'-CGTTTCTGTTgaattCGCCCGGATT-3' (EcoRI); and m9, 5'-CGCCCGGATTggccATCTGGACTT-3' (MscI). A diagnostic restriction site introduced by the mutation is given in parentheses. Sequences of all truncation and substitution mutants were confirmed by DNA sequencing.

Cell culture. KSHV+/EBV– BCBL-1 cells carrying a stably integrated tetracycline-inducible RTA expression vector (TRExBCBL1-Rta) (47) and the precursor (TRExBCBL1) lacking the RTA/ORF50 cDNA were generously provided by John Souvlis and Jae Jung (Harvard Medical School). Both cell lines were maintained in RPMI 1640 medium (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 20% Neugem serum (Gemini Bio-Products, California), 2 mM L-glutamine, and antibiotics. TRExBCBL1-Rta cells were given 100 µg/ml hygromycin B (Invitrogen) to maintain selection of the ORF50 cDNA. Expression of recombinant RTA was induced by addition of 1 µg/ml doxycycline (Dox; BD Biosciences) dissolved in sterile water to the medium. Sodium butyrate (NaB; Sigma) was solubilized in water and added to the culture medium at a final concentration of 3 mM. BC3 cells (a gift of Ornella Flore, New York University School of Medicine) were cultured in the same medium except that the hygromycin was omitted. HeLa cells were maintained in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 2 mM L-glutamine and antibiotics. BCBL1 and BC3 cells were transfected using Lipofectamine 2000 (Invitrogen). Transfection efficiency was determined by transfection of a LacZ expression vector, followed by X-Gal (5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside) staining of fixed cells. Electroporation of HeLa cells (transfection efficiency, ~21%) and luciferase assays were performed as described previously (36).

RNA isolation and Northern blotting. Total RNA was prepared from TRExBCBL1-Rta cells using ULTRASPEC total RNA isolation reagent (Biotect Laboratories, Texas) according to the manufacturer's instructions and resuspended in RNase-free water. Poly(A)+ RNA was purified from total RNA using an Oligotex mRNA purification Kit (QIAGEN). For Northern blot analysis, 0.5 µg of poly(A)+ RNA was resolved on a 1% agarose-18% formaldehyde gel and transferred to a GeneScreen Plus hybridization membrane (NEN Life Science Products) for 12 to 16 h in 10x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). The blot was then UV cross-linked and hybridized with the ORF71/v-FLIP probe in PerfectHyb Plus hybridization buffer (Sigma) at 68°C. After overnight hybridization, the blot was washed with 0.1x SSC-0.1% sodium dodecyl sulfate (SDS) and subsequently exposed to Kodak BioMax MR film. Hybridization probes were radioactively labeled using [32P]dCTP by random priming (Rediprime II; Amersham). The probe fragments used were as follows: v-FLIP (567 bp; KSHV nucleotides 122145 to 122711), K14 (1,165 bp; KSHV nucleotides 128047 to 129211), and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 452 bp) (16). Quantitative analysis of the blots was performed using a STORM 820 PhosphorImager (Molecular Dynamics), and the data were analyzed with ImageQuant software.

Primer extension analysis. Primer LUCPE1 (5'-GCGGATAGAATGGCGCCGGGCC-3') was end labeled with [{gamma}-32P]dATP using T4 polynucleotide kinase (New England Biolabs). For each reaction, 30 µg of total RNA was mixed with 1 ng of 32P-labeled oligonucleotide primer, incubated at 58°C for 20 min, and then slowly cooled to room temperature. Hybridization products were extended with AMV reverse transcriptase (Primer Extension system; Promega). Extension was carried out at 41 to 42°C for 30 min and stopped by ethanol precipitation. Samples were resuspended in loading dye, resolved on an 8% acrylamide-7 M urea denaturing gel, and exposed to Kodak BioMax MR film.

5' RLM-RACE. Poly(A)+ RNA (250 ng) from Dox-treated TRExBCBL1-Rta cells was used as a template for 5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE), according to the manufacturer's protocol (Ambion). After decapping and ligation of the RNA adaptor, cDNA was prepared by random primed reverse transcription (42°C; 1 h), and specific sequences were amplified by nested PCR using oligonucleotide ORF73RACE4 (5'-CCTACAACTTCCTCTCGTTAAGGG-3'; nucleotides 127226 to 127249), followed by oligonucleotide ORF73RACE3 (5'-CAGGCGCATTCCCGGGGGCGCCAT-3'; nucleotides 127274 to 127297), complementary to the beginning of ORF73. Amplification products were subcloned into pGEM-TEasy (Promega) and characterized by DNA sequencing.

Immunoblot analysis. Cell lysed in 2% SDS sample buffer, resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and wet transferred to a nitrocellulose membrane for immunoblotting. Membranes were blocked in 10% nonfat milk before incubation with a polyclonal antibody against RTA (diluted 1:1,000; a kind gift of Gary Hayward, Johns Hopkins University School of Medicine) or monoclonal antibody against the c-myc epitope (clone 9E10, diluted 1:1,000; AbCam). After 60 min at room temperature, blots were washed extensively before incubation with horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G secondary antibodies (Amersham). Bound antibody complexes were detected by enhanced chemiluminescence (SuperSignal, Pierce). For quantitation, chemiluminescence was detected with a ChemiDoc XRS Imaging System (Bio-Rad).


