Identification of new antiviral agents against Kaposi's sarcoma-associated herpesvirus (KSHV) by high-throughput drug screening reveals the role of histamine-related signaling in promoting viral lytic reactivation.

Kaposi's sarcoma-associated herpesvirus (KSHV) causes several human cancers, such as Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). Current treatment options for KSHV infection and virus associated diseases are sometimes ineffective, therefore, more effectively antiviral agents are urgently needed. As a herpesvirus, lytic replication is critical for KSHV pathogenesis and oncogenesis. In this study, we have established a high-throughput screening assay by using an inducible KSHV+ cell-line, iSLK.219. After screening a compound library that consisted of 1280 Food and Drug Administration (FDA)-approved drugs, 15 hit compounds that effectively inhibited KSHV virion production were identified, most of which have never been reported with anti-KSHV activities. Interestingly, 3 of these drugs target histamine receptors or signaling. Our data further confirmed that antagonists targeting different histamine receptors (HxRs) displayed excellent inhibitory effects on KSHV lytic replication from induced iSLK.219 or BCBL-1 cells. In contrast, histamine and specific agonists of HxRs promoted viral lytic replication from induced iSLK.219 or KSHV-infected primary cells. Mechanistic studies indicated that downstream MAPK and PI3K/Akt signaling pathways were required for histamine/receptors mediated promotion of KSHV lytic replication. Direct knockdown of HxRs in iSLK.219 cells effectively blocked viral lytic gene expression during induction. Using samples from a cohort of HIV+ patients, we found that the KSHV+ group has much higher levels of histamine in their plasma and saliva than the KSHV- group. Taken together, our data have identified new anti-KSHV agents and provided novel insights into the molecular bases of host factors that contribute to lytic replication and reactivation of this oncogenic herpesvirus.

A Critical Role of Glutamine and Asparagine γ-Nitrogen in Nucleotide Biosynthesis in Cancer Cells Hijacked by an Oncogenic Virus.

While glutamine is a nonessential amino acid that can be synthesized from glucose, some cancer cells primarily depend on glutamine for their growth, proliferation, and survival. Numerous types of cancer also depend on asparagine for cell proliferation. The underlying mechanisms of the glutamine and asparagine requirement in cancer cells in different contexts remain unclear. In this study, we show that the oncogenic virus Kaposi's sarcoma-associated herpesvirus (KSHV) accelerates the glutamine metabolism of glucose-independent proliferation of cancer cells by upregulating the expression of numerous critical enzymes, including glutaminase 2 (GLS2), glutamate dehydrogenase 1 (GLUD1), and glutamic-oxaloacetic transaminase 2 (GOT2), to support cell proliferation. Surprisingly, cell crisis is rescued only completely by supplementation with asparagine but minimally by supplementation with α-ketoglutarate, aspartate, or glutamate upon glutamine deprivation, implying an essential role of γ-nitrogen in glutamine and asparagine for cell proliferation. Specifically, glutamine and asparagine provide the critical γ-nitrogen for purine and pyrimidine biosynthesis, as knockdown of four rate-limiting enzymes in the pathways, including carbamoylphosphate synthetase 2 (CAD), phosphoribosyl pyrophosphate amidotransferase (PPAT), and phosphoribosyl pyrophosphate synthetases 1 and 2 (PRPS1 and PRPS2, respectively), suppresses cell proliferation. These findings indicate that glutamine and asparagine are shunted to the biosynthesis of nucleotides and nonessential amino acids from the tricarboxylic acid (TCA) cycle to support the anabolic proliferation of KSHV-transformed cells. Our results illustrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.IMPORTANCE We have previously found that Kaposi's sarcoma-associated herpesvirus (KSHV) can efficiently infect and transform primary mesenchymal stem cells; however, the metabolic pathways supporting the anabolic proliferation of KSHV-transformed cells remain unknown. Glutamine and asparagine are essential for supporting the growth, proliferation, and survival of some cancer cells. In this study, we have found that KSHV accelerates glutamine metabolism by upregulating numerous critical metabolic enzymes. Unlike most cancer cells that primarily utilize glutamine and asparagine to replenish the TCA cycle, KSHV-transformed cells depend on glutamine and asparagine for providing γ-nitrogen for purine and pyrimidine biosynthesis. We identified four rate-limiting enzymes in this pathway that are essential for the proliferation of KSHV-transformed cells. Our results demonstrate a novel mechanism by which an oncogenic virus hijacks a metabolic pathway for cell proliferation and imply potential therapeutic applications in specific types of cancer that depend on this pathway.

