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Regulation of Kaposi's Sarcoma-Associated Herpesvirus Reactivation by Dopamine Receptor-Mediated Signaling Pathways

Lee, Shaoying MS*; Deng, Hongyu PhD*†; Yu, Fuqu PhD; Melega, William P PhD; Damoiseaux, Robert PhD§; Bradley, Kenneth A PhD§; Sun, Ren PhD

JAIDS Journal of Acquired Immune Deficiency Syndromes: August 15th, 2008 - Volume 48 - Issue 5 - p 531-540
doi: 10.1097/QAI.0b013e31817fbdcf
Basic Science

Background: Kaposi's sarcoma-associated herpesvirus (KSHV) possesses two distinct life cycles, lytic replication and latency. An immediate early viral protein, Replication and transcription activator (RTA), is responsible for the virus switch from latency to active replication.

Methods: To identify cellular pathways that reactivate KSHV replication, an RTA-responsive viral early promoter, PAN, coupled with an enhanced green fluorescent protein (EGFP) reporter was delivered into a KSHV latently infected B cell line. Five different chemical libraries with defined cellular targets were screened for their ability to induce the PAN promoter as an indication of lytic replication.

Results: We identified seven chemicals that disrupted latency in KSHV latently infected B cells, five being N-acyl-dopamine derivatives. We showed that these chemicals reactivate KSHV through interacting with dopamine receptors, and that KSHV utilizes dopamine receptors and the associated PKA and MAP kinase pathways to detect and transmit stress signals for reactivation.

Conclusion: Our study identified two cellular signaling pathways that mediate KSHV reactivation and provided a chemical genetics approach to identify new endogenous activators with therapeutic potential against herpesvirus associated malignancies.

From the *School of Dentistry, University of California at Los Angeles, Los Angeles, CA; †Institute of Biophysics, Chinese Academy of Sciences, Beijing, P.R. China; and ‡Department of Molecular and Medical Pharmacology, and §Molecular Screening Shared Resource (MSSR), Department of Microbiology, Immunology and Molecular Genetics, University of California at Los Angeles, Los Angeles, CA

Received for publication December 31, 2007; accepted May 7, 2008.

Correspondence to: Dr. Hongyu Deng, CHS53-076, 10833 Le Conte Avenue, Los Angeles, CA 90095 (e-mail:; or Dr. Ren Sun, CHS23-120, 10833 Le Conte Avenue, Los Angeles, CA 90095 (e-mail:

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Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus type 8, was first discovered in Kaposi's sarcoma (KS) tissue taken from patients with AIDS.1 It is closely related to 2 well-known lymphotropic γ-herpesviruses, herpesvirus saimiri and Epstein-Barr virus (EBV). Subsequently, it was also found to be associated with 2 other human lymphoproliferative diseases, primary effusion lymphoma2 and multicentric Castleman disease.3

All herpesviruses, including KSHV, have 2 distinctive phases in their life cycle, lytic replication and latency. During latency, only a limited number of viral genes are expressed in a subset of host cells. Nevertheless, herpesviruses can switch to the lytic replication phase spontaneously or upon additional stimuli.4 Once the virus is activated, it expresses “immediate early,” “early,” and “late” genes sequentially, entering an active replication stage. In EBV, 2 immediate early viral proteins, Z EBV replication activator (ZEBRA) and RTA, are responsible for the regulation of this switch.5-7 These viral proteins control EBV reactivation and act synergistically to activate a series of lytic gene expressions that lead to the production of infectious viruses. Like EBV, KSHV predominantly exists in the latent state in tumor cells and its viral genomes replicate as circular episomes. However, unlike its homologue in EBV, RTA of KSHV alone plays the major role in mediating the switch of viral latency into productive infection.8-12 Additionally, it is the upstream activator of K-bZip, the homologue of EBVZEBRA.9,13-15