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RESULTS
 
Induction of the LT promoter in TRExBCBL1-Rta cells. Until recently, studies of early events in KSHV lytic reactivation have been hampered by the fact that only a minority of latently infected cells in a culture can be induced to reactivate. To overcome this limitation, Jung and colleagues engineered a KSHV-infected primary effusion lymphoma cell line BCBL1 in which a regulated copy of lytic switch protein RTA was stably integrated into the host genome (47). Treatment of the modified cells (termed TRExBCBL1-Rta) with tetracycline or the more stable Dox results in robust expression of the recombinant RTA protein in almost every cell, leading to viral lytic replication in a large fraction of the culture. We thus chose this cell line as a model system to study the behavior of RTA-responsive promoters during early steps in reactivation. TRExBCBL1-Rta cells were transiently transfected with luciferase reporter constructs derived from the early lytic PAN/Nut-1 promoter (pPAN-luc) and the ORF50 promoter (pORF50-luc). As shown in Fig. 1A, the promoters were induced 16.9 and 8.4 fold, respectively, by the addition of 1 µg/ml Dox to the culture medium. Induction of these well-studied lytic promoters confirms the regulated expression of functional RTA in these cells. As controls, we also tested two promoters that were not known to be responsive to RTA. The cellular bFGF-2 promoter (pFGF-2-luc) displayed a low level of constitutive activity and this remained essentially unchanged by the addition of Dox. We also tested a 1.77-kb fragment (nucleotides 127609 to 129375 of the prototype BC-1 KSHV sequence) (53) encompassing the promoter for KSHV ORF71 to -73 latency transcripts (pLT1-luc) (see Fig. 3A). Unexpectedly, this construct was induced by >20 fold with Dox treatment. This was an unexpected result in light of previous studies showing that LT-derived transcripts were relatively unresponsive to stimuli that induce lytic reactivation (9, 12, 48, 64, 65) and thus warranted further investigation.



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  		FIG. 1. The LT promoter can be induced by RTA. (A) A total of 107 TRExBCBL1-Rta cells were transiently transfected using Lipofectamine 2000 with 2 µg of the following luciferase reporter constructs: promoterless vector, pGL3basic-luc; LT promoter, pLT1-luc; PAN promoter, pPAN(–102/+15); ORF50 promoter, pORF50-luc; and human bFGF-2 promoter, pFGF-2-luc. After 24 h, the transfected cells were split into two flasks, one of which was mock treated with sterile water and the other treated with Dox (1 µg/ml). After a further 18 h, cell extracts were prepared and assayed for luciferase activity. Bars represent the mean and standard deviation of three independent transfections. (B) TRExBCBL1 cells were cotransfected with 0.5 µg pPAN(–102/+15) or pLT4-luc (see Fig. 3 for details) and either mock or Dox treated. In addition, parallel cultures we cotransfected with 1.5 µg of an expression vector encoding full-length KSHV RTA (pCGFlag-RTA). (C) The reporter constructs (0.5 µg) used in the results shown in panel A were electroporated into 106 HeLa cells, together with 1 µg of empty expression vector or pCGFlag-RTA. Luciferase activity was measured 24 h after transfection. (D) Comparison of RTA expression in mock-treated (lane 1) or Dox-treated (lane 2) TRExBCBL1-Rta cells and HeLa cells transfected with 2 µg empty expression vector (lane 3) or pCGFlag-RTA (lane 4). Total cell lysates (5 x 104 cell equivalents per lane) were resolved on 15% SDS-PAGE, transferred to a nitrocellulose membrane, and probed with a polyclonal antibody against RTA (71).



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  		FIG. 3. Separation of the constitutive (LTc) and inducible (LTi) promoters. (A) Schematic showing the organization of the three open reading frames (ORF71, -72, and -73) encoded by the LT mRNAs and two open reading frames (K14 and ORF74) transcribed in the opposite direction as a bicistronic mRNA (9, 56, 57, 65). Reporter construct pLT1-luc contains a 1.8-kb fragment of KSHV genomic DNA spanning nucleotides 127609 to 129375 (nucleotide positions refer to sequences for GenBank accession number KSU75698) (53), orientated such that the luciferase gene is placed downstream of the LT promoter. Note that this fragment contains the entire K14 ORF (open arrow). Boundaries of further truncations within this fragment are shown: LT3 (nucleotides 127609 to 128282), LT4 (nucleotides 127609 to 127807), LT5 (nucleotides 127609 to 127992), LT6 (nucleotides 127816 to 129375), LT7 (nucleotides 127816 to 127992), LT8 (nucleotides 127764 to 127992), and LT9 (nucleotides 127609 to 127948). Note: LT2 of this series is only slightly shorter than LT1 and was omitted from the analysis. (B) HeLa cells were transiently transfected with a promoterless reporter (pGL3basic-luc) or plasmids pLT1-luc through pLT9-luc together with an empty expression vector (open bars) or a vector encoding full-length RTA (pCGFlag-RTA) (shaded bars). Luciferase activity was measured after 24 h and displayed as a histogram showing the mean and standard deviation of three independent assays. (C) As in panel B, except that each reporter was cotransfected with either empty vector (open bars) or a vector encoding LANANC (pCGT-LANANC) (shaded bars).