Selective peptide inhibitors of antiapoptotic cellular and viral Bcl-2 proteins lead to cytochrome c release during latent Kaposi's sarcoma-associated herpesvirus infection.

Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with B-cell lymphomas including primary effusion lymphoma and multicentric Castleman's disease. KSHV establishes latency within B cells by modulating or mimicking the antiapoptotic Bcl-2 family of proteins to promote cell survival. Our previous BH3 profiling analysis, a functional assay that assesses the contribution of Bcl-2 proteins towards cellular survival, identified two Bcl-2 proteins, cellular Mcl-1 and viral KsBcl-2, as potential regulators of mitochondria polarization within a latently infected B-cell line, Bcbl-1. In this study, we used two novel peptide inhibitors identified in a peptide library screen that selectively bind KsBcl-2 (KL6-7_Y4eK) or KsBcl-2 and Mcl-1 (MS1) in order to decipher the relative contribution of Mcl-1 and KsBcl-2 in maintaining mitochondrial membrane potential. We found treatment with KL6-7_Y4eK and MS1 stimulated a similar amount of cytochrome c release from mitochondria isolated from Bcbl-1 cells, indicating that inhibition of KsBcl-2 alone is sufficient for mitochondrial outer membrane permiabilzation (MOMP) and thus apoptosis during a latent B cell infection. In turn, this study also identified and provides a proof-of-concept for the further development of novel KsBcl-2 inhibitors for the treatment of KSHV-associated B-cell lymphomas via the targeting of latently infected B cells.

Latent KSHV Infected Endothelial Cells Are Glutamine Addicted and Require Glutaminolysis for Survival.

Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiologic agent of Kaposi's Sarcoma (KS). KSHV establishes a predominantly latent infection in the main KS tumor cell type, the spindle cell, which is of endothelial cell origin. KSHV requires the induction of multiple metabolic pathways, including glycolysis and fatty acid synthesis, for the survival of latently infected endothelial cells. Here we demonstrate that latent KSHV infection leads to increased levels of intracellular glutamine and enhanced glutamine uptake. Depletion of glutamine from the culture media leads to a significant increase in apoptotic cell death in latently infected endothelial cells, but not in their mock-infected counterparts. In cancer cells, glutamine is often required for glutaminolysis to provide intermediates for the tri-carboxylic acid (TCA) cycle and support for the production of biosynthetic and bioenergetic precursors. In the absence of glutamine, the TCA cycle intermediates alpha-ketoglutarate (αKG) and pyruvate prevent the death of latently infected cells. Targeted drug inhibition of glutaminolysis also induces increased cell death in latently infected cells. KSHV infection of endothelial cells induces protein expression of the glutamine transporter, SLC1A5. Chemical inhibition of SLC1A5, or knockdown by siRNA, leads to similar cell death rates as glutamine deprivation and, similarly, can be rescued by αKG. KSHV also induces expression of the heterodimeric transcription factors c-Myc-Max and related heterodimer MondoA-Mlx. Knockdown of MondoA inhibits expression of both Mlx and SLC1A5 and induces a significant increase in cell death of only cells latently infected with KSHV, again, fully rescued by the supplementation of αKG. Therefore, during latent infection of endothelial cells, KSHV activates and requires the Myc/MondoA-network to upregulate the glutamine transporter, SLC1A5, leading to increased glutamine uptake for glutaminolysis. These findings expand our understanding of the required metabolic pathways that are activated during latent KSHV infection of endothelial cells, and demonstrate a novel role for the extended Myc-regulatory network, specifically MondoA, during latent KSHV infection.