RTA is highly conserved among all γ-herpesviruses and functions as a replication and transcription activator. It contains an N-terminal DNA-binding domain and a C-terminal activation domain and regulates lytic viral gene transcription, including that of itself in KSHV.8,9,16-18 The latency in KSHV-infected B cells can be disrupted by the ectopic introduction of KSHV RTA, which leads to the activation of lytic gene expression and the initiation of the KSHV lytic replication cycle.19,20 Additionally, the expression of RTA at both the transcript and protein levels is highly restricted during latency, suggesting that repression of RTA is required to maintain KSHV latency. All of this evidence indicates that RTA plays a critical role in regulating the KSHV life cycle. One of the early viral transcripts, PAN (polyadenylated nuclear) RNA, is tightly controlled by RTA-it is not expressed in latency but is induced by RTA to express to an abundant level during lytic replication. Previously, our group has identified a 31 bp RTA-responsive element (RRE) in the PAN promoter region, located between −69 to −38 nt upstream of the PAN transcription initiation site.21-23 The PAN RRE activity provides a direct indication of KSHV reactivation and therefore a tool to study the basic mechanisms of KSHV reactivation.

Chemicals such as 12-O-tetradecanoylphorbol-13-acetate (TPA), sodium butyrate (NaB), and 5′-azacytidine (a DNA-demethylating agent);24-26 proinflammatory cytokines;27-29 and catecholamines (norepinephrine and epinephrine)30 have been reported to induce KSHV reactivation in cell cultures. To systematically investigate the cellular signaling pathways involved in KSHV reactivation, we undertook 2 independent approaches. One is to screen a comprehensive cDNA library to identify cellular proteins and related pathways responsible for mediating the switch of KSHV from latency to lytic replication.31 The other approach is to apply chemical genetics to screen for chemicals that can reactivate KSHV and to further identify the associated cellular pathways and underlying molecular mechanisms.

We thus conducted a chemical library screening using the BIOMOL chemical libraries. Most of the chemicals in these libraries have known biological targets, facilitating further validation of the potential cellular targets of the hits. Taking advantage of the PAN RRE, we constructed a reporter plasmid, pPAN-69/d2EGFP, and established an in vitro cell screening assay using a KSHV latently infected B cell line. Among the top 7 hits identified in our chemical screening, 5 are different endocannabinoids containing an N-acyl-dopamine moiety. Investigations on the mechanism of KSHV reactivation using these chemicals have demonstrated their binding to dopamine receptors (DRs) and subsequent reactivation of the protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signaling pathways. These results suggest that DR antagonists and/or inhibitors of downstream signaling pathways represent potential therapeutic targets for KS.

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Cell Culture

BC-3 cell line (a gift from Dr. E. Cesarman) was derived from a primary effusion lymphoma latently infected by KSHV. Raji cell line (KSHV negative, EBV positive) and DG-75 cell line (KSHV and EBV negative) were established from patients with Burkitt lymphoma. All the B cell lines were cultured in RPMI 1640 media containing 15% fetal bovine serum at 37°C in the presence of 5% CO2.

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Plasmid Construction and Chemicals

The PAN promoter-driven green fluorescent reporter construct (pPAN-69/d2EGFP) was constructed by cloning the (−69 to +14) region from the KSHV PAN RNA promoter into a pd2EGFP reporter plasmid (Invitrogen Corporation, Carlsbad, CA). The reporter construct pPAN-69-luc was described previously.21 The cellular kinase inhibitor U1026 and H89 were purchased from Calbiochem (San Diego, CA) and Upsate Biotechnology Incorporate (Lake Placid, NY), respectively. The D1-like receptor antagonist, R(+)-SCH-23390 HCL, and the D2-like receptor antagonist, haloperidol, were both purchased from Sigma (St. Louis, MO).

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Chemical Screening of KSHV Reactivation

A BIOMOL chemical library was provided by the Molecular Screening Shared Resource core facility of University of California at Los Angeles. This chemical library includes a total of 504 different chemicals categorized into 5 different classes, which include bioactive lipid, endocannabinoid, ion channel, enzyme inhibitors, and orphan ligand. The complete list of compounds is available at BC-3 cells were transfected with pPAN-69/d2EGFP as a pool, and 5 × 104 cells/well were dispensed into 384-well plates. Liquid drops of the BIOMOL chemicals were added to 384 wells by transferring them from the stock plate with the 384-well pin at 24 hours posttransfection. EGFP expressions were monitored by a fluorescent microscope after 24-hour drug treatment. Each plate contained nearly 250 different chemicals and DMSO as a negative control and TPA as a positive control for KSHV reactivation.