Dox treatment of TRExBCBL1-Rta cells initiates a cascade of KSHV lytic gene expression, which includes expression of a number of virus-encoded regulatory proteins including transcription factors. We next asked whether the Dox-mediated stimulation was a direct consequence of RTA by testing the response of PAN and LT reporters in TRExBCBL1 cells (Fig. 1B). These cells are identical to TRExBCBL1-Rta, except they lack the cDNA encoding recombinant RTA (47). Neither reporter was induced by treatment of Dox, indicating that expression of the recombinant RTA was essential for induction. As a control, we cotransfected a constitutive mammalian expression plasmid encoding full-length RTA (pCGFlag-RTA) and observed a robust activation of both reporters (PAN, 33.7 fold; LT, 15.3 fold). Thus, the activity of both promoters is dependent on expression of the lytic switch protein RTA.

To rule out the involvement of other viral gene products, we examined induction of the LT1-luc reporter in KSHV-negative HeLa cells (Fig. 1C). Each reporter was cotransfected with an empty expression vector or one encoding RTA (pCGFlag-RTA). The LT promoter was strongly induced 11.9 fold by expression of RTA, exceeding that of the ORF50 promoter (3.9 fold). Induction of the PAN promoter was greater (59 fold), due in part to its lower initial activity. From these results, we conclude that activation of the pLT1-luc in TRExBCBL1-Rta cells was most likely a direct consequence of RTA expression and does not require other virus-encoded products.

The pLT1-luc construct was induced to a similar extent in both TRExBCBL1-Rta cells (20.8 fold) (Fig. 1A) and HeLa cells (11.9 fold) (Fig. 1C). We compared the expression of RTA in these two contexts using a polyclonal antibody against RTA to immunoblotting total cell lysates prepared from equal numbers of cells (Fig. 1D). Consistent with previous reports, treatment of TRExBCBL1 cells with Dox induced very high levels of RTA (Fig. 1D, compare lanes 1 and 2). RTA levels were significantly lower in HeLa cells transfected with pCGFlag-RTA (lane 4), although this was due in part to the fact that only 50 to 60% of cells in the culture were transfected as judged by LacZ staining. This result indicates that pLT1-luc does not require extremely high levels of RTA for activation.

LT mRNAs are induced by RTA. The organization of known transcripts in the region of the LT cluster is shown in Fig. 2A. To determine whether the responsiveness of the LT promoter to RTA was limited to the context of a reporter plasmid, we prepared poly(A)+ selected RNA from either mock- or Dox-treated TRExBCBL1-Rta cells and detected specific mRNAs by Northern blot analysis (Fig. 2B). Using a probe spanning ORF71 (v-FLIP), we detected two major transcripts estimated at ~5.3 and ~1.7 kb (Fig. 2B), most likely the principal mRNA species (Fig. 2A) generated by alternative splicing of the primary LT precursor (9, 57, 65). The relative abundance of both transcripts was significantly increased when cells were treated with Dox. Quantitation by phosphorimager analysis indicated a 7.0- and 7.7-fold increase for the 5.3- and 1.7-kb transcripts, respectively. The larger 5.8-kb transcript was not detected in either RNA preparation and may indicate that the majority of LT transcripts are spliced in these cells. We also noticed the appearance of an additional band at ~1.4 kb in the presence of Dox, suggestive of different promoter usage or a change in splice site/termination site usage. As a positive control for lytic reactivation, we reprobed the blot with a fragment spanning the coding sequence of K14, a known RTA-responsive gene transcribed in the opposite direction from the LT cluster (6, 28, 65). The probe was designed to avoid any overlap with the beginning of the previously LT transcript and, as expected, detected a ~2.5-kb mRNA present only in mRNA from Dox-treated cells. Last, the blot was reprobed for cellular GAPDH (Fig. 2B, bottom) to confirm that equal amounts of RNA were loaded. Thus, forced expression of RTA protein in TRExBCBL1-Rta cells leads to increased steady-state levels of long and short forms of LT mRNA.

Identification of separate constitutive and RTA-responsive promoters. Having shown that RTA can induce synthesis of LT mRNAs from the natural context of the latent viral genome, we next sought to narrow down the promoter sequences responsible for this response. The divergent promoter region between ORF73 and K14 is shown schematically in Fig. 3A. Using pLT1-luc as a starting point, we prepared a series of truncations, removing sequences both upstream and downstream of the previously characterized LT initiation sites (labeled LTc) centered on the major initiation site at nucleotide 127880. Each reporter construct was transfected into HeLa cells, together with an empty expression vector or one encoding RTA (Fig. 3B). Truncation to –113 (pLT5-luc; 20.4-fold induction) and –69 (pLT9-luc; 32-fold induction) with respect to the main LT mRNA start site (nucleotide 127880) retained the full response to RTA. Unexpectedly, further truncation beyond the major start sites to + 74 (pLT4-luc; 117 fold) resulted in a more substantial response to RTA and at the same essentially abolished the constitutive (uninduced) activity (22% of pLT1-luc). Additional truncations that removed sequences between +272 and +117 (pLT8-luc) or +272 and +65 (pLT6-luc and pLT7-luc) abolished all responsiveness to RTA. A similar mapping profile was observed with TRExBCBL1-Rta cells in the presence or absence of Dox (data not shown). Thus, sequences both upstream and including the major initiation sites for LT mRNA (nucleotide 127880 with additional starts at 127900 and 127948) (9, 56) were entirely dispensable for responsiveness to RTA, and the minimal responsive promoter (pLT4-luc) mapped to the area between nucleotides 127609 and 127807. For simplicity, we will refer to the previously described constitutive promoter as LTc and the RTA-inducible promoter as LTi.