KSHV targeted therapy: an update on inhibitors of viral lytic replication.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, primary effusion lymphoma and multicentric Castleman's disease. Since the discovery of KSHV 20 years ago, there is still no standard treatment and the management of virus-associated malignancies remains toxic and incompletely efficacious. As the majority of tumor cells are latently infected with KSHV, currently marketed antivirals that target the virus lytic cycle have shown inconsistent results in clinic. Nevertheless, lytic replication plays a major role in disease progression and virus dissemination. Case reports and retrospective studies have pointed out the benefit of antiviral therapy in the treatment and prevention of KSHV-associated diseases. As a consequence, potent and selective antivirals are needed. This review focuses on the anti-KSHV activity, mode of action and current status of antiviral drugs targeting KSHV lytic cycle. Among these drugs, different subclasses of viral DNA polymerase inhibitors and compounds that do not target the viral DNA polymerase are being discussed. We also cover molecules that target cellular kinases, as well as the potential of new drug targets and animal models for antiviral testing.

Dysregulation of KSHV replication by extracts from Carthamus tinctorius L.

Carthamus tinctorius L. (CT) is traditionally used to reduce ailments from diseases of the musculoskeletal system and connective tissue and diseases of blood circulation and the cardiovascular system. Flower extracts from CT are known to have antibacterial activity, anti-inflammatory activity, and to inhibit tumor promotion in mouse skin carcinogenesis. In order to discover new antiviral agents from CT extracts, we tested whether CT extracts contain antiviral activity against gammaherpesvirus infection. This study demonstrated that treatment with CT extracts disrupted KSHV latency in the viral-infected host cells, iSLK-BAC16. n-Hexane and EtOH fractions of CT extracts critically affected at least two stages of the KHSV life-cycle by abnormally inducing KSHV lytic reactivation and by severely preventing KSHV virion release from the viral host cells. In addition to the effects on KSHV itself, CT extract treatments induced cellular modifications by dysregulating cell-cycle and producing strong cytotoxicity. This study demonstrated for the first time that CT extracts have antiviral activities that could be applied to development of new anti-gammaherpesviral agents.

Synthesis and antiviral activities of methylenecyclopropane analogs with 6-alkoxy and 6-alkylthio substitutions that exhibit broad-spectrum antiviral activity against human herpesviruses.

Methylenecyclopropane nucleosides have been reported to be active against many of the human herpesviruses. The most active compound of this class is cyclopropavir (CPV), which exhibits good antiviral activity against human cytomegalovirus (HCMV), Epstein-Barr virus, both variants of human herpesvirus 6, and human herpesvirus 8. CPV has two hydroxymethyl groups on the methylenecyclopropane ring, but analogs with a single hydroxymethyl group, such as the prototypical (S)-synguanol, are also active and exhibit a broader spectrum of antiviral activity that also includes hepatitis B virus and human immunodeficiency virus. Here, a large set of monohydroxymethyl compounds with ether and thioether substituents at the 6 position of the purine was synthesized and evaluated for antiviral activity against a range of human herpesviruses. Some of these analogs had a broader spectrum of antiviral activity than CPV, in that they also inhibited the replication of herpes simplex viruses 1 and 2 and varicella-zoster virus. Interestingly, the antiviral activity of these compounds appeared to be dependent on the activity of the HCMV UL97 kinase but was relatively unaffected by the absence of thymidine kinase activity in HSV. These data taken together indicate that the mechanism of action of these analogs is distinct from that of CPV. They also suggest that they might be useful as broad-spectrum antiherpesvirus agents and may be effective in the treatment of resistant virus infections.

Resveratrol inhibits KSHV reactivation by lowering the levels of cellular EGR-1.

In the field of herpesvirus research, the exact molecular mechanism by which such viruses reactivate from latency remains elusive. Kaposi's sarcoma-associated herpesvirus (KSHV) primarily exists in a latent state, while only 1-3% of cells support lytic infection at any specific time. KSHV reactivation from latency is an exceedingly intricate process mediated by the integration of viral and cellular factors. Previously, our lab has described early growth response-1 (Egr-1) as an essential component for the KSHV reactivation process via its ability to mediate transcription of KSHV ORF50, the gene encoding for replication and transcription activator (RTA), a viral component known to control the switch from latent to lytic infection. In here, electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments revealed that Egr-1 binds KSHV ORF50 promoter (ORF50P) in at least two different GC-rich binding domains. Expression profiles of cellular egr-1 and KSHV-encoded ORF50 follow a similar pattern during de novo KSHV infection. Over-expressing Egr-1, a signaling component downstream of Raf>MEK>ERK1/2, in KSHV-infected cells activates KSHV lytic replication. Through performing more physiologically relevant experiments, we analyzed the effect of a dietary supplement containing resveratrol on KSHV-infected cells. Our results, for the first time, demonstrate resveratrol to act in lowering ERK1/2 activity and expression of Egr-1 in KSHV-infected cells, resulting in the suppression of virus reactivation from latency. Taken together, these findings will undoubtedly contribute to future studies on not only combating KSHV related disease conditions, but also on other herpesviruses-induced pathogenesis.