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Western Blotting

BC-3 cells were treated with either 5 or 10 μM N-acyl-dopamine derivatives. TPA (10 or 20 ng/mL) and DMSO were used as positive and negative controls, respectively. Whole-cell extracts from 105 cells were analyzed by western blotting at either 24 or 48 hours after drug treatment. Blots were first probed with an antibody against K8 (a gift from Dr. J. Jung), a KSHV early protein, and re-probed with a monoclonal antibody against actin (Sigma) as a loading control.

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Transfection and Reporter Assays

BC-3 cells were seeded into 24-well plates (105 cells/well) at 20 hours before transfection. A DNA mixture of 50 ng of pPAN-69-luc, 2 ng of pRL-SV40, and 750 ng of pcDNA3 vector DNA was cotransfected into each well using Lipofectamine 2000 (Invitrogen). Cells were treated with drugs at 24 hours posttransfection and harvested and luciferase activities measured by a Dual-Luciferase assay kit according to the manufacturer's instructions (Promega, Biotech).

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RT-PCR and RT-Q-PCR Analyses

Cellular RNA from cells growing in 6-well plates was isolated using the RNeasy kit with on-column DNA digestion (Qiagen, Valencia, CA). The mRNA was subsequently reverse transcribed into cDNA using SuperScript II RNase H-Reverse Transcriptase (Invitrogen). Expression of DR transcripts was then examined by RT-PCR with primer pairs as follows: D1 receptor primers (forward: 5′-AACACCTCTGCCATGGACG-3′; reverse: 5′-TGATGGCCACAGGGATGTAA-3′), D2 receptor primers (forward: 5′-GCGGACAGACCCCACTACAA-3′; reverse: 5′-AAGGGCACGTAGAAGGAGAC-3′), D2 shot/long isoforms primer (forward: 5′-CCATGCTGTACAATACGCGCT-3′; reverse: 5′-GGCAATCTTGGGGTGGTCTTT-3′), D3 receptor primers (forward: 5′-CCCGCCCACATGCCTACTAT-3′; reverse: 5′-GAAGGCTTTCCGGAACTCGAT-3′), D4 receptor primers (forward: 5′-CCCACCCCAGACTCCACC-3′; reverse: 5′-GAACTCGGCGTTGAAGACAG-3′), and D5 receptor primers (forward: 5′-ACCTGTGCGTCATCAGCGT-3′; reverse: 5′-TGCGATCGAAAGGACCCTC-3′).61 The expression of individual DR transcripts was analyzed by electrophoresis on a 1% agarose gel.

The RT-Q-PCR was carried out using the QuantiTect SYBR Green RT-PCR kit (Qiagen). The primers used for RT-Q-PCR were as follows: PAN (forward: 5′-GCCGCTTCTGGTTTTCATTG-3′; reverse: 5′-TTGCCAAAAGCGACGCA-3′), viral TK (forward: 5′-CGTAGCCGACGCGGATAA-3′; reverse: 5′-TGCCTGTAGATTTCGGTCCAC-3′), and RTA (forward: 5′-CACAAAAATGGCGCAAGATGA-3′; reverse: 5′-TGGTAGAGTTGGGCCTTCAGTT-3′). The PCR product amplified by GAPDH primers (forward: 5′-GAAGGTGAAGGTCGGAGTC-3′; reverse: 5′-GAAGATGGTGATGGGATTTC-3′) was used as an internal control.