It has been shown previously that the constitutively active LT promoter can be stimulated by expression of LANA (25, 27, 50). In our hands, cotransfection of an expression plasmid encoding LANA (LANANC) gave an approximately twofold increase in HeLa cells (Fig. 3C). The truncated promoters responded to LANA to various extents, with the one exception being pLT4-luc (+272 to +74). This fragment showed the lowest constitutive activity and was not activated by LANA (Fig. 3C), yet was fully responsive to RTA (Fig. 3B). These results show that the RTA-inducible LTi promoter is distinct and separable from the LANA-responsive constitutive promoter LTc.

Mapping the transcriptional start site of the inducible LTi promoter. To determine where the RTA-responsive transcripts initiate, we performed primer extension analysis of poly(A)+ RNA isolated from HeLa cells transfected with either pLT1-luc or pLT4-luc in the presence or absence of the RTA expression plasmid (Fig. 4A). Using a 32P-labeled oligonucleotide primer (LUCPE1) complementary to the 5' end of the luciferase gene, we detected two extension products, 82 and 84 bp in length, in cells transfected with pLT1-luc (Fig. 4A, lane 8) and pLT4-luc (lane 10). We believe these extension products correspond to RTA-induced transcripts initiating from LTi because they were not detected in the absence of the RTA expression vector (lanes 7 and 9) or in cells transfected with a reporter plasmid lacking a functional promoter, even in the presence of RTA (lanes 5 and 6). The putative initiation sites mapped to a HindIII site (5'-AAGCTT-3') added by PCR to the end of the KSHV genomic DNA fragment to facilitate subcloning (Fig. 4B). Naturally, this was of some concern; however, we were encouraged by the fact that the putative initiation sites were positioned 33- and 35-bp downstream of an AT-rich sequence (5'-TATATA-3') resembling a consensus TATA box (5'-TAT[A/T][A/T][A/T]-3').



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  		FIG. 4. Structure of the LTi promoter. (A) Mapping of the 5' end of the RTA-inducible transcript using primer extension with 32P-labeled LUCPE1 oligonucleotide annealed to poly(A)+ RNA isolated from transiently transfected HeLa cells. HeLa cells were transfected with promoterless reporter pGL3basic-luc (lanes 5 to 6), pLT1-luc (lanes 7 to 8), and pLT4-luc (lanes 9 to 10) and pCGFlag-RTA where indicated (lanes 6, 8, and 10). To accurately size the extension products, a sequencing ladder generated with the same primer and pLT4-luc plasmid DNA was run along side (lanes 1 to 4). Locations of primers used for primer extension (LUCPE1) or 5'RLM-RACE (RACE3 and RACE4) are shown schematically. Sizes of the principal extension products (dotted line) are given. (B) DNA sequences surrounding the transcriptional start sites of pLT1-luc/pLT4-luc (top) or KHSV genome (bottom). Transcriptional start sites detected by primer extension in transfected HeLa cells or by 5' RLM-RACE from Dox-treated TRExBCBL1-Rta cells are shown, and a TATA box 30 to 35 bp upstream of the initiation sites is highlighted.

To characterize transcripts arising from the LTc and LTi promoters in their natural context of the viral episome, we performed 5' RLM-RACE using nested primers complementary to the coding sequence of ORF73 (nucleotides 127226 to 127297). Briefly, poly(A)+ RNA was isolated from TRExBCBL1-Rta cells that had been treated with Dox for 18 h and dephosphorlyated to remove free 5' phosphates from fragmented or uncapped RNAs and thus prevent subsequent amplification of these RNAs. Next, the mRNA cap structures were removed using tobacco acid phosphatase, revealing a 5' monophosphate necessary for addition of an RNA oligonucleotide adaptor with T4 RNA ligase. Once attached, the pool of tagged RNAs was reverse transcribed using random primers, and the 5' ends of LT-derived cDNAs were PCR amplified using specific primers corresponding to the 5' end of ORF73 and the ligated adaptor. Amplification products were gel purified, subcloned into a plasmid, and characterized by DNA sequencing. Of seven clones analyzed, two were derived from an unspliced RNA that had initiated at either nucleotide 127610 or 127611 (Fig. 4B). These positions were 2 to 4 nucleotides upstream from the initiation sites of the reporter constructs mapped by primer extension in HeLa cells and are 29 to 30 nucleotides downstream of the putative TATA box, a typical distance in mammalian cells (Fig. 4B). Three additional clones extended further upstream, reaching nucleotides 127883 (two clones) and 127888, and all were derived from RNAs that had been spliced (deletion of nucleotides 127556 to 127815). The endpoints of these longer transcripts fell within the cluster of LTc start sites described previously (9, 57). Lastly, we identified two additional sequences corresponding to shorter unspliced products terminating at nucleotides 127548 and 127554, which may represent minor initiation points or partially degraded RNAs. Together, these results indicate that RTA-inducible transcripts initiate from previously unknown initiation sites located ~270 nucleotides downstream of the constitutive promoter LTc (centered around nucleotide 127880) and lie within an intron that is excised from the 5'-untranslated leader of many the constitutive transcripts (Fig. 2A).