Role of the cellular transcription factor YY1 in the latent-lytic switch of Kaposi's sarcoma-associated herpesvirus.

Lytic cycle reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) is initiated by expression of the ORF50 gene. Here we show that YY1 protein specifically binds to the ORF50 promoter (ORF50p) region in vitro and in vivo. After treatment with chemical inducers, including sodium butyrate (SB) and TPA, the levels of YY1 protein are inversely correlated with the lytic induction of KSHV in cells. Overexpression of YY1 completely blocks the ORF50p activation in transient reporter assays, while mutation at the YY1 site in the ORF50p or knockdown of YY1 protein confers an enhancement of the ORF50p activation induced by SB and TPA. YY1 overexpression in a stable cell clone HH-B2(Dox-YY1) also inhibits expression of the ORF50 and its downstream lytic genes. On the other hand, a chimeric YY1 construct that links to its coactivator E1A can disrupt viral latency. These results imply that YY1 is involved in the regulation of KSHV reactivation.

An immunotoxin targeting the gH glycoprotein of KSHV for selective killing of cells in the lytic phase of infection.

Amongst the pathologies associated with infection by Kaposi's sarcoma-associated herpesvirus (KSHV), multicentric Castleman's disease is distinctive for involvement of the lytic phase of the virus replication cycle. This B cell lymphoproliferative disorder has shown clinical responsiveness not only to generalized immunotherapy and cytotoxic chemotherapy, but also to inhibitors of herpesvirus DNA replication, consistent with the involvement of lytic phase of replication. These findings suggest that selective killing of virus-producing cells might represent a novel therapeutic strategy. We designed an immunotoxin, YC15-PE38, containing a single chain variable region fragment of a monoclonal antibody against KSHV glycoprotein H (gH) linked to the effector domains of Pseudomonas aeruginosa exotoxin A. Purified YC15-PE38 displayed highly selective and potent killing of a gH-expressing transfectant cell line (subnanomolar IC(50)). The immunotoxin also strongly inhibited production of infectious KSHV virions from an induced chronically infected cell line, by virtue of selective killing of the virus-producing cells. Combination treatment studies indicated complementary activities between YC15-PE38 and the herpesviral DNA replication inhibitor ganciclovir. These results provide support for the development of anti-KSHV strategies based on targeted killing of infected cells expressing lytic phase genes.

Evaluation of bloodsucking arthropod bite as possible risk co-factor in Human herpesvirus-8 transmission route.

Human herpesvirus-8 non-sexual transmission occurs primarily from mother-to-child. The viral load in saliva is higher than in other human fluids. Moreover, there is evidence that bloodsucking arthropod bites induce an inflammatory/immune response that facilitates viral replication. We aim to explore possible risk factors in mother-to-child HHV-8 transmission associated with traditional methods which involve the use of saliva to relieve the irritation and skin reaction caused by arthropod bites. We administered questionnaires to 2244 children from several African countries and Italy. Descriptive statistics and logistic regression were used in the analysis of the answers to evaluate the relationships between the use of traditional methods and other risk factors. The use of traditional methods is high in Cameroon (63.0%) and Uganda (39.9%), intermediate in Senegal (26.7%) and Italy (21.7%), low in Madagascar (6.7%). Statistical analyses show significant direct relationships between the use of traditional methods, skin reactions to the bite and their duration in Cameroon, Uganda and Senegal. The use of saliva and herbs applied by the mothers on the child's skin, is a common habit in Africa. If this practice plays a role in the HHV-8 transmission, then, it could provide the basis for interventions capable of reducing the health impact of the infection in children in tropical areas.