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Establishing a Reporter Assay for KSHV Reactivation

To systematically analyze the cellular signaling pathways involved in KSHV reactivation, we first established a sensitive cell-based reporter assay. A reporter plasmid pPAN-69/d2EGFP, driven by the PAN promoter, was constructed. The PAN promoter region (69 to +14), which contains an RRE, was amplified from pPAN-69-luc.21 The ability of pPAN-69/d2EGFP to be activated by RTA was confirmed by cotransfection with pCMV/RTA, an RTA expression plasmid, into the DG75 cell line (a KSHV-negative B lymphoma cell line) and monitoring enhanced green fluorescent protein (EGFP) expression 24 hours posttransfection (data not shown). In addition, our previous results have shown that TPA induces RTA expression in KSHV latently infected cell lines and further stimulates PAN production (data not shown). Therefore, we transfected pPAN-69/d2EGFP into a KSHV latently infected cell line, BC-3, and 24 hours later, treated the cells with TPA. Detection of EGFP expression 48 hours posttransfection demonstrated that our reporter assay recapitulated viral reactivation and viral gene expression induced by chemical TPA and thus can be used to screen for other chemical compounds that reactivate KSHV (Figs. 1A, 2, 3).







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Screening for Chemical Compounds That Reactivate KSHV

The Molecular Screening Shared Resource core facility of University of California at Los Angeles provided 5 chemical libraries for our screening purposes. These libraries, purchased from BIOMOL International, L. P. (Plymouth Meeting, PA), represent 5 different categories of chemicals: 204 chemicals from a bioactive lipid library, 60 chemicals from an endocannabinoid library, 84 chemicals from an enzyme inhibitors library, 72 chemicals from an ion channel inhibitors library, and 84 chemicals from an orphan (drug or ligand) library.

To perform the screening, the pPAN-69/d2EGFP plasmid was transfected into BC-3, and 24 hours later, the cells were individually treated with compounds from the BIOMOL libraries. The compounds were used at 0.1, 1, or 10 μM final concentrations according to the manufacturer's recommendations, and TPA was used as a positive control. EGFP expression was monitored under a fluorescence microscope 48 hours posttransfection (Fig. 1A). Seven hits were identified from the chemical libraries by comparing the green fluorescent signals with those from the dimethyl sulfoxide (DMSO) vehicle control (Fig. 1A; data not shown). Among them, 5 hits were N-acyl-dopamine derivatives, which include oleoyl dopamine (OLDA), linoleoyl dopamine (LDA), gamma-linolenoyl dopamine (γLDA), dihomo-gamma-linolenoyl dopamine (DγLDA), and docosatetra-7Z, 10Z, 13Z, 16Z-enoyl dopamine (Table 1). These dopamine derivatives have 2 common features: all of them bear an N-acyl-dopamine moiety and a long lipid chain (Fig. 1B). Because of the unavailability in large quantities of docosatetra-7Z, 10Z, 13Z, 16Z-enoyl dopamine, we chose to continue to investigate the other 4 compounds in further experiments.

Table 1

Table 1

To determine the optimal concentrations for these 4 compounds, we tested the cytotoxicity effects of each of these N-acyl-dopamine derivatives by CellTiter-Glo Assay (Promega; Biotech, Madison, WI). BC-3 cells were treated with each compound at 5 different concentrations ranging from 0.25 to 20 μM, and cell viabilities were examined at 24 hours posttreatment. Adenosine triphosphate (ATP) quantities in treated BC-3 cells were comparable to those in the DMSO control when these compounds were used at 5 or 10 μM (data not shown), and these concentrations were therefore used in further experiments.

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KSHV Reactivation by N-Acyl-Dopamine Derivatives

To confirm our initial screening results, we employed a different reporter assay using a luciferase reporter, which provides a more quantitative measurement than does the green fluorescent protein (GFP) reporter. Previously, our group had generated a luciferase reporter construct, PAN-69-luc, which contains the RTA-responsive element of the PAN promoter. This luciferase reporter has a low basal activity in the absence of RTA but is highly responsive to RTA activation.21 By using this system, we analyzed the induction of the PAN promoter by these 4 different N-acyl-dopamine derivatives to measure KSHV reactivation upon their stimulation. As expected, the positive control TPA activated PAN promoter-driven luciferase expression (Fig. 2A). The 4 N-acyl-dopamine derivatives also induced PAN promoter to different degrees (Fig. 2A). In general, a 4- to 5-fold induction was observed at 48 hours postinduction, higher than that obtained at 24 hours. The treatment of LDA, γLDA, or DγLDA at 5 μM gave slightly higher induction than that at 10 μM, likely due to milder cytotoxicity associated with lower drug concentration.