Fine mapping analysis of the inducible LTi promoter. To map promoter elements necessary for stimulation by RTA, we generated several additional truncations within the pLT4-luc construct (Fig. 5A; shown schematically in Fig. 5D). Truncations to –181 (pLT4.1-luc) and –164 (pLT4.2-luc) with respect to the inducible mRNA start site (+1), remained fully RTA inducible in Dox-treated TRExBCBL1-Rta cells (Fig. 5B) or HeLa cells cotransfected with an RTA-expression plasmid (Fig. 5C). Further truncation of the promoter to –121 (pLT4.3-luc) or beyond (pLT4.4-luc and pLT4.5-luc) essentially abolished the response to RTA in either context. From this, we conclude that sequences between –121 and –164 are important for the RTA responsiveness of the LTi promoter. Careful scrutiny of the DNA sequence for this region highlighted potential binding sites for C/EBP (5'-TTTGAAATGCT-3'), Sp1 (5'-GATGTGTGGG-3'), and CSL (5'-GTGGGAA-3'). These sequence matches are notable because both the cellular C/EBP{alpha} and CSL are known to interact with RTA and facilitate transactivation of a number of KSHV lytic promoters (32, 71).



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  		FIG. 5. Truncation analysis of the LTi promoter. (A) Sequence of the LTi promoter highlighting potential binding sites for the cellular transcription factors Sp1, C/EBP, CSL (CBF1/RBP-J{kappa}), interferon regulatory factor 8 (IRF/ICSBP), and nuclear factor 1 (NF1). A candidate TATA is located at –30 relative to the transcriptional start sites (+1) of the newly mapped LTi promoter. Three RTA-responsive elements (A, B, and C) used by the K14 promoter are indicated, and binding of CSL to RRE-A and C has been demonstrated (33). Sequences present in pLT4-luc are shown in boldface uppercase letters. Upstream end points for additional promoter truncations (LT4.1 to LT4.5) are indicated by arrows below the sequence. (B) Luciferase assays of the promoter truncations tested in TRExBCBL1-Rta cells in presence (+) or absence (–) of Dox. (C) Luciferase assays of the truncations in HeLa cells in the presence (+) or absence (–) of transfected RTA expression plasmid (pCGFlag-RTA). (D) Schematic showing the distribution of candidate binding sites and known RTA-response elements relative to truncations (LT4 to LT4.5).

Identification of RTA-responsive elements. To more precisely identify sequences elements important for RTA-mediated activation of LTi, we introduced nucleotide substitution mutations (m1 to m9) into pLT4-luc, targeting several of the candidate binding site sequences. In addition to those identified by computer, we mutated clusters of nucleotides within three RTA response elements (termed RRE-A, -B, and -C) identified by Liang and Ganem that are critical for activation of the divergent K14/v-GPCR promoter (33). Mutation m4 falls within RRE-C, m5 and m6 fall within RRE-B, and m8 falls within RRE-A (Fig. 4A). Each mutation was assayed with or without Dox treatment of TRExBCBL1-Rta cells (Fig. 6B) and with or without cotransfected pCGFlag-RTA in HeLa cells (Fig. 6C). In comparison to the wild type (pLT4-luc), m1 had no effect on promoter activity in the presence or absence of RTA expression in TRExBCBL1-Rta cells and increased activity slightly in HeLa cells. In contrast, m2, m3, m5, and m7 showed intermediate levels of induction (approximately 40 to 80% of the wild type). Mutations m4, m6, and m8 targeted RRE-C, -B and -A, respectively, and in both assays showed a major decrease in the extent of induction. In HeLa cells, m9, which targeted the putative TATA box, also had a significant effect on induction but interestingly, this was more modest in TRExBCBL1-Rta cells. We imagine the different behaviors of individual mutations between the two assays reflect differences in cellular transcription factors or possibly differences in the abundance or posttranslational modification status of RTA itself. In summary, these results show that the three RTA-responsive elements (RRE-A, -B, and -C) utilized by the K14/v-GPCR are also important for RTA responsiveness of the LTi promoter.



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  		FIG. 6. The LTi and K14 promoters use common elements to respond to RTA. (A) Schematic showing candidate transcription factor binding sites targeted for mutagenesis (m1 to m9). (B) Wild-type and mutant versions of pLT4-luc were transfected into TRExBCBL1-Rta cells and induced with Dox. (C) As in panel B, except that each reporter was cotransfected with the pCGFlag-RTA expression plasmid.

The LTi promoter is unresponsive to sodium butyrate. Latent KSHV can be induced to enter lytic replication by exposure to a variety of chemicals, and this property has been exploited by numerous studies seeking to characterize the kinetics of gene and protein expression during reactivation (48, 64). Given our data from TRExBCBL1-Rta cells, we were puzzled by the lack of evidence for induction of LT mRNAs in previous studies (25). One reason might be that we are inducing high levels of RTA through a direct mechanism, rather than by using broad-spectrum chemical inducing agents that also bring about numerous changes in the cell. To address this, we asked whether the LTi promoter was responsive to short-chain fatty acid NaB, a potent inducer of the KSHV lytic cycle (42, 43, 76). TRExBCBL1-Rta cells were transfected with pLT4-luc or early lytic PAN (pPAN-luc) reporter genes and treated with combinations of Dox and/or NaB (Fig. 7A). As expected, Dox was sufficient to activate the promoter (21.7 fold) whereas NaB had only a minor effect (3 fold). Simultaneous treatment with both agents reduced the level of induction to about approximately half that of Dox alone (10.2 fold). The PAN promoter provided an excellent control for the efficacy of NaB in this experiment. For this promoter, NaB alone gave a robust stimulation (36.5 fold), similar to that achieved with Dox (31.2 fold). Moreover, treatment with both agents gave a slight increase in activation compared to either agent alone (45.6 fold). Thus, the LTi promoter appears to be selectively unresponsive to NaB, and even the induction achieved by Dox can be suppressed in the presence of NaB. A similar experiment was repeated in KSHV+/EBV– BC3 cells (1), using a cotransfected expression vector to supply RTA (Fig. 7B). Again, pLT4-luc was readily induced by expression of ectopic RTA, although the magnitude of the induction was much greater (107 fold) due to a lower level of basal activity. RTA-mediated induction was suppressed by simultaneous treatment with NaB (29 fold). On its own, NaB was a comparatively poor stimulus for LTi (12.1 fold). Induction of the PAN promoter by NaB was comparatively poor (40.4 fold) compared to the RTA expression vector (147.3 fold), a contrast to TRExBCBL1-Rta cells, where NaB and forced RTA expression were similarly effective (Fig. 7A). This result shows that NaB selectively inhibits LTi in multiple PEL lines and is independent of the Dox-regulated expression system.