KSHV manipulates Notch signaling by DLL4 and JAG1 to alter cell cycle genes in lymphatic endothelia.

Increased expression of Notch signaling pathway components is observed in Kaposi sarcoma (KS) but the mechanism underlying the manipulation of the canonical Notch pathway by the causative agent of KS, Kaposi sarcoma herpesvirus (KSHV), has not been fully elucidated. Here, we describe the mechanism through which KSHV directly modulates the expression of the Notch ligands JAG1 and DLL4 in lymphatic endothelial cells. Expression of KSHV-encoded vFLIP induces JAG1 through an NFkappaB-dependent mechanism, while vGPCR upregulates DLL4 through a mechanism dependent on ERK. Both vFLIP and vGPCR instigate functional Notch signalling through NOTCH4. Gene expression profiling showed that JAG1- or DLL4-stimulated signaling results in the suppression of genes associated with the cell cycle in adjacent lymphatic endothelial cells, indicating a role for Notch signaling in inducing cellular quiescence in these cells. Upregulation of JAG1 and DLL4 by KSHV could therefore alter the expression of cell cycle components in neighbouring uninfected cells during latent and lytic phases of viral infection, influencing cellular quiescence and plasticity. In addition, differences in signaling potency between these ligands suggest a possible complementary role for JAG1 and DLL4 in the context of KS.

Lytic induction of Kaposi's sarcoma-associated herpesvirus in primary effusion lymphoma cells with natural products identified by a cell-based fluorescence moderate-throughput screening.

Kaposi's sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi's sarcoma primary effusion lymphoma (PEL), and multicentric Castleman's disease. Intentional lytic induction of gammaherpesviruses in the presence of antiviral drugs is thought to be an effective treatment option for gammaherpesvirus-related tumors. In this study, we used a cell-based fluorescence bioassay system in which a KSHV-infected PEL cell line was stably transfected with a potent viral-promoter-driven reporter gene to identify effective non-toxic reagents capable of inducing latent KSHV. Among 400 plant extracts screened, three extracts increased reporter gene expression in a dose-dependent manner. Furthermore, the three extracts activated the RTA promoter and induced expression of lytic genes in the endogenous viral genomes of KSHV-infected tumor cells. Together, our results demonstrate the effectiveness of a moderate-throughput screening system to identify natural products capable of inducing KSHV reactivation, thereby facilitating the development of novel therapeutic agents for KSHV-associated malignancies.

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions.

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

Licking latency with licorice.

Numerous viruses cause latent infections in humans, and reactivation often results in pain and suffering. While vaccines for several of these viruses are available or currently being studied in clinical trials, and antiviral therapies have been successful in preventing or treating active infection, therapy to eradicate latent infection has lagged behind. A new study reported in this issue of the JCI shows that treatment of cells latently infected with Kaposi sarcoma-associated herpesvirus (KSHV) with glycyrrhizic acid, a component of licorice, reduces synthesis of a viral latency protein and induces apoptosis of infected cells. This finding suggests a novel way to interrupt latency.

Polyadenylylated nuclear RNA encoded by Kaposi sarcoma-associated herpesvirus.

A newly recognized gamma herpesvirus known as Kaposi sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) is present in Kaposi sarcomas and body-cavity-based lymphomas. Here we identify a novel abundant 1.2-kb RNA, polyadenylated nuclear RNA (PAN RNA), encoded by the virus. The majority of cDNAs produced from poly(A)-selected RNA isolated from a human body cavity lymphoma cell line 48 hr after butyrate induction of KSHV lytic replication represented PAN RNA. Within PAN RNA were two 9 and 16 nt stretches with 89% and 94% identity to U1 RNA. A third stretch of 14 nt was 93% complementary to U1. The 5' upstream region of PAN RNA contained both proximal and distal sequence elements characteristic of regulatory regions of U snRNAs, whereas the 3' end was polyadenylylated. PAN RNA was transcribed by RNA polymerase II, lacked a trimethylguanosine cap, and did not associate with polyribosomes. PAN RNA formed a speckled pattern in the nucleus typical of U snRNAs and colocalized with Sm protein. Therefore, PAN represents a new type of RNA, possessing features of both U snRNA and mRNA.