To further confirm the results from reporter systems, we directly measured PAN gene expression from the endogenous viral genome upon N-acyl-dopamine derivative stimulation by reverse transcription quantitative polymerase chain reaction (RT-Q-PCR). Fold induction of PAN gene expression was normalized to the housekeeping gene, GAPDH, and calculated by comparison with the DMSO control for each treatment (Fig. 2B). At 24 hours posttreatment, OLDA induced PAN expression around 90-fold, whereas the other 3 N-acyl-dopamine derivatives each induced PAN expression to 30- to 50-fold (Fig. 2B). The fold induction of PAN transcripts treated with LDA was lower than that with γLDA (Fig. 2B), whereas the fold induction of the PAN promoter reporter treated with LDA was higher than with γLDA (Fig. 2A). This is due to the different measurements we took: the PAN-69-luc reporter only contains the RTA response element in the promoter region and served as a readout for RTA activation by chemical treatment. However, expression of the endogenous PAN transcript from the viral genome is under control of a longer promoter and involves other response elements in addition to the RTA response element.

We also analyzed the transcripts of 2 other viral lytic genes, the immediate early gene RTA and another early gene thymidine kinase (TK), and observed their upregulation by these N-acyl-dopamine derivative treatments. They increased approximately 40-fold by OLDA stimulation and 15-fold by the stimulation of other 3 N-acyl-dopamine derivatives (data not shown). Although the fold induction of these 2 transcripts by N-acyl-dopamine derivatives was not as significant as that of the PAN transcript, the relative induction levels of PAN, RTA, and TK by N-acyl-dopamine derivatives were similar to those by TPA treatment (data not shown).

We next investigated gene expression from the endogenous viral genome at the protein level. We examined, upon dopaminergic stimulation, the expression level of the KSHV early protein, K8, by western blot analysis after 24 or 48 hours of induction. Expression of actin was used as a loading control. DMSO vehicle control showed a low basal level of K8 protein production in BC-3 cells (Fig. 2C, lane 11), and the level was elevated after TPA treatment (Fig. 2C, lanes 1 and 2). Production of K8 was also increased by all N-acyl-dopamine derivative treatments (Fig. 2C, lanes 3-10). K8 expression was detected as early as 24 hours and also at 48 hours, although K8 expression levels varied in response to different N-acyl-dopamine derivatives. In addition, K8 was expressed to a higher level with 5 μM LDA, γLDA, or DγLDA treatment than with 10 μM, consistent with the results from the reporter assay.

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DRs Mediate KSHV Reactivation

Among the 7 chemicals that we have identified from the screen, 5 are from the endocannabinoids library. This group of chemicals contains an array of 10 different fatty acids with 10 different N-acyl groups. Only those with an N-acyl-dopamine polar group of fatty acids disrupted KSHV latency, whereas the same fatty acids with other N-acyl groups did not reactivate KSHV. This result suggested that these chemicals reactivated KSHV through receptor signaling pathways other than the cannabinoid receptor (CB) signaling pathway, a common receptor of endocannabinoids which is mostly involved in the pain pathway.

Interestingly, only 5 of 10 endocannabinoids with N-acyl-dopamine reactivated KSHV in our in vitro chemical screening assay. For example, OLDA reactivated KSHV, whereas N-arachidonoyldopamine (NADA) did not activate PAN promoter activities in our screening. OLDA and NADA are both chemicals classified as endocannabinoids. Endogenous OLDA and NADA have recently been identified as a new class of brain neurotransmitters, which behave as potent agonists for the vanilloid receptor (VR), a receptor involved in pain signaling.32,33

Therefore, we analyzed the specificity of receptor-ligand interaction in KSHV reactivation by using a receptor antagonist. Palmitoyl dopamine (PLDA), an endogenous VR antagonist, was used in our assay. This chemical was also included in our primary screening and showed no induction of KSHV reactivation. As shown in Figure 3A, OLDA induced PAN transcription by 28-fold, whereas PLDA treatment did not elevate the level of PAN transcripts. The fold induction by OLDA was lower than that observed in Figure 2 due to the later passage of the BC-3 cell line, which gave higher background of reactivation at the time of experiment. KSHV latently infected cells are very sensitive to cell culture conditions, and this in turn affects the frequency of spontaneous viral reactivation as well as how they respond to different chemicals. Nonetheless, 1-hour pretreatment with PLDA did not change the induction level of PAN transcripts by OLDA, suggesting that OLDA disrupts KSHV latency through receptors other than VR (Fig. 3A).