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  		FIG. 7. LTi promoter is not induced by sodium butyrate. (A) TRExBCBL1-Rta cells were transfected with pLT4-luc or pPAN-luc reporters, cultured for 24 h, and then treated with Dox, sodium butyrate (NaB), or a combination of Dox and NaB. After a further 36 h, extracts were prepared and assayed for luciferase activity. (B) BC3 cells transfected with 0.5 µg pLT4-luc or pPAN-luc reporters, together with 1.5 µg of pCGFlag-RTA (+RTA) or empty expression vector (–RTA). After 24 h, NaB was added to the medium, and extracts were prepared 36 h later.

Activation of KSHV lytic replication leads to many changes in the infected cell and ultimately loss of cell viability, and we were concerned that simultaneous treatment with Dox and NaB might accelerate the lytic program, resulting in the decreased activity of pLT4-luc at the 36-h time point when activity was measured. To address this, we performed a time course experiment in which luciferase activity was measured at various times after addition of Dox, NaB, or a combination of the two (Fig. 7B). Consistent with the previous result, NaB did not activate the LTi promoter at any time (filled triangles), whereas promoter activity rose steadily in cells treated with Dox alone, reaching a maximum at 36 h. Interestingly, simultaneous treatment with Dox and NaB gave a very different profile, with a maximum activity at 16 h followed by a steady decline. By 36 h, pLT4-luc activity had fallen to less than half that of Dox alone, consistent with the previous experiment (Fig. 7A). The PAN promoter was assayed in parallel and showed a steady increase in luciferase activity when cells were treated with Dox or with NaB. Combination of the two agents resulted in a strong but transient synergy, similar to that seen with the LTi reporter; however, at the 36 h time point, activity levels were indistinguishable from those with Dox and NaB alone. Thus, it seems highly unlikely that the inhibitory effect of NaB on Dox-mediated activation of the pLT4-luc reporter was due to loss of cell viability and suggests instead that NaB blocks the ability of this particular promoter to respond to RTA.

Lastly, we examined the levels of RTA protein by immunoblotting (Fig. 8C and D). First, we looked selectively at the Dox-regulated recombinant RTA (rRTA) using the c-myc epitope tag added to the inducible copy of the ORF50 gene (Fig. 8C). Blotting of each time point under the different inducing conditions confirmed that NaB was unable to induce rRTA by itself but interestingly, significantly increased steady-state levels of the protein in the presence of Dox. It should be noted that the panels are intentionally underexposed to illustrate the difference in rRTA abundance between Dox alone and Dox plus NaB. Quantitation of the immunoblot signal indicated that from 6 h onwards, rRTA levels were ~8-fold higher in the presence of NaB. The same lysates were also probed using a polyclonal antibody against RTA, allowing us to follow both virus encoded and recombinant proteins. Multiple bands were detected and probably correspond to different posttranslational modification states (20). A band that is present in uninduced cells, and might represent a cross-reacting cellular protein or an inactive form of RTA, is shown in Fig. 8D. A modest accumulation of the virus-encoded RTA is detectable in cells treated with NaB alone and is presumably responsible for the induction of the PAN promoter. Simultaneous treatment with Dox and NaB gives much higher levels of total RTA than with Dox alone, although it is unclear what fraction of this protein corresponds to recombinant RTA. This experiment demonstrates that RTA levels are high at late time points, contrasting the significantly reduced activation of the LTi promoter.



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  		FIG. 8. Delayed suppression of the LTi promoter by sodium butyrate. (A and B) Time course analysis of TRExBCBL1-Rta cells transfected with pLT4-luc (A) and pPAN-luc (B). Time 0 h corresponds to addition of inducing agents: mock (filled squares), Dox (filled circles), NaB (filled triangles), or a combination of Dox and NaB (open squares). (C) Dox-dependent expression of myc-tagged RTA in TRExBCBL1-Rta cells detected by immunoblotting using mouse monoclonal antibody 9E10. Cells were lysed in SDS-sample buffer, and proteins were resolved by 10% SDS-PAGE. The time shown indicates hours after addition of inducing agents. (D) As in panel C, except that protein extracts were separated by 5% SDS-PAGE and immunoblotted with an {alpha}-RTA polyclonal antibody. A band that is present in uninduced cells and might represent a cross-reacting cellular protein or an inactive form of RTA is shown, indicated by open arrowheads.