Because CB and VR did not seem to be the main receptors that mediate these endocannabinoid N-acyl-dopamine derivatives to reactivate KSHV, we reasoned that these dopamine-related compounds may exert their effects on cells through interactions with DRs. There are 2 types of DRs. The D1-like receptors are D1 and D5 receptors, and the D2-like receptors include D2, D3, and D4.33 As a first step to investigate the role of these DRs in KSHV reactivation, we examined the expression patterns of the DRs in BC-3 cell line. As controls, the presence of DRs in an EBV-positive B cell line, Raji, and a KSHV- and EBV-negative B cell line, DG-75, was also analyzed. We used primers specific for each of the known DR transcripts and performed RT-PCR to analyze the expression patterns of the 2 types of DRs in these cell lines. D5 (D1-like) and D2 and D4 (D2-like) transcripts were expressed in all 3 cell lines (Fig. 3B; lanes 6, 12, and 18: D5; lanes 2, 8, and 14: D2; lanes 5, 11, and 17: D4), whereas the D1 receptor transcripts were not detected in any of them (Fig. 3B; lanes 1, 7, and 13). The D3 receptor transcripts were present in DG75 and Raji cells (Fig. 3B; lanes 4 and 10).

To study the involvement of N-acyl-dopamine derivatives-DR interactions in reactivation of KSHV, we used 2 DR antagonists, R(+)-SCH-23390 HCL, which is specific for D1-like receptors, and haloperidol, which is specific for D2-like receptors. We pretreated BC-3 cells with either antagonist for 1 hour before incubating the cells with the various N-acyl-dopamine-related compounds. Twenty-four hours later, we analyzed the PAN transcript level by RT-Q-PCR to compare the effects of DR antagonists on the induction stimulated by individual chemical treatments. PAN transcript levels dropped more significantly with the pretreatment of the D1-like receptor antagonist but were also downregulated by the D2-like receptor antagonist pretreatment when compared with the nonantagonist control (Fig. 3C). This result, combined with those from the VR antagonist pretreatment experiments, suggests that the N-acyl-dopamine derivatives reactivate KSHV through interactions with DRs, which are mainly the D1-like receptors in BC-3 cells.

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The Signaling Pathways That Mediate KSHV Reactivation Induced by Dopamine Derivatives

Dopamine-responsive receptors are G protein-coupled receptor proteins. Upon receptor activation, 2 distinct signaling pathways are activated (Fig. 4A). D1-like DRs initiate a cascade of intracellular events that includes the activation of adenylate cyclase and formation of cyclic adenosine monophosphate (cAMP),34-36 and also the activation of cAMP-dependent protein kinase (PKA),36 a PKA-dependent pathway involving mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases (ERKs).37 In contrast, D2-like DRs act by inhibiting adenylate cyclase and regulating phospholipases but also have a parallel intervening signal pathway that involves the activation of MAPK phosphorylation, which goes through the Ras/Raf/MEK/ERK pathway.38,39

We thus investigated the downstream events after binding of the dopamine derivatives to DRs. We pretreated cells with a specific cellular protein inhibitor for 1 hour before adding N-acyl-dopamine derivatives and evaluated the induction of PAN transcripts. As shown in Figure 4B, pretreatment with the PKA inhibitor, H89, markedly reduced induction of PAN transcripts with all treatments, and the MEK inhibitor, U1026, had smaller inhibitory effects. This result indicated that PKA and, to a lesser degree, MAPK pathways are involved in KSHV reactivation induced by these N-acyl-dopamine derivatives. However, these compounds have different affinities toward DRs, and the utilities of cellular signaling pathways are different as well. Because the PKA pathway is activated by ligand binding to DR1, but not DR2, the result also suggested that the D1R receptor signaling pathway plays a major role in N-acyl-dopamine derivative-mediated KSHV reactivation, consistent with the data obtained using the DR antagonists (Fig. 3C).