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DISCUSSION
 
We have shown that the KSHV lytic switch protein RTA can activate a previously unknown promoter (termed LTi) located immediately upstream of ORF73 encoding LANA. The new promoter is distinct from the previously characterized constitutive promoter (Fig. 2A and 3A), LTc (also known as LANAp) and lies within an intron that is removed from the majority of LT mRNAs in latently infected cells (9, 25, 27, 56, 57, 65). Further mapping of LTi revealed extensive overlap with the K14/v-GPCR promoter, oriented in the opposite direction and responsible for inducible expression of a bicistronic mRNA encoding the important signaling molecules v-Ox (K14) and v-GPCR (ORF74) (6, 25, 28, 33, 65). Discovery of an inducible promoter downstream of the well-characterized constitutive LTc promoter adds another layer of complexity to the already elaborate arrangement of spliced and unspliced mRNAs that crisscross this important region of the KSHV genome. It is notable that all of the genes in 12.5-kb region between K12 (encoding kaposins A, B, and C) and ORF74 (encoding v-GPCR) are involved in virus-host interactions and that all are thought to contribute to KSHV pathogenesis (5, 10, 23, 44, 68). By incorporating multiple levels of control, KSHV may be better able to tailor expression of these proteins to specific cellular contexts and thereby optimize colonization.

By Northern blotting, we showed that forced expression of RTA can induce the expression of ~5.3-kb and ~1.7-kb LT transcripts that are virtually indistinguishable in size from the constitutive transcripts. The results of primer extension and 5' RLM-RACE reveal, however, that the inducible transcripts differ with respect to the site of initiation. Induction of LT transcripts following Dox treatment of TRExBCBL1-Rta cells has also been observed with gene array technology (47). Probes specific to ORF71 or ORF72 detect a rapid accumulation of mRNA within the first 8 to 16 h after induction of RTA, with kinetics comparable to the viral ORF50 gene and several other virally encoded transactivators or regulatory proteins. An increase in ORF73-containing mRNA was also clearly evident in the microarray study, although the rate of accumulation appeared to be more gradual than for RNAs detected by the ORF71 and ORF72 probes.

Our results contrast with previous induction studies using the chemical agents TPA and butyrate, in which LT mRNAs remain relatively constant compared to the marked accumulation of conventional lytic mRNAs (9, 48, 56, 65). This suggests that the choice of induction cue (e.g., chemical inducing agents versus direct expression of RTA) can have a significant influence on the subsequent program of gene expression. Of course, none of these methods can be considered truly physiological, but these observations do highlight the potential for a reactivating virus to tailor its lytic reactivation program to better suit the immediate environment. A reasonable caveat to this hypothesis is that the levels of RTA protein produced by direct expression (Dox mediated) are almost certainly higher than with the chemical inducers and thus might induce nonphysiological targets. While additional studies will be needed to rigorously rule this out, this seems an unlikely explanation for the differential response because the PAN promoter is activated to a similar extent by butyrate or Dox treatment, suggesting that RTA is not limiting. In addition, we show that when combined with Dox treatment, butyrate has an antagonistic effect of LTi induction, even though it elevates steady-state levels of RTA protein. This antagonism is specific to the PEL cell environment because in HeLa cells, RTA and butyrate show a strong synergy for activation of both the LTi and PAN promoters.

Whether the LTi promoter is responsible for induction of shorter mRNAs encoding ORF71 and ORF72 is not clear. By Northern blotting, we observed an additional 1.4-kb species in the presence of Dox (Fig. 2B) but this data cannot distinguish between alternative promoter usage and changes in splice site (or termination site) selection. As mentioned above, previous gene array studies of Dox-treated TRExBCBL1-Rta cells found a difference in the rates of mRNA accumulation detected with probes specific to ORF71 or ORF72 compared to ORF73 (47). Although this discordance might arise through differences in the stability of the long and short LT mRNAs or from changes in the frequency at which ORF73 coding sequences are removed from the larger precursor by differential splicing, it is also consistent with the presence of transcripts initiating at an inducible promoter near the 3' end of ORF73. This region is already known to contain an inducible promoter responsible for a ~2.8-kb spliced K12 mRNA (31). In murine herpesvirus 68 (mHV68), ORF72 is expressed as an early late (leaky-late) gene in infected murine fibroblasts and only weakly during latency (39, 61, 69); it is worth noting that ORF72 and ORF73 are separated by another gene (ORF M11 encoding v-bcl-2). M11 is transcribed in the opposite orientation, and this arrangement may necessitate the use of separate promoters for ORF72 and ORF73. It is possible that production of tricistronic mRNAs initiating at LTc or LTi in KSHV has only recently evolved. Expression of mHV68 ORF72 as part of the lytic rather than latent program is also consistent with the phenotypes of mutant viruses lacking v-cyclin, which establish latency but are defective for reactivation (24, 66). It is also worth noting than in mHV68, ORF73 mRNA is most abundant during lytic replication and behaves as a cycloheximide-insensitive immediate-early gene (52).

Why does the LT cluster respond to RTA? Regulatory proteins encoded by the LT cluster are likely to be important in the first few minutes or hours of de novo infection. During this narrow window of time, the virus must seize control of the cell, establishing latency and preventing the innate antiviral response from triggering apoptosis. Expression profiling of newly infected primary human dermal microvascular endothelial (HMVEC-d) cells and human foreskin fibroblast cells showed that a surprising mixture of lytic and latency-associated genes are expressed at early times (29). In HMVEC-d cells, high levels of ORF50 mRNA can be detected shortly after exposure to virus, peaking at 120 min before declining rapidly as latency is established. ORF73 mRNA accumulates steadily during the early phase of infection before leveling off and in both cell types reaches a maximum after that of ORF50 mRNA. Discovery of the RTA-inducible LTi promoter suggests that this initial burst of ORF50 mRNA expression facilitates colonization of the cell and (ultimately) latency by helping to raise intracellular levels of LANA v-Cyc and v-FLIP, which are implicated in preventing apoptosis and activating the cell proliferation machinery. How the virus achieves selective activation of these RTA-responsive genes without initiating the full-blown lytic cascade represents an important challenge to our understanding of the regulatory network. There is evidence that LANA itself counters transactivation of certain lytic promoters by RTA, but it is hard to see how this would operate at early times when RTA is more abundant (30, 58). Alternatively, environmental factors unique to the naïve cell might prevent many of lytic promoters from responding to RTA at early times.