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In this study, we designed a sensitive reporter assay and screened a chemical library for compounds that reactivate KSHV from a latently infected cell line. We identified 5 N-acyl-dopamine derivatives and confirmed that 4 of them disrupt KSHV latency by monitoring endogenous viral gene expression at both the transcript and the protein levels. Using antagonists, we showed that these dopamine derivatives exert their effect through interacting with DRs. We further dissected the downstream cellular signaling pathways and demonstrated that the PKA pathway and, to a lesser degree, the MAPK pathway are responsible for the reactivation of KSHV by these compounds.

Dopamine, together with epinephrine and norepinephrine, belongs to catecholamines, a group of important molecules involved in the response to stress.40 Stress-induced virus reactivation has been reported for other herpesviruses, including human cytomegalovirus41 and herpes simplex virus.42 Here our results suggest that KSHV uses cellular signaling pathways to detect environmental stress signals and reactivates from a latent stage to lytic replications upon activation of DR-mediated pathways. Downstream signaling of DRs is expressed through either the D1- or the D2-like receptor pathways. When the D1-like receptor pathway is activated, cAMP level increases, leading to the PKA signaling pathway43-45 and subsequent increase in PKA pathway downstream proteins including cAMP response element-binding protein, transducer of regulated cAMP response element-binding protein, p38 MAPK, and c-jun amino-terminal kinase.36,37,46,47 Activation of the D2-like receptor pathway activates the MAPK pathway and phospholipase C (PLC β1) phosphorylation.38,48-52 We summarized the potential dopaminergic pathways that may be involved in KSHV reactivation in Figure 4A.

Interestingly, similar downstream signaling pathways (PKA and Ras) that reactivated KSHV were identified by a cDNA library screening of 26,000 mammalian cDNA clones in an independent study from our laboratory.31 Signaling pathways involved in norepinephrine/epinephrine or TPA-mediated KSHV reactivation were described in previous studies.13,30,53 In addition, PKCδ, a molecule involved in D1R signaling pathway, has been shown to be involved in TPA-mediated KSHV reactivation as well.53 In Figure 4A, we have combined results from both chemical and cDNA library screens, highlighting the overlapping pathways that are involved in KSHV reactivation. Molecules in red are the ones that have been identified from the cDNA library screening: PKA, Ras, and Ets-1 are the top hits from the screen. The involvement of PKA signaling in KSHV reactivation has been previously reported,30 and the same phenomenon was also shown in a different subfamily of herpesvirus, herpes simplex virus-1.42 Our group has further demonstrated that the immediate early gene RTA expression can be triggered by the Ras/Raf/MEK/ERK/Est-1 pathway. Ets-1 has an essential role by directly activating RTA promoter.31 Therefore, our results from these 2 screens illustrate consistency and complementarities of genetic and chemical genomic screening approaches.

Our results have shown the effect of N-acyl-dopamine derivatives on KSHV reactivation through signaling pathways downstream of both DRs. However, the signaling pathways downstream of D1-like receptors may contribute more to KSHV reactivation by activating not only the PKA pathway but also some MAPKs. Several molecules that are involved in Ras/Raf/MEK/ERK/Est-1 pathway, such as ERK, Ras, and MAPK MEK, have been found to be activated through a PKA-dependent pathway or a cAMP-dependent mechanism (Fig. 4A).35,37,54,55 This phenomenon is consistent with our results, which showed that the PKA inhibitor, H89, and the D1R antagonist act more potently than do the MEK inhibitor, U1026, and the D2R antagonist. H89 and the D1R antagonist might inhibit not only PKA but also PKA-dependent ERK, PKC, and other downstream molecules in the MAPK pathway. We compared these results with those from the D2-like antagonist pretreatment experiments and found that there are smaller inhibition effects caused by D2-like antagonist pretreatment. Treating the BC-3 cells with U1026 disrupted the Ras activation of the MAPK pathway31 and particularly inhibited the dopamine derivative-mediated KSHV reactivation. Multiple downstream effectors of DR signaling pathways interconnected through distinct or partially overlapping pathways. These results revealed the complexity of DR signaling pathways and suggested that D1R-like and D2R-like signaling pathways have some common downstream effectors that converge the signals together and reactivate KSHV.

Because our library contained only a limited number of chemicals, only a small number of ligands of DRs have been identified. Therefore, we may have underestimated the number of physiological ligands for DRs that lead to KSHV reactivation. In fact, DRs respond to a variety of dopamine derivatives produced naturally in the body. Among these 5 chemicals we screened out, only OLDA has been characterized physiologically.33 The other 4 molecules are various lipid derivatives of the dopamine scaffold. In the DR antagonist treatment experiments (Fig. 3C), we observed that the lipid moiety of the molecule modulates KSHV reactivation potency. However, some endocannabinoid amides from the same pool of chemicals that bear the same alkyl chain but are not N-acyl-dopamine did not activate the KSHV lytic cycle. Similarly, several other N-acyl-dopamine derivatives in the library with different alkyl moieties did not reactivate KSHV (Table 1). This result might be attributed to the diverse affinities between the ligands and the DRs or to the different stabilities of the chemicals. This class of lipid molecules is a combination of fatty acid amides and small polar groups. There is increasing evidence showing that fatty acid amides (cannabinoids) play an important role in cell signaling and also in the regulation of pain and inflammatory responses.56 Along this line, it is interesting to note that Δ9-tetrahydrocannabinol, a major active compound of marijuana, has recently been shown to stimulate KSHV reactivation and enhance replication in the infected cells.57 By means of advanced mass spectrometry and high-performance liquid chromatography technologies, more lipid molecules are being discovered and characterized. Eventually, we may be able to identify more natural ligands of DRs that mediate KSHV reactivation and may be involved in KS development.

The development of KS has been etiologically linked to KSHV infection in patients with AIDS. The involvement of dopamine in the progression of AIDS dementia complex in patients with HIV-specific neurological disorders has been clearly demonstrated.58 It is also suggested that the excess of dopamine caused by drug abuse, which directly stimulates the dopaminergic synapse, might modulate vulnerability to AIDS. Therefore, by understanding other stress responses induced by dopamine and dopamine-related compounds, we will be able to identify the relationship between dopamine-related signaling and replications of these 2 viruses as their pathogeneses are closely related. Developing therapeutic reagents and strategies for herpesviruses-associated diseases would be the ultimate goal of understanding how herpesviruses switch between lytic replication and latency. Two different therapeutic approaches can be applied to malignancies associated with this tumorigenic virus. One way is to prevent the disease progression by blocking viral replication at the early stage. Therefore, we need to further evaluate the potential of DR antagonists to serve as drugs to impede the progression of KS development in patients with AIDS through blocking the downstream signaling pathways and preventing virus spreading. The other approach is to destroy tumor by administering drugs that reactivate KSHV lytic gene expression in the tumor lesion in combination with antiviral drugs such as ganciclovir. Ganciclovir will be phosphorylated by viral early proteins TK and phosphotransferase, leading to inhibition of DNA replication. The additional “bystander” killing effects caused by metabolized ganciclovir may lead to the destruction of neighboring tumor cells.59,60 Additionally, the large amount of viral lytic protein expression at the tumor site will induce strong immune responses to clear the infected cells. Our study provides a potential platform of using chemical and genetic approaches to identify new therapeutic targets for KS.

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We thank Dr. E. Cesarman for providing BC-3 cell lines and Dr. J. Jung for antibody against K8 protein; Gus Lee and Yuri Shindo for editing; and all members in the Sun lab for helpful discussions and inputs. This study was supported by NIH grants DE15623 to H.D.; DE14153, CA 091797, and Burroughs Welcome Fund to R.S.

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KSHV; herpesvirus reactivation; dopamine; signal transduction

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