Regulation of the LT cluster by Notch signaling pathway. While this study was in progress, Liang and Ganem reported the identification of three RTA-responsive elements termed RRE-A, -B, and -C required for induction of the K14/v-GPCR promoter (33). Sites A and C correspond to binding sites for the cellular transcription factor CSL (also known as CBF1 or RBP-J{kappa}) and confer RTA-responsiveness when placed upstream of an unrelated promoter. Although important for RTA responsiveness of both the K14/vGPCR and LTi promoters, RRE-B does not function in the same manner as RRE-A or -C. The sequence itself shows no obvious similarity to known CSL or RTA binding sites, and this is corroborated by in vitro binding assays (33). Moreover, when tested alone, RRE-B did not confer RTA responsiveness on another promoter, suggesting it requires neighboring sequences or a particular promoter context to function.

Identification of CSL binding sites in the LTi promoter raises the possibility that expression of the LT cluster can be upregulated by extracellular signals. CSL is the end point of the Notch signaling pathway and has been targeted by multiple viral regulatory proteins including the Epstein-Barr virus EBNA2 and EBNA3 proteins and the 13S isoform of adenovirus E1A (22, 45). In its ground state, CSL is a transcriptional repressor, recruiting a corepressor complex that contacts the basal transcription machinery and promotes formation of repressive chromatin. This repressor function would account for the low constitutive activities of the LTi and K14/v-GPCR promoters in the absence of RTA, although it should be noted that mutation of individual sites did not lead to an obvious derepression of the LTi promoter in our transient assay.

Activation of the Notch receptor in the plasma membrane leads to release of the cytoplasmic domain (termed NICD or NotchIC), which translocates to the nucleus and promotes the exchange of the CSL corepressor complex for a coactivator complex. Liang and Ganem raised the interesting idea that KSHV promoters containing CSL sites might respond directly to Notch signaling and perhaps provide a context in which transforming proteins such as v-GPCR can be expressed in the absence of lytic replication (33). Identification of the LTi promoter increases the number of key growth control genes encoded by the virus that are potentially responsive to Notch-mediated signals.

Differential response of the LTi promoter to RTA and butyrate. How deacetylase inhibitors such as butyrate trigger reactivation of latent KSHV has not been rigorously explored. Butyrate is best known as a potent inhibitor of lysine deacetylases, and there is a wealth of data showing that acetylation of the N-terminal tails of the core histones often leads to gene activation. Thus, it is assumed that butyrate stimulates the expression of activator proteins such as RTA by derepressing the genes that encode them and may be relevant to the ORF50 gene of latent viral episomes, which are incorporated into chromatin (35). It should be kept in mind that histone acetylation is only one facet of this important posttranslational modification (3). The list of nonhistone proteins shown to undergo reversible acetylation is growing rapidly and includes general and specific transcription factors, nonhistone chromosomal proteins, nuclear import factors, and even nonnuclear proteins such as {alpha}-tubulin. Butyrate could therefore act at multiple levels within a latently infected cell. Our blotting data showed significant elevation in steady-state levels of rRTA protein in the presence of butyrate and Dox compared to Dox alone, but this was probably due to stimulation of the de-repressed CMV promoter (data not shown). Synergy between Dox and butyrate at early but not late times might be explained by a lag in the accumulation of acetylation events that somehow modulate RTA function.

It is known that other commonly used inducers of herpesvirus reactivation can also bring about changes in levels of protein acetylation. Phorbol esters, such as TPA, are known to stimulate protein kinase C, initiating a cascade of kinase-mediated signaling that can elevate the expression of the acetyltransferases p300 and PCAF (40, 41). This may help to explain why LTi is not induced to any significant level by TPA treatment (data not shown). In conclusion, it is likely that the bidirectional LTi-K14 promoter represents a very useful model system to explore the important relationship between different lytic reactivation cues and RTA function.


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ACKNOWLEDGMENTS
 
We thank John Souvlis and Jae Jung for sharing their TRExBCBL1-Rta cell line. Ornella Flore, Gary Hayward, Randy Luciano, Tatsushi Matsumura, and LaiYee Wong kindly provided antibodies, phage clones, and plasmids. We are indebted to senior members of the KSHV research community for helpful suggestions regarding the nomenclature of the various viral transcripts discussed in this study.

This work was supported by a Lymphoma Research Foundation Junior Faculty Award (A.C.W.), American Heart Association grant 0151213T (N.T.), NIH grant GM61139-04 (A.C.W.), and the Center for AIDS Research. S.M. is a postdoctoral fellow of the Rett Syndrome Research Foundation (RSRF).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, New York University School of Medicine, 550 First Ave., New York, NY 10016. Phone: (212) 263-0206. Fax: (212) 263-8276. E-mail: angus.wilson{at}med.nyu.edu. Back


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Journal of Virology, July 2005, p. 8493-8505, Vol. 79, No. 13
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.13.8493-8505.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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